Adherent cells from adipose or placenta tissues and use thereof in therapy

[0001] The invention relates to the treatment of ischemia using adherent cells from placental tissues propagated by means of three-dimensional 3D cell culture.

[0002] In the growing medical world, there is an increasing demand for large quantities of adult stem cells for cell inoculation and tissue engineering. In addition, adult stem cell therapy is still being developed to treat various conditions such as hematopoietic disorders, heart disease, Parkinson's disease, Alzheimer's disease, stroke, burns, muscular dystrophy, autoimmune diseases, diabetes and arthritis.

[0003] In recent years, considerable activity has focused on the therapeutic potential of mesenchymal stromal cells (MSC) for various medical applications involving tissue repair of damaged organs such as brain, heart, bone and liver and in supporting bone marrow transplants transplantations - BMT). MSC, heterogeneous population of cells obtained from e.g. bone marrow, adipose tissue, placenta and blood, is able to differentiate into different types of mature mesenchymal cells (e.g., endothelial retinal cells, fibroblasts, adipocytes, osteogenic precursor cells) depending on the effects of various bioactive factors. Thus, MSCs have been widely studied in regenerative medicine as a basis for building new tissues such as bone, pancreas and fat for repairing trauma or replacing pathological tissues and as a therapy for genetic and acquired diseases. [Fibbe and Noort, Ann NY Acad Sci (2003) 996: 235-44; Hor1243-PAT-EP-E

EP2200622 witz et al., Cytotherapy (2005) 7 (5): 393-5; Zimmet and Hare,

Basic Res Cardiol (2005) 100 (6): 471-81]. Furthermore, the multipotent abilities of MSCs, their easy isolation and breeding as well as their high ex vivo expansion potential make them an attractive therapeutic tool [Fibbe and Noort, above; Minguell et al. Exp Biol Med (Maywood) (2001) 226 (6): 507-20].

[0004] Placental MSCs display many markers common to MSCs isolated from other tissues, e.g., CD105, CD73, CD90 and CD29, and lack expression of markers specific for hematopoietic, endothelial and trophoblastic cells. Adipogenic, osteogenic and neurogenic differentiation was obtained after cultured from MSC placenta under appropriate conditions [Yen et al., Stem Cells (2005) 23 (1): 3-9]. Furthermore, MSCs isolated from placenta and cultured in vitro have been shown to be immunologically privileged as are MSCs. Thus, the placenta provides an ethically uncontroversial and easily accessible MSC source for experimental and clinical applications [Zhang et al., Exp Hematol (2004) 32 (7): 657-64].

[0005] The present inventors have previously developed three-dimensional (3D) culture conditions suitable for the expansion of placental MSCs (WO 2007/108003).

[0006] The leading clinical uses of MSC are summarized below. Ischemia

Peripheral arterial disease (PAD) [0007] Peripheral artery disease (PAD) is a chronic disease that progressively restricts blood flow to the limbs, which can lead to serious medical complications. This disease is often associated with other clinical conditions, including hypertension, cardiovascular disease, hyperlipidemia, diabetes,

PAT-1243-EP-E

EP2200622 obesity and stroke. Critical Limb Ischemia (Critical Limb

Ischemia - CLI) is used to describe patients with pain induced by chronic ischemia, ulcers, tissue loss or gangrene of the limb. CLI represents the final stage of PAD patients who need extensive therapy by vascular surgery or a vascular specialist. Unlike coronary artery and cerebral artery disease, peripheral artery disease (PAD) remains an underestimated condition that, although severe and very prevalent, is rarely diagnosed and less frequently treated. As a result, CLI often leads to amputation or death, and mortality in PAD patients exceeds mortality in patients with heart attack and stroke.

[0008] Various adult stem cells have been used in the treatment of ischemic conditions. Thus, co-cultivation of adipose derived stromal cells (ADSC) and endothelial cells (EC) resulted in a significant increase in viability, migration and tube formation by EF mainly by the secretion of VEGF and HGF. Four weeks after stromal cell transplantation to mouse ischemic hind limb, oncogenic values ​​improved [Nakagami et al., J Atheroscler Thromb (2006) 13 (2): 77-81]. Moon et al. [Cell Physiol Biochem. (2006) 17: 279-90] studied the ability of adipose derived stem cells (ADSC) to treat limb ischemia in immunodeficient mice and showed a significant increase in perfusion index in the Doppler laser study in the ADSC group .

[0009] In addition, when umbilical cord blood (UCB) derived mesenchymal stem cells were transplanted 1243-PAT-EP-PL

For four men with Buerger's disease who had already undergone treatment and surgery before, ischemic resting pain suddenly disappeared from their affected limbs [Kim et al., Stem Cells (2006) 24 (6): 1620-6]. Furthermore, transplantation of human mesenchymal stem cells isolated from fetal membranes of term placenta (FMhMSC) to rat hearts after infarction was associated with increased capillary density, normalization of left ventricular function, and significant reduction of scar tissue, which was increased when stem cells were preconditioned with a mixed hyaluronan ester with butyric acid and retinoic acid [Ventura et al. (2007) J. Biol. Chem., 282: 14243-52].

Stroke [0010] Stroke is one of the leading causes of death in the world, causing about 9% of all deaths and consuming about 2-4% of total healthcare expenditure. Although stroke mortality continues to decline in developed countries, probably due to improved control of stroke risk factors (especially high blood pressure, diabetes and smoking), stroke still leads to permanent damage (e.g. tissue damage, neurological damage).

[0011] New treatment regimens for stroke include stem cell therapy. Transplantation of stem or progenitor cells to a damaged site, either locally or intravenously to replace non-functional cells, increase the proliferation and / or differentiation of endogenous stem or progenitor cells and provide the needed immune modulators has been considered and is the main cell-based strategy. Potential sources of stem / progenitor cells for

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Strokes include fetal neuronal stem cells, embryonic stem cells, neuroteratocarcinoma cells, non-haematopoietic umbilical cord stem cells, bone marrow stem cells and placental mesenchymal stem cells [Andres et al., Neurosurg Focus (2008) 24 (3) -4): E16].

[0012] In a recent study, Koh et al. [Koh et al., Brain Res.

(2008)] studied the neuroprotective effects and mechanisms of implanted umbilical-derived mesenchymal stem cells (hUC-MSC) in a rat ischemic stroke model. Twenty days after induction of neuronal differentiation in vitro, hUC-MSC exhibited morphological characteristics of neurons and expressed neuronal markers and neuronal factors (e.g., neurotrophic factor derived from glial cell line, brain derived neurotrophic factor). Furthermore, in-vivo implantation of hUC-MSC into the injured hemisphere of immunosuppressed rats after ischemic stroke improved neurobehavioural function and reduced stroke volume compared to control rats. Three weeks after implantation, hUC-MSC was present in the damaged hemisphere and expressed neuron-specific markers, however, these cells did not become functionally active neuronal cells.

Orthopedic applications [0013] Various conditions and pathologies require regeneration and / or repair of connective tissue (e.g. bone, tendon and ligament). These include, for example, bone fractures, burns, burn wounds, deep wounds, degenerated bone, various cancers associated with connective tissue loss (e.g. bone cancer, osteosarcoma, bone metastases), and cartilage defects.

[0014] Use of autologous BM-MSC to promote healing

PAT-1243-EP-E

EP2200622 bone has been described for veterinary orthopedic applications in humans and includes percutaneous bone marrow injection for ligament healing (Carstanjen et al., 2006), treatment of bone defects by autografts or bone marrow allografts at an orthopedic clinic (Horwitz et al., 1999, Horwitz et al., 2002), regeneration of critical bone defects in dogs using allogeneic [Arinzeh TL, et al., J Bone Joint Surg Am.

2003, 85-A (10): 1927-35] or autologous [Bruder SP, et al., J Bone Joint Surg Am. 1998 Jul; 80 (7): 985-96] Bone marrow MSC charged on a ceramic cylinder composed of tricalcium hydroxyapatityphosphate, or in a rabbit using allogeneic peripheral blood MSCs (Chao et al., 2006), and extensive bone formation using implantation Baboon MSC (Livingston et al., 2003).

[0015] In the field of horse orthopedics, mesenchymal stem cells from BM and fat sources have been experimentally used for the surgical treatment of subconjunctival bone cysts, bone fracture repair [Kraus and Kirker-Head, Vet Surg (2006) 35 (3): 232-42] and cartilage repair [Brehm et al., Osteoarthritis Cartilage (2006) 14 (12): 1214-26; Wilke et al., J Orthop Res (2007) 25 (7): 913-25] and clinically in the treatment of overload-induced tendon damage in horses. Furthermore, various therapeutic approaches have been used to promote healing of the hanging ligament in horses (Herthel, 2001). Herthel (2001) demonstrated a new biological approach to facilitate healing of the posterior ligament, which includes injection to damage autologous stem cells and associated bone marrow components to stimulate the natural regeneration of ligaments.

[0016] Rabbit models for injured tendons showed that

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EP2200622 treated MSC tissues were stronger and more rigid than tissues repaired naturally (Gordon et al., 2005). In addition, inoculation of cultured MSCs into a tendon gap resulted in a significant improvement in repair biomechanics (Young et al., 1998,

Osiris Therapeutics,<a href="http://www.osiris.com/"> www.osiris.com)</a>.

[0017] Osiris Chondrogen (adult mesenchymal stem cells) is tested in patients to assess safety and efficacy. In MSC-treated animals, the surgically removed meniscus tissue was regenerated and the cartilage surface was protected, and reduced joint damage was observed compared to control animals. These benefits persist in animal models for at least one year (Osiris Therapeutics,<a href="http://www.osiris.com/"> www.osiris.com)</a>

SUMMARY OF THE INVENTION [0018] The invention is directed to adherent placental cells for the treatment of ischemia, said cells being propagated by means of three-dimensional (3D) cell cultures.

[0019] Described is the treatment of a medical condition requiring regeneration and / or repair of connective tissue in an individual in need thereof by administering to the subject a therapeutically effective amount of adherent cells from a tissue selected from the group consisting of placenta and adipose tissue, thereby treating a medical condition requiring regeneration of connective tissue and / or individual repair.

[0020] The use of adherent cells from placenta in the production of a medicament specified for the treatment of ischemia has been described.

[0021] The use of adherent cells from tissue selected from the group consisting of placenta and adipose tissue for the production of a medicament specified for the treatment of a medical condition requiring tissue regeneration and / or repair is described.

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EP2200622 [0022] A product comprising packaging material has been described that includes a label for use in the treatment of ischemia, the packaging material wraps a pharmaceutically effective amount of adherent cells from a tissue selected from the group consisting of placenta and adipose tissue.

[0023] A product comprising packaging material has been described that includes a label for use in treating a medical condition requiring tissue regeneration and / or repair, the packaging material wraps a pharmaceutically effective amount of adherent cells from a tissue selected from the group consisting of placenta and adipose tissue.

[0024] According to some embodiments of the invention, the adherent cells are capable of suppressing the immune response in a subject.

[0025] According to some embodiments of the invention, at least 10% of the adherent cells are in the proliferative phase.

[0026] According to some embodiments of the invention, ischemia is a peripheral artery disease (PAD).

[0027] According to some embodiments of the invention, the peripheral artery disease (PAD) is critical limb ischemia (CLI).

[0028] According to some embodiments of the invention, the ischemia comprises central nervous system (CNS) ischemia.

[0029] According to some embodiments of the invention, the ischemia is selected from the group consisting of peripheral arterial disease, ischemic vascular disease, ischemic heart disease, ischemic brain disease, ischemic kidney disease, and placental ischemia.

[0030] Adherent cells are obtained from three-dimensional (3D) culture.

[0031] According to some embodiments, three-dimensional (3D)

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EP2200622 culture includes a 3D bioreactor.

[0032] According to some embodiments of the invention, the cell culture in 3D culture is perfused.

[0033] According to some embodiments of the invention, the culture conditions for the three-dimensional culture include an adherent material selected from the group consisting of polyester and polypropylene.

[0034] According to some embodiments of the invention, the cell culture is carried out for at least 3 days.

[0035] According to some embodiments of the invention, the cell culture is carried out until at least 10% of the cells proliferate. [0036] According to some embodiments of the invention, the adherent cells comprise expressing a positive marker selected from the group consisting of CD73, CD90, CD29 and CD105.

[0037] According to some embodiments of the invention, the adherent cells comprise the expression of a negative marker selected from the group consisting of CD3, CD4, CD45, CD80, HLA-DR, CD11b, CD 14, CD 19, CD34 and CD79.

[0038] According to some embodiments of the invention, the adherent cells comprise an expression profile essentially as described herein.

[0039] According to some embodiments of the invention, the adherent cells include cells containing a stromal stem cell phenotype. [0040] According to some embodiments of the invention, the stromal stem cell phenotype comprises T cell suppression activity.

[0041] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, appropriate methods and materials.

EP2200622 are described below. In the event of a conflict, the patent description and definitions are the deciding factor. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

BRIEF DESCRIPTION OF THE FIGURES [0042] The invention is described herein, by way of example only, with reference to the accompanying figures. Referring specifically to the figures, it should be emphasized that the data are shown only as an example and for purposes of illustrative discussion of embodiments of the invention, and are provided to provide what is believed to be the most useful and easy to understand description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show the structural details of the invention in more detail than is necessary to understand the invention, the description together with the figures makes it clear to those skilled in the art how several embodiments of the invention can be made in practice.

[0043] In the figures:

FIG. 1A-G show a bone-like microenvironment created in a bioreactor system containing 3D carriers. Figures 1A-B are electron micrographs showing a comparison of natural bone (Figure 1A) and PluriX ™ 3D carrier structure 7 days after inoculation of adherent cells imitating bone microenvironment (Figure 1B). Figures 1C-F are electron micrographs showing a PluriX ™ 3D matrix inoculated with adherent cells, made from bone marrow, 20 days (Figures 1C-D, X magnification 150 and 250, respectively) and 40 days (Figures 1EF, X 350 magnification and 500, respectively) after vaccination. Figure 1G is a schematic diagram of a Plurix 3D piston flow bioreactor with separate parts determined by numbers: reservoir pod1243-PAT-EP-PL

EP2200622 culture bed (1), gas mixture supply (2), filter (3), injection site (4), column in which there is a separation container (7), cell growth analyzers (8); peristaltic pump (9), sampling site (10), dissolved O2 electrode (11), pH electrode (12), control system (13), fresh culture media (14), complex culture media (15).

FIG. 2 is a graph depicting various production batches of adherent cells (batches 5-8) derived from placenta, grown under 3D growth conditions within bioreactor systems. Adherent cells (2 X 10⁶) was seeded in a bioreactor at a density of 10,000 - 15,000 cells / carrier. After cultured for 12 days, adherent 3D cells reached a density between 150,000-250000 cells / carrier or 22.5-37.5 X 10⁶ in a bioreactor containing 150 media.

FIG. 3A-B are histograms showing differences in expression levels of expressed membrane markers in placental derived adherent 3D cells (dark purple) compared to membrane markers in placental cells cultured under conventional 2D (light purple) culture conditions. Adherent cells were cultured for 4-6 weeks in flasks (2D) or for 2-3 weeks in a bioreactor system, on polystyrene carriers (3D). After harvesting from flasks or carriers, the cells were incubated and bound to a panel of monoclonal antibodies (MAb) that recognize membrane markers characteristic of adherent cells (Figure 3A), or hematopoietic cells (Figure 3B). Note significantly higher expression of MSC membrane markers in cells grown in 2D as shown for membrane markers CD90, CD 105, CD73 and CD29, compared to membrane markers expressed in adherent1243-PAT-EP-PL

EP2200622 cells grown in 3D, especially CD105, which showed 56% expression in cells grown in 3D versus 87% in cells grown in 2D (Figure 3A). Adherent cells of both 2D and 3D cultures did not express any hematopoietic membrane markers (Figure 3B).

FIG. 4A-D are histograms showing a comparison of protein levels in adherent cells produced from placenta grown in 2D and 3D conditions or in conditioned media from the same. Figures 4A-C show levels of Flt-3 ligand (Figure 4A), IL-6 (Figure 4B) and SCF (Figure 4C) in pg / ml, normalized for 1 X 10⁶ cells / ml, analyzed by ELISA, in conditioned media of adherent cells grown in 2D and 3D. The results show one of three independent experiments. Figure 4D shows the expression levels of various cellular proteins, analyzed by mass spectrometry with comparison of protein samples labeled with iTRAQ reagents. Protein samples were taken from adherent cells grown under 2D (white bars) and 3D (gray bars) conditions. The figure shows one of two replicates of the experiment. Note the differences in the level of expression of some proteins in cells and conditioned media in 2D and 3D culture conditions.

FIG. 5A-D are micrographs showing the ability to differentiate in vitro of placental adherent 3D cells into osteoblasts. Adherent human placental derived cells were cultured in osteogenic induction medium (DMEM containing 10% FCS, 100 nM dexamethasone, 0.05 mM ascorbic acid 2-phosphate, 10 mM B-glycerophosphate) for a period of 3 weeks. Figures 5A-B show cells expressing the calcified matrix, as indicated by Alizzarin Red S. staining. Figures 5C-D show cells

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Control EP2200622, which were not treated with osteogenic induction medium and retained a fibroblast-like phenotype and showed no mineralization.

FIG. 6 is a graph showing the percentage of human CD45 + cells detected in the bone marrow (BM) of NOD-SCID mice treated with chemotherapy (25 mg / kg intraperitoneal injections of busulfan for two consecutive weeks) 3.5 weeks after transplantation. CD34 + cells (100,000) purified from umbilical cord blood-derived mononuclear cells were transplanted alone (5 mice, a) or transplanted together with 0.5 X 10⁶ adherent placental cells cultured under 2D conditions (2D-adherent cells; 2 mice, b), or adherent cells derived from placenta cultured under 3D conditions (3D-adherent cells), in the pluriX ™ bioreactor (5 mice, c). BM was then collected from the femur and tibia of mice. Human cells in BM were detected by flow cytometry. The percentage of human CD45 expressing cells was determined by incubating the cells with anti-human CD45FITC. It should be noted a higher percentage of human cells (hCD45 +) in the bone marrow of mice co-transplanted with adherent 2D (b) as well as adherent 3D (c) cells compared to HSC (a) alone. Better implantation seen in mice treated with adherent cells grown in 3D compared to mice treated with adherent cells grown in 2D indicates a higher therapeutic advantage unique to adherent cells grown in 3D.

FIG. 7A-B are FACS analyzes of human CD45 + cell transplants in mice transplanted with CD34 + cells alone (Figure 7A) compared to CD34 + cells together with adherent adipose tissue cells (Figure 7B). It should be noted familiar with 1243-PAT-EP-PL

EP2200622 a significantly higher percentage of the human hematopoietic population (hCD45 +) (7A - 29%) in a mouse co-transplanted with adherent adipose tissue cells compared to a mouse treated with human CD34 + alone (7B-12%).

FIG. 8A is a histogram showing the reaction of mixed lymphocytes carried out between human mononuclear cord blood cells (CB), and equal amounts of irradiated (3000 Rad) umbilical cord blood cells (iCB), monocytes derived from human peripheral blood (PBMC), grown in 2D (2D) or cultured in 3D (3D) derived from placental adherent cells, or a combination of PBMCs and cultured in 2D and 3D derived from placental adherent cells (PBMC + 2D and PBMC + 3D). Pop size₃ CB cell lation is represented by retrieval ³H-thymidine (measured in CPM) that was measured in the last 18 hours of culture. An increase in stimulated CB cell proliferation indicates a higher level immune response. It should be noted the lower level of immune response exhibited by cells incubated with adherent cells, and in particular, the reduction of CB immune response to PBMCs when they are incubated together with adherent cells. Three replicates of each reaction were done.

FIG. 8B is a block diagram showing the production of adherent 3D cells from placenta using Celligen ™ (referred to as PLX-C cells).

FIG. 8C is a drawing of the Celligen ™ bioreactor vessel and ports adapted from The New Brunswick Scientific website.

FIG. 9A-B show cell cycle analysis for the production of adherent 3D cells by Plurix (referred to as PLX, Figure

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EP2200622

9B) and by Celligen (referred to as PLX-C, Figure 9A). Cells were fixed in 70% EtOH overnight, centrifuged and resuspended in propidium iodide (PI) solution and then analyzed by FACS.

FIG. 10A-C show expression of typical fibroblast markers but not expression of endothelial typical markers on PLX-C. Figure 10A shows negative expression of the CD31 endothelial marker; Figure 10B shows the negative expression of the KDR endothelial marker; and Figure 10C shows positive expression of human fibroblast marker (D7-FIB). Note that the red histograms for the IgG1 Isotype (FITC) represent a negative control while the blue histograms represent positively stained cells.

FIG. 11A-D depict the expression of stimulating and co-stimulatory molecules on PLX-C cells. Figure 11A shows expression of CD80 in PLX-C; Figure 11B shows the expression of CD86 in PLX-C; Figure 11C shows the expression of CD40 in PLX-C; and Figure 11D shows HLA-A / B / C expression in PLX-C. Negative controls were prepared with the appropriate isotypes of fluorescent molecules. It should be noted that red histograms indicate a population of cells showing markers iPLX-C, blue histograms a population of cells showing markers of bone marrow (BM) and green histograms indicate a population of cells showing markers of mononuclear cells (MNC).

FIG. 12A-B depict inhibition of lymphocyte proliferation by₅

PLX-C. Figure 12A shows MLR tests carried out with 2 x 10⁵derived from peripheral blood (PB) MNC (donor A) stimulated with an equal amount of irradiated (3000 Rad) derived from PB MNC (donor B) followed by increasing amounts

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EP2200622 PLX-C cells for culture. Three replicates of each group were selected on 96-well plates. The proliferation rate was measured after₃ by incorporation [³H] thymidine incorporation; Figure 12B shows ConA stimulated MNC derived peripheral blood (PB) (1.5 mg / ml). Increasing amounts of PLX-C cells were added to homology. Three replicates of each group were inoculated into 96-well plates. Speed₃ proliferation was measured by incorporation [³H] thymidine incorporation;

FIG. 13A-C depict PLX-C regulation of pro-inflammatory and anti-inflammatory cytokine secretion after co-culture with peripheral blood cells. Figures 13A-B show the secretion of IFNy (Figure 13A) and TNFa (Figure 13B) after co-culture with human-derived MNC (isolated from peripheral blood) stimulated with ConA with PLX-C; Figure 13C shows the secretion of IFNγ, TNFα and IL10 after co-culture with human-derived MNC (isolated from peripheral blood) stimulated LPS with PLX-C. Supernatants were collected and subjected to cytokine analysis using ELISA.

FIG. 14 shows a luciferase expression vector used to infect PLX-C cells. The expression vector Lv33 from OmicsLink was used. The luciferase gene was cloned into ORF.

FIG. 15 shows high luciferase expression by infected PLX-C cells. Cells were infected with the luciferase expression vector and visualized using the IVIS system 48 hours after infection. It should be noted that the cells showed high levels of luciferase expression.

FIG. 16A-D show a 2 x 10 injection⁶ PLX-C cells expressing luciferase to SCID / Beige mice. One mouse was administered IM and one IV. After injection, the mice were monitored using the IVIS system to assess in vivo biodistribution of PLX-C. The IVIS results are shown for day 1 (Figure 16A), day 4 (Figure 16B), day 6 (Figu1243-PAT-EP-PL

EP2200622 ra 16C) and on day 22 (Figure 16D).

FIG. 17 is a graph showing increased perfusion in the hip and foot of mice treated with the adherent cells of the invention (referred to as PLX-C). The figure shows the median percent perfusion in the hip and foot of mice. Blood flow in the hip and foot was measured using a non-contact Doppler laser on both sides on days 0, 6, 9, 14 and 21 after surgery (measurements shown on day 21). The results are expressed as the ratio of the ischemic limb blood flow to the normal limb during the experiment. FIG. 18 is a graph depicting the in vivo assessment of limb function and ischemia. The evaluation was carried out sequentially using the following evaluation system: 3 = foot trapping, 2 = no trimming but no plantar flexion, 1 = plantar flexion, and 0 = flexion of the fingers to resist mild tail traction.

Figures 19A-C show the increased density of capillaries after PLX-C treatment. Figure 19A shows capillary density in PBS treated mice; Figure 19B shows the density of capillaries in mice treated with PLX-C cells; Figure 19C is a histogram showing the number of capillaries per muscle cell. It should be noted that increased capillary density was observed in PLX-C treated mice but not in control mice, after induced limb ischemia demonstrated by specific capillary staining of the capillary.

(Figures 20A-B show reduced oxidative stress and endotheliitis after PLX-C administration. Fig.) 20A is a bar graph showing oxidative stress (nitrotyrosine staining); and Figure 20B is a histogram depicting inflammation

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EP2200622 endothelium (VCAM assessment). Note the noted reduced oxidative stress and endothelium in treated mice

PLX-C.

DESCRIPTION OF EMBODIMENTS OF THE INVENTION [0044] The invention relates to an increase in tissue angiogenesis and the treatment of ischemia using adherent placental cells propagated by means of three-dimensional 3D cell cultures.

[0045] The principles and operation of the invention may be better understood with reference to the figures and accompanying descriptions.

[0046] Before explaining at least one embodiment in detail, it is to be understood that the invention is not limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it should be understood that the phraseology and terminology used herein is for the purpose of description and should not be seen as limiting.

[0047] In bringing the invention into practice, the present inventors have found that adherent placental cells propagated by means of three-dimensional 3D cell cultures are very effective in increasing angiogenesis and in treating ischemia.

[0048] As illustrated here below and in Examples 1-8 of the next Examples, the present inventors have been able to expand adipose-derived fat and placental adherent cells that have the properties of stromal stem cells. Appropriately expanded cells proved viable after cryopreservation, as demonstrated by adherence and repopulation assays (see Example 1). Analysis by placental flow cytometry of adherent cells showed

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EP2200622 expression pattern of specific markers (see Figures 3A-B). As further shown in Example 6 in the next section Examples, implantation of placenta-derived adherent cells significantly induced hip and foot blood flow (Figure 17) of arterial ligation mice (hind limb ischemia model), significantly improving hind limb function (Figure 18) , increased capillary density (Figures 19A-C) and reduced oxidative stress and endothelium (Figures 20AB).

[0049] Thus, one aspect of the invention relates to an increase in tissue angiogenesis carried out by contacting the tissue with adherent placental cells propagated by three-dimensional 3D cell cultures thereby increasing tissue angiogenesis.

[0050] As used herein, the term "increase in angiogenesis in tissue" refers to an increase (induction, increase) in the process of generating new blood capillaries in tissue.

[0051] As used herein, the term "adherent cells" refers to a homogeneous or heterogeneous population of cells that are dependent on anchorage, i.e., require attachment to the surface to grow in vitro.

[0052] As used herein, the term "adipose tissue" refers to connective tissue that contains fat cells (adipocytes).

[0053] As used herein, the term "placental tissue" refers to any part of the female organ in mammals that surrounds a fetus during pregnancy to which it is connected by an umbilical cord. After delivery, the placenta is excreted (and is referred to as

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EP2200622 postpartum placenta). In an exemplary embodiment, the bearing refers to the entire bearing.

[0054] Adherent cells derived from placenta or adipose tissue can be propagated using two-dimensional or three-dimensional culture conditions.

[0055] Conditions for propagating adherent cells in 2D culture are described below and in the following examples.

[0056] As used herein, the term "three-dimensional culture" refers to a culture in which the cells are exposed to conditions that are suitable for cell growth while allowing the cells to grow in more than one layer. It is appreciated that the in situ environment of the cell in a living organism (or tissue) is in three-dimensional architecture. Cells are surrounded by other cells. They are maintained in a complex network of nanoscale fibers of the extracellular matrix, which allows the determination of various microenvironments. Their extracellular ligands not only mediate attachment to the basement membrane but also access to a number of blood and lymphatic vessels. Oxygen, hormones and nutrients are supplied to the cells and unnecessary products are discharged. The conditions in the three-dimensional culture of the invention are designed to mimic such an environment as further exemplified below.

[0057] It will be appreciated that the three-dimensional culture conditions are such as to allow expansion of adherent cells.

[0058] As used herein, the terms "expanding" and "expansion" refer to maintaining essentially no cell differentiation and ultimately cell growth, i.e., increasing the cell population (e.g., at least 2 times) without differentiation accompanying such an increase.

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[0059] As used herein, the terms "maintainer" and "maintenance" refer to cell renewal substantially without cell differentiation, i.e., substantially stationary cell population without differentiation associated with such stationarity.

[0060] As mentioned, adherent cells of this aspect of the invention are obtained from placenta.

[0061] Placental cells can be obtained from a full-term or pre-delivery placenta. The placenta is collected after bleeding. The placenta is perfused for a period of time sufficient to remove residual cells. The term "perfuse" or "perfusion" as used herein refers to the act of pouring or passing fluid onto or through an organ or tissue. Placental tissue can be from any mammal; for example, placental tissue is human. A convenient source of placental tissue is the placenta after delivery (e.g. 1-6 hours), however, the source of placental tissue or cells or the method of isolating placental tissue is not critical to the invention.

[0062] Placental adherent cells can be obtained from both the fetal (i.e. allantoic or internal parts of the placenta, see Example 1) and maternal (ie temporal sub-primary and temporal wall) parts of the placenta. Tissue samples are washed in physiological buffer [e.g. phosphate buffered saline - phosphate-buffered saline (PBS) or Hank's buffer]. Single cell suspensions are prepared by treating the tissue with digestive enzyme (see below) and / or disintegrating or passing a portion of the tissue through a nylon filter or gentle pipetting (Falcon, Becton, Dickinson, San Jose, CA) with wash buffer.

[0063] Adipose cells derived from adipose tissue can be isolated by a number of methods known to those skilled in the art.

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EP2200622 field. For example, such methods are described in Pat. US No.

6,153,432. Adipose tissue can come from the net / visceral, nipple, gonadal, or other places of fat. One source of adipose tissue is fat net. In humans, adipose tissue is typically isolated by liposuction.

[0064] Isolated adherent cells from adipose tissue can be obtained by treating the tissue with a digestive enzyme such as collagenase, trypsin and / or dispase; and / or effective hyaluronidase or DNAse concentrations; and ethylenediaminetetraacetic acid (EDTA); at temperatures between 25 - 50 ° C, for periods between 10 minutes to 3 hours. The cells can then be passed through a nylon mesh or gauze filter between 20 microns and 1 mm. The cells are then subjected to differential centrifugation directly in media or on a gradient from Ficoll or Percoll or other molecules. Cells are centrifuged at a speed between 100 to 3000 xg for periods of between 1 minute to 1 hour at temperatures between 4-50 ° C (see US Pat. No. 7,078,230).

[0065] In addition to adherent cells derived from placenta or adipose tissue, the description of the invention also contemplates the use of adherent cells from other sources that are characterized by the stromal stem cell phenotype (as will be described hereinafter). Sources of tissues from which adherent cells can be obtained include, without limitation, cord blood, scalp, hair follicles [e.g. as described in Report. Stalemate. US 20060172304], nuclei [e.g. as described in Guan K., et al., Nature. 2006 Apr

27; 440 (7088): 1199-203], human olfactory mucosa [e.g. As described in Marshall, CT., Et al., Histol Histopathol. 2006 Jun; 21

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EP2200622 (6): 633-43], embryonic yolk sac [e.g. as described in

Geijsen N, Nature. 2004 Jan 8; 427 (6970): 148-54] and amniotic fluid [Pieternella et al. (2004) Stem Cells 22: 1338-1345], all known to contain mesenchymal stem cells. Adherent cells from these tissue sources can be isolated by culturing the cells on an adherent surface, thereby isolating the adherent cells from other cells in the original population.

[0066] Regardless of origin (e.g., placenta or adipose tissue), cell acquisition is carried out under sterile conditions. After obtaining isolated cells, they are allowed to adhere to adhering material (e.g. configured as a surface) to thereby isolate adherent cells. Culturing can take place under 2D conditions as described in Example 4 of the Examples section and the cells can be further transferred to 3D conditions.

[0067] As used herein, "adherent material" refers to a synthetic, naturally occurring or combination thereof of a non-cytotoxic (ie, biologically compatible) material having a chemical structure (eg, charged surface-exposed groups) that can retain cells on the surface. [0068] Examples of adherent materials that can be used in accordance with this aspect of the invention include, without limitation, polyester, polypropylene, polyalkylene, polyfluorochloroethylene, polyvinyl chloride, polystyrene, polysulfone, cellulose acetate, fiberglass, ceramic molecule, matrix gel, extracellular matrix component (e.g. fibronectin, chondronectin, laminin), collagen, poly L lactic acid and passive metal fiber. [0069] Further purification or enrichment steps for cells

PAT-1243-EP-E

The stem stromal EP2200622 can be carried out using methods well known in the art (such as by FACS using expression of stromal cell markers as further described below).

[0070] Non-limiting examples of basic useful media in culture according to the invention include Minimum Essential

Medium Eagle, ADC-1, LPM (free of bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with and without Fitton-Jackson modification), Basal Medium Eagle (BME- with the addition of Earle's basic salts), Dulbecco's Modified Eagle

Medium (DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium (GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199 (M199E- with Earle's basic salts), Medium M199 (M199H- with Hank's salt base), Minimum Essential Medium Eagle (MEM-E- with Earle's basic salts), Minimum Essential

Medium Eagle (MEM-H- with basic Hanks salts) and Minimum Essential Medium Eagle (MEM-NAA with non-essential amino acids), among many others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261 , MAB 8713, DM 145, Williams' G, Neuman &amp; Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB

401, MCDB 411, MDBC 153. A preferred substrate for use in the invention is DMEM. These and other useful media are available from GIBCO, Grand Island, NY, USA and Biological Industries, Bet HaEmek, Israel, among others. A number of these media are summarized in Methods in Enzymology, vol. LVIII, "Cell Culture", p.

62 72, edited by William B. Jakoby and Ira H. Pastan, published by Academic Press, Inc.

[0071] The medium may be supplemented with a serum such as fetal bovine or other serum, and optionally or alternatively with growth factors, vitamins (e.g. ascorbiic acid1243-PAT-EP-PL

EP2200622 new), cytokines, salts (e.g. B-glycerophosphate), steroids (e.g. dexamethasone) and hormones e.g. growth hormone, erythropoietin, thrombopoietin, interleukin 3, interleukin 6, interleukin

7, macrophage colony stimulating factor, c-kit / stem cell factor, osteoprotegerin ligand, insulin, insulin-like growth factors, epithelial growth factor, fibroblast growth factor, nerve growth factor, cilia neurotropic factor, platelet derived factor and bone morphogenetic protein at concentrations between picogram / ml to milligram / ml levels.

[0072] It is further known that additional ingredients may be added to the culture medium. Such ingredients may be antibiotics, anti-fungal substances, albumin, amino acids and other ingredients known in the art of cell culture. In addition, ingredients can be added to increase the differentiation process (see below).

[0073] It will be appreciated that when the adherent cells for use according to the invention are administered to a human subject, the cells and culture medium (e.g. with the medium additives described) should be substantially free of xenon-type substances, i.e. free of any animal contamination e.g. mycoplasma. For example, the culture medium may be supplemented with serum substitute, human serum and / or synthetic factors or produced by recombinant techniques.

[0074] As mentioned, after obtaining adherent cells, they can be passaged in two-dimensional or three-dimensional systems (see Examples 1 and 4 of the next section Examples). However, it should be appreciated that the cells can be transferred to the 3D matrix immediately after isolation or alternatively can be passaged

PAT-1243-EP-E

EP2200622 to a three-dimensional system after two-dimensional conditions (as mentioned above).

[0075] Thus, the adherent material for use in accordance with this aspect of the invention is configured for 3D culture thus providing a growth matrix that substantially increases the available attachment surface for cell adhesion so as to mimic tissue infrastructure (e.g., placenta).

[0076] For large scale production, cultivation can be carried out in a 3D bioreactor.

[0077] Examples of such bioreactors include, without limitation, a piston flow bioreactor, a continuously stirred bioreactor, a stationary bed bioreactor, a CelliGen Plus® bioreactor system (New Brunswick Scientific (NBS) or a BIOFLO 310 bioreactor system (New Brunswick Scientific (NBS ).

[0078] As shown in Example 4, Examples, the Celligen bioreactor is capable of expanding 3D adherent cells under controlled conditions (e.g., pH, temperature and oxygen levels) and with continuous perfusion of cell culture medium. Furthermore, cell culture can be directly monitored for glucose, lactate, glutamine, glutamate and ammonium levels. The rate of glucose consumption and the rate of lactate formation of adherent cells allows measuring the rate of cell growth and determining the time of collection.

[0079] Other 3D bioreactors that can be used with the invention include, without limitation, a continuously stirred bioreactor, where the culture medium is constantly supplied to the bioreactor and the product is constantly selected to keep the solid state constant throughout the reactor. A mixed storage bioreactor with a fibrous bed is available at

PAT-1243-EP-E

EP2200622 example from New Brunswick Scientific Co., Edison, NJ), Fixed bed bioreactor, bioreactor with an air liquid lift, where air is typically introduced into the bottom of the central suction tube flowing upwards forming bubbles, and releasing exhaust gas at the top of the column] , inoculating perfusion bioreactor with Polyactive foams [as described in Wendt, D. et al., Biotechnol Bioeng 84: 205-214, (2003)] tubular poly-L-lactic acid (PLLA) porous skeletons in a radial-flow perfusion bioreactor [as described in Kitagawa et al., Biotechnology and

Bioengineering 93 (5): 947-954 (2006). Other bioreactors that can be used in accordance with the invention are described in Pat. US No. 6,277,151, 6,197,575, 6,139,578, 6,132,463, 5,902,741 and 5,629,186.

[0080] Cell seeding is preferably carried out at 100,000-1500000 cells / mm per seed. In an exemplary embodiment, a total of 150 ± 30 x 10 are seeded⁶, vaccinated 3-5 x 10⁶ cells / g carrier or inoculated 0.015-0.1 x 10⁶ cells / ml.

[0081] Cells can be harvested when at least about 10% of the cells proliferate while avoiding uncontrolled senescence.

[0082] The culture is carried out for at least about 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, a month or even longer. It should be appreciated that culturing in a bioreactor can extend this period. Cultivation of adherent cells in 3D culture can be carried out with continuous flow of the culture medium. Passaging can also be carried out to increase the number of cells. It should be appreciated that the culture medium can be changed to extend and improve the culture conditions.

PAT-1243-EP-E

EP2200622 [0083] Adherent cells of some embodiments of the invention contain at least 10%, 28%, 30%, 50%, 80% or more proliferating cells (which can be assessed by FACS monitoring of S and G2 / M phases).

[0084] Adherent cells of some embodiments of the invention may contain at least one "stromal stem cell phenotype". As used herein, one "stromal stem cell phenotype" refers to a structural or functional phenotype typical of a bone marrow derived stromal cell. (i.e. mesenchymal).

[0086] As used herein, the term "stem cell" refers to a cell that is not ultimately differentiated [0087] For example, the cells may have a spindle shape. Alternatively or additionally, the cells may express a marker or set of markers (e.g. surface markers) typical of stromal stem cells. Examples of stromal stem cell surface markers (positive and negative) include, but are not limited to, CD105 +, CD29 +, CD44 +, CD73 +, CD90 +, CD3-, CD4-, CD34-, CD45-, CD80-, CD 19-, CD5-, CD20-, CD11B-, CD 14-, CD19-,

CD79-, HLA-DR-, and FMC7-. Other stromal stem cell markers include, but are not limited to, tyrosine hydroxylase, nestin and H-NF.

[0088] Adherent placental tissue cells generated according to current science have a gene expression profile essentially as described in Example 4 of the next section Examples.

[0089] Examples of functional phenotypes typical of stromal stem cells and include, but are not limited to, suppression of T-cell activity (do not stimulate T-cells and contrary suppress them), hematopoietic cell support activity ma1243-PAT-EP-PL

EP2200622 even as well as any of adipogenic, hepatogenic, osteogenic and neurogenic differentiation.

[0090] Any of these structural or functional features can be used to determine the cells of the invention (see Examples 4 of the next section Examples).

[0091] Cell populations generated according to current science have a unique protein expression profile as shown in Example 1 of the Examples section. For example, adherent placental or adipose tissue cells generated according to current science are capable of expressing and / or secreting high levels of selected factors. For example, such cells express or secrete SCF, Flt-3, H2A, histone family (H2AF) or aldehyde dehydrogenase X (ALDH X) at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or even 12 times higher than those expressed or secreted by adherent placental cells or adipose tissue cultured in 2D culture. Additionally or alternatively, the cell population of the invention secretes or expresses IL-6, eukaryotic translation elongation factor 2 (EEEF2), reticulocalbine 3, EF-hand calcium binding domain (RCN2) or calponin 1 basic smooth muscle (CNN1) at a level of at least 2, 3 or higher than those expressed or secreted by adherent placental cells or adipose tissue cultured in 2D culture. Additionally or alternatively, the cell population of the invention is characterized by a lower expression level of various other proteins compared to cells grown in 2D. Thus, for example, they secrete or express less than 0.6, 0.5, 0.25 or 0.125 expression levels of heterogeneous H1 nuclear ribonucleoprotein (Hnrph1), precursor of CD44 antigen isoform 2, isoforms and synthase 2 3 phosphoadenosine 5 phosphosulfate (Papss2) or ribosomal L7a protein (rpL7a)

PAT-1243-EP-E

EP2200622 expressed or secreted by adherent placental cells or adipose tissue cultured in 2D culture.

[0092] As shown in Examples 3-4 of the next Examples section, it was found that adherent cells, and especially adrent herent 3D cells, abolish the immune response of mononuclear human blood cord cells in a mixed lymphocyte reaction (MLR) assay, yes thus they show biological activities that can be advantageously used in the clinic (e.g. T cell suppression activity, hematopoietic stem cell support activity).

[0093] According to one embodiment of the invention, the adherent cells of the invention are capable of suppressing the immune response in a subject.

[0094] As used herein, the term "suppression of an immune response in a subject" refers to a reduction or inhibition of an immune response that occurs in a subject in response to an antigen (e.g., a foreign cell or portion thereof). An immune response that can be suppressed by adherent cells includes humoral and cellular immune responses that include specific recognition of pathogen antigens by antibodies and T lymphocytes (T cell proliferation), respectively.

[0095] According to one embodiment of the invention, the adherent cells for use according to the invention have higher immunosuppressive activity than adherent placental or adipose tissue cells cultured in two-dimensional (2D) culture.

[0096] According to one embodiment of the invention, immunosuppressive activity includes a reduction in T cell proliferation.

[0097] As mentioned above and described in Example 6 on 1243-PAT-EP-PL

The following examples, adherent cells for use according to the invention induced angiogenesis in vivo (e.g. blood flow in the hip and leg), significantly improved the function of the limb of animals subjected to arterial ligation, increased capillary density, and reduced oxidative stress and endothelium. Furthermore, as described in detail in Example 7 of the next Examples section, adherent cells for use according to the invention significantly improved post-stroke condition in a rat model.

[0098] Thus, the invention relates to the treatment of ischemia in a subject in need thereof with a therapeutically effective amount of adherent cells for use according to the invention, thereby treating ischemia in the subject.

[0099] The term "ischemia" as used herein refers to any pathology (disease, condition, syndrome or disorder) characterized or associated with insufficient angiogenesis. Examples include, but are not limited to, peripheral arterial disease (PAD) such as limb ischemia and critical limb ischemia (CLI), ischemic heart disease, ischemic brain disease (e.g. stroke), delayed wound healing, delayed healing of ulcers, reproductive disorders, atherosclerosis, ischemic vascular disease, ischemic heart disease, myocardial ischaemia, coronary artery disease (CAD), cardiovascular atherosclerosis, left main coronary artery disease arterial occlusive disease, peripheral ischemia, peripheral vascular disease, renal vascular disease, peripheral arterial disease, limb ischemia, lower limb ischemia, brain hypoxia, cerebrovascular disease, retinopathy, retinal repair, remodel disorder1243-PAT-EP-PL

EP2200622, von Hippel-Lindau syndrome, hereditary hemorrhagic telangiectasia, ischemic vascular disease, Buerger's disease, ischemic kidney disease, and placental ischemia.

[0100] As used herein, the term "treatment" refers to inhibiting or arresting the progression of pathology (e.g., ischemia) and / or causing a reduction, remission or regression of the pathology. Those skilled in the art will understand that different methodologies and assays can be used to assess the development of pathology, and similarly, different methodologies and assays can be used to reduce, remission or regression pathologies. The term "treatment" may also refer to alleviating or reducing a symptom associated with pathology.

[0101] As used herein, the term "subject in need thereof" refers to any individual (e.g., a mammal), such as a human individual, who is diagnosed or suffers from pathology.

[0102] As mentioned above and described in Example 8 in the Examples section that will follow, adherent cells are capable of regenerating and / or repairing connective tissue.

[0103] Thus, an additional aspect relates to the treatment of a medical condition requiring regeneration and / or repair of connective tissue in a subject in need thereof with a therapeutically effective amount of adherent cells.

[0104] The term "connective tissue" refers to supportive matrix tissue containing collagen bands, elastic fibers (eg, between and around muscles and blood vessels) and straight cells. Examples of connective tissues include, without limitation, dense connective tissue (e.g., ligament, tendon, periodontal ligament), reticulate connective tissue (e.g., with protein fibers such

PAT-1243-EP-E

EP2200622 (collagen and elastin), reticulated connective tissue, adipose tissue, blood, bone, cartilage, skin, intervertebral disc, tooth pulp, dentin, gum, cells forming extracellular matrix (ECM), loose connective tissue and smooth muscle cells .

[0105] As used herein, the term "medical condition requiring regeneration and / or repair of connective tissue" refers to any pathology characterized by connective tissue damage (i.e., non-functional tissue, tissue with cancer or pre-cancerous condition, broken tissue, broken tissue, fibrotic tissue or ischemic tissue) or loss (e.g., after trauma, infectious disease, genetic disease, and the like). Non-limiting examples of such pathologies include bone fracture, bone cancer (e.g. osteosarcoma, bone cancer metastases), burn wounds, joint cartilage defects, and deep wound.

[0106] The term "administration to a subject" refers to the introduction of cells according to the invention into target tissue. Cells may be from the recipient or from an allogeneic or xenogeneic donor. This term also includes "transplantation", "replacement of cells" or "implantation" of cells of the invention into a subject.

[0107] The subject may be any mammal in need of regeneration and / or repair of connective tissue including, e.g., human or domesticated animals including, but not limited to, horses (i.e. equines), cattle, goats, sheep, pigs, dogs, cats, camels, alpaca, llama and yak.

[0108] Adherent cells can be used to treat conditions including sub-cartilage bone cysts, bone fractures, osteoporosis, osteoarthritis, bone degeneration, various

PAT-1243-EP-E

EP2200622 cancers associated with connective tissue loss (e.g. bone cancer, osteosarcoma, bone metastases), cartilage damage, joint bone defects, disc degenerative disease, osteogenesis imperfecta (OI), burns, burn wounds, deep wounds, delayed wound healing, damaged ligaments and damaged tendons, e.g. tendon damage caused by overloading in horses and other individuals in need (as stated above).

[0109] Cells that can be administered in accordance with this aspect of the description include the above-described adherent cells that can be cultured in three-dimensional systems, as well as mesenchymal and non-mesenchymal their partially or ultimately differentiated derivatives.

[0110] Methods for obtaining cells of a specific line from stromal stem cells described herein are well known in the art. See, for example, US Pat. No.

5,486,359, 5,942,225, 5,736,396, 5,908,784 and 5,902,741.

[0111] Cells can be naive or genetically modified to derive an interesting line from them (see U.S. Pat.

No. 20030219423).

[0112] The cells may be from an autologous or non-autologous (i.e. allogeneic or xenogeneic) source from fresh or frozen preparations (e.g. cryopreserved).

[0113] Depending on the medical condition, subjects may receive additional chemical drugs (e.g., immunomodulatory, chemotherapy) or cells.

[0114] Because non-autologous cells can induce an immune response when administered to the body, several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include suppression of the recipient's immune system

PAT-1243-EP-E

EP2200622 or encapsulation of non-autologous cells in immuno-isolating semipermeable membranes prior to transplantation.

[0115] Encapsulation techniques are generally classified as micro-encapsulation, including small spherical carriers and macro-encapsulation including larger flat and hollow fiber membranes (Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64).

[0116] Methods for preparing microcapsules are well known in the art and include, for example, those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alphaphenoxycinnamylidene-acetylated poly (allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly (allylamine alpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

[0117] For example, microcapsules are prepared by complexing the modified collagen with a thermopolymer coating with

2-hydroxyethyl methyl acrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), giving a capsule thickness of 2-5 μη. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer coatings to impart a negatively charged smooth surface and minimize absorption of plasma proteins (Chia, SM et al. Multi-layered microcapsules for cell encapsulation Biomaterials. 2002 23: 849-56).

[0118] Other microcapsules are based on alginate, marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Technol. Ther. 2003, 5: 665-8) or its po1243-PAT-EP-PL

EP2200622 mining. For example, microcapsules can be prepared by polyelectrolyte complexation of sodium alginate polyanions and sodium cellulose sulfate with polycation poly (methylene co-guanidine) hydrochloride in the presence of calcium chloride.

[0119] It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, quality control, mechanical stability, diffusion properties, and in vitro activity of the encapsulated cells improved when the capsule size was reduced from 1 mm to 400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater Sci Polym Ed 2002; 13: 783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, matched surface chemistry and precise microarchitectures have been successfully immuno-insulated for micro-environments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device Technol. 1999, 10: 6-9; Desai,

TA Microfabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

[0120] Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulfasalazopyrin), gold salts, D-penicillamine, leflunomide, azathioprine, infracimat, EDEX TNF.alpha blockers, a biological agent that targets an inflammatory cytokine, and nonsteroidal anti-inflammatory drugs (NSAIDs). Examples of NSAIDs include, but are not limited to, acetylsalicylic acid, magnesium choline salicylate, diflunisal, magnesium salicylate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen,

PAT-1243-EP-E

EP2200622 nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol.

[0121] In any of the methods described herein, the cells can be administered either as such or as part of a pharmaceutical composition that further comprises a pharmaceutically acceptable carrier.

[0122] As used herein, "pharmaceutical composition" refers to a preparation of adherent cells for use according to the invention (ie, adherent placental cells that are obtained from three-dimensional culture), with other chemical components such as pharmaceutically suitable carriers and excipients. The purpose of pharmaceutical compositions is to facilitate administration of cells to a subject.

[0123] Hereinafter, the "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not cause significant irritation to an individual and does not abrogate the biological activity and properties of the compound administered. Examples, without limitation, of the carriers are propylene glycol, saline, emulsions and mixtures of organic solvents with water.

[0124] The term "excipient" refers to a passive substance added to a pharmaceutical composition to further facilitate administration of the compound. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. [0125] According to a preferred embodiment of the invention, the pharmaceutical carrier is an aqueous saline solution.

[0126] Techniques for formulation and administration of drugs can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

PAT-1243-EP-E

EP2200622 [0127] The pharmaceutical composition may be administered in a systemic manner (as described herein above). Alternatively, the pharmaceutical composition may be administered topically, for example, by injecting the pharmaceutical composition directly into the tissue area of ​​the patient.

[0128] Pharmaceutical compositions can be produced by processes well known in the art, e.g., by conventional mixing, dissolving, granulating, dragee-making, wet-milling and sedimentative fractionation processes, emulsifying, encapsulating, capturing or lyophilizing. [0129] Pharmaceutical compositions for use in accordance with the invention may thus be formulated in a conventional manner using one or more physiologically acceptable carriers including excipients and auxiliaries that facilitate processing of the active ingredients into preparations which can be used pharmaceutically. The appropriate formulation depends on the chosen route of administration.

[0130] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, saline buffer or freezing medium containing cryopreservatives. For transmucosal administration, penetrants appropriate to the barrier to be overcome are used in the formula. Such penetrants are generally known in the art.

[0131] For each formulation used in the invention, a therapeutically effective amount or dose can be originally evaluated from in vitro and cell culture assays. Preferably, the dose is formulated in an animal model to obtain the desired concentration or

PAT-1243-EP-E

EP2200622 titer. Such information can be used to more accurately determine useful doses in humans.

[0132] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or in experimental animals.

[0133] Data obtained from these assays in in vitro cell cultures and from animal studies can be used in formulating a range of doses for human use. The dose may vary depending on the dosage form used and the route of administration used. The exact formula, route of administration and dose can be selected by the physician according to the patient's condition (see, e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics", chapter 1 p. 1). For example, a patient with Parkinson's may be monitored for symptoms to improve motor function indicating a positive response to treatment.

[0134] For injection, the active ingredients of the pharmaceutical composition may be formulated in solutions, preferably in physiologically suitable buffers, preferably such as Hank's solution, Ringer's solution, or buffered saline.

[0135] The amount of dose and interval may be adjusted individually to levels of the active ingredient that are sufficient to effectively regulate the synthesis of neurotransmitters by implanted cells. The doses needed to achieve the desired effect will depend on the individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0136] Depending on the severity and reaction of the condition to be treated, the dosage may be a single or a series of administrations, with a duration of therapy lasting from several days to several weeks or up to

PAT-1243-EP-E

EP2200622 achieving a reduction in the disease state.

[0137] The amount of composition to be administered will of course depend on the subject being treated, the severity of the discomfort, the method of administration, the opinion of the treating physician, etc. The dose and duration of administration will respond to careful and continuous monitoring of the subject's changing condition. For example, a patient treated with Parkinson will receive a sufficient amount of cells to alleviate the symptoms of the disease based on monitoring indications.

[0138] Ligament injury models include, but are not limited to, anterior cruciate ligament reconstruction model using mesenchymal stem cells [Jit-Kheng et al.,

Arthroscopy (2004) 20 (9): 899-910], a goat model for using long-term bioresorptive skeletons to repair the anterior cruciate ligament [Altman et al., J Am Acad Orthop

Surg. (2008) 16 (4): 177-187]. Tendon repair models include, but are not limited to, the New Zealand White rabbit model of tendon repair involving autologous mesenchymal stem cells [Awad et al., Tissue Eng. (1999) 5 (3): 26777]. Bone repair models have been described in, e.g., Stem Cells in Endo crinology, Humana Press (2005) 183-206, describing the manipulation of mesenchymal stem cells for bone repair.

[0139] After transplantation, the cells for use according to the invention preferably survive in the disease area for a period of time (e.g. about 1 month), so that a therapeutic effect is observed.

[0140] Compositions comprising a formulation formulated in a compatible pharmaceutical carrier may also be prepared, placed in a suitable container, and labeled for the treatment of an indicated condition.

PAT-1243-EP-E

EP2200622 [0141] The compositions may, if desired, be presented in a package or dispensing device, such as an FDA approved kit, which may contain one or more unit doses containing the active ingredient. The package may, for example, contain metal or plastic foil, such as a blister pack. The packaging or dispensing device may be accompanied by administration instructions. The packaging or dispensing device may also be accompanied by a container-related note in the form recommended by a government agency regulating the production, use or sale of pharmaceuticals, which note reflects the agency's approval of the composition or administration to humans or animals. Such a note, for example, may be the US Food and Drug Administration's approval mark for prescription drugs or approved product introduction.

[0142] It will be appreciated that the adherent cells for use according to the invention are capable of inducing immunosuppression and / or tolerance in a subject. Methods of use for treating any condition in need of immunosuppression and / or tolerance are described. Such conditions include, but are not limited to, autoimmune diseases and inflammatory diseases (including acute and chronic inflammatory diseases) including, but not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, skin diseases, liver diseases, neurological diseases, diseases muscle, kidney disease, reproductive disease, connective tissue disease, and systemic disease. [0143] Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al., Lupus. 1998; 7 Suppl 2: S135), myocardial infarction (Vaarala O.

PAT-1243-EP-E

EP2200622

Lupus. 1998; 7 Suppl 2: S132), thrombosis (Tincani A. et al., Lupus 1998; 7 Suppl 2: S107-9), Wegener's granulomatosis, Takayasu arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien

Klin Wochenschr 2000 Aug 25; 112 (15-16): 660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost. 2000; 26 (2): 157), necrotizing inflammation of small vascular, microscopic vasculitis, Churg-Strauss syndrome, immunological focal necrosis and sickle glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000

May; 151 (3): 178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171), antibody-induced heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17; 83 (12A): 75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun; 14 (2): 114; Semple JW. Et al., Blood 1996 May

15; 87 (10): 4245), autoimmune hemolytic anemia (Efremov DG. Et al., Leuk Lymphoma 1998 Jan; 28 (3-4): 285; Sallah S. et al., Ann Hematol 1997 Mar; 74 (3 ): 139), cardiac autoimmunity in Chagas disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15; 98 (8): 1709) and autoimmunity against helper T lymphocytes (Caporossi AP. Et al., Viral Immunol 1998; 11 (1): 9).

[0144] Examples of autoimmune rheumatoid diseases include, without limitation, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15 (3): 791; Tisch R, McDevitt HO. Proc Natl Acad Sci USA 1994 Jan 18; 91 (2): 437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).

[0145] Examples of autoimmune gland diseases include, without limitation, pancreatic disease, type I diabetes, thyroid disease, Graves disease, thyroiditis, spontaneous au1243-PAT-EP-PL

EP2200622 to immunological thyroiditis, Hashimoto's thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostate inflammation and autoimmune multi-glandular syndrome type I. Diseases include, without limitation, autoimmune pancreatic disease and Diabetes Eisenbarth GS. Ann. Rev. Immunol. 8: 647; Zimmet P. Diabetes Res Clin Pract 1996 Oct; 34 Suppl: S125), autoimmune thyroid disease, Graves disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000

Jun; 29 (2): 339; Sakata S. et al., Mol Cell Endocrinol 1993 Mar; 92 (1): 77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12): 7262), inflammation Hashimoto thyroid (Toyoda N. et al., Nippon Rinsho 1999 Aug; 57 (8): 1810), idiopathic mucosal edema (Mitsuma T. Nippon Rinsho. 1999 Aug; 57 (8): 1759), ovarian autoimmunity (Garza KM et al., J Reprod Immunol 1998 Feb; 37 (2): 87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar; 43 (3): 134), autoimmune prostatitis (Alexander RB. Et al., Urology 1997 Dec; 50 (6): 893) and autoimmune multi-glandular syndrome type I (Hara T. et al., Blood. 1991 Mar 1; 77 (5): 1127). [0146] Examples of autoimmune gastrointestinal diseases include, without limitation, chronic inflammatory bowel disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan; 23 (1): 16), celiac disease (Landau YE. And Shoenfeld Y. Harefuah

2000 Jan 16; 138 (2): 122), colitis, colitis, Crohn's disease.

[0147] Examples of autoimmune skin diseases include, without limitation, autoimmune bullous skin diseases

PAT-1243-EP-E

EP2200622 such as, but not limited to, pemphigus vulgaris, bullous pemphigoid, and deciduous pemphigus.

[0148] Examples of autoimmune liver disease include, without limitation, hepatitis, autoimmune chronic hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar; 54 (3): 382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov; 91 (5): 551; Strassburg CP. Et al., Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6): 595) and autoimmune hepatitis (Manns MP. J Hepatol 2000

Aug; 33 (2): 326).

[0149] Examples of autoimmune neurological diseases include, without limitation, multiple sclerosis (Cross AH. Et al., J Neuroimmunol 2001 Jan 1; 112 (1-2): 1), Alzheimer's disease (Oron L. et al., J Neural Transm Suppl. 1997; 49: 77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999; 18 (12): 83; Oshima M. et al., Eur J Immunol 1990 Dec; 20 (12): 2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci.

2000 May; 7 (3): 191); Guillain-Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr; 319 (4): 234), myasthenia gravis, myasthenic syndrome Lambert-Eaton (Takamori M. Am J Med Sci. 2000 Apr; 319 (4) : 204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, and general stiffness syndrome (Hiemstra HS. et al., Proc Natl Acad Sci USA 2001 Mar 27; 98 (7): 3988); non-paraneoplastic general stiffness syndrome, progressive cerebellar atrophy, encephalitis, Rasmussen encephalitis, amyotrophic lateral sclerosis, Sydeham's chorea, Gilles de la Tourette syndrome and autoimmune poliendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1): 23); dysimmune neuropathies (Nobi1243-PAT-EP-PL

EP2200622 le-Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl

1999; 50: 419); acquired neuromyotonia, congenital stiffness of the joints (Vincent A. et al., Ann NY Acad Sci. 1998 May 13; 841: 482), neuritis, optic neuritis (Soderstrom M. et al.,

J Neurol Neurosurg Psychiatry 1994 May; 57 (5): 544) and neurodegenerative diseases [0150] Examples of autoimmune muscle diseases include, without limitation, myositis, autoimmune myositis, primary Sjogren's syndrome (Feist E. et al., Int Arch Al10 lergy Immunol 2000 Sep; 123 (1): 92) and smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999

Jun; 53 (5-6): 234).

[0151] Examples of autoimmune kidney disease include, without limitation, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug; 1 (2): 140). [0152] Examples of autoimmune reproduction-related diseases include, without limitation, repeated fetal loss (Tincani A. et al., Lupus 1998; 7 Suppl 2: S107-9).

[0153] Examples of autoimmune connective tissue diseases include, without limitation, ear diseases, autoimmune ear diseases (Yoo TJ. Et al., Cell Immunol 1994 Aug; 157 (1): 249) and autoimmune inner ear diseases (Gloddek B. and et al., Ann NY Acad Sci 1997 Dec 29; 830: 266).

[0154] Examples of autoimmune systemic diseases include, without limitation, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (1-2): 49) and systemic scleroderma (Renaudineau Y. el al., Clin Diagn Lab Immunol. 1999 Mar; 6 (2): 156); Chan OT. et al., Immunol Rev 1999

Jun; 169: 107).

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EP2200622 [0155] Furthermore, adherent cells can be used to treat diseases associated with transplant transplantation, including but not limited to transplant rejection, chronic transplant rejection, subacute transplant rejection, excess transplant rejection, acute transplant rejection and graft versus host disease .

[0156] As used herein, the term "about" refers to ±

%.

[0157] Additional objects, advantages and new features of the invention will become apparent to those of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. In addition, each of the various embodiments and aspects of the invention as set forth herein above and as claimed in the portion of the claim below finds experimental support in the following examples.

EXAMPLES [0158] Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non-limiting manner.

[0159] Generally used nomenclature and laboratory procedures used in the invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al. (1989);

"Current Protocols in Molecular Biology" vol. I-III Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley &amp; Sons, New York (1988); Watson et al. ., "Recombinant DNA", Scien1243-PAT-EP-PL

EP2200622 tific American Books, New York; Birren et al. (Ed) "Genome Analysis: A Laboratory Manual Series", vol. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as described in Pat. US No. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", vol. I-III Cellis, JE, ed. (1994); "Current Protocols in Immunology" vol. I-III Coligan JE, ed. (1994); Stites et al. (ed.), "Basic and Clinical Immunology" (8th edition), Appleton &amp; Lange, Norwalk, CT (1994); Mishell and Shiigi (ed.), "Selected Methods in Cellular Immunology," WH Freeman and Co., New York (1980);

3,839,153;

3,879,262;

3,850,752;

3,901,654;

<td colspan="3">patens widely described in literature</td>

<td>e.g,</td><td>Stalemate. US No.</td><td> 3,791,932;</td>

<td> 3,850,578;</td><td> 3,853,987;</td><td> 3,867,517;</td>

<td> 3,935,074;</td><td> 3,984,533;</td><td> 3,996,345;</td>

4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; 'Oligo1984); "Nucleic Acid (1985);

nucleotide Synthesis ”Gait, MJ, ed.

Hybridization, "Hames, BD, and Higgins SJ, red." Transcription and Translation "Hames, BD, and Higgins SJ, ed. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated herein by reference as if they were fully given herein. Other general links are provided throughout the document. It is believed that the described procedures are well known in the art and are provided for the convenience of the reader. All contained

PAT-1243-EP-E

EP2200622 in them, the information is incorporated herein by reference.

EXAMPLE 1

PRODUCTION AND CULTURE OF ADHERENT CELLS FROM BONE Marrow, BEARING AND FAT TISSUE [0160] Adherent cells were cultured in a bioreactor system containing 3D carriers to produce adherent 3D cells characterized by the expression profile of specific cell markers. Growth efficiency was tested by cell counting. The ability to differentiate these cells was tested by cultivating on differentiating media.

Experimental materials and procedures [0161] Adherent bone marrow cells - Adherent bone marrow (BM) cells were obtained from the aspirated marrow of hematologically healthy donors undergoing open-heart surgery or BM biopsy. Marrow aspirates were diluted 3-fold in Hank's Balanced Salts Solution (HBSS; GIBCO

BRL / Invitrogen, Gaithersburg MD) and subjected to Ficoll-Hypaque density gradient centrifugation (Robbins Scientific Corp. Sunnyvale, CA). Mononuclear bone marrow cells (&lt;1.077 gm / cm were then collected³), washed 3 times with HBSS and suspended in growth media [DMEM (Biological Industries, Beit Ha'emek, Israel) supplemented with 10% FCS (GIBCO BRL), 10^-4 M mercaptoethanol (Merck, White House Station, NJ), Pen-Strep-Nystatin mixture (100 U / ml: 100 μg / ml: 1.25 U / ml; Beit Ha'Emek), 2 mM L-glutamine (Beit Ha 'Emek)]. Cells from individual donors were incubated separately in tissue culture flasks (Coming, Acton, MA) at 37 ° C (5% CO2) with a change of culture medium every week. Cells were separated every 3-4 days using 0.25% trypsin-EDTA (Beit Ha'Emek). After 2-40 passages, when 60-80% confluence was achieved, chambers 1243-PAT-EP-PL

EP2200622 k were collected for analysis or for cultivation in bioreactors.

[0162] Adherent placental cells - Internal parts of a placental pregnancy (Bnei Zion medical center, Haifa, Israel) were cut under sterile conditions, washed 3 times with Hank's buffer and incubated for 3 hours at 37 ° C with 0.1% collagenase (1mg / ml tissue; Sigma-Aldrich, St. Lewis, MO). Using gentle pipetting, the suspended cells were then washed with DMEM supplemented with 10% FCS, a Pen-Strep-Nystatin mixture (100 U / ml: 100 μg / ml: 1.25 U / ml) and 2 mM L-glutamine, inoculated into 75 cm flasks² and incubated at 37 ° C in a tissue culture incubator under humid conditions with 5% CO2. Then, the cells were allowed to adhere to the plastic surface for 72 hours after which the medium was changed every 3-4 days. After reaching 60-80% confluence (usually 10-12 days), cells were detached from the culture flask using 0.25% trypsin-EDTA and inoculated into new flasks. The cultured cells were then harvested for analysis or for culture in bioreactors.

[0163] Adherent fat-derived cells - Adherent cells were obtained from human adipose tissue from liposuction (Rambam Haifa, Israel). Adipose tissue was extensively rinsed with equal volumes of PBS and digested at 37 ° C for 30 minutes with collagenase (20 mg / ml). Cells were then washed with DMEM containing 10% FCS, Pen-Strep-Nystatin (100

U / ml: 100 μg / ml: 1.25 U / ml) and L-glutamine and centrifuged at 1200 rpm for 10 minutes at room temperature (RT), suspended in lysis solution (1:10; Biological Industries, Beit Ha'emek, Israel (to remove red blood cells) was centrifuged and suspended in DMEM containing 10% FCS, a mixture of Pen-Strep-Nystatin (100 U / ml: 100 μg / ml: 1.25 U / ml) and L-glutamine . Cells washed out

PAT-1243-EP-E

EP2200622 was then inoculated into a sterile flask with culture medium

3-10 X 10⁷ cells / flask. The next day, cells were washed with PBS to remove residual RBC and dead cells. Cells were kept at 37 ° C in a tissue culture incubator in humid conditions with 5% CO2. The medium was changed every 3 to 4 days. At 60-80% confluence, cells were detached from the culture flask using 0.25% trypsin-EDTA and inoculated into new flasks. After 2-40 passages, when the cells reached 60-80% confluence, the cells were harvested for analysis or for culture in bioreactors.

[0164] PluriX ™ Plug Flow Bioreactor - PluriX ™ Plunger ™ Bioreactor with plunger flow (Pluristem, Haifa, Israel; as illustrated in Figure 1G, see also US Pat. No. 6,911,201), charged with 1-100 ml packaged 3D porous carriers (4 mm in diameter) ) made of a matrix of non-woven polyester material. These carriers allow the proliferation of large amounts of cells in a relatively small volume. The glass parts were designed and manufactured by Pluristem (Pluristem, Haifa, Israel). The bioreactor was maintained in a 37 ° C incubator, with valve flow control and monitoring (6a in Figure 1G), and a peristaltic pump (9 in Figure 1G). The bioreactor contains a sampling and injection site (4 in Figure 1G), allowing subsequent cell inoculation. Culture medium was supplied at pH 6.7-7.4 from the tank (1 in Figure 1G). A filtered gas mixture (2, 3 in Figure 1G) was supplied to the tank, containing air / CO2 / O2 in various proportions, depending on the cell density in the bioreactor. The O2 proportions were adjusted to the level of dissolved O2 at the exit from the bioreactor, as determined by the monitor (6 in Figure 1G). The gas mixture was delivered to the tank using silicone tubing or a diffuser (Degania Bet, Emek Hayar1243-PAT-EP-PL

EP2200622 den, Israel). The culture medium was passed through a separating container (7 in Figure 1G) allowing collection of circulating, non-adherent cells. Circulation of the medium was achieved by a peristaltic pump (9 in Figure 1G). The bioreactor was additionally equipped with an additional point (10 in Figure 1G) and containers for continuous substrate change.

[0165] Production of adherent 3D cells - Non-confluent primary human cultures of adherent 2D cells, cultured as described above, were trypsinized, washed, suspended in DMEM supplemented with 10% FBS, Pen-Strep-Nystatin mixture (100 U / ml: 100 μg / ml: 1 , 25 U / ml) and 2 mM L-glutamine, and inoculated (10³-10⁵cells / ml) via the injection point into 3D carriers in a sterile piston flow bioreactor (see Figure 1G). Before inoculation, the bioreactor was filled with PBS-Ca-Mg (Biological Industries, Beit Ha'emek, Israel), autoclaved (120 ° C, 30 min) and washed with Dulbecco growth medium containing 10% heat inactivated fetal calf serum and PenStrep-Nystatin mixture (100 U / ml: 100 μg / ml: 1.25 units / ml). The flow rate was maintained at 0.1-5 ml / min. The inoculation process involved stopping circulation for 2-48 hours, which allowed cells to settle on carriers. The bioreactor was kept under controlled temperature (37 ° C) and pH (pH = 6.7-7.4); using an incubator supplied with sterile air and CO2 as needed. Growth medium was changed 2-3 times a week. The circulating medium was replaced with fresh DMEM medium every 4 hours. up to 7 days. At a density of 1 X 10⁶-1 X 10⁷ cells / ml (after 12-40 days of growth), the total volume of medium was removed from the bioreactor and the bioreactor and the carriers were washed 3-5 times with PBS. Adherent 3D cells were then detached from trypsin-EDTA carriers; (Biological

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Industries, Beit Ha'emek, Israel; 3-15 minutes with gentle shaking (1-5 times), and then suspended in DMEM and cryopreserved.

[0166] Biological quality assays of adherent 3D cells Cryopreserved adherent 3D cells were thawed and counted.

₅

To assess cell viability, 2 X 10 was seeded⁵ cells in col₂ tissue culture tissue 150 cm² and their ability to adhere and repopulate within 7 days after vaccination was evaluated. The phenotype of markers of adherent 3D cells was then analyzed using a flow cytometer (Beckman Coulter, Fullerton, CA) with fluorescent monoclonal antibodies.

[0167] Comparison between membrane marker profile of cultured in

3D and 2D adherent cells using flow cytometry assays 100,000 - 200,000 adherent cells from 2D culture and flow culture 3D were suspended in 0.1 ml culture medium in a 5 ml tube and incubated (4 ° C, 30 minutes, in the dark) with saturating concentrations of each of the following MAb: FITC conjugated anti-human CD90 (Chemicon International Inc. Temecula, CA), PE-conjugated anti-human CD73 (Bactlab Diagnostic, Ceasarea, Israel), PE-conjugated anti-human CD105 (eBioscience, San Diego, CA), FITC conjugated anti-human CD29 (eBioscience, San Diego, CA), Cy7-PE conjugated anti-human CD45 (eBiosience), PE conjugated anti-human CD19 (IQProducts, Groningen, Netherlands), PE conjugated anti-human CD14 MAb (IQProducts), FITC conjugated anti-human CD11b (IQProducts) and PE conjugated anti-human CD34 (IQProducts) or FITC conjugated anti-human HLA-DR MAb (IQProducts). After incubation, cells were washed twice with ice-cold PBS containing 1% heat inactivated FCS, suspended in 500 μΐ

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EP2200622

0.5% formaldehyde and analyzed using FC-500 flow cytometer (Beckman Coulter, Fullerton, CA).

[0168] Comparison between the protein profile of 3D and 2D cultured adherent cells using mass spectrometry analysis - Adherent cells derived from 2D and 3D culture procedures were produced from placenta as described above. Briefly, 2D cultures were obtained by culturing 0.3-0.75 X 10⁶ cells in 175 cm flasks² for 4 days in a humid atmosphere with 5% CO2 at 37 ° C, up to 60 80% confluence. 3D cultures were obtained by inoculation 2-10 x 10⁶ cells / gram in a bioreactor containing 2000 carriers, and grown for 18 days. After harvesting, the cells were washed (X 3) to remove all serum, centrifuged and frozen. Proteins were isolated from sediments [using the Tri Reagent kit (Sigma, Saint Louis, USA) and digested with trypsin and labeled with iTRAQ reagent (Applied Biosciences, Foster City, CA)] according to the manufacturer's protocol. Briefly, iTRAQ reagents are non-polymeric, isobaric labels. The peptides in each sample are labeled with one of four isobaric encoded labels by their N-terminal and / or lysine side chains. Four labeled samples are mixed and peptides are analyzed by mass spectrometry. After peptide fragmentation, each label releases a separate ion mass reporter; the ratio of four reporters therefore gives the relative abundance of a given peptide in the sample (information:<a href="http://www.docs.appliedbiosystems.com/pebiodocs/00113379.pdf">www.docs.appliedbiosystems.com/pebiodocs/00113379.pdf)</a>.

[0169] Proteomic analysis of 2D cultures versus 3D cultures derived from placental adherent cells was performed in Smoler proteomic medium (Department of Biology, Technion, Haifa, Israel) using LC-MS / MS on QTOF-Premier (Waters, San Francisco, CA), with identification and analysis using software 1243-PAT-EP-PL

EP2200622 Pep-Miner [Beer, I., et al., Proteomics, 4, 950-60 (2004)] to the human portion of the database. Proteins analyzed: heterogeneous H1 nuclear ribonucleoprotein (Hnrph1 GenBank Accession No. NP_005511), H2A histone family (H2AF, GenBank Accession No.

NP_034566.1), eukaryotic translation elongation factor 2 (EEEF2, GenBank Accession No. NP_031933.1), reticulocalbin 3, EF-hand calcium binding domain (RCN2, GenBank Accession No. NP_065701), precursor of isoform 2 of CD44 antigen (NP Accession No. Gen1313B44) calponin 1 basic smooth muscle (CNN1, Access No.

GenBank NP_001290), phosphoadenosine 5 phosphosulfate synthase 3 isoform 2 (Papss2, GenBank Accession No. NP_004661), L7a ribosomal protein (rpL7a, GenBank Accession No. NP_000963) and aldehyde dehydrogenase X (ALDH X, Gen38 Accession No. 37). Each experiment was carried out twice. Due to the nature of the analysis, each protein was analyzed for the number of peptides that appeared in the sample (2-20 protein appearances in each analysis) [0170] Comparison between secreted proteins in 3D and 2D cultured adherent cells using ELISA - cultured in under 2D and 3D conditions, adherent placental cells were produced as described above, with 3D cultures for a period of 24 days. Subsequently, conditioned media was collected and analyzed for the presence of Flt-3 ligand, IL-6, thrombopoietin (TPO) and stem cell factor (SCF) using ELISA (R&amp;D Systems, Minneapolis, MN) in three independent experiments. The results were normalized to 1 X 10⁶ cells / ml.

[0171] Osteoblast differentiation medium - Osteogenic differentiation was evaluated by culturing cells in an osteoblast differentiating medium composed of DMEM supplemented with 10% FCS, 100 nM dexamethasone, 0.05 mM ascorbic acid 2-phosphate, 10 mM B1243-PAT-EP-PL

EP2200622 glycerophosphate, for a period of 3 weeks. Matrix calcification was demonstrated by Alizzarin Red S staining and alkaline phosphatase was detected using an alkaline phosphatase detection kit (all reagents from Sigma-Aldrich, St. Lewis, MO)

Experimental results [0172] The Bioreaktor PluriX ™ system creates a physiological-like microenvironment.

[0173] To achieve efficient culture conditions for adherent cells, a physiological-like environment (shown in Figure 1A) was artificially created using a PluriX Bioreactor (Pluristem, Haifa, Israel; the carrier is shown in Figure 1G and shown before inoculation in Figure 1B). As shown in Figures 1C-F, adherent 3D cells produced from bone marrow were successfully cultured and expanded on a 3D matrix,

twenty days (Figures 1B-C, magnification X 150 and 250 respectively) and 40 days (Figures 1C-D, enlargement X 350 and 500 respectively) after inoculation.

[0174] Cells grown in the PluriX Bioreactor system have expanded significantly - Various production batches of placental adherent 3D cells have been grown in the PluriX bioreactor system. Inoculation density was 13300 cells / vehicle (2 X 10 in total)⁶ cells). Fourteen days after inoculation, the cell density increased 15-fold to reach about 200,000 cells / vehicle (Figure 2), or 30 X 10⁶ in a bioreactor with 150 carriers. In another experiment, the cells were seeded into a density bioreactor

1.5 X 10⁴ cells / ml and 30 days after inoculation, the carriers contained over 50 times higher number of cells, i.e. approx. 0.5 X 10⁶ cells / carrier, or 0.5 X 10⁷ cells / ml. The cell density on the supports at different levels of the growth column was similar, indica 1243-PAT-EP-PL

EP2200622 for the homogeneous transfer of oxygen and nutrients to the cell. In this way, it has been proven that the 3D culture system provides conditions that support the growth and prolonged maintenance of high density mesenchymal cell culture that can be efficiently cultured to an amount sufficient to support implantation and successful transplantation.

[0175] Adherent 3D cells exhibit the unique features of membrane markers - To determine the differences in the secretion profile of dissolved molecules and protein production caused by a 3D imitating bone culture procedure, FACS analysis was performed. As shown in Figure 3A, FACS analysis of cell markers shows that adherent 3D cells have different marker expression than adherent cells grown under 2D conditions. Cells grown in 2D expressed significantly higher levels of membrane markers

CD90, CD105, CD73 and CD29 compared to cells cultured in

3D. For example, CD105 showed 56% expression in cells grown in 3D versus 87% in cells grown in 2D. Adherent cells of both 2D and 3D placental cultures did not express any markers of hematopoietic membranes (Figure 3B).

[0176] Adherent 3D cells exhibit a unique soluble factor profile - The hematopoietic niche includes cells that support cells that produce excess cytokines, chemokines and growth factors. To further determine the difference between 2D and 3D cultured adherent cells, profiles of four major secreted hematopoietic proteins in conditioned 2D and 3D culture media of adherent cells were tested by ELISA. Figures 4A-C show that cells cultured in 3D conditions conditioned media with higher levels of Flt-3 ligand (Figure 4A), IL-60 (Figure 4B), and SCF (Figure 4C), and ni1243-PAT-EP-PL

EP2200622 IL-6 levels, and in the conditioned culture media of 2D a close to zero level of Flt-3 ligand and SCF. The production of thrombopoietin (TPO) was very low and equal in both cultures.

[0177] Adherent 3D cells exhibit a unique protein profile in mass spectrometry analysis - To further determine the difference between 2D and 3D cultured adherent cells, the protein profiles of these cells were examined by mass spectrometry. Figure 4D shows that adherent cells grown in 2D and 3D show strikingly different protein expression profiles. As shown in the Table

1 below, cells grown in 3D show a much higher level of H2AF and ALDH X expression (more than 9 and 12 times higher, respectively) and a higher level of EEEF2, RCN2 and CNN1 proteins (about 3, 2.5 and 2 times, respectively). In addition, cells grown in 3D show about half the expression levels of Hnrph1 proteins and the CD44 antigen isoform 2 precursor and about one-third expression levels of Papss2 and rpL7a.

Table 1

<td>protein</td><td colspan="3">Protein level (relative to group iTRAQ)</td><td>reporter</td>

<td></td><td>adherent</td><td>cell</td><td colspan="2">adherent cells</td>

<td></td><td>bred in</td><td>2D</td><td>grown</td><td>in 3D</td>

<td></td><td>av</td><td>SD</td><td>av</td><td>SD</td>

<td>Hnrph1</td><td> 1,434493</td><td> 0,260914</td><td> 0,684687</td><td> 0,197928</td>

<td>H2AF</td><td> 0,203687</td><td> 0,288058</td><td> 1,999877</td><td> 0,965915</td>

<td>EEEF2</td><td> 0,253409</td><td> 0,130064</td><td> 0,799276</td><td> 0,243066</td>

<td>RCN2</td><td> 0,54</td><td> 0,25</td><td> 1,34</td><td> 0,26</td>

<td>Isoform precursor 2</td><td> 1,68</td><td> 0,19</td><td> 0,73</td><td> 0,17</td>

<td>CD44 antigen</td><td></td><td></td><td></td><td></td>

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<td>CNN1</td><td> 0,77</td><td> 0, 15</td><td> 1,55</td><td> 0, 17</td>

<td>Papss2</td><td> 1,48352</td><td> 0,314467</td><td> 0,45627</td><td> 0,137353</td>

<td>rpL7a</td><td> 1,22</td><td> 0,24</td><td> 0,43</td><td> 0, 05</td>

<td>ALDH X</td><td> 0,15847</td><td> 0,22411</td><td> 1,986711</td><td> 0,212851</td>

[0178] Adherent 3D cells have the ability to differentiate into osteoblasts - To further characterize adherent 3D cells, the cells were cultured in osteoblast differentiating medium for a period of

3 weeks. Then calcium precipitation was carried out. Differentiated cells have been shown to produce calcium (shown in red in Figures 5A-B) while control cells have established a fibroblast-like phenotype and showed no mineralization (Figures 5C-D). These results show that placental derived 3D cells have the ability to differentiate in vitro into osteoblast cells.

EXAMPLE 2

ASSESSMENT OF THE ABILITY OF ADHERENT 3D CELLS BEARING TO IMPROVE HSC IMPLEMENTATION [0179] Support by adherent 3D cells for HSC implantation was assessed based on the level of human hematopoietic cells (hCD45 +) detected in sublethally irradiated or chemothermal-treated mouse materials [0180] Isolation of CD34 + cells - Cord blood samples were taken under sterile conditions during delivery (Bnei Zion Medical Center, Haifa, Israel) and mononuclear cells were fractionated using Lymphoprep density gradient centrifugation (Axis-Shield PoC As, Oslo, Norway). Thawed mononuclear cells were washed

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EP2200622 and incubated with anti-CD34 antibodies and isolated using MACS midi (Miltenyl Biotech, Bergish Gladbach, Germany). Cells from more than one sample were combined to obtain the desired amount (50,000-100,000 cells).

[0181] Detection of transplanted cells in irradiated mice - Seven weeks old male and female NOD-SCID mice (NOD-CB 17-Prkdcscid / J; Harlan / Weizmann Inst., Rehovot Israel) were housed in sterile cages in an open system, administered sterile diets and autoclaved acid water. Mice were irradiated irradially with (350 cGy), and then (48 hours after irradiation) 50,000-100,000 hCD34 cells were transplanted⁺, with or without additional adherent cells (0.5 x 10⁶ - 1 x 10⁶) derived from placenta or adipose tissue (3-7 mice in each group), by intravenous injection into the lateral tail vein. Four to six weeks after transplantation, mice were killed by dislocation and BM collected by flushing both femurs and tibias with FACS buffer (50 mL PBS, 5 mL FBS, 0.5 mL 5% sodium azide). Human cells in mouse BM were detected by flow cytometry, and the percentage of human and mouse cells expressing CD45 hematopoietic markers in treated NOD-SCID mice were determined by incubating cells with anti-human CD45-FITC (IQ Products, Groningen, The Netherlands). The lowest threshold value for unequivocal human implantation was determined to be 0.5%.

[0182] Detection of transplanted cells in chemotherapy-treated mice - 6.5 week old male NOD-SCID mice (NOD.CB17 / JhkiHsd-scid; Harlan, Rehovot Israel), kept as described above for irradiated mice, intraperitoneally administered Busulfan (25 mg / kg- for 2 consecutive days). Two days after the second Busulfan injection, the mice were injected with cells alone

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CD34 +, or together with 0.5 X 10⁶ adherent cells produced from placenta. 3.5 weeks after transplantation, mice were killed and the presence of human hematopoietic cells was determined as described above for irradiated mice.

Experimental results [0183] Adherent 3D cells improved HSC implantation in irradiated mice - Human CD34 + hematopoietic cells from adherent cells derived from placenta or adipose tissue were co-transplanted into irradiated NOD-SCID mice. Implantability was assessed 4 weeks after co-transplantation and compared with mice transplanted with HSC alone. As shown in Table 2, co-transplantation of adherent UCB cells and CD34 + cells gave significantly higher implantation rates and higher levels of human cells in BM of recipient mice compared to mice treated with UCB CD34 + cells alone.

Table 2

<td>Transplanted cells</td><td>Medium h-CD45</td><td>STDEV</td>

<td>CD34</td><td> 3,8</td><td> 7,9</td>

<td>CD34 + adherent 3D cells with</td><td></td><td></td>

<td>bearing</td><td> 5,1</td><td> 12,2</td>

<td>CD34 + adherent 3D cells with</td><td></td><td></td>

<td>body fat</td><td> 8,7</td><td> 9,6</td>

[0184] Adherent 3D cells improved HSC implantation in chemotherapy-treated mice - Human hematopoietic cells

CD34 + was co-transplanted from 500,000 adherent 2D cells or adherent 3D cells derived from placenta into NOD-SCID mice previously treated with chemotherapy. Implantation efficiency

PAT-1243-EP-E

EP2200622 was evaluated 3.5 weeks after co-transplantation and compared with mice transplanted with HSC alone. As shown in Table 3 and Figure 6, co-transplantation of adherent CD34 + UCB cells and cells gave higher implantation rates and higher levels of human cells in the BM of recipient mice compared to mice treated with UCB CD34 + cells alone. Furthermore, as shown in Table 3, the average level of implantation was higher in placental co-transplanted mice adherent with cells grown in the PluriX bioreactor system (adherent 3D cells) than in mice co-transplanted with cells from the same donor grown under conventional static 2D culture conditions ( flask).

Table 3

<td>Transplanted cells</td><td>Medium h-CD45</td><td>STDEV</td>

<td>CD34</td><td> 0,9</td><td> 1,1</td>

<td>CD34 + conventional 2D cultures with</td><td> 3,5</td><td> 0,2</td>

<td>profit</td><td></td><td></td>

<td>CD34 + adherent 3D cells from placenta</td><td> 6,0</td><td> 7,9</td>

[0185] The results of the FACS analysis shown in Figures 7A-B show the advantage of co-transplanting adherent cells from hHSC (Figure 7B), and the ability of adherent cells to improve recovery of the hematopoietic system after HSC transplantation.

[0186] All in all, these results show that adherent cells can serve as supportive cells to improve hematopoietic recovery after HSC (autologous or allogenic) transplantation. The ability of adherent 3D cells to enhance the implantation of hematopoietic stem and / or progenitor cells after HSC transplantation may be due to the adherent ability of ko1243-PAT-EP-PL

EP2200622 3D cells for the secretion of HSC-supporting cytokines that may improve self-targeting, self-renewal and the ability to proliferate transplanted cells, or from the ability of these cells to rebuild a damaged hematopoietic microenvironment needed for self-guidance and proliferation of transplanted HSCs.

EXAMPLE 3

SUPRESSION OF LYMPH RESPONSE BY 2D AND 3D CULTURED ADHERENT CELLS [0187] Adherent cells, and in particular adherent 3D cells, have been found to suppress the immune response of mononuclear human blood cord cells in the MLR assay

Experimental materials and procedures [0188] Assay of mixed lymphocyte reaction (MLR) - Immunosuppressive and immuno-privileged properties derived from 2D and 3D culture processes of adherent cells produced from placenta, were performed using MLR assay that measures histocompatibility at the HLA locus . demonstrated by the proliferation rate of incompatible lymphocytes in mixed cultures of reacting (proliferating) and stimulating (non-proliferating) cells. Human cells unite₅ drow cord blood (CB) (2 x 10⁵) were used as responsive cells and stimulated by co-cultivation with equals ₅ quantities (10⁵) irradiated (3000 Rad) human peripheral blood derived Monocytes (PBMC), or with 2D or 3D cultured adherent cells, produced from placenta, or a combination of adherent cells and PBMCs. Each determination was repeated three times. Cells were co-cultivated for 4 days in RPMI 1640 medium (containing 20% ​​FBS in conditions 1243-PAT-EP-PL

EP2200622 humid atmosphere with 5% CO2 (37 ° C), in plate 96₃ well. Plates were administered a pulse of 1μ0 H-thymidine during the last 18 hours of culture. Cells were then harvested on a glass fiber filter and thymidine uptake was determined using a scintillation counter.

Experimental results [0189] Figure 8A shows the CB cell immune response represented by the increased proliferation of these cells when they are stimulated by PBMC, which, without a desire to be limited by theory, is probably associated with T cell proliferation in response to HLA incompatibility. However, these cells showed a significantly lower level of immune response when incubated with the adherent cells of the invention. Furthermore, the CB immune response to PBMCs was significantly reduced when they were co-incubated with these adherent cells. Thus, in a similar manner to MSC, it has been found that adherent cells have the potential to reduce T cell proliferation from a donor cell, typical of GvHD. Although both cultures, 2D and 3D, reduced the immune response of lymphocytes, and according to the other advantages of adherent 3D cells described above, adherent 3D cells were much more immunosuppressive.

EXAMPLE 4

3D ADHERENT CELLS PRODUCED BY PLURIX COMPARED TO 3D ADHERENT CELLS PRODUCED BY CELLIGEN [0190] To provide large scale adherent 3D cells, a new production system called Celligen was used. Experimental Materials and Methods [0191] PluriX ™ Bioreactor Piston Flow - As described in Example 1, above.

PAT-1243-EP-E

EP2200622 [0192] Production of adherent 3D cells by Plurix (cells

PLX) - As described in Example 1 above.

[0193] Celligen ™ Piston Flow Bioreactor - Production of adherent cells by Celligen^™ (PLX-C cells) is composed of several major steps as illustrated in Figure 8B. The process begins with the collection of placenta from a planned caesarean section in pregnancy.

[0194] Adherent cells are then isolated from whole placenta grown in tissue culture flasks (2D cultures), collected and stored in liquid nitrogen as 2D-Cell Stock (2DCS stock of 2D cells), appropriate amounts of 2DCS are thawed, washed and inoculated on carriers in bioreactors for further expansion as a 3D culture. After 1-3 weeks of growth in bioreactors, the cells are harvested and cryopreserved in the liquid nitrogen gas phase as PLX-C.

Reception of human tissue [0195] All obtained placenta were obtained from the maternity ward with the consent of the Helsinki Committee of the medical institution. Thus, donors of all placenta signed an informed consent form and donor screening and testing (IPC1) was performed. Immediately after taking the placenta from a donor (during Caesarean section), they were placed in a sterile plastic bag and then in a polystyrene box with ice compresses. The placenta was delivered and immediately placed in the quarantine until released for use by Quality Control (QC) and Quality Assurance (QA). All the following production steps were carried out in quarantine, in a clean room to obtain QC approval after the results of the mycoplasma tests and the cells were released for 2D cell growth.

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EP2200622

Recovery and processing of adherent cells [0196] To initiate the process, the entire placenta was cut under aseptic conditions in a laminar chamber, rinsed with Hank's solution and incubated for 3 hours at 37 ° C with 0.1% collagenase (1 mg collagenase / ml tissue). 2D medium was added to the cells (2D medium containing DMEM supplemented with 10% FBS, 0.25 μg / ml fungizone and 50 μg / ml gentamycin) and the digested tissue was coarsely filtered through a sterile metal sieve, collected in a sterile beaker and centrifuged (10 minutes, 1200 rpm, 4 ° C). Using gentle pipetting, suspended cells were washed with 2D medium supplemented with antibiotics, inoculated into 80 cm flasks² and incubated in a 37 ° C tissue culture incubator under humid conditions with 5% CO2. After 2-3 days, during which the cells were allowed to adhere to the surface of the flask, they were washed with PBS and 2D medium was added.

Cell growth in two dimensions (2D) [0197] Before the first passage, growth medium samples were combined with 10% of the total number of quarantine flasks and used for mycoplasma (IPC2) testing. If the cells were found to be negative for Mycoplasma (EZ-PCR Mycoplasma kit, Biological

Industries, Israel), cells were released from quarantine. After 1-2 additional passages, the cells were transferred to a clean 2D production room (2DP). In Room 2DP, culture was continued for another 3-5 passages. After passage 4, an IPC-3 sample was taken for the immune phenotype. During the whole process, the cultures were grown in a 2D medium without antibiotics in a tissue culture incubator in humid conditions with 5% CO2 at 37 ° C. After a total of 6-8 passages (9-16 cell doublings), the cells were harvested and cryopreserved as 2D-Cell Stock (2DCS).

[0198] The first passage was usually carried out after 10-15 days.

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EP2200622

Starting from passage 2 and continuing to passage 6-8, the cells were passaged when the culture reached 70-80% confluence, usually 3-5 days (1.5-2 doubles). Cells were detached from the flasks using 0.25% trypsin-EDTA (4 minutes at 37 ° C) and seeded with a culture density of 3 ± 0.2 x 10³ cells / cm². The size of tissue culture flasks increased with successive passages. The breeding process begins₂ poured into a tissue culture flask 80 cm² was continued in

175 cm²and then at 500 cm² (Triple flask) and finally the cells were inoculated into the Cell Factory 10 tray (6320 ₂ cm²).

[0199] Before cryopreservation at the end of the 2DCS growth period, the growth medium was collected and a sample prepared for sending to a recognized GLP laboratory for the Mycoplasma test (IPC 4). Cryopreservation Procedure for 2D-Cell-Stock [0200] For 2DCS cryopreservation, 2D cultured cells were harvested under aseptic conditions using 0.25% trypsin-EDTA. Cells were then centrifuged (1200 rpm, 10 ', 4 ° C), counted and resuspended in medium.

[0201] For freezing, the cell suspension was diluted 1: 1 with 2DFreezing Mixture (freezing mixture, final concentrations were 10% DMSO, 40% FBS and 50% 2D medium). About 1.5 - 2.5 x 10⁹ cells were produced from one placenta. 4 ml of cells were stored at a final concentration of 10 x 10⁶/ ml in 5ml polypropylene vials for cryopreservation. The vials were labeled and transferred to a freezer at a controlled rate for a gradual temperature reduction process (1 ° C / min), after which they were transferred to the gas phase of the liquid nitrogen freezer placed in a cold room. This material was designated as a 2D-Cell Stock lot (2DCS).

Initiation of three-dimensional (3D) breeding procedures

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EP2200622 [0202] To start the 3D culture, the appropriate number of cells were thawed (150 ± 30 x 10⁶) with 2DCS in a 2DP room and washed with 3D medium (DMEM with 10% FBS and 20 Mm Hepes) to remove DMSO before seeding into previously prepared bioreactor systems. The contents of each 2DCS vial were pipetted and diluted 1: 9 with preheated 3D medium (37 ° C). The cells were centrifuged (1200 rpm, 10 ', 4 ° C) and resuspended in 50-100 ml pre-heated 3D medium (37 ° C) in a sterile 250 ml bottle. A sample was taken and cells were counted using Trypan Blue dye to determine cell number and viability. The cell suspension was transferred in a laminar chamber to a 0.5 L seed bottle. From the inoculation bottle, the cell suspension was transferred via sterile tubes to the bioreactor by gravity.

Production of adherent 3D cells in the Celligen Bioreactor, description

Bioreactor (PLX-C) [0203] The 3D growth phase was carried out using the CelliGen Plus® or BIOFLO 310 automated bioreactor system [(New Brunswick Scientific (NBS)] shown in Figure 8C. The bioreactor system was used for cell culture in which the conditions were suitable for high cell concentrations The culture process was carried out using a bioreactor in perfusion mode. The laboratory-scale bioreactor was constructed of two main systems - the control system and the bioreactor itself (container and accessories). Process parameters were monitored and controlled through a control console that included probe connectors, motor and pumps, control loops for Dissolved Oxygen (DO), pH, perfusion and mixing (with motor), gas control system, water circulation and system heating element for temperature control and operator interface. Controlled parameters of procedures (such as temperature 1243-PAT-EP-PL

EP2200622 ra, pH, DO etc.) could be shown on the operator interface and monitored by the controller.

Cell culture procedure in bioreactors [0204] As noted in the section above, 150 ± 30 x 10⁶ 2DCS cryopreserved cells were thawed, washed and seeded in a sterile bioreactor. The bioreactor contained 30-50 grams of carriers (FibraCel® discs, NBS) made of polyester and polypropylene and 1.5 ± 0.1 L 3D substrate. The growth medium in the bioreactor was kept under the following conditions: 37 ° C, 70% dissolved oxygen (DO) and pH 7.3. Filtered gases (air, CO2, N2 and O2) were supplied as determined by a control system to maintain the DO value at 70% and the pH value at 7.3. For the first 24 hours, the medium was shaken at 50 rpm (rpm) and increased to 200 rpm by day 2. For the first 2-3 days, the cells were batch grown. Perfusion was initiated when the glucose concentration in the medium dropped below 550 mg / liter. The medium was pumped from the delivery container into the bioreactor using sterile plastic tubes. All tube connections were made under laminar using sterile connectors. Perfusion was adjusted daily to maintain a stable glucose level of about 550 ± 50 mg / liter. A sample of growth medium was taken every 1-2 days for the determination of glucose, lactate, glutamine, glutamate and ammonium (BioProfile 400 analyzer, Nova Biomedical). The glucose consumption rate and lactate formation rate of the cell culture allowed measurement of the cell growth rate. These parameters were used to determine the collection time based on accumulated experimental data.

Collecting 3D PLX-X cells from the Bioreactor [0205] The cell harvesting process started at the end of the phase

PAT-1243-EP-E

EP2200622 growth (4-10 days). Two growth medium samples were collected. One sample was prepared for sending to the recognized GLP laboratory for Mycoplasma testing according to USP and EU standards, and the other was transferred to a controlled speed freezer for a gradual temperature reduction process (1 ° C / min), after which they were transferred to gas freezer storage liquid nitrogen placed in a cold storage room in case you need to repeat the Mycoplasma test. These substrate samples were considered part of the final product tests on Mycoplasma and the results were considered part of the product release criteria.

[0206] The 3D-grown culture was collected in a Class-100 laminar space in a 3DP room as follows:

[0207] The bioreactor vessel was emptied using gravity through the tubes into the waste container. The vessel was opened by removing the lid and the carriers were aseptically transferred using sterile tweezers from the basket to the upper basket mesh (see Figure 8C). The bioreactor vessel was then closed and filled

1.5 L pre-heated PBS (37 ° C). Mixing speed increased to 150 rpm. for 2 minutes. PBS was drained through tubes by pressure or gravity into a waste bottle. The rinsing procedure was repeated twice.

[0208] To release the cells, 1.5 L of pre-heated 37 ° C trypsin-EDTA (0.25% trypsin, 1 mM EDTA) was added to the bioreactor vessel and the carriers shaken for 5 minutes at 150 rpm RPM, 37 °. The cell suspension was collected in a 5 L sterile container containing 250 ml FBS. The cell suspension was divided into 4,500 ml sterile centrifuge tubes and a sample was taken for the Mycoplasma test. Closed centrifuge tubes were transferred by active 3DP passage to the classroom filling room

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EP2200622

10000 (FR1) in which the cells were aseptically filled and cryopreserved as PLX-C.

[0209] Cell cycle analysis - PLX-C cells obtained by Celligen and PLX cells obtained by Plurix were fixed with 70% EtOH overnight, centrifuged and suspended in a solution of propidium iodide (PI) containing 2 μg / ml PI (Sigma), 0.2 mg / ml Rnase A (Sigma) and 0.1% (v / v) Triton (Sigma) for 30 minutes. The cell cycle was analyzed by FACS.

[0210] Gene expression matrix (microarray) - Adherent cells were obtained from human placenta from full-term pregnancies and expanded with Plurix or Celligen. Three different batches of cells were obtained from each of the expansion methods for further study.

[0211] RNA was extracted from cells (Qiagen-Rneasy micro kit) and applied to an Affymetrix matrix for expression of the entire genome. GeneChip® Human Exon 1.0 ST Array (Affymetrix, Santa Clara, California, USA) was used.

[0212] FACS analysis of membrane markers - cells were stained with monoclonal antibodies as described previously. Briefly, 400,000-600,000 cells were suspended in 0.1 ml flow cytometry buffer in a 5 ml tube and incubated for 15 minutes at room temperature (RT), in the dark, with each of the following monoclonal antibodies (MAbs): FITC conjugated anti-human CD29 MAb (eBioscience), PE conjugated anti-human CD73 MAb (Becton Dickinson), PE conjugated anti-human CD105 MAb (eBioscience), PE conjugated anti-human CD90 MAb (Becton Dickinson), conjugated with FITC anti-human CD45 MAb (IQProducts), PE conjugated anti-human CD19 MAb (IQProducts), PE conjugated anti-human CD14 MAb (IQProducts), conjugated with FITC anti-human HLA-DR MAb (IQProduct), conjugated with PE anti-1243-PAT-EP-PL

EP2200622 human CD34 MAb (IQProducts), FITC conjugated anti-human

CD31 MAb (eBioscience), FITC conjugated anti-human KDR MAb (R&amp;D systems), anti-human fibroblast marker (D7-FIB)

MAb (ACRIS), FITC conjugated anti-human CD80 Mab (BD), FITC conjugated anti-human HLA-ABC MAb (BD), IgG1 conjugated FITC (IQ Products), IgG1 conjugated PE isotype (IQ Products) ).

[0213] Cells were washed twice with flow cytometry buffer, resuspended in 500 µl flow cytometry buffer and analyzed by flow cytometry using FC-500 Flow Cytometer (Beckman Coulter). Negative controls were prepared with properly separated molecules of the corresponding isotype of fluorescence molecules.

Mixed lymphocyte reaction (MLR) [0214] 2 x 10⁵ derived from peripheral blood (PB) MNC (from a donor

A) stimulated with an equal amount of irradiated (3000 Rad) derived from PB MNC (from donor B). Increasing amounts of PLX-C were added to the culture. Three replicates of each group were inoculated into plates

96-well. Cells were cultured in RPMI 1640 medium containing 20₃ % FBS. Plates were administered a pulse of 1 pC H-thymidine over 18 hours

5 days old breeding. Cells were harvested on a glass fiber filter and thymidine uptake was determined using a scintillation counter.

[0215] For CFSE staining, PB-MNC cells were stained for CFSE (Molecular Probes) to measure proliferation before culture. Cells were harvested after 5 days and the intensity of CFSE staining was detected by flow cytometry.

ELISA [0216] The ELISA was performed as described previously. Briefly the MNC

PAT-1243-EP-E

EP2200622 (isolated from peripheral blood) was stimulated with 5 μg / ml ConA (Sigma),

0.5 μg / ml LPS (SIGMA), or 10 μg / ml PHA (SIGMA) in the presence of PLXC in a humidified atmosphere with 5% CO2 at 37 ° C. Supernatants were collected and used for cytokine analysis using IFN? (DIACLONE), TNF? (DIACLONE) and IL-10 (DIACLONE) ELISA kits.

Experimental results [0217] Changes in the production of Celligen compared to Plurix gave several major differences (summarized in Table 4 below).

Table 4: Comparison between the Plurix system and the Celli10 gen. System

<td>Parameter</td><td>Cell growth by Example 1</td><td>Teachings according to invention</td><td>Improvement</td>

<td>Working volume (ml)</td><td> 280</td><td> 1500</td><td>Larger scale of the process. Higher level of production in current science (2-8 population doubles)</td>

<td>Media weight (Gr)</td><td> 1,4</td><td> 30</td><td>Larger scale of the process.</td>

<td>Configuration deposit</td><td>Conical, 50 ml column</td><td>Cylindrical packed bed</td><td>Current learning better flow substrates and nutrients. Learning Example 1 - Inefficient flow due to</td>

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EP2200622

<td></td><td></td><td></td><td>narrow outlet of conical structure. Better homogeneity substrate.</td>

<td></td><td></td><td></td><td>Channel effect in</td>

<td></td><td></td><td></td><td>example teachings</td>

<td></td><td></td><td></td><td> 1.</td>

<td>Cell concentration</td><td>3 x 10⁶ ventricular</td><td>5 x 10⁶ ventricular</td><td>Better interaction</td>

<td>a hand for</td><td>rek / gr no-</td><td>rek / gr no-</td><td>cell to cell</td>

<td>trunk (cells</td><td>SNIK</td><td>SNIK</td><td>in current science</td>

<td>/ gr media)</td><td></td><td></td><td></td>

<td>Cell concentration</td><td rowspan="2">0.015 x 10⁶</td><td rowspan="2">0.1 x 10⁶ co-</td><td>Better interaction</td>

<td rowspan="2">a hand for</td><td rowspan="2">cell to cell</td>

<td></td><td></td>

<td>trunk (cells</td><td>cells / ml</td><td>cell / ml</td><td>in current science</td>

<td>/ ml)</td><td></td><td></td><td></td>

<td></td><td></td><td></td><td>Teach Example 1</td>

<td></td><td></td><td></td><td>- heterogeneous solutions</td>

<td></td><td>Inoculation</td><td></td><td>breeding house</td>

<td></td><td>in low</td><td></td><td>li cells in the bed</td>

<td></td><td>volume of</td><td></td><td>media. Too small</td>

<td></td><td>beds for 24</td><td>Inoculation</td><td>volume of ground in</td>

<td>The procedure includes</td><td>h. And for-</td><td>in volume</td><td>within the first</td>

<td>tacking</td><td>then add-</td><td>working with</td><td>24 h przebie-</td>

<td></td><td>teak ground</td><td>shaking</td><td>gu. Leads to</td>

<td></td><td>to final</td><td></td><td>inadequate</td>

<td></td><td>volume of</td><td></td><td>working conditions</td>

<td></td><td>SIDE</td><td></td><td>(acidic environment</td>

<td></td><td></td><td></td><td>Cubic)</td>

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EP2200622

<td>Duration production phase</td><td>14-21 days</td><td>4-10 days</td><td>Better product quality. Efficient collection process. Better performance. Lower process cost in current sciences</td>

<td>Way</td><td>repeated</td><td>Perfusion</td><td>Current</td>

<td>actions</td><td>Party change</td><td>Speed</td><td>sciences - moderate</td>

<td></td><td>ground two</td><td>fit in with</td><td>changes in conditions</td>

<td></td><td>times a week</td><td>depending on</td><td>on composition</td>

<td></td><td>day</td><td>changes in</td><td>ground over time</td>

<td></td><td></td><td>glucose</td><td>Running. Still</td>

<td></td><td></td><td>(subsoil</td><td>removing factors</td>

<td></td><td></td><td>changed</td><td>toxic produ</td>

<td></td><td></td><td>at concentration</td><td>forged by</td>

<td></td><td></td><td>glucose 550 ±</td><td>Morka. In mode</td>

<td></td><td></td><td>50 mg / L)</td><td>batch - lower</td>

<td></td><td></td><td></td><td>concentrations necessary</td>

<td></td><td></td><td></td><td>ingredients</td>

<td></td><td></td><td></td><td>nutrients (active</td>

<td></td><td></td><td></td><td>limitations</td>

<td></td><td></td><td></td><td>ce) Less</td>

<td>Collection procedure</td><td>Collecting in test tubes 50 ml, 3 cycles trypsinized</td><td>Collecting in bioreactor 1 trypsinization cycle</td><td>Current Teachings - More efficient process, collection is carried out in closed</td>

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<td></td><td></td><td></td><td>system. 1 cycle trypsinization better cell quality.</td>

<td>Mixing</td><td>Ground circulation between tank to the column using a peristaltic pump</td><td>Drive rotor for lifting cells</td><td>Current teachings The substrate flows by packed deposit. Better nutrient delivery and oxygen. Homogeneity of the substrate</td>

<td></td><td></td><td></td><td>Improves other control loops (temp., DO, pH)</td>

<td>Temperature control</td><td>production carried out inside incubator. indirect temperature control (incubator chambers). Transfer heat on the air interface</td><td>direct constant control. Heat transfer through coat thermal</td><td>Current teachings - more accurate temperature measurement breeding. Fast reaction. Short time to reach set point.</td>

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EP2200622

<td></td><td>Manually. Intermediate</td><td>Direct</td><td>Current science better monitoring</td>

<td>monitoring</td><td>tored</td><td>monitoring</td><td>not and control</td>

<td>temperature</td><td>temperature</td><td>online.</td><td>process. Fast</td>

<td></td><td></td><td></td><td>response to disorders</td>

<td></td><td>water.</td><td></td><td>neighing.</td>

<td></td><td></td><td></td><td>Current teachings</td>

<td></td><td></td><td></td><td>more extensive monitoring</td>

<td></td><td></td><td>Direct</td><td></td>

<td>monitoring</td><td>Lack</td><td>monitoring</td><td>and process control</td>

<td>DOWN</td><td></td><td>online.</td><td>su. Fast reply</td>

<td></td><td></td><td></td><td>copper on disorder</td>

<td></td><td></td><td></td><td>her.</td>

<td></td><td></td><td>On-line without</td><td></td>

<td></td><td></td><td>intermediate</td><td>Current Teachings -</td>

<td></td><td></td><td>control</td><td>better control</td>

<td></td><td>Lack. Only</td><td>specific</td><td>DO level. Better</td>

<td>DO control</td><td>introduction of</td><td>steady</td><td>maintaining the period</td>

<td></td><td>air</td><td>point of use</td><td>conditions</td>

<td></td><td></td><td>saying</td><td>actions</td>

<td></td><td></td><td>third, O2 and</td><td></td>

<td></td><td></td><td>N2.</td><td></td>

<td></td><td>Only visual</td><td></td><td></td>

<td></td><td>ne monitor</td><td>Control and</td><td>Current Teachings -</td>

<td>Monitoring and</td><td>wanie (Cz-</td><td>monitoring</td><td>better control</td>

<td>pH control</td><td>phenol wreath</td><td>online.</td><td>pH level</td>

<td></td><td>as an ingredient</td><td></td><td></td>

<td></td><td>backing)</td><td></td><td>Better holds</td>

<td></td><td></td><td></td><td>not specified</td>

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EP2200622

<td></td><td></td><td></td><td>operating conditions</td>

<td></td><td></td><td>Overlay</td><td>Teach Example 1 - aeration</td>

<td></td><td>Only</td><td>(device</td><td>through the device</td>

<td>Aeration</td><td>work injury</td><td>spraying</td><td>sprinkler creates</td>

<td></td><td>ly</td><td>as an opportunity)</td><td>foam that would could damage cell.</td>

[0218] Changes in the production process gave changes in the characteristics of the resulting adherent 3D cells. These differences are summarized below.

[0219] Analysis of the PLX cell cycle produced by Plurix versus PLX-C produced by Celligen - PLX-C cells obtained by Celligen were compared with PLX cells obtained by Plurix to study cell distribution between different phases of the cell cycle. As is clear from Figure 9A-B, PLX-C cells expanded by Celligen showed a typical proliferative profile (distribution of cells between different phases of the cell cycle). Specifically, 28% of the cells were in S and G2 / M phases (Figure 9A). These results indicate that the cells were harvested during proliferation and that the Celligen bioreactor conditions supported cell growth.

[0220] Microarray comparison between cells obtained with Plurix and Celligen - matrices for analysis of gene expression allowed for simultaneous tracking of expression profiles in the whole genome of adherent cells derived from human placenta from full-term pregnancy expanded with Plurix (PLX) or Celligen ( PLX-C). These results allowed the assessment of mecha1243-PAT-EP-PL

The molecularism underlying the phenotypic variation between cells obtained using these different culture methods (see Table 5 below).

Table 5: Gene expression in Celligen cells compared to Plurix cells

<td>Gene</td><td>Celligen relative to Plurix (fold change)</td><td>P value (treat)</td>

<td>interferon induced protein from tetratrikopeptide repeats</td><td> 17,52</td><td> 0,0401812</td>

<td>aldehyde dehydrogenase 1 family, member of A1</td><td> 16,76</td><td> 0,00145807</td>

<td>leukocyte-derived arginine aminopeptidase</td><td> 13,99</td><td>3.88-06</td>

<td>keratin 27 pseudogen 27</td><td> 12,25</td><td> 0,000224998</td>

<td>similar to keratin, type I cytoskeletal 18 (cytokeratin)</td><td> 11,83</td><td> 0,000304949</td>

<td>G protein-coupled receptor, family C, group 5, member A</td><td> 10,35</td><td>3.39-05</td>

<td>integrin, alpha 6</td><td> 9,84</td><td> 0,0411667</td>

<td>receptor connected to G 126 protein</td><td> 8,73</td><td> 0,00197635</td>

<td>coagulation factor III (thromboplastin, tissue factor)</td><td> 7,36</td><td> 0,012192</td>

<td>Rho GDP dissociation inhibitor (GDI) beta</td><td> 7,36</td><td> 0,00200066</td>

<td>signal peptide, CUB domain, EGF-</td><td> 7,20</td><td> 0,0255115</td>

PAT-1243-EP-E

EP2200622

<td>like 3</td><td></td><td></td>

<td>interferon induced protein from tetratrikopeptide repeats</td><td> 7,09</td><td> 0,0139777</td>

<td>homologue 1 dickkopf (Xenopus laevis)</td><td> 7,06</td><td>3.06-07</td>

<td>NAD (P) H dehydrogenase, quinone I</td><td> 6,63</td><td> 0,000282423</td>

<td>keratin 18</td><td> 6,46</td><td> 0,000514523</td>

<td>receptorlike opioid growth factor 1</td><td> 5,96</td><td> 0,00114551</td>

<td>mal, Tlike cell differentiation protein</td><td> 5,95</td><td> 0,00664216</td>

<td>neurofilament, medium polypeptide 150kD</td><td> 5,86</td><td> 0,0190611</td>

<td>containing DEP domain 1</td><td> 5,82</td><td> 0,000370513</td>

<td>cathepsin C.</td><td> 5,72</td><td> 0,00532262</td>

<td>MUSTACHE</td><td> 5,47</td><td> 0,00178153</td>

<td>serpin peptidase inhibitor, clade B (ovalbumin), member</td><td> 5,44</td><td> 0,0190218</td>

<td>carrier of dissolved substances family 7, (cationic transporter acid</td><td> 5,33</td><td> 0,00688017</td>

<td>interferon induced protein from tetratrikopeptide repeats</td><td> 5,18</td><td> 0,00357376</td>

<td>component of the kinetochore complex NUF2, NDC80, homologue (S. cere</td><td> 5,05</td><td> 0,00276524</td>

<td>SHC protein 1 binding SH2-1 domain</td><td> 4,95</td><td> 0,00430878</td>

<td>thioredoxin reductase 1</td><td> 4,86</td><td> 0,000197486</td>

<td>protein associated with metastatic cancer spit</td><td> 4,85</td><td> 0,00148024</td>

PAT-1243-EP-E

EP2200622

<td>Rho GTPase activating protein 29</td><td> 4,85</td><td> 0,0466211</td>

<td>20 cycle cell division homologue</td><td> 4,80</td><td> 0,00514206</td>

<td>him (S. cerevisiae)</td><td></td><td></td>

<td>family with sequence similarity</td><td> 4,63</td><td> 0,000125819</td>

<td>111, member B</td><td></td><td></td>

<td>PDZ binding kinase</td><td> 4,54</td><td> 0,00784983</td>

<td>homologue 2 to establish cohesion 1 (S.</td><td> 4,53</td><td> 0,000773033</td>

<td>cerevisiae)</td><td></td><td></td>

<td>guanylate binding protein 4</td><td> 4,47</td><td> 0,000215944</td>

<td>lipase A, lysosomal acid ester</td><td> 4,42</td><td> 0,0167385</td>

<td>for cholesterol (Wolman chor</td><td></td><td></td>

<td>member of the 20A kinesin family</td><td> 4,39</td><td> 0,00582352</td>

<td>KIAA0101</td><td> 4,28</td><td> 0,0105909</td>

<td>cyclin dependent kinase inhibitor</td><td> 4,25</td><td> 0,000732492</td>

<td>3 (double associated with CDK2</td><td></td><td></td>

<td>thymidylate synthetase</td><td> 4,23</td><td> 0,00685584</td>

<td>chromosome 13 open reading frame 3</td><td> 4,18</td><td> 0,000548296</td>

<td>Aurora A kinase</td><td> 4,16</td><td> 0,00632571</td>

<td>VIII-like 3 endonuclease (E. coli)</td><td> 4,14</td><td> 0,00115606</td>

<td>centrosome 55kDa protein</td><td> 4,13</td><td> 0,0021952</td>

<td>oxidized lipoprotein receptor I</td><td> 4,11</td><td> 0,0205198</td>

<td>low density (similar to lect</td><td></td><td></td>

<td>ny)</td><td></td><td></td>

<td>denticleless homolog (Drosophila)</td><td> 4,05</td><td> 0,00141153</td>

<td>anillin, actin-binding protein</td><td> 4,01</td><td> 0,010923</td>

<td>ribonucleotide reductase polypeptide</td><td> 3,98</td><td> 0,00834059</td>

<td>M2</td><td></td><td></td>

<td>domain of 1 repetition of ankyrin (muscle</td><td> 3,93</td><td> 0,00911953</td>

PAT-1243-EP-E

EP2200622

<td>cardiac)</td><td></td><td></td>

<td>transcription factor 19 (SC1)</td><td> 3,89</td><td> 0,00109627</td>

<td>keratin 18</td><td> 3,89</td><td> 0,000112551</td>

<td>condensin and non-SMC complex, G subunit</td><td> 3,88</td><td> 0,00537097</td>

<td>E2 cyclin</td><td> 3,87</td><td> 0,000203389</td>

<td>trypsinogen C.</td><td> 3,86</td><td> 0,00416276</td>

<td>small nucleated RNA, C.</td><td> 3,81</td><td> 0,0334484</td>

<td>strict protein 2 junction (zona occludens 2)</td><td> 3,81</td><td> 0,00012562</td>

<td>member of the 18A kinesin family</td><td> 3,78</td><td> 0,00134108</td>

<td>member of the 2C kinesin family</td><td> 3,77</td><td> 0,0059888</td>

<td>shugoshin-like 1 (S. pombe)</td><td> 3,76</td><td> 0,00101318</td>

<td>polo-like kinase 1 (Drosophila)</td><td> 3,75</td><td> 0,0140309</td>

<td>Thymidine kinase 1, soluble</td><td> 3,73</td><td> 0,00124134</td>

<td>transcription factor 19 (SC1)</td><td> 3,73</td><td> 0,00124327</td>

<td>transcription factor 19 (SC1)</td><td> 3,73</td><td> 0,00124327</td>

<td>Class homologue (Xenopus laevis)</td><td> 3,71</td><td> 0,00683624</td>

<td>subunit1 GINS complex (homologation Psfl)</td><td> 3,69</td><td> 0,00104515</td>

<td>microsomal glutathione S-transferase 1</td><td> 3,67</td><td> 0,041701</td>

<td>arylacetamide-like deacetylase 1</td><td> 3,67</td><td> 0,000902645</td>

<td>homologue of a component of the kinetochore complex SPC25, NDC8 (S. ce</td><td> 3,65</td><td> 0,00568662</td>

<td>integrin alpha 4 (CD49D antigen, alpha 4 VLA-4 subunit</td><td> 3,62</td><td> 0,0158411</td>

<td>catenin (cadherin-related protein</td><td> 3,57</td><td>7.46-05</td>

PAT-1243-EP-E

EP2200622

<td>n), alpha-like 1</td><td></td><td></td>

<td>discs, large homologue 7 (Drosophila)</td><td> 3,56</td><td> 0,0317074</td>

<td>viral oncogen v-myb homolog myeloblastosis (avian) -lik</td><td> 3,55</td><td> 0,0043878</td>

<td>serglycin</td><td> 3,54</td><td> 0,0443487</td>

<td>centromere protein N</td><td> 3,53</td><td> 0,000540143</td>

<td>A2 scraper</td><td> 3,53</td><td> 0,00965934</td>

<td>protein 8 heat shock 22kDa</td><td> 3,52</td><td> 0,0219583</td>

<td>sema domain, immunoglobulin domain (Ig), short alkaline domain</td><td> 3,49</td><td> 0,008548</td>

<td>11A protein activating Rho GTPase</td><td> 3,49</td><td> 0,00834174</td>

<td>Fanconi anemia, group I complementation on</td><td> 3,43</td><td> 0,00464532</td>

<td>BUB1 homolog 1 budding not inhibited by benzimidazole (yeast</td><td> 3,42</td><td> 0,0108258</td>

<td>acid specific for the ovary</td><td> 3,42</td><td> 0,00334641</td>

<td>cholinergic, muscarinic receptor 2</td><td> 3,41</td><td> 0,0320078</td>

<td>cell division cycle 2, G1 to S and G2 to M.</td><td> 3,41</td><td> 0,0017111</td>

<td>protein cytokinesis regulator 1</td><td> 3,39</td><td> 0,0325664</td>

<td>component of the minichromosome maintenance complex</td><td> 3,38</td><td> 0,00475504</td>

<td>sperm-associated antigen 5</td><td> 3,37</td><td> 0,00906321</td>

<td>maternal embryonic kinase with a slider leucine</td><td> 3,34</td><td> 0,00908391</td>

<td>small nucleated RNA, C.</td><td> 3,33</td><td> 0,0298703</td>

PAT-1243-EP-E

EP2200622

<td>carnitine palmitoyltransferase 1A (liver)</td><td> 3,33</td><td> 0,00170894</td>

<td>similar to the enzyme conjugating ubiquitin E2S (ubiquitous</td><td> 3,33</td><td> 0,000415822</td>

<td>member of the kinesin family I 1</td><td> 3,33</td><td> 0,00915145</td>

<td>NIMA (never in mitosis gene a) related kinase 7</td><td> 3,33</td><td> 0,00159114</td>

<td>ADAM metallopeptidase with type motif 1 thrombosponde,</td><td> 3,32</td><td> 0,0102751</td>

<td>transforming protein 3 containing sour coiled-coil</td><td> 3,31</td><td> 0,0014577</td>

<td>cyclin B1</td><td> 3,29</td><td> 0,0103092</td>

<td>MAD2 mitotic arrest deficient-like I (yeast)</td><td> 3,28</td><td> 0,00488102</td>

<td>dihydrofolate reductase</td><td> 3,28</td><td> 0,00178879</td>

<td>containing a NIPA-like domain 3</td><td> 3,27</td><td> 0,00164708</td>

<td>associated with the cell division cycle2</td><td> 3,26</td><td> 0,0122226</td>

<td>apolipoprotein B mRNA editing enzyme, catalytic polypeptide</td><td> 3,26</td><td> 0,00308692</td>

<td>B2 scraper</td><td> 3,25</td><td> 0,016544</td>

<td>containing the endonuclease 1 domain</td><td> 3,24</td><td> 0,000429245</td>

<td>pseudogen of dihydrofolate reductase</td><td> 3,23</td><td> 0,00141306</td>

<td>ATPase, Na +</td><td> 3,23</td><td> 0,000381464</td>

<td>replication factor C (activator 1) 3, 38kDa</td><td> 3,23</td><td> 0,00109668</td>

<td>repeating WD domain 76</td><td> 3,22</td><td> 0,0023531</td>

<td>plexstrin 2</td><td> 3,17</td><td> 0,0304429</td>

PAT-1243-EP-E

EP2200622

<td>Protein I activating GTPase Rac</td><td> 3,</td><td> 17</td><td> 0,</td><td> 00381613</td>

<td>PHD finger protein 19</td><td> 3,</td><td> 17</td><td> 0,</td><td> 000177604</td>

<td>deleted in lymphocytic leukemia</td><td> 3,</td><td> 15</td><td> 0,</td><td> 0109528</td>

<td>nej, 2</td><td></td><td></td><td></td><td></td>

<td>centromere protein I</td><td> 3,</td><td> 15</td><td> 0,</td><td> 0106816</td>

<td>BRCA1 related domain 1 RING</td><td> 3,</td><td> 14</td><td> 0,</td><td> 000540414</td>

<td>4 G signaling regulator</td><td> 3,</td><td> 13</td><td> 0,</td><td> 00781061</td>

<td>similar to STAM 1 binding protein</td><td> 3,</td><td> 11</td><td> 0,</td><td> 0181743</td>

<td>sulfiredoxin 1 homologue (S. cerevi</td><td> 3,</td><td> 10</td><td> 5,</td><td>14E-05</td>

<td>SIAE)</td><td></td><td></td><td></td><td></td>

<td>chromosome 15 open reading frame</td><td> 3,</td><td> 08</td><td> 0,</td><td> 000147331</td>

<td> 23</td><td></td><td></td><td></td><td></td>

<td>TTK protein kinase</td><td> 3,</td><td> 08</td><td> 0,</td><td> 0112171</td>

<td>non-SMC condensate II complex,</td><td> 3,</td><td> 08</td><td> 0,</td><td> 0130322</td>

<td>unit G2</td><td></td><td></td><td></td><td></td>

<td>vilina 2 (ezine)</td><td> 3,</td><td> 07</td><td> 0,</td><td> 0131934</td>

<td>stomatyna</td><td> 3,</td><td> 06</td><td> 0,</td><td> 00387095</td>

<td>protein containing a domain similar to</td><td> 3,</td><td> 06</td><td> 0,</td><td> 0419644</td>

<td>tyrosine phosphatase A</td><td></td><td></td><td></td><td></td>

<td>serpin protease inhibitor, clade</td><td> 3,</td><td> 05</td><td> 0,</td><td> 0030439</td>

<td>B (ovalbumin), member</td><td></td><td></td><td></td><td></td>

<td>member of the 4A kinesin family</td><td> 3,</td><td> 05</td><td> 0,</td><td> 0114203</td>

<td>the hypothetical DKFZp762E1312 protein</td><td> 3,</td><td> 05</td><td> 0,</td><td> 00726778</td>

<td>enzyme conjugating ubiquitin E2S</td><td> 3,</td><td> 04</td><td> 0,</td><td> 00118205</td>

<td>similar to hydroxy-dehydrogenase</td><td> 3,</td><td> 03</td><td> 3,</td><td>71E-05</td>

<td>steroid 2</td><td></td><td></td><td></td><td></td>

<td>ATPase family containing the domain</td><td> 3,</td><td> 01</td><td> 0,</td><td> 00415258</td>

<td>AAA 2</td><td></td><td></td><td></td><td></td>

PAT-1243-EP-E

EP2200622

<td>TPX2, associated with microtubules, ho-</td><td> 3,00</td><td> 0,0253137</td>

<td>molog (Xenopus laevis)</td><td></td><td></td>

<td>histone cluster 1, H4d</td><td> 3,00</td><td> 0,030183</td>

<td>member of the kinesin family 23</td><td> 2,99</td><td> 0,00790585</td>

<td>70kDa heat shock protein 2</td><td> 2,99</td><td> 0,0215102</td>

<td>origin recognition complex, sub-</td><td> 2,99</td><td> 0,00207753</td>

<td>1-like unit (yeast)</td><td></td><td></td>

<td>dihydrofolate reductase</td><td> 2,98</td><td> 0,00307793</td>

<td>hia- motility receptor</td><td> 2,97</td><td> 0,00467816</td>

<td>luronianu</td><td></td><td></td>

<td>(RHAMM)</td><td></td><td></td>

<td>3'-phosphoadenosine 5'- synthase 2</td><td> 2,97</td><td>1.43-05</td>

<td>phosphosulfate</td><td></td><td></td>

<td>glycerol-3- dehydrogenase 2</td><td> 2,95</td><td> 0,00211969</td>

<td>phosphate (mitochondrial)</td><td></td><td></td>

<td>protein 1 associated with the nucleolus and</td><td> 2,95</td><td> 0,00520875</td>

<td>spindle</td><td></td><td></td>

<td>homologue 3 diaphanous (Drosophila)</td><td> 2,95</td><td> 0,00107709</td>

<td>member of the kinesin family 14</td><td> 2,94</td><td> 0,00947901</td>

<td>histone cluster 1, H1b</td><td> 2,93</td><td> 0,0470898</td>

<td>guanine nucleotide binding protein G</td><td> 2,92</td><td> 0,00184597</td>

<td>(protein G), alpha inhi</td><td></td><td></td>

<td>minichromosome maintenance complex</td><td> 2,92</td><td> 0,000841489</td>

<td>component 8</td><td></td><td></td>

<td>cancer sensitivity candidate 5</td><td> 2,92</td><td> 0,0330594</td>

<td>Leukotriene 12-hydroxydehydrogenase</td><td> 2,92</td><td> 0,000685452</td>

<td>B4</td><td></td><td></td>

<td>glutamate-cysteine ​​ligase, subunit</td><td> 2,91</td><td> 0,00378868</td>

PAT-1243-EP-E

EP2200622

<td>modifying unit</td><td></td><td></td>

<td>forkhead box M1</td><td> 2,91</td><td> 0,0203154</td>

<td>protein associated with adipocyte differentiation</td><td> 2,90</td><td> 0,000331751</td>

<td>containing a membrane-associated domain O-acyltransferases 1</td><td> 2,90</td><td> 0,01185</td>

<td>E2T ubiquitin conjugating enzyme (putative)</td><td> 2,90</td><td> 0,00741886</td>

<td>associated with the cell division cycle 3</td><td> 2,89</td><td> 0,006289</td>

<td>integrin, alpha 3 (CD49C antigen, alpha 3 VLA-3 subunit</td><td> 2,88</td><td> 0,00574148</td>

<td>coagulation factor XIII, polypeptide B</td><td> 2,88</td><td> 0,0294465</td>

<td>RAD51 homolog (RecA homolog, E. coli) (S. cerevisiae)</td><td> 2,87</td><td> 0,000854739</td>

<td>ATP binding cassette, subfamily C (CFTR</td><td> 2,87</td><td> 0,00382491</td>

<td>family with sequence similarity 29, member A</td><td> 2,85</td><td> 0,00111165</td>

<td>domain containing SH2 4A</td><td> 2,84</td><td> 0,0323646</td>

<td>membrane protein, palmitoylated 1, 55kD</td><td> 2,84</td><td> 0,000396285</td>

<td>1B kinase regulatory subunit protein CDC28</td><td> 2,84</td><td> 0,0107391</td>

<td>protein interacting with PSMC3</td><td> 2,84</td><td> 0,00766442</td>

<td>interfacer 2 elastin microfibers</td><td> 2,84</td><td> 0,0192072</td>

<td>topoisomerase (DNA) II alpha 170kDa</td><td> 2,83</td><td> 0,0321109</td>

<td>106C transmembrane protein</td><td> 2,82</td><td> 0,000214223</td>

PAT-1243-EP-E

EP2200622

<td>histone cluster 1, H3b</td><td> 2,80</td><td> 0,0304598</td>

<td>chromosome 18 open reading frame 24</td><td> 2,80</td><td> 0,00347442</td>

<td>substrate 8 of the epithelial growth factor receptor pathway</td><td> 2,79</td><td> 0,0194949</td>

<td>high mobility group binding domain 2 nucleosomes</td><td> 2,78</td><td> 0,0030536</td>

<td>SCL</td><td> 2,78</td><td> 0,00390288</td>

<td>hect domain and RLD 4</td><td> 2,78</td><td> 0,00679184</td>

<td>ASF1 anti-silencing function 1 homologue B (S. cerevisiae)</td><td> 2,77</td><td> 0,00543408</td>

<td>thyroid hormone receptor 13 interactor</td><td> 2,76</td><td> 0,0118319</td>

<td>associated with the cell division cycle 8</td><td> 2,75</td><td> 0,00619878</td>

<td>member of the C1 kinesin family</td><td> 2,74</td><td> 0,00821937</td>

<td>high mobility group binding domain 2 nucleosomes</td><td> 2,73</td><td> 0,00384071</td>

<td>ornithine decarboxylase 1</td><td> 2,73</td><td> 0,00144868</td>

<td>viral oncogen v-myb homolog myeloblastosis (avian) -like 2</td><td> 2,71</td><td> 0,00989416</td>

<td>KIT ligand</td><td> 2,70</td><td> 0,00641955</td>

<td>with dual specificity regulated by tyrosine- (Y) -k phosphorylation</td><td> 2,70</td><td> 0,0234606</td>

<td>approval for inter-shipment transport 80 (Chlamydomonas)</td><td> 2,70</td><td> 0,0247286</td>

<td>transmembrane protein 48</td><td> 2,69</td><td> 0,00458248</td>

<td>EBNA 1 binding protein 2</td><td> 2,69</td><td> 0,00296292</td>

PAT-1243-EP-E

EP2200622

<td>ZW 10 interactor</td><td> 2,</td><td> 69</td><td> 1,</td><td>88E-05</td>

<td>exonuclease 1</td><td> 2,</td><td> 68</td><td> 0,</td><td> 00739393</td>

<td>transketolase (Wernicke syndrome</td><td> 2,</td><td> 68</td><td> 1,</td><td>92E-05</td>

<td>Korsakoff)</td><td></td><td></td><td></td><td></td>

<td>somatostatin receptor 1</td><td> 2,</td><td> 68</td><td> 0,</td><td> 0144901</td>

<td>isocitrate dehydrogenase 3</td><td> 2,</td><td> 67</td><td> 0,</td><td> 00297129</td>

<td>(NAD +) alpha</td><td></td><td></td><td></td><td></td>

<td>cytoskeleton associated protein 2</td><td> 2,</td><td> 67</td><td> 0,</td><td> 0030499</td>

<td>Minichromosome maintenance complex</td><td> 2,</td><td> 67</td><td> 0,</td><td> 00342054</td>

<td>component 4</td><td></td><td></td><td></td><td></td>

<td>DNA1 binding inhibitor, dominant</td><td> 2,</td><td> 66</td><td> 0,</td><td> 036485</td>

<td>negative helix-loop-helium</td><td></td><td></td><td></td><td></td>

<td>1B kinase regulatory subunit</td><td> 2,</td><td> 66</td><td> 0,</td><td> 0145263</td>

<td>protein CDC28</td><td></td><td></td><td></td><td></td>

<td>keratin 18</td><td> 2,</td><td> 66</td><td> 8,</td><td>40E-05</td>

<td>CD97 molecule</td><td> 2,</td><td> 66</td><td> 0,</td><td> 00994045</td>

<td>chromosome 6 open reading frame</td><td> 2,</td><td> 64</td><td> 0,</td><td> 00222408</td>

<td> 173</td><td></td><td></td><td></td><td></td>

<td>containing BTB domain (POZ) 3</td><td> 2,</td><td> 62</td><td> 0,</td><td> 0166824</td>

<td>autosomal dominant deafness 5</td><td> 2,</td><td> 62</td><td> 0,</td><td> 00235481</td>

<td>protein KIAA0286</td><td> 2,</td><td> 62</td><td> 0,</td><td> 00130563</td>

<td>Fanconi anemia, complementary group</td><td> 2,</td><td> 61</td><td> 0,</td><td> 0281405</td>

<td>D2</td><td></td><td></td><td></td><td></td>

<td>4 polo-like kinase (Drosophila)</td><td> 2,</td><td> 60</td><td> 0,</td><td> 00209633</td>

<td>ribonucleotide reductase polypeptide</td><td> 2,</td><td> 60</td><td> 0,</td><td> 000170076</td>

<td>M1</td><td></td><td></td><td></td><td></td>

<td>apple enzyme 1, dependent on NADP (+),</td><td> 2,</td><td> 59</td><td> 0,</td><td> 0435444</td>

<td>cytosolowy</td><td></td><td></td><td></td><td></td>

PAT-1243-EP-E

EP2200622

<td>condensin and non-SMC complex,</td><td rowspan="2"> 2,59</td><td rowspan="2"> 0,0216752</td>

<td>unit H</td>

<td>S 100 calcium binding protein A3</td><td> 2,58</td><td> 0,0324073</td>

<td>enzyme conjugating ubiquitin E2L 3</td><td> 2,57</td><td> 0,00343347</td>

<td>BUB 1 budding not inhibited by benzimidazole 1 homologue beta</td><td> 2,56</td><td> 0,0166047</td>

<td>glycerol kinase</td><td> 2,55</td><td>2,66-05</td>

<td>TAF9B RNA polymerase II, TATA box binding protein (TBP) -as</td><td> 2,54</td><td> 0,0170365</td>

<td>TAF9B RNA polymerase II, TATA box binding protein (TBP) -as</td><td> 2,54</td><td> 0,0170365</td>

<td>histone cluster 1, H2bg</td><td> 2,52</td><td> 0,000180822</td>

<td>high mobility group box 2</td><td> 2,52</td><td> 0,0196872</td>

<td>NIMA (never in mitosis gene a) related kinase 2</td><td> 2,50</td><td> 0,00289469</td>

<td>rich in proline 11</td><td> 2,50</td><td> 0,0357125</td>

<td>miopaladyna</td><td> 2,49</td><td> 0,0255088</td>

<td>containing brix domain 1</td><td> 2,49</td><td> 0,00471977</td>

<td>associated with the cell division cycle 5</td><td> 2,49</td><td> 0,01021</td>

<td>fucosidase, alpha-L-2, plasma</td><td> 2,49</td><td> 0,00540929</td>

<td>cyclin-dependent kinase 2</td><td> 2,49</td><td> 0,00250724</td>

<td>L-receptor B</td><td> 2,49</td><td> 0,000151784</td>

<td>Hypoxanthine 1 phosphoribosyl transferase (Lesch-Nyhan syndrome</td><td> 2,49</td><td> 0,000634057</td>

<td>containing the three-part recital 25</td><td> 2,47</td><td> 0,0456344</td>

<td>proteasome subunit (prosom, macropain), type beta, 9 (lar</td><td> 2,46</td><td> 0,0202595</td>

<td>proteasome subunit (prosom, ma-</td><td> 2,46</td><td> 0,0202595</td>

PAT-1243-EP-E

EP2200622

<td>sprinkle), beta type, 9 (lar</td><td></td><td></td>

<td>proteasome subunit (prosom, macropain), type beta, 9 (lar</td><td> 2,46</td><td> 0,0202595</td>

<td>sphingomyelin 2 synthase 2</td><td> 2,46</td><td> 0,0020701</td>

<td>transmembrane protein 62</td><td> 2,45</td><td> 0,00761064</td>

<td>glucose-6-phosphate dehydrogenase</td><td> 2,44</td><td> 0,00278311</td>

<td>protein I with PHD fingers</td><td> 2,44</td><td> 0,010191</td>

<td>retinoblastoma-like 1 (p107)</td><td> 2,44</td><td> 0,00319946</td>

<td>KIAA1524</td><td> 2,43</td><td> 0,0380688</td>

<td>ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,</td><td> 2,43</td><td> 0,00830766</td>

<td>caffeine 2 (muscles)</td><td> 2,43</td><td> 0,0459235</td>

<td>hypothetical protein LOC201725</td><td> 2,42</td><td> 0,000313319</td>

<td>cell division cycle, homologue A (S. pombe)</td><td> 2,42</td><td> 0,000341692</td>

<td>breast cancer 1, early onset</td><td> 2,41</td><td> 0,0180553</td>

<td>transaldolase 1</td><td> 2,41</td><td> 0,00199537</td>

<td>rotation of 4 homologue mRNA (S. cerevisiae)</td><td> 2,41</td><td> 0,00373104</td>

<td>glucosaminyl (N-acetyl) transferase 1, core 2 (beta-1,6-N-</td><td> 2,41</td><td> 0,0197148</td>

<td>rich in transmembrane cysteine BMP regulator 1 (cordine-like)</td><td> 2,41</td><td> 0,0267286</td>

<td>tissue factor pathway inhibitor (associated with lipoprotein</td><td> 2,40</td><td> 0,0356227</td>

<td>chromosome 16 open reading frame 59</td><td> 2,40</td><td> 0,00185191</td>

<td>glycogenin 1</td><td> 2,39</td><td> 0,0224317</td>

<td>transmembrane protein 154</td><td> 2,39</td><td> 0,0045589</td>

PAT-1243-EP-E

EP2200622

<td>tubulointerstitial inflammation antigen-like kidneys</td><td> 2,39</td><td> 0,00510812</td>

<td>CTP synthase</td><td> 2,38</td><td>8,80-05</td>

<td>phenylalanyl-tRNA synthetase, beta subunit</td><td> 2,38</td><td> 0,000245973</td>

<td>Geminin, a DNA replication inhibitor</td><td> 2,38</td><td> 0,00167629</td>

<td>Lamin B 1</td><td> 2,37</td><td> 0,0477748</td>

<td>SPC24, component of the NDC80 kinetochore complex, homologue (S. ce</td><td> 2,36</td><td> 0,00287227</td>

<td>glutathione reductase</td><td> 2,36</td><td> 0,00353875</td>

<td>ribosomal L22-like protein 1</td><td> 2,36</td><td> 0,00335381</td>

<td>fumarylacetoacetate hydrolase (fumarylacetoacetase)</td><td> 2,36</td><td>3.88-05</td>

<td>small nucleated RNA, C.</td><td> 2,35</td><td> 0,0188991</td>

<td>family with sequence similarity 64, member A</td><td> 2,35</td><td> 0,0019785</td>

<td>oncogene transforming sequence 2 epithelial cell</td><td> 2,35</td><td> 0,000571152</td>

<td>polymerase (guided by DNA), epsilon 2 (p59 s subunit)</td><td> 2,34</td><td> 0,00479612</td>

<td>glycerol kinase</td><td> 2,34</td><td>3.37-06</td>

<td>M2 glutathione S-transferase (muscles)</td><td> 2,33</td><td> 0,0402076</td>

<td>elongation factor, polymerase II RNA, 2</td><td> 2,33</td><td> 0,0130017</td>

<td>thioredoxin</td><td> 2,33</td><td> 0,009636</td>

<td>polymerase (guided by DNA) alpha 2 (70kD subunit)</td><td> 2,32</td><td> 0,0033903</td>

PAT-1243-EP-E

EP2200622

<td>breast cancer 2, early onset</td><td> 2,32</td><td> 0,00586847</td>

<td>CDC45 45-like cell division cycle (S. cerevisiae)</td><td> 2,32</td><td> 0,00735977</td>

<td>histone H2A family, member of Z</td><td> 2,32</td><td> 0,0129697</td>

<td>transporter 1, ATP binding cassette, subfamily B (MDR</td><td> 2,31</td><td> 0,0164234</td>

<td>transporter 1, ATP binding cassette, subfamily B (MDR</td><td> 2,31</td><td> 0,0164234</td>

<td>transporter 1, ATP binding cassette, subfamily B (MDR</td><td> 2,31</td><td> 0,0164234</td>

<td>homologue 3 associated with the nuclear complex (S. cerevisiae)</td><td> 2,30</td><td> 0,000373346</td>

<td>ATPase, transporting Ca ++, membrane plasma 4</td><td> 2,30</td><td> 0,023011</td>

<td>Minichromosome maintenance complex component 7</td><td> 2,30</td><td> 0,0457691</td>

<td>Protein that interacts with TIMELESS</td><td> 2,29</td><td> 0,00771062</td>

<td>1 von Hippel-Lindau binding protein</td><td> 2,28</td><td> 0,00329061</td>

<td>ras-related 2 toxin substrate C3 botulinum (rho family, sma</td><td> 2,28</td><td> 0,0292466</td>

<td>thymopoietin</td><td> 2,28</td><td> 0,0223176</td>

<td>peptidylrolyl F isomerase (cyclophilin F)</td><td> 2,28</td><td> 0,00093846</td>

<td>activated leukocyte cell adhesion molecule</td><td> 2,27</td><td> 0,00242163</td>

<td>polycomb ring finger group 5</td><td> 2,27</td><td> 0,000294142</td>

<td>Protein 1 activating GTPase Ran</td><td> 2,27</td><td>9,68-05</td>

PAT-1243-EP-E

EP2200622

<td>replication factor C (activator 1) 4, 37kDa</td><td> 2,26</td><td> 0,00164152</td>

<td>tubulin, beta 2C</td><td> 2,26</td><td> 0,000346744</td>

<td>minichromosome maintenance complex component 10</td><td> 2,26</td><td> 0,0037925</td>

<td>histone H2B family, member S</td><td> 2,25</td><td> 0,000885505</td>

<td>gamma-glutamyl hydrolase (conjugate, folylpolygammaglutamyl</td><td> 2,25</td><td> 0,0195219</td>

<td>transcription termination factor, RNA polymerase II</td><td> 2,25</td><td> 0,000393489</td>

<td>polymerase (guided by DNA), delta 2, regulatory subunit 50k</td><td> 2,25</td><td> 0,0123823</td>

<td>transporter 1, ATP binding cassette, subfamily B (MDR</td><td> 2,25</td><td> 0,00859077</td>

<td>transporter 1, ATP binding cassette, subfamily B (MDR</td><td> 2,25</td><td> 0,00859077</td>

<td>transporter 1, ATP binding cassette, subfamily B (MDR</td><td> 2,25</td><td> 0,00859077</td>

<td>histone cluster 1, H2bf</td><td> 2,25</td><td> 0,0124279</td>

<td>eukaryotic factor of initiation 1A translation, conjugated to X</td><td> 2,24</td><td> 0,00330183</td>

<td>phosphoglucomutase 2</td><td> 2,24</td><td> 0,00818204</td>

<td>peroxisomal D3, D2enoyl-CoA isomerase</td><td> 2,24</td><td> 0,00148722</td>

<td>interferon induced protein from tetratrikopeptide repeats</td><td> 2,24</td><td> 0,0177928</td>

<td>expressed in phase G-2 and S 1</td><td> 2,23</td><td> 0,0241887</td>

PAT-1243-EP-E

EP2200622

<td>minichromosome maintenance complex component 2</td><td> 2,23</td><td> 0,0021347</td>

<td>family with sequence similarity 72, member A</td><td> 2,23</td><td> 0,00143248</td>

<td>RMI1, associated with RecQ instability genome 1, homologue (S.</td><td> 2,23</td><td> 0,00294705</td>

<td>FLJ20105 protein</td><td> 2,23</td><td> 0,0127979</td>

<td>multiple clotting factor deficiency 2</td><td> 2,22</td><td> 0,0116892</td>

<td>phytoceramidase, alkaline</td><td> 2,22</td><td> 0,0157729</td>

<td>containing coiled-coil 68 domain</td><td> 2,22</td><td> 0,00227586</td>

<td>dedicator of cytokinesis 11</td><td> 2,21</td><td> 0,00697577</td>

<td>platelet derived polypeptide alpha growth factor</td><td> 2,21</td><td> 0,00176418</td>

<td>N-acyl phosphingosine amidohydrolase (non-lysosomal complexion</td><td> 2,20</td><td> 0,00728536</td>

<td>protein 2 associated with S phase kinase (P45)</td><td> 2,20</td><td> 0,00230153</td>

<td>polymerase (RNA) III (targeted through DNA) G polypeptide (32kD)</td><td> 2,20</td><td> 0,0298794</td>

<td>protein 1 interacting with ribosylation factor ADP-like 6</td><td> 2,20</td><td> 0,00139745</td>

<td>histone cluster1, H2bh</td><td> 2,19</td><td> 0,0377748</td>

<td>origin recognition complex, 5-like subunit (yeast)</td><td> 2,19</td><td> 0,049697</td>

<td>regulatory kinase 2 subunit protein CDC28</td><td> 2,19</td><td> 0,0128024</td>

<td>histone cluster1, H4c</td><td> 2,19</td><td> 0,0112695</td>

PAT-1243-EP-E

EP2200622

<td>hypothetical protein LOC729012</td><td> 2,19</td><td> 0,000446087</td>

<td>DEAD (Asp-Glu-Ala-Asp) box polypeptide 39</td><td> 2,19</td><td> 0,000340561</td>

<td>assembly factor for chromatin 1, subunit B (p60)</td><td> 2,18</td><td> 0,0119687</td>

<td>protein interacting with MLF1</td><td> 2,18</td><td> 0,0177203</td>

<td>microtubule-bound serine</td><td> 2,18</td><td> 0,00536974</td>

<td>polypeptide-related sequence Class I MHC</td><td> 2,18</td><td> 0,0165406</td>

<td>shugoshin-like 2 (S. pombe)</td><td> 2,18</td><td> 0,000852557</td>

<td>COP9 continental homologue subunit 6 (Arab</td><td> 2,18</td><td> 0,000793512</td>

<td>methylene tetrahydrofolate dehydrogenase (NADP + dependent)</td><td> 2,18</td><td> 0,00119726</td>

<td>chromosome 6 open reading frame 167</td><td> 2,18</td><td> 0,95</td>

<td>pituitary-transforming tumor 1</td><td> 2,17</td><td> 0,0485166</td>

<td>H2 ribonuclease, subunit A</td><td> 2,17</td><td> 0,00669936</td>

<td>complementary X-ray repair repair defect in Chinese chom</td><td> 2,16</td><td> 0,0369865</td>

<td>membrane protein, palmitoylated 5 (MAGUK p55 membrane subfamily</td><td> 2,16</td><td> 0,00211873</td>

<td>caryoferrin alfa 2 (RAG cohort 1, alpha importin 1)</td><td> 2,16</td><td> 0,000650645</td>

<td>containing the homology domain of pleckstrin, family A (phosphoi</td><td> 2,15</td><td> 0,0256434</td>

<td>ribosomal L39-like protein</td><td> 2,15</td><td> 0,00429384</td>

<td>caryoferrin alfa 2 (RAG cohort 1,</td><td> 2,15</td><td> 0,000700649</td>

PAT-1243-EP-E

EP2200622

<td>alpha importin 1)</td><td></td><td></td>

<td>binding to the precursor protein</td><td> 2,15</td><td> 0,00201004</td>

<td>amyloid beta (A4), family B, m</td><td></td><td></td>

<td>minichromosome maintenance complex</td><td> 2,14</td><td> 0,0018389</td>

<td>component 3</td><td></td><td></td>

<td>histone cluster 1, H2ai</td><td> 2,14</td><td> 0,0129155</td>

<td>chromosome 13 open reading frame</td><td> 2,14</td><td> 0,000702936</td>

<td> 34</td><td></td><td></td>

<td>RAD18 homolog (S. cerevisiae)</td><td> 2,14</td><td> 0,0016685</td>

<td>protein 1 binding WD and DNA</td><td> 2,13</td><td> 0,0034833</td>

<td>HMG-box</td><td></td><td></td>

<td>quinone sulfide reductase</td><td> 2,13</td><td> 0,0473641</td>

<td>(yeast)</td><td></td><td></td>

<td>chromosome 16 open reading frame</td><td> 2,12</td><td> 0,000804179</td>

<td> 63</td><td></td><td></td>

<td>Phase 1 Phosphote protein M</td><td> 2,12</td><td> 0,0271814</td>

<td>minichromosome maintenance complex</td><td> 2,12</td><td> 0,0161279</td>

<td>component 6</td><td></td><td></td>

<td>homeobox A9</td><td> 2,11</td><td> 0,00520942</td>

<td>fibroblast growth factor 9</td><td> 2,10</td><td> 0,0475844</td>

<td>(activating factor)</td><td></td><td></td>

<td>cell division cycle, homologue C</td><td> 2,10</td><td> 0,0169914</td>

<td>(S. pombe)</td><td></td><td></td>

<td>chromosome 9 open reading frame 64</td><td> 2,10</td><td> 0,0265979</td>

<td>1U2AF kinase homology motif (UHM)</td><td> 2,09</td><td> 0,0255167</td>

<td>replication factor C (activator 1)</td><td> 2,09</td><td> 0,00768959</td>

<td>2, 40kDa</td><td></td><td></td>

<td>hypothetical protein LOC440894</td><td> 2,09</td><td> 0,0103358</td>

PAT-1243-EP-E

EP2200622

<td>16kDa polypeptide small nuclear fish</td><td> 2,09</td><td> 0,0334665</td>

<td>D1 nucleoprotein</td><td></td><td></td>

<td>CSE1 1-like chromosome segregation</td><td> 2,09</td><td> 0,0013662</td>

<td>(yeast)</td><td></td><td></td>

<td>biosynthesis of phosphoino glycan anchor</td><td> 2,09</td><td> 0,0151967</td>

<td>zytolu, class W</td><td></td><td></td>

<td>centromere protein</td><td> 2,09</td><td> 0,00397056</td>

<td>family with sequence similarity</td><td> 2,09</td><td> 0,00460031</td>

<td>20, member B</td><td></td><td></td>

<td>the hypothetical FLJ40869 protein</td><td> 2,09</td><td> 0,00444509</td>

<td>guanine nucleotide binding protein</td><td> 2,08</td><td> 0,00140559</td>

<td>(protein G), gamma 11</td><td></td><td></td>

<td>calcine binding protein</td><td> 2,08</td><td> 0,00524566</td>

<td>ATP binding cassette, subfamily E</td><td> 2,08</td><td> 0,00454751</td>

<td>(OABP), member 1</td><td></td><td></td>

<td>CD44 molecule (Indian blood group)</td><td> 2,08</td><td> 0,000651436</td>

<td>component 8 of the exosome</td><td> 2,08</td><td> 0,00132017</td>

<td>family with sequence similarity</td><td> 2,08</td><td> 0,025743</td>

<td>102, member B</td><td></td><td></td>

<td>histone cluster 2, H3d</td><td> 2,07</td><td> 0,0102932</td>

<td>family with sequence similarity</td><td> 2,07</td><td> 0,000318673</td>

<td>33, member A</td><td></td><td></td>

<td>Fanconi anemia, complementary group</td><td> 2,07</td><td> 0,000255109</td>

<td>tation B</td><td></td><td></td>

<td>member of the kinesin family 22</td><td> 2,07</td><td> 0,0192406</td>

<td>histone cluster 1, H2ai</td><td> 2,07</td><td> 0,0161621</td>

<td>Vaccinia kinase 1 related</td><td> 2,06</td><td> 0,0233182</td>

<td>subunit integrator complex 7</td><td> 2,06</td><td> 0,000841371</td>

PAT-1243-EP-E

EP2200622

<td>endonuclease 1 specific for</td><td> 2,06</td><td> 0,006882</td>

<td>flap structures</td><td></td><td></td>

<td>the hypothetical FLJ25416 protein</td><td> 2,06</td><td> 0,000177531</td>

<td>ecotropic virus integration site</td><td> 2,06</td><td> 0,0171408</td>

<td>2B</td><td></td><td></td>

<td>retinitis pigmentosa 2 (recessive</td><td> 2,05</td><td> 0,0264185</td>

<td>coupled with X)</td><td></td><td></td>

<td>centromere L protein</td><td> 2,05</td><td> 0,000880856</td>

<td>cofactor required to activate tran</td><td> 2,04</td><td> 0,00141809</td>

<td>scripting Sp1, taken by</td><td></td><td></td>

<td>chromosome 20 open reading frame</td><td> 2,04</td><td> 0,0146323</td>

<td> 121</td><td></td><td></td>

<td>family with sequence similarity</td><td> 2,04</td><td> 0,00162905</td>

<td>72, member A</td><td></td><td></td>

<td>family with sequence similarity</td><td> 2,04</td><td> 0,00165234</td>

<td>72, member A</td><td></td><td></td>

<td>eukaryotic factor of initiation</td><td> 2,04</td><td> 0,00520549</td>

<td>1A translation, conjugated to X</td><td></td><td></td>

<td>elongation factor, polymerase II</td><td> 2,03</td><td> 0,0458007</td>

<td>RNA, 2</td><td></td><td></td>

<td>ATPase, Na +</td><td> 2,03</td><td> 0,0189108</td>

<td>histone cluster1, H3a</td><td> 2,03</td><td> 0,0244273</td>

<td>containing brix domain 1</td><td> 2,03</td><td> 0,00981178</td>

<td>containing sushi domain 1</td><td> 2,03</td><td> 0,0258164</td>

<td>ecto- triphosphohydrolase</td><td> 2,03</td><td> 0,00423628</td>

<td>nucleoside 6 (supposed</td><td></td><td></td>

<td>fructosamine kinase 3</td><td> 2,03</td><td> 0,00470972</td>

<td>Bloom syndrome</td><td> 2,02</td><td> 0,0209259</td>

PAT-1243-EP-E

EP2200622

<td>tubulin, alpha 1c</td><td> 2,01</td><td> 0,00862586</td>

<td>E2F transcription factor 2</td><td> 2,01</td><td> 0,0496479</td>

<td>component of the exosome 2</td><td> 2,01</td><td> 0,00649147</td>

<td>member of the kinesin family 22</td><td> 2,01</td><td> 0,0242075</td>

<td>LTV 1 homologue (S. cerevisiae)</td><td> 2,01</td><td> 0,00812652</td>

<td>Dihydrolipamide S-acetyltransferase (E2 component pyruvate</td><td> 2,01</td><td> 0,00179011</td>

<td>B-cell leukemia homologue of the monkey v-ral (related to races</td><td> 2,01</td><td> 0,012225</td>

<td>finger ring and WD domain repeat 3</td><td> 2,01</td><td> 0,0013797</td>

<td>Annexin A1</td><td> 2,01</td><td> 0,0173578</td>

<td>2 elaC homolog (E. coli)</td><td> 2,00</td><td> 0,00266504</td>

<td>alcohol dehydrogenase family 9, member of A1</td><td> 2,00</td><td> 0,00911609</td>

<td>tubulin, alpha 4a</td><td> 2,00</td><td> 0,0435427</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,00</td><td> 0,00111223</td>

<td>okulomedyna</td><td> -2,01</td><td> 0,00778869</td>

<td>similar to PI-3 kinase — bound SMG-1 kinase</td><td> -2,01</td><td> 0,0356628</td>

<td>Golgi autoantigen, subfamily and Golgin-like pseudogen</td><td> -2,01</td><td> 0,00770626</td>

<td>containing spectrine repetition, nuclear envelope 1</td><td> -2,01</td><td> 0,00438469</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,01</td><td> 0,00117582</td>

<td>sushi, nidogen and EGF-like domain 1</td><td> -2,01</td><td> 0,00161129</td>

PAT-1243-EP-E

100

EP2200622

<td>integrin, alpha V (Vitamin Receptor</td><td rowspan="2"> -2,02</td><td rowspan="2"> 0,00252702</td>

<td>nectin, alpha polypeptide</td>

<td>cyclin dependent kinase inhibitor 2B (p 15, inhibits CDK4)</td><td> -2,04</td><td> 0,0150268</td>

<td>Lysine-like oxidase 4</td><td> -2,04</td><td> 0,0120148</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,04</td><td> 0,000213956</td>

<td>calcium</td><td> -2,04</td><td> 0,00657494</td>

<td>calsyntenin 3</td><td> -2,04</td><td> 0,00300887</td>

<td>cell adhesion molecule 1</td><td> -2,05</td><td> 0,0261129</td>

<td>carrier of dissolved substances family 22 (organic cation transporter),</td><td> -2,05</td><td> 0,0137275</td>

<td>containing the RUN domain and FYVE 3</td><td> -2,05</td><td> 0,00387265</td>

<td>glucosidase, alpha; acid (disease Pompe Golga, a storage disease glycogen</td><td> -2,05</td><td> 0,000418401</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,05</td><td> 0,00988632</td>

<td>nuclear receptor 1 co-activator rich proline 1</td><td> -2,06</td><td> 0,0039587</td>

<td>membrane metal endopeptidase</td><td> -2,06</td><td> 0,0152684</td>

<td>PHD finger 21A protein</td><td> -2,06</td><td> 0,00980401</td>

<td>RHO GTPase activating protein</td><td> -2,06</td><td> 0,00705186</td>

<td>homeobox B6</td><td> -2,06</td><td> 0,00301714</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,07</td><td> 0,00032839</td>

<td>phospholipase A2 receptor 1, 180kDa</td><td> -2,07</td><td> 0,00069343</td>

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<td rowspan="2">interacting protein nuclear pore plexus</td><td>from com-</td><td rowspan="2"> -2,08</td><td rowspan="2"> 0,000352007</td>

<td></td>

<td>homologue 3 slit (Drosophila)</td><td></td><td> -2,08</td><td> 0,02844</td>

<td>interacting protein nuclear pore plexus</td><td>from com-</td><td> -2,09</td><td> 0,000414309</td>

<td>Cyclin dependent kinase 6</td><td></td><td> -2,09</td><td> 0,0456892</td>

<td>dynamin 1</td><td></td><td> -2,09</td><td> 0,00139674</td>

<td>jumonji, AT-rich domain C 1B</td><td>interactivity</td><td> -2,09</td><td> 0,00861002</td>

<td colspan="2">calcium binding and coiled-coil domain 1</td><td> -2,09</td><td> 0,00370041</td>

<td colspan="2">insulin-like growth factor receptor 1</td><td> -2,09</td><td> 0,00114467</td>

<td>interacting protein nuclear pore plexus</td><td>from com-</td><td> -2,10</td><td> 0,000377834</td>

<td>CD82 molecule</td><td></td><td> -2,10</td><td> 0,0175517</td>

<td>bromodomain adjacent to zinc finger, 2B</td><td>domain</td><td> -2,10</td><td>9.88-05</td>

<td> ---</td><td></td><td> -2,10</td><td> 0,00666187</td>

<td>synaptotagmin XI</td><td></td><td> -2,11</td><td> 0,0129428</td>

<td>KIAA 1546</td><td></td><td> -2,11</td><td> 0,000255634</td>

<td>jun B proto-oncogen</td><td></td><td> -2,12</td><td> 0,0120169</td>

<td>CXXC finger 6</td><td></td><td> -2,12</td><td> 0,0277527</td>

<td>interacting protein nuclear pore plexus</td><td>from com-</td><td> -2,14</td><td> 0,00282604</td>

<td>Cdon homolog (mouse)</td><td></td><td> -2,15</td><td> 0,0350357</td>

<td>cell B CLL</td><td></td><td> -2,15</td><td> 0,00343507</td>

<td>interacting protein nuclear pore plexus</td><td>from com-</td><td> -2,15</td><td> 0,00263888</td>

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<td>homologue 1 of Abelson's v-abl oncogene murine leukemia</td><td> -2,16</td><td> 0,0136688</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,16</td><td> 0,00583397</td>

<td>homologue 1 of tumor suppressor FAT (Drosophila)</td><td> -2,18</td><td> 0,0158766</td>

<td>transformer-2 alpha</td><td> -2,18</td><td> 0,012256</td>

<td>chimeric (chimeric) 1</td><td> -2,18</td><td> 0,0287031</td>

<td>milk fat globule protein -EGF factor 8</td><td> -2,18</td><td> 0,000987073</td>

<td>vitamin D receptor (1,25- dihydroxyvitamin D3)</td><td> -2,19</td><td> 0,000192208</td>

<td>neuroblastoma carcinogenicity suppressor, 1</td><td> -2,20</td><td> 0,00090639</td>

<td>containing the jumonji domain 1A</td><td> -2,20</td><td> 0,0188513</td>

<td>WNK protein kinase 1 with lysine deficiency</td><td> -2,21</td><td>1.57-05</td>

<td>protocoladherin beta 14</td><td> -2,21</td><td> 0,0103892</td>

<td>cortactin binding protein 2</td><td> -2,21</td><td>2.28-05</td>

<td>transcription regulator 1 containing WW domain</td><td> -2,22</td><td> 0,0379899</td>

<td>cyclin L1</td><td> -2,22</td><td> 0,00831474</td>

<td>nuclear factor of activated T cells, cytoplasmic, calcium</td><td> -2,22</td><td> 0,00786451</td>

<td>homologue 1 pellino (Drosophila)</td><td> -2,23</td><td> 0,00939357</td>

<td>golgi autoantigen, golgin subfamily a-like pseudogen</td><td> -2,24</td><td> 0,00603583</td>

<td>chromosome 7 open reading frame 10</td><td> -2,26</td><td> 0,00738442</td>

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<td>Golgi autoantigen, Golgi subfamily</td><td rowspan="2"> -2,27</td><td rowspan="2"> 0,00320764</td>

<td>ny a-like pseudogen</td>

<td>Small body specific RNA 17 Cajal</td><td> -2,27</td><td> 0,0301336</td>

<td>latent factor binding protein transforming growth beta 2</td><td> -2,29</td><td>4.08-05</td>

<td>Golgi autoantigen, Golgin a subfamily, 8A</td><td> -2,29</td><td> 0,0111179</td>

<td>inhibin, beta A (activin A, activin AB polypeptide alpha)</td><td> -2,29</td><td> 0,00877271</td>

<td>solute carrier family 41, member 2</td><td> -2,30</td><td> 0,00453672</td>

<td>forkhead box P1</td><td> -2,30</td><td> 0,0463138</td>

<td>matrix metallopeptidase 14 (inserted into membrane)</td><td> -2,31</td><td>1.93-05</td>

<td>transcription factor 4</td><td> -2,31</td><td> 0,0367869</td>

<td>oncogen jun</td><td> -2,32</td><td>7.21-05</td>

<td>gene 1 neuroepithelial cell transforming factor</td><td> -2,33</td><td> 0,0109689</td>

<td>asporyna</td><td> -2,33</td><td> 0,000659873</td>

<td>v-fos viral oncogene homolog FBJ of mouse osteosarcoma</td><td> -2,35</td><td> 0,0138624</td>

<td>Ephrins-B2</td><td> -2,36</td><td> 0,00611474</td>

<td>WD repeat and SOCS box - containing 1</td><td> -2,36</td><td> 0,0387851</td>

<td>similar to dJ402H5.2 (new protein similar to</td><td> -2,36</td><td> 0,00621503</td>

<td>PX domain containing serine</td><td> -2,38</td><td> 0,000927628</td>

<td>collagen type VII, alpha 1 (epider-</td><td> -2,38</td><td> 0,00109233</td>

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<td>molysis bullosa, dist</td><td></td><td></td>

<td>AE1 binding protein</td><td> -2,39</td><td> 0,000105628</td>

<td>peroxidazine homolog (Drosophila)</td><td> -2,40</td><td> 0,00219049</td>

<td>voltage dependent calcium channel, type L, alpha 1C vol</td><td> -2,41</td><td> 0,0189661</td>

<td>Prader-Willi syndrome chromosome region 1</td><td> -2,45</td><td> 0,0415526</td>

<td>midline 1 (Opitz</td><td> -2,45</td><td> 0,00130803</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,45</td><td> 0,00354416</td>

<td>chromosome 1 open reading frame 54</td><td> -2,47</td><td> 0,0186089</td>

<td>16A transmembrane protein</td><td> -2,48</td><td> 0,0481085</td>

<td>containing the basic helix-loop helix domain, class B, 2</td><td> -2,49</td><td> 0,00270257</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,50</td><td> 0,00316496</td>

<td>runt related transcription factor I (acute myeloid white</td><td> -2,50</td><td> 0,000607387</td>

<td>zinc finger protein 292</td><td> -2,50</td><td> 0,029832</td>

<td>fibronectin leucine-rich protein transmembrane 2</td><td> -2,51</td><td> 0,0135122</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,51</td><td> 0,00283418</td>

<td>potassium gated channel, subfamily G, member 1</td><td> -2,54</td><td> 0,0244306</td>

<td>interleukin 19</td><td> -2,54</td><td> 0,0310328</td>

<td>transforming growth factor, beta 3</td><td> -2,54</td><td> 0,0287865</td>

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<td>dihydropyrimidinase-like 3</td><td> -2,55</td><td> 0,0165203</td>

<td>Golgi autoantigen, Golgin subfamily a, 8B</td><td> -2,56</td><td> 0,0121417</td>

<td>hypothetical protein PR02012</td><td> -2,57</td><td> 0,00756704</td>

<td>SATB homeobox 2</td><td> -2,57</td><td> 0,039781</td>

<td>t-complex 11 (mouse) -like 2</td><td> -2,57</td><td> 0,0324227</td>

<td>ring finger protein 122</td><td> -2,57</td><td> 0,0236621</td>

<td>chromosome 8 open reading frame 57</td><td> -2,59</td><td> 0,00261522</td>

<td>ADAM metallopeptidase with thrombospondin type 1 motif,</td><td> -2,60</td><td> 0,0113968</td>

<td>sushi, von Willebrand factor typ A, EGF and pentraxin house</td><td> -2,63</td><td>2,23-05</td>

<td>ST6 alpha-2,6-sialyltransferase 2beta-galaktozoamidu</td><td> -2,64</td><td> 0,0216987</td>

<td>receptor 2 containing related to sortiliny VPS10 domain</td><td> -2,65</td><td> 0,00936311</td>

<td>protocoladherin beta 9</td><td> -2,66</td><td> 0,0285124</td>

<td>chromosome 5 open reading frame 13</td><td> -2,67</td><td> 0,00410172</td>

<td>Enah</td><td> -2,68</td><td> 0,0077547</td>

<td>containing the pyridoxal dependent domain of decarboxylase 2</td><td> -2,69</td><td> 0,00683647</td>

<td>similar to a protein interacting with a nuclear pore complex</td><td> -2,70</td><td> 0,0187322</td>

<td>protein interacting with the nuclear pore complex</td><td> -2,70</td><td> 0,00368967</td>

<td>transmembrane protein 119</td><td> -2,70</td><td> 0,00801387</td>

<td>chromosome 14 open reading frame 37</td><td> -2,70</td><td> 0,0182453</td>

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<td>protein containing sushi-repeat coupled with X 2</td><td> -2,71</td><td> 0,0253856</td>

<td>RING finger 3 containing the PDZ domain</td><td> -2,71</td><td> 0,00931014</td>

<td>collagen, type XII, alpha 1</td><td> -2,72</td><td> 0,000204664</td>

<td>associated with mother remodeling</td><td> -2,72</td><td> 0,000317637</td>

<td>s 5</td><td></td><td></td>

<td>collagen, type V, alpha 1</td><td> -2,72</td><td> 0,0166427</td>

<td>dystrophin related protein 2</td><td> -2,72</td><td> 0,0137557</td>

<td>ATP binding cassette, subfamily A</td><td> -2,73</td><td> 0,00131361</td>

<td>(ABC1), member 1</td><td></td><td></td>

<td>Trofinina</td><td> -2,77</td><td> 0,00298044</td>

<td>homologue 3 cornichon (Drosophila)</td><td> -2,78</td><td> 0,0261738</td>

<td>1-like formin binding protein</td><td> -2,78</td><td> 0,00290401</td>

<td>brain and acute leukemia, cytoplasma</td><td> -2,78</td><td> 0,0476919</td>

<td>tic</td><td></td><td></td>

<td>tyrosine protein phosphatase, type re</td><td> -2,80</td><td> 0,0270428</td>

<td>ceptora, U</td><td></td><td></td>

<td>the hypothetical protein MGC24103</td><td> -2,82</td><td> 0,0346673</td>

<td>induced by domain interferon</td><td> -2,83</td><td> 0,0024839</td>

<td>1 helicase C.</td><td></td><td></td>

<td>phospholipid transfer protein</td><td> -2,84</td><td> 0,00999206</td>

<td>direct early response 3</td><td> -2,87</td><td> 0,0152127</td>

<td>direct early response 3</td><td> -2,87</td><td> 0,0152127</td>

<td>ADAM metallopeptidase domain 12 (mel-</td><td> -2,87</td><td> 0,000870288</td>

<td>tryna alpha)</td><td></td><td></td>

<td>glycoprotein 2A synap-</td><td> -2,88</td><td> 0,00704212</td>

<td>tycznych</td><td></td><td></td>

<td>chromosome 9 open reading frame 3</td><td> -2,88</td><td> 0,00410177</td>

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<td>protein interacting with thioredoxin</td><td> -2,90</td><td> 0,0135494</td>

<td>early growth response 1</td><td> -2,93</td><td> 0,000425035</td>

<td>small nucleated RNA, C.</td><td> -2,94</td><td> 0,00666866</td>

<td>small nucleated RNA, C.</td><td> -2,95</td><td> 0,00765575</td>

<td>direct early response 3</td><td> -2,99</td><td> 0,0167309</td>

<td>Related protein Low-density proteins</td><td> -2,99</td><td>4.26-05</td>

<td>density 1 (alpha-2-macrogl</td><td></td><td></td>

<td>bicaudal C homolog 1 (Drosophila)</td><td> -2,99</td><td> 0,0347162</td>

<td>homeobox B2</td><td> -3,03</td><td> 0,00665994</td>

<td>small nucleated RNA, C.</td><td> -3,10</td><td> 0,0274043</td>

<td>small nucleated RNA, C.</td><td> -3,10</td><td> 0,0274043</td>

<td>matrix metallopeptidase 2 (gelatine-</td><td> -3,13</td><td>5,59-05</td>

<td>naza A, 72kDa</td><td></td><td></td>

<td>gelatinase, KIAA1641</td><td> -3,14</td><td> 0,00659194</td>

<td>collagen, type VI, alpha 3</td><td> -3,14</td><td>2.09-06</td>

<td>homeobox A2</td><td> -3,15</td><td> 0,0435423</td>

<td>SH3 and PX domain 2B</td><td> -3,15</td><td> 0,0244357</td>

<td>collagen, type VI, alpha 2</td><td> -3,16</td><td> 0,0149554</td>

<td>chromosome 9 open reading frame 3</td><td> -3,21</td><td> 0,0233723</td>

<td>small nucleated RNA, C.</td><td> -3,24</td><td> 0,0104491</td>

<td>small nucleated RNA, C.</td><td> -3,24</td><td> 0,0104491</td>

<td> ---</td><td> -3,27</td><td> 0,00488845</td>

<td>UDP-N-acetyl-alpha-D-</td><td> -3,35</td><td> 0,00964109</td>

<td>galactosomine: N-acetyllog polypeptide</td><td></td><td></td>

<td>Cholesterol 25-hydroxylase</td><td> -3,38</td><td> 0,0445558</td>

<td>KIAA1641</td><td> -3,40</td><td> 0,013175</td>

<td>ring finger protein 144</td><td> -3,40</td><td> 0,0135334</td>

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<td>wersikan</td><td> -3,41</td><td> 0,023885</td>

<td>angiopoietin-like 2</td><td> -3,42</td><td> 0,0245161</td>

<td>KIAA1641</td><td> -3,44</td><td> 0,0170531</td>

<td>homologue B of the FBJ viral oncogene mouse osteosarcoma B</td><td> -3,54</td><td> 0,00025573</td>

<td>similar to the RIKEN 1110018M03 cDNA</td><td> -3,59</td><td> 0,00516476</td>

<td>early growth response 2 (Krox-20 homolog, Drosophila)</td><td> -3,62</td><td> 0,00821813</td>

<td>dachsous 1 (Drosophila)</td><td> -3,63</td><td> 0,00697244</td>

<td>member of the 26B kinesin family</td><td> -3,64</td><td> 0,00363199</td>

<td>distal-less homeobox 5</td><td> -3,66</td><td> 0,000640157</td>

<td>similar to the KIAA0220 protein</td><td> -3,69</td><td> 0,0302619</td>

<td>insulin-like growth factor receptor 1</td><td> -3,71</td><td>3.42-05</td>

<td>tyrosine phosphatase protein, typ receptor, N</td><td> -3,77</td><td> 0,0294569</td>

<td>KIAA1641</td><td> -3,85</td><td> 0,0191782</td>

<td>protein containing a repeat of sushi, conjugated to X</td><td> -3,85</td><td> 0,00370941</td>

<td>protein 2 bound to microfibers</td><td> -3,91</td><td> 0,0152901</td>

<td>complement 1 component, subcomponent s</td><td> -3,97</td><td> 0,0395863</td>

<td>CD24 molecule</td><td> -3,99</td><td> 0,0340122</td>

<td>homeobox B3</td><td> -4,02</td><td> 0,0354368</td>

<td>I-naso-finger syndrome</td><td> -4,02</td><td> 0,00557712</td>

<td>sequence of the Kallmann team 1</td><td> -4,04</td><td> 0,000548703</td>

<td>containing leucine-rich repetition 17</td><td> -4,09</td><td> 0,0263961</td>

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<td>containing the plexin domain 2</td><td> -4,32</td><td> 0,031799</td>

<td>PTK7 tyrosine protein kinase 7</td><td> -4,42</td><td> 0,000116114</td>

<td>Superwilina</td><td> -4,43</td><td> 0,0412717</td>

<td>protein with zinc fingers 521</td><td> -4,58</td><td> 0,00668815</td>

<td>calbinidine 2,29kDa (calretinin)</td><td> -4,77</td><td> 0,0290743</td>

<td>ras gene homolog family, member</td><td> -4,79</td><td> 0,00197982</td>

<td>J</td><td></td><td></td>

<td>integrin, alpha 11</td><td> -4,80</td><td> 0,000390317</td>

<td>from, Oz</td><td> -5,05</td><td> 0,00172671</td>

<td>F-box 32 protein</td><td> -5,52</td><td> 0,0212957</td>

<td>family member of 2 raftlyn family</td><td> -5,72</td><td> 0,0260454</td>

<td>Clusterin</td><td> -5,74</td><td> 0,0303973</td>

<td>neurotrimina</td><td> -5,79</td><td>3.78-06</td>

<td>protein 1 signaling pathway inducible</td><td> -5,86</td><td> 0,000672342</td>

<td>WNT1</td><td></td><td></td>

<td>growth factor binding protein 5</td><td> -6,34</td><td> 0,011614</td>

<td>good for insulin</td><td></td><td></td>

<td>sulfatase 2</td><td> -6,34</td><td>5,88-05</td>

<td>protein 4 associated with microfibrils</td><td> -6,93</td><td> 0,00155578</td>

<td>2 junctional adhesion molecule</td><td> -7,07</td><td> 0,0306758</td>

<td>containing 1 fibronectin domain</td><td> -7,29</td><td> 0,0334696</td>

<td>type III</td><td></td><td></td>

<td>sarcoglycan, delta (35kDa associated with</td><td> -7,37</td><td> 0,000881984</td>

<td>glycoprotea dystrophin</td><td></td><td></td>

<td>hefaestyna</td><td> -7,53</td><td> 0,0123141</td>

<td>serpin peptidase inhibitor, clade F</td><td> -7,66</td><td> 0,00362941</td>

<td>(alpha-2 anti-plasmas</td><td></td><td></td>

<td>cystatin SN</td><td> -7,96</td><td> 0,0496433</td>

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<td>hemicentyna1</td><td> -8,18</td><td> 0,0461603</td>

<td>tenascin C (hexabrachion)</td><td> -8,32</td><td>8.26-05</td>

<td>biglikan</td><td> -8,62</td><td> 0,00161284</td>

<td>transmembrane, andro-induced RNA</td><td> -11,20</td><td> 0,000100935</td>

<td>prostate gene</td><td></td><td></td>

<td>carboxypeptidase E</td><td> -11,22</td><td> 0,00738131</td>

[0221] Marker expression on PLX-C cells - surface antigens expressed by PLX-C were tested using monoclonal antibodies. The results showed that PLX-C cells were characterized by positive markers: CD73, CD29 and CD105 and negative markers: CD34, CD45, CD 19, CD14 and HLA-DR (data not shown). The immunological phenotype test specifications were set as:&gt; 90% for all positive markers and &lt;3% for all negative markers.

[0222] Furthermore, as shown in Figures 10A-B, PLX-C cultures did not express endothelial markers as shown by negative staining for the two markers CD31 and KDR. However, PLX-C expression of a fibroblast-typical marker was evident (D7fib expression, Figure 10C).

[0223] Immunogenicity and immunomodulating properties of cells

PLX-C - as PLX-C consists of adherent cells obtained from placenta, it is expected to express HLA type I, which is expressed by all body cells, and is known to induce an alloreactive immune response. HLA ty20 pu II and other co-stimulatory molecules are typically expressed only on the surface of Antigen Presenting Cells (APC).

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EP2200622 [0224] To study the immunogenicity of the obtained PLX-C cells, the expression of co-stimulatory molecules on the surface of membranes of these cells was examined. FACS analysis showed the absence of CD80, CD86 and CD40 on PLX-C cell membranes (Figures 11A-C). Furthermore, PLX-C expressed low HLA class I levels detected by HLA A / B / C staining (Figure 11D). Expression of stimulatory and co-stimulatory molecules was similar to MCS derived from bone marrow (BM) (as shown in Figures 11A-D).

[0225] To further study the immunogenicity as well as the ability to immuno-modulate PLX-C cells, mixed lymphocyte reaction (MLR) tests were performed. As shown in Figure 12A-B, PLX-C cells also escape allorrecognition and reduce T cell response as measured by thymidine incorporation. Furthermore, the decrease in lymphocyte proliferation (assessed by CPM measurement) increased with an increase in the number of PLX-C cells (in a dose-dependent manner). PLX-C also reduced leukocyte proliferation after mitogenic stimuli such as concavalin A (Con A, Figure 12B) and phytohemagglutinin (PHA), and non-specific stimulation by anti-CD3, anti-CD28 (data not shown).

[0226] To study the mechanism of action by which PLX-C immunomodulates lymphocyte proliferation, and to determine whether this action is mediated by cell-cell interactions or cytokine secretion, PB-derived mononuclear cells (MNCs) were stimulated with PHA using trans well method (which prevents runs cell-cell contacts but allows cytokine diffusion between two compartments). The results showed that inhibition of proliferation was maintained even with inhibition of cell-cell contacts (data not shown).

[0227] Cytokine secretion - as outlined herein above, PLX1243-PAT-EP-PL

112

EP2200622

C lower the rate of lymphocyte proliferation, probably by soluble factors. Further studies of lymphocyte secreted cytokines in response to PLX-C were performed to clarify the mechanism of action of PLX-C. As shown in the Figures

13A-B, culturing mononuclear cells from PLX-C slightly reduces secretion of the pro-inflammatory cytokine INFy and dramatically reduces TNFα secretion (even in the presence of small amounts of PLXC). In addition, after stimulation with lipopolysaccharide (LPS), IL-10 secretion by PB-derived MNCs increased in the presence of PLX-C, while TNFα secretion decreased in a dose-dependent manner (Figure 13C).

EXAMPLE 5

BIX STRUCTURE

Experimental materials and methods

Transfection of PLX-C cells with a luciferase expression vector [0228] PLX-C cells were stably infected with a lentiviral construct expressing the luciferase gene under the CMV promoter (Figure 14).

Production of infectious virus [0229] 293TN producing cells were cultured in DMEM medium (Gib20 co) supplemented with serum and antibiotics for 2-3 days (50-70% confluence) before transfection. A mixture of 10 μg packaging plasmid and 2 μg expression construct and 20 μL Plus ™ Reagent (Invitrogen) was added to 400 μL DMEM without supplements. The mixture was incubated 15 min at room temperature (RT) and Lipo-fectamine ™ was added (30 ml diluted in 400 ml DMEM was added). The mixture was incubated at RT for 15 min. 293TN cells were washed and transferred to 2% serum media and transfection mixture was added. Cells were incubated in a CO2 incubator at 37 ° C overnight and medium was harvested 24 - 60 hours. after infection. Maximal

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EP2200622 virus production was obtained after 48 hours. The medium was harvested and centrifuged at 3000 rpm at room temperature for 5 minutes to pellet cell debris. After centrifugation, the supernatant was filtered through Millex-HV 0.45 pm PVDF filters (Millipore, Cat.

# SLHVR25LS).

PLX-C infection [0230] PLX-C cells were inoculated in a 24-well plate with dense₅ 0.6-1 x 10 cut⁵ cells per well in complete medium 24 hours before virus infection. After 24 hours, 0.5 ml of virus suspension (diluted in Polybrene medium at a final concentration of 5-8 pg / ml) was added. The cells were incubated for 24 hours, then the medium was replaced with full DMEM medium and the cells were incubated at 37 ° C with 5% CO2 overnight. On day 4, the culture reached confluence and was split 1: 3 to 1: 5, the cells were allowed to grow for 48 hours in full DMEM then the cells were analyzed for luciferase expression.

[0231] Infection yields were close to 100%. Luminescence evaluation in live cells and in live mice was performed using the Lumina IVIS imaging system, which includes a highly sensitive CCD camera that captures luciferase luminescence signal.

[0232] Two weeks after infection, 2 x 10 was injected⁶ IM or IV cells into SCID / Beige, NOD / SCID, SCID and Balb / C mice. Injected cells were tracked using the described IVIS system

Experimental results [0233] As can be seen from the results, CXL cells continued to divide after injection and luciferase expression levels in growing cells remained strong and stable (Figure 15).

[0234] Following injection of PLX-C cells into Balb / C mice were examined

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EP2200622 biodistribution formula. As can be seen from the results, the cells disappeared 72 hours. after IM injection (data not shown). However, cells

PLX-C maintained consistent high levels of luciferase expression, in vitro, for more than three weeks (data not shown).

[0235] As shown in Figures 16A-D, cells injected with IM into SCID / Beige immunodeficient mice persisted for up to 5 days at the injection site and were then not observed. IV injected CXL cells into SCID / Beige mice migrated after 24 hours. to the lungs, then to the injection site (probably wandering intentionally to the site of injury). Then the cells gradually disappeared and were not observed after 3-4 weeks.

EXAMPLE 6

ADHERENT CELLS ARE AVAILABLE FOR THE TREATMENT OF LIMB ARCHITAMIS IN VIVO [0236] To determine whether placement of placental adherent cells can reduce ischemic damage and improve clinical and motor function, the hind limb ischemia model was used as follows.

Materials and experimental methods [0237] Hindlimb ischemia model - Hindlimb ischemia was induced in 20 male Balb / c mice that are not immunodeficient, 8-10 weeks of age, body weight about 25 g ± 20%. The animals were housed according to the National Institute of

Health (NIH) and Association for Assessment and Accreditation of

Laboratory Animal Care (AAALAC). Animals were kept under standard laboratory conditions. The animals were kept in a climate-controlled environment. The temperature range was between 20-24 ° C and the relative humidity (RH) was between 30-70% with a cycle of 12 hours of light and 12 hours of darkness.

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EP2200622 [0238] Animals were randomized using a computer generated "Research Randomizer" randomization program and divided into 2 groups of 10 animals. One group received 1 x 10 intramuscular (IM) injection⁶ placenta-derived adherent cells (PLX-C) cells and the second group served as control and received PBS injections.

[0239] Surgical procedures - A 1-1.5 cm cut was made in the skin in the inguinal region. The femoral artery was ligated twice with 6-0 silk and cut distally to the ligature. The wound was closed with 3-0 silk and the mice were allowed to recover. Five hours after surgical resection of one femoral artery, mice received 1 x 10 IM injections⁶ placental adherent cells (PLX-C) in a total volume of 50 μl at 2 injection sites. Control animals received an identical injection with PBS (Gibco), see Table 6 below.

Table 6: Pilot study in PLX-C in a mouse model of hind limb ischemia

<td>Group</td><td>treated</td><td>Number of mice</td><td>Dose</td><td>Party</td><td>Time to kill after</td>

<td>test</td><td>Wani</td><td>per group</td><td>cell</td><td></td><td>a dose of 21 days</td>

<td> 1</td><td>PLX-C</td><td>n = 10</td><td>1x10⁶</td><td>CG13</td><td>n = 10</td>

<td></td><td>them</td><td></td><td></td><td> .0</td><td></td>

<td> 2</td><td>PBS</td><td>n = 10</td><td> 0</td><td>ON</td><td>n = 10</td>

[0240] Further examination - Blood flow on both sides of the legs was measured 3 times using a non-contact Doppler laser immediately after surgery and on days 6, 9, 14 and 21 after surgery, and is expressed as the ratio of flow in the ischemic limb to that in the normal limb [ Tokai. J. et al.].

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EP2200622 [0241] Macroscopic ocean of ischemia severity - The ischemic limb was evaluated macroscopically on days 1, 6, 9, 14, 21, to complete the study using morphological graded scales for the necrosis area; class 0: no necrosis, class I: necrosis limited to toes (loss of toes), class II: necrosis extending to the back of the foot (loss of foot), class III: necrosis extending to crus (shaving) (loss of knee), class IV : necrosis extending to the thigh (complete loss of hind limb) [Tokai. J. et al.].

[0242] In vivo assessment of limb function and ischemic injury - A semi-quantitative ocean of impairment of ischemic limb use was to be performed serially as follows: 3 = foot dragging, 2 = no dragging but no plantar flexion, 1 = plantar flexion, and 0 = bending toes oppose the gentle tail traction. (Rutherford et al.,

1997).

[0243] Molecular and biochemical analysis - In addition to clinical evaluation, molecular and biochemical samples were obtained on day 21 and are currently being analyzed to better understand the molecular mechanisms underlying improved healing in the placental adherent cell (PLX-C) injection group.

Experimental results [0244] Implantation of placental derived adherent cells significantly improves blood flow in the hip and foot of the hind limb ischemia model - To test the performance of adherent cells in vivo, the mice underwent arterial ligation followed by intramuscular injection of adherent cell derived placenta and blood flow measured in hips and foot (both sides of the body) using a non-contact Doppler laser from above

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EP2200622 a fixed period of time after treatment. As shown in Figure 17, PLX-C injection clearly improved blood flow (BF) to the injured limb, as determined by assessing blood flow, increasing limb function, increasing capillary density, reducing oxidative stress, and endothelial damage. In terms of blood flow, the effect was demonstrated 9 days after injection and was observed throughout the study. In the PLX-C treated group, BF increased from 24 ± 2.3 to 80 ± 4.7%, while in the control group treated with BF it was in the range of 35 ± 2 to 54 ± 4.5% - in the hip / implantation area (day 0 relative to day 21, respectively). Similar to the hip area, but to a lesser extent, an increase in BF was also demonstrated in the paw region of PLX-C-treated mice. Thus, in the BF excipient group, it increased from 12 ± 0.6 to 46 ± 4.9%, while in the PLX-C BF group, it increased from 10 ± 0.7 to 52 ± 5.5% (day 0 versus day 21 respectively) as shown in Figure 17.

[0245] Adherent cells are able to improve limb function in vivo - To further evaluate the in vivo effects derived from placenta of adherent cells, limb function in treated mice was evaluated using the evaluation system described in Materials and Experimental Methods above. As shown in Figure 18, mice treated with adherent cells showed a significant improvement in limb function (2.5 ± 0.2 versus 2.1 ± 0.2 control versus PLX-C group, respectively, a significant effect should be noted on day 21 post-treatment ). However, the degree of improvement was comparable, suggesting that PLX-C did not show a large change in function recovery under the conditions of the current study.

[0246] Macroscopic evaluation of ischemic severity showed that at

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To control vehicle treated group, finger-restricted necrosis was observed in two animals on day 6. In the PLX-C treated group, finger-restricted necrosis was only shown in one animal and only after 14 days. Post mortem immunohistochemical analyzes of PLX-C-treated limbs showed a significant increase in the number of new capillary vessels (vessels) supplying the limb and suggesting that PLX-C have the ability to promote angiogenesis (Figure 19).

[0247] Finally, reduced oxidative stress and reduced endothelial inflammation (which was a surrogate parameter for improved endothelial function) was observed in treated animals noticed in PLX-C treated mice (Figures 20A-B). This was probably the result of increased oxygen supply in PLX-C-treated mice, but not in PBS-treated control mice.

[0248] In conclusion, compared to the control, PBS injected mice, none of the PLX-C injected mice showed any adverse clinical symptoms in response to intramuscular (im) cell administration. Thus, PLX-C induces an increase in blood flow, probably due to angiogenesis supported by histological assessment of the injured limb. In addition, the delay in necrosis development and the difference in the number of animals affected suggest a clinical response.

Implantation of placental derived adherent cells [0249] Another efficacy study in Balb / C mice including safety endpoints (i.e. coarse necropsis and histopathological analysis of selected organs) was performed as described in the materials and methods section above.

[0250] Seven groups of mice were used in this study, each composite

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EP2200622 from 10 male Balb / c mice (hind limb ischemia) as given in Table 7 below. Ischemia was not induced in one group of 10 mice (to test the overall safety and tolerability of PLX-C cells in normal healthy animals). After induction of ischemia, control buffer or PLX-C cells were administered to the affected limb, and the mice were observed for up to 1 month after dosing. A single group of mice received two independent injections into the affected limb, separated by 1 week (Days 1 and 8). Blood flow was monitored by Doppler analysis and the severity of ischemia was assessed macroscopically and behaviorally up to 30 days after dosing and mice were killed and tissues saved for histological analysis.

Table 7 Study of PLX-C efficacy in hind limb ischemia model in mice

<td>Group</td><td>Treatment</td><td>Quantity</td><td>number</td><td>Party</td><td>Time to kill</td>

<td>tested</td><td></td><td>cell</td><td>treatments</td><td></td><td>after dose</td>

<td>No.</td><td></td><td>(dose)</td><td></td><td></td><td>niu 30 days</td>

<td> 1</td><td>PLX-C</td><td>1x10⁶</td><td> 1</td><td>CG13.0</td><td>10 rf</td>

<td> 2</td><td>PLX-C</td><td>1 x 10⁶</td><td> 1</td><td>CG25.0</td><td>10 rf</td>

<td> 3</td><td>PLX-C</td><td>1 x 10⁶</td><td> 2</td><td>CG25.0</td><td>10 rf</td>

<td> 4</td><td>PLX-C</td><td>0.5 x 10⁶</td><td> 1</td><td>CG13.0</td><td>10 rf</td>

<td> 5</td><td>PLX-C</td><td>0.1 x 10⁶</td><td> 1</td><td>CG13.0</td><td>10 rf</td>

<td> 6</td><td>Control</td><td>ON</td><td> 1</td><td>ON</td><td>10 rf</td>

<td></td><td>substrate for</td><td></td><td></td><td></td><td></td>

<td></td><td>freezing</td><td></td><td></td><td></td><td></td>

<td> 7*</td><td>PLX-C</td><td>1 x 10⁶</td><td> 1</td><td>CG13.0</td><td>10 rf</td>

<td></td><td></td><td></td><td></td><td>CG25.0</td><td></td>

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EP2200622 [0251] In this study, different batches of PLX-C were administered at three concentrations. The results showed that 0.1 x 10⁶ and 0.5 x 10⁶ PLX-C had a small therapeutic effect. A noticeable improvement in blood flow was observed up to day 29 (end of experiment) in 1 x 10 treated animals⁶. This improvement in blood flow was significant (p &lt;0.05) in the 2M group (G.C25 lot) compared to vehicle-injected control mice. In addition, a second injection of the same batch of cells significantly improved BF on day 15 compared to a single injection (55 ± 24 versus 31 ± 12.9 and 27 ± 12.5%, respectively). Macroscopic evaluation of ischemic severity showed that there was a trend to improve in the 1 x 10 groups⁶ (1M &amp; 2M) compared to the control vehicle treated group (6M).

In total, these results demonstrate the efficacy of adherent cells in the induction of vascularization (e.g. blood flow) and improvement of limb function in a mouse hind limb ischemia model, and suggests the use of these cells (e.g., derived from placenta adherent cells) for the treatment of limb ischemia diseases .

EXAMPLE 7

PLX-C FOR THE TREATMENT OF UDAR [0253] The purpose of this study was to evaluate the therapeutic efficacy of systemic (intravenous) transplantation of human PLX-C derived from placenta adherent cells for the treatment of stroke.

Experimental materials and methods

Subjects, surgery and transplantation [0254] Male rats with spontaneous hypertension, suffering from hypertension, hypercholesterolemia, diabetes and microangiopathy were used. The animals were kept under constant conditions for temperature, humidity and light / dark cycle. Individuals were randomly assigned to experimental groups (see

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Table 8, below).

Table 8: Rat treatment groups in stroke therapy

<td>Group No.</td><td>Treatment</td><td>Number of animals</td>

<td> 1</td><td>PLX-C, lot 1 one pass</td><td>N = 8</td>

<td> 2</td><td>PLX-C, lot 1 two applications</td><td>N = 7</td>

<td> 3</td><td>PLX-C, lot 2 one pass</td><td>N = 8</td>

<td> 4</td><td>PLX-C, lot 2 two applications</td><td>N = 7</td>

<td> 5</td><td>Control - excipient solution</td><td>N = 12</td>

[0255] Animals received a single or double dose of 1x10⁶ PLX-C from different lots. All transplantation procedures were carried out intravenously. The two injection group was transplanted 10 and 24 hours after cerebral ischemia, while single transplants were carried out 24 hours after the stroke. All transplanted cells were pre-labeled with fluorescent PKH26 dye.

[0256] Experimental cerebral ischemia was performed by permanent occlusion of the right cerebral artery. It should be noted that one animal died after anesthesia.

Magnetic resonance imaging (MRI) studies [0257] MRI lesion development was performed on days 1, 8, 29 and 60 using a 1.5T scanner (Philips). Stroke volume and brain atrophy were measured and calculated as mean values ​​obtained by the three working people without knowing the animals' assignment to study groups using the T2 coronary sequence.

Behavioral tests [0258] Functional changes were measured using two dependent test situations. The Beam Walk test is after 1243-PAT-EP-PL

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EP2200622 a universal test used for the quantitative determination of sensory-motor deficiencies. The rats were conditioned to run through a horizontal mounted beam with a rat home cage at the end. Transit time was measured five times and recorded as an average daily value. Hanging on the crossbar was assessed after 20 seconds and falling after 30 seconds. Measurements were carried out daily during the first week and every seven days until the end of the observation period.

[0259] The second test, modified neurological focus score (mNSS) contained additional sensory, motor and reflex aspects. The mNSS score was expressed as a score between 1 and 18, while points between 1 and 6 indicated mild, 7 to 12 moderate and 13 to 18 severe damage. Assessment of mNSS score was performed on days 1, 4, 7, 14, 21, 28, 35, 42, 49 and 56 after cerebral ischemia.

Histology [0260] At the end of the experimental period, all rats were perfused through the heart with a 4% formalin solution. The brains excised were cryopreserved and cut into sections of 30 pm thickness. To assess the glia reaction, an immunohistochemical study with primary anti-GFAP antibody was performed. A 750 pm (semi-quantitative) area near the stroke limit was examined for GFAP + cell density. To study astroglia activity, 15 regions were included with average spaces of 0.6 mm between them.

Statistics [0261] All collected MRI analysis and histological data were checked for Gaussian distribution and analyzed for statistically significant differences using the test

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ANOVA and ANOVA ranking test, respectively.

[0262] The data collected in the Beam Walk and mNSS tests were subjected to detailed statistical analysis, including repeated measurements of individuals as well as individual development of individuals over time (stratified analysis). A random uptake model was used to compensate for differences between individuals regarding the degree of brain damage. Therefore, the data collected with the Beam Walk test had to be transformed into a category system. Here, time values ​​less than 5 seconds were considered as category (0), 5 to 10 seconds as category (1), 10 to 15 seconds as category (2), 15 to 20 seconds as category (3), overhang as category (4) ) and falling as a category (5).

Experimental results

Weight [0263] Periodic weighing allowed a good assessment of the subject's overall health. Initial weight loss due to anesthesia and surgical intervention was observed in all groups (data not shown). Then rapid weight normalization and stable performance were observed until the end of the experiment on day 60 (data not shown). Experimental groups showed homogeneous body weight progression.

Beam Walk Test [0264] All experimental groups showed a significant decrease in the Beam Walk category during the experiment (data not shown).

A significantly lower decrease in the Beam Walk category was observed in experimental group 1 (PLX-C batch 1 single administration) compared to the control group (-0.01247 versus -0.02931, respectively). There was no evidence of statistically significant differences between experimental group 3 (PLX-C lot 2 one administration) and group kon1243-PAT-EP-PL

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EP2200622 trainer (data not shown).

Modified Neurological Severity Score (mNss) [0265] All experimental groups showed a significant decrease in neurological scores (data not shown). Comparison of mNSS results of PLX-2-treated subjects (PLX-C double administration) showed statistically significant superiority compared to the control group. Double transplantation of lot 2 (group 3) showed a significant improvement in the mNSS test compared with a single injection of the same lot (data not shown).

Measurement of stroke volume [0266] Magnetic resonance imaging is a highly complex method of assessing the degree of brain damage and lost tissue in vivo. Taking into account inter-individual variations, stroke volume was determined as a percentage of stroke volume on Day 1, individually. The stroke volume on day 1 did not differ significantly between the experimental groups. Overall stroke volume development showed an approximate 50% decrease between day 1 and day 8. This was mainly the result of retrogression of initial brain edema. An in vivo injury development study using MRI showed that group 4 individuals (PLX-C batch 2 double administration) showed a significantly reduced stroke ratio on day 60 (0.48 ± 0.02 versus 0.60 ± 0.03, respectively). results not shown).

Taken together, these results indicate that intravascular administration of PLX-C gave a significant improvement in functional recovery in both functional tests in the treatment of stroke. Furthermore, significant and statistically significant superiority of PLX-C double transplants was observed compared to the corresponding single injection.

[0268] Confirmation of observed behavioral improvement for po1243-PAT-EP-PL

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EP2200622 MRI power was evident in subjects treated twice with PLX-C. In addition, significant reduction in stroke volume and brain atrophy was observed at the end of the experiment. Moreover, in both functional tests, stable improvement of functional recovery after double-dose PLX-C transplantation was observed compared to controls and unverifiable effects with single injections.

COMPARATIVE EXAMPLE 8

TREATMENT OF PATHOLOGIES REQUIRING REGENERATION AND / OR REPAIR OF JOINT TISSUE

Treatment of pathologies requiring bone regeneration and / or repair using the adherent cells of the invention [0269] Animal models (e.g., white mature New Zealand rabbits) were used to study the effects of adherent cells of the invention (which are derived from placenta or adipose tissue and are obtained from 3D cultures e.g. PLX-C cells) for the healing of critical segmental lesions in the femur. Animals were randomly assigned to one of three groups. Group A animals were injected with 1-10 x 10⁶ adherent cells (PLX-C cells) in place of damage. Group B animals were injected with PBS. Group C animals were not treated with the defect. Radiographs were prepared immediately after the operation and at intervals of one week. After 12 weeks, the animals were killed, the affected femurs were removed, and uncalcified histological sections were prepared from defects and adjacent bones. Mechanical, histological and histomorphometric tests are performed to examine the healing and bone formation at and around the defect site. In addition, reverse transcription-polymerase chain reaction (RT-PCR) is performed to detect type-I and type-II collagen mRNA.

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Treatment of pathologies requiring tendon regeneration and / or repair using the adherent cells of the invention [0270] Animal models (e.g. skeletal mature New Zealand white rabbits) are used to study the effect of the adherent cells of the invention (e.g. PLX-C cells) on tendon healing. Tendon hallucis longus travels to the calcaneal tunnel with a diameter of 2.5mm. Bone tunnels are treated with or without PLX-C. Animals were randomly assigned to one of three groups. Group A animals were injected with 1-10 x 10⁶ cell

PLX-C to the site of damage or IV. Group B animals were injected with PBS. In group C animals, the defect was not treated. Three specimens from each group were collected 2, 4, and 6 weeks after surgery and evaluated for morphological features of tendon healing at the bone interface by using conventional histology and immunohistochemical localization of Type I, II, and III collagen.

Treatment of pathologies requiring cartilage regeneration and / or repair using the adherent cells of the invention [0271] Animal models (e.g. skeletal mature New Zealand white rabbits) are used to study the effect of the adherent cells of the invention (e.g. PLX-C cells) on healing cartilage. Full thickness damage to the articular cartilage of the patellar furrow of the left distal femur is carried out. A lobe of about 6 mm is removed from the fascia on the quadriceps and sutured to the periphery of the artificial defect with catgut 6-0. The animal was randomly assigned to one of three groups. Group A animals were injected with 1-10 x 10⁶ PLX-C cells to a defect site or IV. Group B animals were injected with PBS. In group C animals, the defect was not treated. The animals were killed. Fourteen weeks after implantation of PLX-C cells to osteochondral defect1243-PAT-EP-PL

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EP2200622 go, distal femurs are resected and histological evaluation is performed and samples are semi-quantitatively classified based on the main nature of the repaired tissue, matrix staining, surface regularity, repair thickness, application between repaired cartilage and surrounding normal cartilage, no signs of degeneration in repaired tissue and no degenerative changes in the surrounding normal cartilage.

Treatment of pathologies requiring ligament regeneration and / or repair using the adherent cells of the invention [0272] Animal models (e.g. skeletal mature New Zealand white rabbits) are used to study the effect of the adherent cells of the invention (e.g. PLX-C cells) on healing ligaments. One-sided circular defects with a diameter of 8 mm will be carried out. Animals were randomly assigned to one of three groups. Group A animals were injected with 1-10 x 10⁶ PLX-C cells to the site of injury or IV. Group B animals were injected with PBS. In group C animals, the defect was not treated. Animals are killed fourteen weeks after implantation of PLX-C cells for ligament damage. Histological evaluation is performed and the samples are semi-quantitatively classified based on the dominant nature of the repaired tissue.

[0273] It will be appreciated that certain features of the invention, which for clarity are described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, certain features of the invention, which for brevity are described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0274] Although the invention has been described in connection with its specific embodiments, it is evident that many alternatives, fashions

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EP2200622 ficitions and variants will be clear to those skilled in the art.

REFERENCES (Additional references are cited in the text) [0275]

Bauer, Thomas W., Muschler, George F., Bone Graft Materials: An Overview of the Basic Science. Clinical Orthopedics &amp; Related Research. 371: 10-27, February 2000.

Carstanjen B, Desbois C., Hekmati M, and Behr L. Successful engraftment of cultured autologous mesenchymal stem cells in a surgically repaired soft palate defect in an adult horse. Can J Vet Res. 2006 April; 70 (2): 143-147.

Bruder SP, et al. 1998 The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am. 80 (7): 985-96

Chao Wan, Qiling He, Gang Li, 2006. Allogenic peripheral blood derived mesenchymal stem cells (MSCs) enhance bone regeneration in rabbit ulna critical-sized bone defect model. Journal of Orthopedic Research 24 (4) 610-618.

Herthel DJ 2001, Enhanced Suspensory Ligament Healing in 100 Horses by Stem Cells and Other Bone Marrow Components. AAEP PROCEEDINGS / Vol. 47.

Gordon et al., Tendon Regeneration Using mesenchymal stem cells. Mp313-320 in Tendon Injuries. Springer London. , 2005.

Horwitz et al., 1999. Transplantability and therapeutic effects of bone marrow derived mesenchymal cells in children with osteogenesis imperfecta. Nat. Med. 5: 309-313.

Horwitz et al., 2002. Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with

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EP2200622 osteogenesis imperfecta: Implications for cell therapy of bone. PNAS 99 (13) 8932-8937.

Livingston, TL 2003 Mesenchymal stem cells combined with biphasic calcium phosphate ceramics promote bone regeneration.

Journal of Materials Science: volume 14 (3): 211-218.

Young et al. 1998. Use of mesenchymal stem cells in a collagen matrix for Achilles tendon repair. J Orthop Res. 16 (4): 406-13.

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