Mesenchymal cells and osteoblasts from human embryonic stem cell

Mesenchymal cells and osteoblasts derived from human embryonic stem cells

[0001] This application is the international application number PCT/US 02/20998, the international filing date is July 3, 2002, the application number that entered the Chinese national phase is 02813555. 5. The name is "Mesenchyme from Human Embryonic Stem Cells" Divisional application for the invention patent application of "plasma cells and osteoblasts".

[0002] Reference for related applications

[0003] The present invention claims priority of pending US provisional patent application 60/202,732 filed on July 6, 2001. For the sake of implementation in the United States and other permitted jurisdictions, the priority application is incorporated herein by reference in its entirety and W001/51616.

Technical field

[0004] The present invention generally relates to the field of cell biology of embryonic cells and mesenchymal progenitor cells. More specifically, the present invention relates to the directed differentiation of human pluripotent stem cells into osteoblasts and other cell types, using special culture conditions and selection techniques.

Background technique

[0005] Regenerative drugs are an important new beginning for the biotechnology industry. Methods have been developed to produce cultures of special cells that are planned to promote tissue repair and cure diseases that were previously unsatisfactory with medications.

[0006] An area of interest is the use of cultured cells to improve or repair bone tissue. There are some published reports on osteoblast progenitor cells and mesenchymal stem cells under development.

[0007] U.S. Patent Nos. 5, 691, 175, 5, 681, 701 and 5, 693, 511 (Mayo Foundation) describe immortalized normal human fetal osteoblasts, which express the temperature of 40 ΔΤ antigen of simian virus- Sensitive mutant. US Patent No. 5,972, 703 (Michigan) reported that bone precursor cells that are not hematopoietic cells can differentiate into osteoblasts when exposed to bone growth factors and deposit calcium into the extracellular matrix. US Patent 6,200,602 (Du Puy Orthopedics) reports the isolation of cartilage or bone precursor cells from hematopoietic and non-hematopoietic cells and suggests their use for bone and cartilage regeneration.

[0008] International patent publication WO95/22611 (Michigan) reports a method for in situ transfer of nucleic acid to bone cells for stimulating bone progenitor cells. The use of type II collagen and osteogenic genes to promote bone growth, repair and regeneration in animal models has been studied. International patent publication WO99/30724 (Oregon) suggests the use of osteoblast progenitor cells to treat bone defects. Cells can be transformed to express bone morphogenetic proteins, such as BMP-2.

[0009] The topic of bone stem cells is reviewed by J. E. Aubin (J. Cell Biochem. Suppl. 30/31:73, 1998). Stem and primitive osteoprogenitor cells and related mesenchymal precursors help to replace osteoblasts in bone renewal and fracture healing. The article proposes the hypothesis that the phenotype of mature osteoblasts is heterogeneous with the subset of osteoblasts expressing known osteoblast marker subtypes, which increases the possibility of multiple parallel differentiation pathways and different progenitor cell banks.

[0010] Joyner et al. (Bone21:1, 1997) reported the use of differentiation stage-specific monoclonal antibodies to identify and enrich human osteogenic cells. The selection of a specific antigen is based on its reactivity with bone marrow culture and its immunohistochemical localization in fetal tissues in the progenitor cell area near osteoblasts. In immunopanning, antibodies select colony-forming units of mesenchymal fibroblasts (CFU-F). Thies et al. (Endocrinology130:1318, 1992) reported that bone morphogenetic protein-2 induces osteoblasts in mesenchymal cell lines Differentiate, increase alkaline phosphatase activity in a dose-dependent manner without affecting cell growth

CN 101696397 Β

Colonize.

[0011] Liechty et al. (Nature Med. 6:1282, 2000) reported human mesenchymal stem cell (MSC) transplantation and proved the location-specific differentiation after sheep uterine transplantation. Obtain MSC populations from normal human donors through Osaki inhalation, and transplant them into sheep before producing immune activity. The transplanted cells lasted for 13 months in multiple tissues. The article reports that they undergo location-specific differentiation into chondrocytes, adipocytes, muscle cells, cardiomyocytes, bone marrow mesenchymal cells and thymic mesenchyme.

[0012] US Patent 5,908,784 (Case Western Reserve) relates to obtaining human MSCs by taking bone marrow cells, growing them in BGL medium with fetal bovine serum and identifying them with monoclonal antibodies. Inducing chondrocytes in vitro involves contacting the compacted cell pellet with a cartilage inducer. US Patent 5,486,359 (Osiris Therapy) reports the isolation of human mesenchymal stem cells that can differentiate into bone, cartilage, muscle or bone marrow mesenchyme. International patent publication W097/40137 (Osiris) proposes a system that uses mesenchymal stem cells to regenerate and increase bone. The composition includes MSCs or fresh bone marrow cells combined with ceramic materials or resorbable biopolymers.

[0013] It is not clear whether any of the cell preparations as examples in these publications can be produced in sufficient quantities to be marketed in large quantities as a bone repair therapeutic composition.

[0014] Undifferentiated pluripotent stem cells from embryos

[0015] Different medical research fields involve stem cells that do not produce any specific lineage of offspring. Many recent discoveries have raised expectations that embryonic cell lines can be a source of useful cells and tissues in regenerative drugs, which are used in a variety of degenerative situations. Embryonic stem cells are described as pluripotent because they are believed to be able to differentiate into multiple cell types (RA Pedersen, Scientif. Am. 280 (4): 68, 1999) 0

[0016] The work of early embryonic stem cells uses the inbred mouse Lu line as a model (Robertson, Meth. Cell Biol. 75:173, 1997; Pedersen, Reprod. Fertil. Dev. 6:543, 1994 review). However, compared to mouse ES cells, monkey and human pluripotent cells proved to be much more fragile and not responsive to the same culture conditions. The factors that affect their persistence in culture and subsequent differentiation are significantly different. However, it has recently been discovered that primate embryonic cells can be cultured in vitro.

[0017] Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the first to successfully culture embryonic stem cells from primates using rhesus monkeys and marmosets as models. They subsequently obtained a human embryonic stem (hES) cell line (Science282:1145, 1998) from human blastocysts and co-cultured with mouse embryonic fibroblasts to support their maintenance and growth. hES cells have the long-sought characteristics of pluripotent stem cells: they can proliferate in vitro without differentiation, they maintain a normal karyotype, and they maintain the ability to differentiate to produce a variety of mature cell types.

[0018] Geron company has developed a new tissue culture environment that enables pluripotent stem cells to proliferate continuously in an environment essentially without feeder cells. See Australian Patent AU729377 and International Patent Publication W001/51616. A system capable of culturing stem cells in an environment without feeder cells is provided, in which a cell composition that meets the needs of regulating human treatment can be produced.

[0019] To understand the potential of pluripotent stem cells in human health and disease control, new paradigms must now be developed to promote these cells into groups of therapeutically important tissue types.

Summary of the invention

[0020] The present invention provides a system for efficiently producing primate cells, which differentiate from pluripotent cells into mesenchymal mass spectrometry cells.

[0021] One embodiment of the invention is an isolated cell or cell population in in vitro culture, obtained by differentiating primate pluripotent stem (pPS) cells. The cell population can include at least ~10%, ~30% or ~60% different types of mesenchymal cells, with

CN 101696397 Β

Properties listed elsewhere in this publication. For example, osteoblasts and their precursors can express osteocalcin, type 1 collagen and alkaline phosphatase. Mature osteoblasts can express osteocalcin and can form calcium-containing extracellular matrix.

[0022] These cells can be obtained from a human embryonic stem (hES) cell line and therefore share the same genome as the cell line from which they are derived, with any induced genetic changes. In one embodiment of the invention, mesenchymal cells are obtained by differentiating PPS cells in a culture medium containing bone morphogenetic protein (BMP), ligand for human TGF-B receptor, or ligand for human vitamin D receptor. body. The medium may further include dexamethasone, ascorbic acid-2-phosphate, and calcium and phosphoric acid sources. If necessary, the cells of the present invention can be genetically modified to increase the proliferation capacity: for example, an expression vector for telomerase reverse transcriptase. Cells can also be genetically altered to express bone morphogenetic protein.

[0023] Another embodiment of the invention is a method of screening compounds for mesenchymal cell or osteoblast toxicity or modulation, wherein the compound binds to the cell or cell population of the present invention to determine any mesenchymal cytotoxicity or modulation from the compound .

[0024] A further embodiment of the invention is that a medicament comprising the cell population of the invention is used to treat the human or animal body. The drug may optionally contain or accompany additional ingredients, such as a matrix or ceramic carrier, calcium or bone morphogenetic protein.

[0025] The composition of the present invention can be used to regenerate tissues in need of repair. For example, bone tissue can be repaired by contacting bone tissue with osteoblasts or precursor cell populations of the present invention. In a similar manner, the composition can be used to reconstruct or supplement musculoskeletal cell function in an individual. The composition can also be used to increase mobility in human patients by implanting a prosthetic device in the patient or incorporating a splint of the cell population of the invention.

[0026] These and other embodiments of the invention will be apparent from the following description. The compositions, methods, and techniques described in this disclosure can be used in diagnostic, drug screening, and therapeutic applications.

[0027] Attached drawings

[0028] FIG. 1 is a reproduction of a photomicrograph showing the expression of markers detected by immunocytochemistry of undifferentiated human embryonic stem (hES) cells. The culture is grown according to the conventional method of mouse embryo feeder cells, or in a feeder-free environment containing extracellular matrix Matrigei® or laminin in conditioned medium. The hES cells grown in feeder-free culture have phenotypic markers similar to those of hES grown on the primary mouse fibroblast feeder cell layer. [0029] Figure 2 shows the characteristics of a human cell line named HEF1, which is differentiated from hES cell. Group A is a copy of the phase-contrast photomicrograph, showing that the HEF1 cell line has the morphological characteristics of fibroblasts. Group B (below) is a copy of the results of the TRAP test, showing that HEF1 cells transduced with a retroviral vector of telomerase reverse transcriptase (hTERT) obtained telomerase activity.

[0030] FIG. 3 is a reproduction of a photomicrograph showing the expression of markers in a cell line that undergoes differentiation to produce bone precursor cells and osteoblasts. The culture medium was replaced with osteoblast induction medium (0.1M), and then differentiated for 11 days. 0.1M was prepared from mesenchymal cell growth medium, and was supplemented with 0.1 μM dexamethasone, 5 μM ascorbic acid-2-phosphate, 10 mM B-glycerophosphate, and 100 ng/mL BMP-4_oThe cells used were hES cell line H1, telomerized hES-derived differentiated cell line HEF1, human mesenchymal stem cells and BJ5ta fibroblasts.

[0031] Groups A and B show immunocytochemistry for labeling osteocalcin and collagen T. Panel C shows staining for alkaline phosphatase activity. These features are cellular features of the osteoblast lineage, indicating that hES cells and HEF1 cells produce osteoblasts when subjected to appropriate differentiation operations in vitro.

Detailed ways

[0032] The technology provided by the present invention can be used to prepare and characterize some types of mesenchymal cells, including cells involved in bone renewal and repair.

CN 101696397 Β

[0033] If pPS cells can be differentiated in an undirected manner, the obtained heterologous cell population expresses many different tissue types of markers (W001/51616; Shamblott et al., Proc. Natl. Acad. Sci. USA98: 113, 2001). An important problem in using pPS cells for therapeutic purposes or for studying specific cell types in vitro is to obtain a cell population containing a large number of subpopulations with relatively uniform characteristics. The review article in the background section of this disclosure does not teach or provide methods for obtaining osteoblasts or their precursors from any kind of embryonic stem cells.

[0034] It has now been discovered that a substantially homologous mesenchymal mass spectrum cell population can be obtained by culturing pluripotent embryonic cells under conditions optimized for such cells. Example 2 (below) illustrates how human embryonic stem (hES) cells can differentiate into early mesoderm cell lines. The HES cells are caused to form embryo-like bodies, which are subsequently plated under conditions suitable for selection of cell lines with phenotypic characteristics of mesodermal cells. The isolated cell line was then transduced with telomerase reverse transcriptase to increase the proliferation capacity. This cell line has the ability to renew itself and form progeny of different mature mesenchymal cell types.

[0035] In Example 3, cells that sequentially caused the mesenchymal cell line to differentiate into the osteoblast lineage were identified by staining collagen-1 osteocalcin and alkaline phosphatase activity. Example 3 also clarifies that cells with characteristics of osteoblasts can also be directly obtained by culturing human embryonic stem (hES) cells in a suitable culture environment. Specifically, the cells were cultured in a commercially available mesenchymal cell growth medium for 11 days, supplemented with 0.1 μM dexamethasone, 5 μM ascorbic acid-2-phosphate, glycerophosphate, and 100 ng/mL BMP-4_o

[0036] Some cell populations obtained according to the method of the present invention contain a high proportion of osteoblasts and their precursors. It is not known whether the culture conditions induce hES cells to adopt the osteoblast phenotype, whether they promote the growth of such cells or whether they inhibit the growth of other types of cells-indeed it is likely that some of these mechanisms work together to enrich the desired types of cells. Of course, the mechanism that causes cell enrichment of the osteoblast lineage is of interest, but it is not necessary to understand the mechanism in order to practice the invention.

[0037] The remarkable unity and functional properties of the cells produced according to this system make them useful for the development of new treatment modalities and as a tool for the study of mesenchymal tissue in vitro.

[0038] Definition

[0039] The prototype "primate pluripotent stem cells" (pPS cells) are pluripotent cells that have the characteristics of being able to produce offspring of different cell types under suitable conditions. The cell types are all three germ layers (endoderm, mesoderm). And ectoderm) derivatives, which are based on standard technically accepted tests such as the ability to form teratomas in SCID mice at 8-12 weeks or the ability to form identifiable cells in all three germ layers in tissue culture.

[0040] The definition of pPS cells includes different types of embryonic cells, examples are embryonic stem cells from other primates such as rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995) and velvet Monkey stem cells (Thomson et al., Biol. Reprod. 55:254, 1996). Other types of pluripotent cells are also included in this term. This includes any primate-derived cell that can produce offspring of all three germ layer derivatives. pPS cells are not obtained from malignant sources. Ideally (but not always necessary) the cell karyotype is normal.

[0041] When most of the stem cell populations and their derivatives in the population exhibit the morphological characteristics of undifferentiated cells, pPS cell culture is described as "undifferentiated", and they are clearly distinguished from differentiated cells of embryonic or mature origin. Undifferentiated pPS cells can be easily identified by those skilled in the art, and they usually appear in cell colonies with a high nucleus/cytoplasm ratio and prominent nucleoli in two-dimensional microscopic observations. It should be understood that undifferentiated cell colonies in a population are usually surrounded by adjacent differentiated cells. [0042] For the purpose of the present disclosure, "mesenchymal cells" may be terminally differentiated cells or proliferative precursor cells that are stereotyped to form mesenchymal tissue cells, such as bone, dental tissue, cartilage, tendon, bone marrow mesenchyme, hematopoietic Pedigree or muscle. Mesenchymal stem cells are included in the term, as well as terminally differentiated (post-mitotic) cells and more committed replication-competent cells such as osteoblast precursor cells. The property of mesenchymal cells is that they terminally differentiate or limit the formation of mesenchymal cells in the mesenchymal mass spectrum.

CN 101696397 Β

The descendants of mass spectrometry or their precursors. They do not form endoderm or ectoderm cells unless they are subjected to nuclear transfer or other reprogramming.

[0043] In terms of cell ontogeny, the adjective "differentiated" is a relative term. A "differentiated cell" is a cell that compares a cell's developmental pathway further down. Therefore, pluripotent embryonic stem cells can differentiate into lineage-restricted precursor cells (such as mesenchymal stem cells), which in turn can differentiate into other types of precursor cells (such as osteoblast precursors) that are lower in the pathway. Then become the final stage of differentiated cells that play a characteristic role in certain tissue types, and they may or may not retain the ability to further proliferate.

[0044] As used in the present disclosure, "differentiation agent" refers to one of the collections of compounds used in the culture system of the present invention to produce differentiated cells (including precursor cells and terminal differentiated cells) of the mesenchymal mass spectrum. There is no restriction on the mode of action of the compound. For example, the agent can assist in the differentiation process by inducing or assisting phenotypic changes, promoting the growth of cells with a specific phenotype or hindering the growth of other cells, or acting in synergy with other agents through unknown mechanisms.

[0045] Unless explicitly stated otherwise, the technology of the present invention has no limitation on any type of progenitor cells that can differentiate into bone.

[0046] The term "feeder cell" or "feeder cell" is used to describe one type of cell that is co-cultured with another type of cell to provide an environment in which the second type of cell can grow. If the cells grow for at least one round after division, and no fresh feeder cells are added to support the growth of pPs, then the pPs cell population is called "essentially free" feeder cells.

[0047] "Growth environment" is an environment in which cells of interest proliferate, differentiate or mature in vitro. Environmental characteristics include the medium in which the cells are cultured, any growth factors or differentiation-inducing factors that may be present, and supporting structures (if present, such as substrates on a solid surface).

[0048] When the polynucleotide is transferred into the cell by any appropriate manual operation method or the cell is the offspring of the original altered genetic polynucleotide, the cell is called "genetic alteration". Polynucleotides usually include transcribable sequences encoding the protein of interest, allowing cells to express the protein at elevated levels. If the offspring of the changed cell have the same change, the genetic change is called "heritable".

[0049] As used in this disclosure, the term "antibody" refers to polyclonal and monoclonal antibodies. The term scope includes not only complete immunoglobulin molecules, but also fragments and derivatives of these immunoglobulin molecules (such as single-chain Fv constructs, dimer molecules and fusion constructs), which can be prepared and maintained by techniques known in the art. Requires antibody binding specificity.

[0050] General Technology

[0051] To further illustrate the general techniques useful in the practice of the present invention, the operator can refer to standard textbooks and reviews of cell biology, tissue culture, and embryology.

[0052] Regarding tissue culture and embryonic stem cells, readers can refer to "Teratocarcinomas and embryonic stem cells: A practical approach" (Edited by EJ Robertson, IRL Press Ltd. 1987); "Teratocarcinomas and embryonic stem cells: A practical approach); Guide to Techniques in Mouse Development (P. M. Wasserman et al., Academic Press 1993); "Embryonic Stem Cell Differentiation in Vitro" (M. V. Wiles, Meth. Enzymol. 225:900, 1993); "Properties and uses of Embryonic stem cells: Prospects for Application to Human Biology and Gene Therapy) ( PD Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998) 0 [0053] The general principles of preparation and culture of bone cells and bone damage repair can be found in "Bone: The Osteoblast and Osteocytes" (Bone: The Osteoblast and Osteocyte) (B. K. Hall, CRC Pressl990); "Differentiation and Morphogenesis of Bone" (B. K. Hall, CRC Pressl994); "Bone

CN 101696397 Β

Principles of Bone Bidogy (JP Bilezikian et al., Academic Press 1996); The Cellular and Molecular Basis of Bone Formation and Repair (V. Rosen&S. Thies, RG Landes Co. 1995) Other books of interest include "The Bone People" (K. Hulme, Viking Pressl986); "Bone Appetit" (B. E. Romano, West Coast) Media Group1998)0

[0054] Molecular genetics and genetic engineering methods are described in "Molecular Cloing: A Laboratory Manual", 2nd Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait, ed., 1984); "Animal Cell Culture" (RI Freshney, ed., 1987); "Methods in Enzymology" series (Academic Press); "Gene transfer vector for mammalian cells "(Gene Transfer Vector for Mammalian Cell) (JM Miller&M. P. Calos, 1987); "New Molecular Biology Experiment Guide" and "Refined Molecular Biology Experiment Guide" (Current Protocols in Molecular Biology and Short Protocols in Molecular Biology 1), 3rd edition (FM Ausubel et al. eds., 1987 & 1995); "Recombinant DNA Methodology Π" (R. Wu eds., 1995). The genetic manipulation reagents, cloning vectors and kits referred to in this disclosure come from commercial suppliers such as BioRad, Stratagene, Invitrogen and ClonTecho

[0055] Source of Stem Cells

[0056] The present invention can be carried out with different types of pluripotent stem cells, specifically stem cells with characteristics that can produce offspring of all three germ layers.

[0057] An example is embryonic stem cells used as existing cell lines.

[0058] Propagation of pPS cells in an undifferentiated state

[0059] pPS cells can be continuously propagated in culture, and the culture conditions used promote proliferation but not differentiation. Example: Serum-containing ES medium uses 80% DMEM (e.g., DMEM, Gibco excluded), 20% determined fetal bovine serum (FBS, Hyclone) or serum substitute (W098/30679), 1% non-essential amino acids, 1mM L-gluten Aminoamide and 0.1πιΜ β-hang-based ethanol made. Before use, add human bFGF to 4ng/mL (W099/20741, Geron Corp.).

[0060] Conventionally, ES cells are cultured on a feeder cell layer, and fibroblasts are usually obtained from embryonic or fetal tissues. Embryos were collected from CF1 mice on the 13th day of pregnancy, transferred to 2mL trypsin/EDTA, finely minced and incubated at 37°C for 5 minutes. 10% FBS was added to precipitate the debris, and the cells were multiplied in 90% DMEM, 10% FBS and 2mM glutamine. To prepare the feeder cell layer, the cells were irradiated to inhibit proliferation but allow to support factor synthesis by ES cells (~4000 radya-radiation). The culture plate was coated with 0.5% gelatin overnight, and the plate was cultured with 375,000 irradiated mEFs per well, and the plate was used for 5 hours to 4 days after culture. The medium was replaced with fresh hES medium before inoculation of pPS cells.

[0061] Scientists at Geron found that pPS cells can be maintained in an undifferentiated state even without feeder cells (WO01/51616). The environment without feeder cell culture includes suitable culture substrates, specifically extracellular matrix such as Matrigel® or laminar adhesion protein. pPS cells are more than 15,000 cells cm-² (90, 000^ to [70, 000 one? Best) Plate culture. Generally, enzyme digestion is stopped before the cells are completely dispersed (for example, with collagenase IV for ~5 to 20 minutes). Then the block of -10-2000 cells was plated directly on the substrate without further dispersion.

[0062] The culture without feeder cells is supported by a nutrient medium, and usually conditioned culture irradiated primary mouse embryonic fibroblasts, telomerized mouse fibroblasts or fibroblast-like cells derived from pPS cells. The medium can be cultured feeder cells in a serum-free medium such as K0 DMEM at a density of ~5-6×10'cnf? The serum-free medium is supplemented with 20% serum substitute and 4ng/mL bFGF. The culture medium in a normal state for 24 hours is filtered through a 0.2 P m membrane, further

CN 101696397 Β

Add ~8ng/mL bFGF step by step and used to support pPS cell culture for 1-2 days.

[0063] Under the microscope, ES cells have a high nucleus/cytoplasm ratio, prominent nucleoli, and dense cluster formation with poorly resolved cell connections. Primate ES cells can express one or more stage-specific embryonic antigens (SSEA) 3 and 4, and detectable markers with antibodies named Tra-1-60 and Tra-1-81 (Thomsom et al. ,Science282:1145, 1998)_oUndifferentiated hES cells also usually express Oct-4 and TERT as detected by RT-PCR. In vitro differentiation of hES cells usually results in the loss of these markers (if present) and increased SSEA-1 expression.

[0064] Materials and steps for preparing mesenchymal cells and osteoblasts

[0065] The cells of the present invention can be obtained by culturing, differentiating or reprogramming stem cells in a special growth environment, which is enriched for cells with the desired phenotype (by growing the desired cells or by inhibiting or killing other cells). Types of). These methods can be applied to many types of stem cells, including the primate pluripotent stem (pPS) cells described in the previous section.

[0066] Differentiation can optionally be initiated by the formation of embryo-like bodies or aggregates: for example, by overgrowing a donor pPS cell culture or culturing pPS cells in suspension in a culture vessel that allows EB The formation of a substrate with low adhesion properties. PPS cells were collected by brief collagenase digestion, separated into pieces, and plated in non-adherent cell culture plates. Aggregates are fed every few days and then collected after a suitable period of time, usually 4-8 days. In addition or in addition, the differentiation process can be initiated by cultivating non-specific differentiation paradigms: by including retinoic acid (RA) or dimethylsulfoxide (DMSO) in the medium; or by removing it from common extracellular matrix, Cells are cultured on extracellular matrix. See U.S. Patent Application 60/213,740 and International Patent Publication W001/51616.

[0067] Produce a relatively homogenous population of mesenchymal cells, specifically an osteoblast lineage that can be obtained by culturing pPS cells (undifferentiated, or after the initiation of differentiation) in a growth environment, where the growth environment contains beneficial to these cells Factors, such as one or more of the following factors:

[0068] Bone morphogenetic proteins, examples are BMP-2, BMP-3, BMP-4, BMP-6 and BMP-7.

[0069] «TGF-β, examples are TGF-B 1, TGF-B 2, and TGF-B 3 and their analogs, and other members of the TGF-6 superfamily that bind to the TGF-B receptor

[0070] Vitamin D receptor ligand. An example is 1,25-dihydroxyvitamin D3. Other analogs are known (see, for example, Tsugawa et al., Biol. Pharm. Bull. 23:66, 2000)

[0071] It is recognized that antibodies specific to the receptors of any of these factors are functionally equivalent ligands that can be used to replace (or in addition to) the listed factors. Other additives that can be used include:

[0072] Other morphogens, such as fibroblast growth factors like basic FGF

[0073] Glucocorticoid

[0074] Dexamethasone or other small molecule osteoblast maturation factors

[0075] Ascorbic acid (or its analogues, such as ascorbic acid-2-phosphate), which is a cofactor for proline hydroxylation during collagen synthesis.

[0076] · Glycerophosphate or other substrates of alkaline phosphatase during mineralization

[0077] Calcium source (may or may not be present in the alkaline medium at a sufficient concentration)

[0078] The cells can also be supported on a substrate coated with an appropriate substance that contributes to the growth of the desired cell phenotype, or cultured in a medium containing such substance components.

[0079] Matrigel®, laminin, collagen (especially type I collagen), glycosaminoglycans, osteocalcin and osteonectin are all suitable as extracellular matrix by themselves or in different combinations. Osteoblast lineage cells that are also suitable for growth are gel-derived glass, silica gel, and sol-derived titanium dioxide (Saravanapavan et al., J. Biomed Mater.

CN 101696397 Β

Res. 54:608, 2001; Dieudonne et al., Biomaterials34:3041, 2002).

[0080] The cells obtained according to the present invention can be characterized according to some phenotypic criteria. Relatively undifferentiated mesenchymal cells can be identified by their characteristic mononuclear oval, star or spindle shape, with round to elliptical nuclei and poorly defined cell boundaries. Elliptical elongated nuclei usually have a mixture of prominent nuclei and heterochromatin. These cells have little cytoplasm but many thin protrusions that seem to extend from the nucleus. They usually stain one, two, three or more of the following markers: CD106 (VCAM), CD166 (ALCAM), CD29, CD44, GATA-4 and alkaline phosphatase, and for hematopoietic lineage cell markers (CD14 or CD45) Is feminine. Mesenchymal stem cells can also express STROT.

[0081] Under suitable conditions, early mesenchymal cells can be further differentiated into many mature connective tissue cell types, such as fibroblasts, chondroblasts, osteoblasts, odontoblasts, reticular cells or adipocytes. Therefore, mesenchymal stem cells can be identified by their ability to form one or more special mesenchymal mass spectrometry lines.

[0082] Osteoblasts and bone precursor cells usually have at least one of the following characteristics (generally at least three or five characteristics):

[0083] · Density between -1.050 and ~1.090g cnf3

[0084] Positive for osteonectin (positive in osteoblasts and precursors)

[0085] Osteocalcin positive (specific to mature osteoblasts)

[0086] Cell diameter of ~8 to ~70 μπι

[0087] Cubic shape

[0088] Positive regulation produces alkaline phosphatase, especially in response to the presence of BMP

[0089]-Positive for type I collagen (procollagen) or vimentin

[0090] · Osteoblast specific markers are positive, such as BMP receptor, PTI receptor or CD105 (endoglin)

[0091] The ability to mineralize the external environment or synthesize calcium-containing extracellular matrix

[0092] The skilled person knows that chondrocytes usually express type II collagen, aggrecan, or proteoglycan stained with ici blue. In the mature form, chondrocytes are less than 1% positive for elastin, type I collagen, type X collagen, or calcineurin. Hematopoietic cell populations and their precursors carry such markers such as re CD45, CD34, CD13, AC133, hemoglobin , Surface antibodies and Class II histocompatibility antigens. Where appropriate, replicating hematopoietic cells form colonies in the hematopoietic colony forming unit (CFU) test. Cardiomyocytes and their precursors usually express cardiac troponin I (cTnl), cardiac troponin T (cTnT), atrial natriuretic factor (ANF) and alpha cardiac myosin heavy chain (MHC). Fibroblasts have an easily identifiable morphology and usually express tissue inhibitors of collagenase I and metalloproteinase I (TIMPT). Striated muscle cells usually express contractile proteins such as skeletal alpha-actin, skeletal myosin heavy and light chains, and tropomyosin. The early myogenic markers are myoD and myogenin. Tendon and ligament tissues are stained with type I collagen in unidirectional fiber arrangement. Early tendon and chondrocyte progenitors usually express scleraxiso. Adipocytes are usually stained with Oil Red 0, which shows lipid accumulation, and express peroxisome proliferation-activated receptor Y2 (PPARY2), lipoprotein lipase (LPL), and fatty acids. Binding protein (aP2).

[0093] Tissue-specific markers can be detected by any suitable immunological technique-such as flow immunocytochemistry or affinity absorption cell surface markers, immunocytochemistry for intracellular or cell surface markers (for example, fixed cells or tissue sections) , Western blot analysis of cell extracts and enzyme-linked immunoassay for cell extracts or products secreted into the culture medium. If a clearly detectable amount of antibody binds to the antigen in standard immunocytochemistry or flow cytometry assays, the antigen expression of the cell is called "antibody-detectable", optionally after cell fixation and optionally using a labeled second Diabodies or other conjugates (such as biotin-avidin conjugates) to expand the labeling.

[0094] The expression of tissue-specific gene products can also be detected at the mRNA level by Northern blot analysis, dot blot analysis, or reverse transcriptase-initiated polymerase chain reaction (RT-PCR), using standard amplification methods. Sequence-specific primers.

CN 101696397 Β

See U.S. Patent No. 5,843,780 for general technical details and International Patent Publication WO99/339724 for osteoblast-specific PCR primers. The sequence data of other markers listed in this disclosure can be obtained from public databases such as GenBank (URL www^ ncbi. nlm. nih. gov:80/entrez) It can be clear if it is generated on a cell sample according to standard procedures in a typical control experiment The differentiated hybridization or amplification product, mRNA level expression according to one of the tests described in this disclosure is called "detectable". If the level is at least 2 times higher than control cells such as undifferentiated pPS cells or other unrelated cell types, preferably 10 or 50 times higher, the tissue-specific marker expression detected at the protein or mRA level is considered positive.

[0095] As stated by the manufacturer (Vector Laboratories, Burlingame CA), detection of the presence of alkaline phosphatase activity can be achieved by fixing the cells with 4% paraformaldehyde and then using Vector Red as a substrate for visualization. Intracellular calcium accumulation and matrix protein deposition can be measured by incubating, washing and reculturing in *Ca++, and then confirming that there is any radioactivity in the cells or deposits in the extracellular matrix (US Patent 5, 972, 703); or by using Ca** test kit (Sigma Kit#587) Determines the culture substrate used for mineralization.

[0096] Once the markers are identified on the cell surface of the desired phenotype, they can be used for immunoselection to further enrich the cell population by techniques such as immunopanning or antibody-advocated fluorescence-activated cell sorting.

[0097] Since it has been demonstrated that mesenchymal cells and osteoblasts can be generated from pPS cells, it is within the reader's scope to adjust the differentiation paradigm described in this disclosure to suit their own purposes. Readers can test the suitability of some culture conditions, for example by culturing pPS under test conditions parallel to cells obtained according to the present disclosure and other control cell types (such as primary human mesenchymal stem cells, hepatocytes, or fibroblasts) Cells or their derivatives, then compare the cell phenotypes obtained according to the markers listed above. Adjusting culture and cell separation conditions to include, remove or replace specific components is a matter of routine optimization normally expected in the present invention, and does not deviate from the inventive spirit of the claims.

[0098] Genetic changes in differentiated cells

[0099] Cells are required to have the ability to replicate and provide stocks for the production of mesenchymal cells and osteoblasts in some drug screening and therapeutic applications. The cells of the present invention may optionally be telomerized to increase their ability to replicate before or after they progress to restricted developmental lineage cells or terminally differentiated cells. Telomerized pPS cells can be taken out from the lower part of the above differentiation pathway or differentiated cells can be directly telomerized.

[0100] Cells are telomerized by genetic changes, transfected or transduced with a suitable vector, homologous recombination or other suitable techniques, so that they express the telomerase catalytic component (TERT), usually under a heterologous promoter Telomerase expression exceeds the expression produced under the endogenous promoter. Specifically suitable is the human telomerase catalytic component (hTERT) provided by the international patent application W098/14592. For some applications, homologs like mouse TERT (W099/27113) can also be used. The transfection and expression of telomerase in human cells are described in Bodnar et al., Science279:349, 1998 and Jiang et al., Nat. Genet. 21:111, 1999_OIn another example, the hTERT clone (W098/14592) was used as the source of the hTERT coding sequence, spliced into the EcoRI site of the PBBS212 vector under the control of the MPSV promoter or spliced into a commercially available one under the control of the LRT promoter EcoRI site of PBABE retroviral vector.

[0101] Differentiated or undifferentiated pPS cells are genetically altered with a supernatant-containing vector for more than 8 to 6 hours, and then exchanged into growth medium for 2 days. The genetically altered cells were selected with 0.5-2. 5 Pg/mL Pterinomycin and recultured. Then their hTERT expression, telomerase activity (TRAP test), immunocytochemical staining of hTERT or replication ability can be assessed by RT-PCR. The following test kits are commercially available for research purposes: TRAPeze®XL Telomerase Detection Kit (Cat. No. s7707; Intergen Co., Purchase NY); and TeloTAGGG Telomerase PCR ELISAplus (Cat. No. 2, 013, 89; Roche Diagnostics, Indianapolis IN) The LightCycler TeloTAGGG hTERT quantitative kit (catalog number 3, 012, 344; Roche Diagnostics) is purchased commercially for research purposes.

CN 101696397 Β

Colonies that continue to replicate are enriched by further culturing under conditions that support proliferation, and cells with the desired phenotype can optionally be cloned by limiting dilution.

[0102] In some embodiments of the invention, pPS cells are differentiated into pluripotent or definitive mesenchymal cells and then genetically altered to express TERT. In other embodiments of the invention, pPS cells are genetically altered to express TERT, and then Differentiate into osteoblast precursors or terminally differentiated cells. The successful modification to increase the expression of TERT can be determined by the TRAP test, or by determining whether the cell's replication ability is improved.

[0103] Depending on the application, other immortalization methods can also be used, such as transforming cells with DNA encoding myc, SV40 large T antigen or MOT-2 (U.S. Patent 5,869,243, International Patent Applications W097/32972 and W001 /23555) _oWhen cells are used for therapeutic purposes, transfection with oncogene or oncogenic virus products is not suitable. The telomerized cells are of particular interest in the application of the present invention. Among them, cells that can proliferate and maintain their karyotype have advantages-for example, in drug screening and treatment protocols, administering differentiated cells to individuals to enhance musculoskeletal function.

[0104] The cells of the present invention can also be genetically modified to increase their ability to participate in tissue regeneration or deliver therapeutic genes to the site of administration. The vector is designed with a known coding sequence for the desired gene, and is operably linked to a pan-specific or specifically active promoter in the differentiated cell type. Of particular interest are cells genetically altered to express bone morphogenetic proteins such as BMP-2 or BMP-4. See W099/39724. The production of these or other growth factors at the site of administration can increase the beneficial effects of the administered cells or increase the proliferation or activity of host cells adjacent to the treatment site.

[0105] Use of mesenchymal stem cells, osteoblast precursors and terminally differentiated cells

[0106] The present invention provides methods for generating large numbers of precursor cells and mature cells. These cell populations can be used for some important research, development and commercial purposes.

[0107] The cells of the present invention can be used to prepare a cDNA library, which is relatively uncontaminated with cDNA preferentially expressed in cells from other lineages. For example, mesenchymal progenitor cells or osteoblasts are collected by centrifugation at 1000 rpm for 5 minutes, and then mRNA is prepared from the pellet by standard methods (Sambrook et al., supra). After reverse transcription into cDNA, the preparation can subtract cDNA from undifferentiated pPS cells, other progenitor cells, or end-stage cells from osteoblasts or any other developmental pathway.

[0108] The differentiated cells of the present invention can also be used to prepare antibodies specific for mesenchymal cells, osteoblasts and intermediate precursors. Polyclonal antibodies can be prepared by injecting cells of the invention into vertebrates in an immunogenic form. The production of monoclonal antibodies is described in standard references such as U.S. Patent Nos. 4,491,632,4,472,500 and 4,444,887 and Methods in Enzymology 73B: 3 (1981) also available Specific antibody molecules are produced by contacting a library of immunocompetent cells or virus particles with the target antigen and growing a positive selection clone. See Marks et al., New Eng. J. Med. 335:730, 1996 and McGuiness et al., Nature Biotechnol. 14:1449, 1996_O Another option is to reassemble random DNA fragments into the antibody coding region as described in EP Patent Application 1, 094, 108A.

[0109] The required specificity can be obtained by using the positive selection of the pPS of the present invention and the negative selection of stem cells carrying a wider distribution of antigens (such as differentiated embryonic cells) or mature sources. Antibodies can be used in turn to identify or rescue mesenchymal cells with the desired phenotype from a mixed cell population, for purposes such as co-staining with tissue samples in immunodiagnosis and isolating precursor cells derived from terminally differentiated osteoblasts and Cells of other lineages.

[0110] The cells of the present invention are also of interest in identifying expression patterns of transcripts and newly synthesized mesenchymal cell-specific proteins, and can help guide differentiation pathways or promote cell-cell interactions. Obtain the expression patterns of differentiated cells and compare them with control cell lines, such as undifferentiated pPS cells, other types of committed precursor cells (such as pPS cells that differentiate into other lineages), or terminally differentiated cells.

CN 101696397 Β

Π/page 15

[0111] The use of microarrays in the analysis of gene expression is reviewed by Firtz et al. Science 288:316, 2000; "Microarray Biochip Technology", L Shi, www. Gene-Chips, com. The demonstration method is performed with a genetic microsystem array generator and Axon GenePix'M scanner. To prepare the microarray, firstly, amplify the cDNA fragments encoding the marker sequence to be analyzed and directly spot them on the glass slide. To compare mRNA preparations from two cells of interest, one preparation was converted to Cy3-labeled cDNA, and the other was converted to Cy5-labeled cDNA. The two cDNA preparations were hybridized to the microarray slide at the same time, and then washed to remove non-specific binding. The slide is then scanned at a wavelength appropriate for each label, the resulting fluorescence is quantified, and the results are arranged to indicate the relative abundance of mRNA for each label on the array.

[01Drug Screening

[0113] The mesenchymal cells and osteoblasts of the present invention can be used to screen for factors that affect these cells and their different progeny (such as solvents, small molecule drugs, peptides, oligonucleotides) or environmental conditions (such as culture environment or operation) .

[0114] In some applications, pPS cells (undifferentiated or differentiated) are used to screen mesenchymal cell precursors or terminally differentiated cells that promote maturation to later stages, or factors that promote the proliferation and maintenance of these cells in long-term culture . For example, candidate maturation factors or growth factors are tested by adding them to cells in different wells, and then determining any resulting phenotypic changes based on the required criteria for further culturing and using the cells. In one example, PPS-derived cells with an early mesenchymal phenotype are used to screen factors for the ability to direct differentiation into specific cell types, such as muscle cells, cartilage, or adipocytes.

[0115] Other screening applications of the present invention relate to the effect of test drug compounds on the maintenance or repair of musculoskeletal tissue. In one example, PPS-derived cells with osteoblast characteristics are used to screen the ability of factors to affect calcium deposition. Screening can be performed because the compound is designed to have a pharmacological effect on cells, or because a compound designed to have other effects can have unexpected side effects on cells of this tissue type. Any of the precursor cells or terminally differentiated cells of the present invention can be used for screening.

[0116] Readers generally refer to the standard textbook "In vitro Methods in Pharmaceutical Research" (In vitro Methods in Pharmaceutical Research), Academic Press, 1997 and US Patent 5,030,015. Evaluating the activity of a candidate drug compound generally includes combining the differentiated cells of the invention and the candidate compound alone or in combination with other drugs. The researchers determined any changes in cell morphology, labeled phenotype, or functional activity (compared to untreated cells or cells treated with an inert compound) attributable to the compound, and then correlated the compound's effect with the observed change.

[0117] Cytotoxicity can be determined by the effect on cell viability, survival, morphology, and expression of some markers and receptors in the first example. The effect of drugs on chromosomal DNA can be determined by measuring DNA synthesis and repair. The incorporation of Hirota or BrdU, especially at the end of the cell cycle or higher than the level required for cell replication, is consistent with the drug's effect. Unwanted effects can also include abnormal rates of sister chromatid exchange determined by metaphase expansion. Readers can refer to A. Vickers ("In vitro Methods of Drug Research", pages 375-440, Academic Press, 1997) for further clarification.

[0118] Any standard test can be used to evaluate the effect of cell function to observe the phenotype or activity of mesenchymal or osteoblast-like cells, such as receptor binding, matrix deposition or calcium processing-in cell culture or in a suitable animal model .

[0119] Therapeutic use

[0120] The present invention also provides the use of mesenchymal cells or osteoblasts to improve tissue maintenance or repair of the musculoskeletal system for any sensory needs, such as congenital defects in metabolic function, effects of disease conditions, or significant damage. result.

[0121] To determine the suitability of the cell composition for therapeutic administration, the cells can first be tested in a suitable animal model. The ability of cells to survive and maintain their phenotype can be assessed at one level. The cell composition is administered to immunodeficient animals (such as nude mice, or animals exhibiting chemical immunodeficiency or through radiation). After growing for a period of time, the tissues were collected to evaluate whether the cells derived from pPS still exist.

CN 101696397 Β

[0122] This can be achieved by administering cells expressing detectable markers (such as green fluorescent protein or β-galactase) and pre-labeled (such as with BrdU or "Tian-Chest and Back), or by subsequent detection of constitutive cell markers (such as Use human-specific antibodies) to assess the presence and phenotype of the administered cells can be determined by immunohistochemistry or ELISA using human-specific antibodies, or by RT-PCR with primers and based on published sequence data to cause specific human polynucleotides Hybridization conditions for amplification.

[0123] Suitability can also be determined by evaluating the degree of recovery resulting from treatment with a mesenchymal cell population. For example, the regenerative capacity of bone and cartilage can be determined using a rat calvarium defect model (US Patent 6,200,606). There are established animal models for the treatment of mandibular defects, maxillary alveolar clefts and bone resections in rabbits, dogs and monkeys (W099/39724). X-ray analysis and other techniques can be used to monitor bone deposition into the model damage. The reconstructed bone tissue can be evaluated for function using standard biomechanical tests. See Minamide et al., Spine24: 1863, 1999; Takahashi et al., J. Neurosurg. 90 (4suppl.): 224, 1999; Helm et al., J. Neurosurg. 88: 354, 1997.

[0124] After appropriate tests, the differentiated cells of the present invention can be used for tissue reconstruction or regeneration in human patients or other subjects in need of such treatment. Cells are administered in such a way that they are transplanted or moved to the desired tissue site and reconstruct or regenerate functionally defective areas. Medical instructions for this treatment include regenerative musculoskeletal defects, fracture repair, spinal cord restoration, prosthesis installation, and repair of osteoporosis-related injuries.

[0125] The composition application depends on the musculoskeletal site to be repaired. For example, osteogenesis can be promoted in accordance with surgical procedures to reshape tissue or insert lobes or prosthetic devices such as medullary substitutes. In other cases, without invasive surgery, the composition can be administered by injection or (to repair the spine) with a guided endoscope.

[0126] The mesenchymal cells and osteoblasts of the present invention can be provided in the form of pharmaceutical compositions, including isotonic excipients prepared under sufficiently sterile conditions for human administration. For the general principles of medicinal preparations, readers can refer to "Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy" (Cell Therapy: Stem Cell Transplantation, Gene Therapy, and Cellular Immunotherapy), edited by G. Morstyn & W. Sheridan, Cambridge University Press , 1996; "Hematopoietic Stem Cell Therapy", ED Ball, J. Lister & P. Law, Churchill Livingstone, 2000. The selection of cell excipients and any accompanying composition components varies according to the device used for administration.

[0127] If desired, cell preparation may further include or be co-administered with supplementary biologically active factors, such as synthetic glucocorticoids like dexamethasone, or bone morphogenetic proteins like BMP-2 or BMP-4. Other possible concomitant ingredients include inorganic sources of calcium or phosphoric acid suitable for assisting in bone regeneration (W000/07639). If desired, cell preparation can be applied to a carrier matrix or material to provide improved tissue regeneration. For example, the material may be granular ceramics or biopolymers such as gelatin, collagen, osteonectin, fibrinogen, or osteocalcin. The porous matrix can be synthesized according to standard techniques (e.g. Mikos et al., Biomaterials14: 323, 1993; Mikos et al., Polymer 35:1068, 1994; Cook et al., J. BioMed. Mater. Res. 35: 513, 1997).

[0128] The composition can optionally be packaged in a suitable container and used for the desired purpose according to written instructions, such as rebuilding the function of mesenchymal cells to improve some musculoskeletal deformities.

[0129] The following examples are provided as further non-limiting illustrations of specific embodiments of the invention.

[0130] Embodiment

Example 1: Propagation of embryonic stem cells without feeder cells

[0132] The established line of undifferentiated human embryonic stem (hES) cells is maintained in a culture environment essentially free of feeder cells.

[0133] The culture without feeder cells was maintained with a conditioned medium, and the conditioned medium was prepared with primary mouse embryonic fibroblasts isolated according to standard procedures (W001/51616). Fibroblasts are passed without Ca+VMg++

CN 101696397 Β

Wash once with PBS and incubate in 1.5-2 mL trypsin/EDTA (Gibco) for ~5 minutes to collect from the ΊΊ50 flask. After the fibroblasts were separated from the flask, they were collected in mEF medium (DMEM+10% FBS). Cells are irradiated with 4000 rads, counted and seeded at ~55,000 cells cm''_mEF medium (525,000 cells/well 6-well plate).

[0134] At least 4 hours later, the medium was exchanged with SR, and the SR contained ES medium (80% DMEM (Gibco BRL, Rockville MD) removed, 20% removed serum substitute (Gibco), 1% non-essential amino acid (Gibco) , ImML-glutamine (Gibco) and 0.1MMB-base ethanol (Sigma, St.Louis, MO), added with 4ng/mL recombinant human basic fibroblast growth factor (bFGF; Gibco)₀About 0.3-0. 4mL of culture medium per cm2 of the surface area of the plate as a condition. Before adding hES culture, the conditioned medium was supplemented with 4ng/mL human bFGF.

[0135] The plate for culturing ES cells was coated with Matrigel® (Becton-Dickinson, Bedford MA), which was achieved by diluting the stock solution in cold K0 DMEM~1:30 at 0.75-1.0 mL per 9.6 cmz well Disperse and incubate at room temperature for 4 hours or 4°C overnight.

[0136] The HES culture was passaged by incubating in ~200U/mL collagenase IV at 37°C for about 5T0 minutes. Collect cells by removing individual colonies or scraping under the microscope with PipetmarT, then gently separate them into small pieces in conditioned medium, and then inoculate them on Mangel®-coated plates. About 1 week after inoculation, the cultures were confluent and could be passaged. Cultures maintained under these conditions for more than 180 days continuously exhibited ES-like morphology.

[0137] Immunocytochemistry was performed by culturing the sample wells with the first antibody of SSEA-4 (1:20), Tra-1-60 (1:40) and TraT-81 (1:80), and in the elimination of DMEM 37 °C diluted for 30 minutes. The cells were washed with gentle knockout DMEM and fixed in 2% paraformaldehyde for 15 minutes, and then incubated with PBSo cells with 5% goat serum in PBS for 30 minutes at room temperature, followed by FITC-conjugated goat anti-mouse IgG (1:25 ) (Sigma) incubate for 30 minutes. The cells were washed, stained with DAPI and mounted.

[0138] Cells were also tested for alkaline phosphatase expression, which is a marker for undifferentiated ES cells. This is done by culturing the cells on cavity slides, fixing them in 4% paraformaldehyde for 15 minutes and then washing them with PBS. The cells were then incubated with alkaline phosphatase substrate (Vector Laboratories, Inc., Burlingame CA) for 1 hour at room temperature in the dark. Rinse the slides in 100% ethanol for 2-5 minutes before mounting.

[0139] FIG. 1 shows the expression of markers on hES cells detected by histochemistry. As seen in cells on feeder cells, SSEA-4, Tra-1-60 and Tra-1-81 and alkaline phosphatase are expressed by hES colonies-but not by differentiated cells between colonies.

[0140] The expression of undifferentiated hES cell markers was analyzed by reverse transcriptase PCR amplification. To quantify individual gene products relative to radioactivity, use QuantumRNATM Alternate18s internal standard primers (Ambion, Austin TX, USA) according to the manufacturer's instructions. Briefly, determine the linear range of the specific primer pair amplification, and then use the appropriate alternate 18s primer: competitor ( The mixture of competimer) is co-amplified to produce PCR products with a consistent linear range. Before adding AmpllTaqTM (Roche) to the PCR reaction, pre-incubate the enzyme with TaqStartRProMega according to the manufacturer's instructions. The radioactive PCR reaction was analyzed on a 5% non-denaturing polyacrylamide gel, dried and exposed to a phosphoimage screen (Molecular Dynamics) for 1 hour. The screen was scanned with Molecular Dynamics Storm860 and the band intensity was quantified with ImageOuant'M software. The results are expressed as the ratio of radioactivity incorporated into the hTERT or Oct-4 band, normalized to the radioactivity incorporated into the 18s band. The primer sequence used in this experiment can be found in PCT publication WO01/51616.

[0141] The transcription factor Oct-4 is usually expressed in undifferentiated hES cells and negatively regulates differentiation. Cells maintained on Matrigel® in conditioned medium for 21 days express hTERT and Oct-4_oTelomerase activity is measured by the TRAP test (Kim et al., Science 266: 2011, 1997; Weinrich et al., Nature Genetics 7: 498, 1997).

CN 101696397 Β

Cells in a cell culture environment showed positive telomerase activity after being cultured for more than 40 days.

[0142] The pluripotency of undifferentiated cells cultured without feeder cells was determined by forming embryo-like bodies in suspension culture for 4 days, and then culturing on polyornithine-coated plates for 7 days. Immunocytochemistry showed a staining pattern consistent with neuronal and cardiomyocyte lineages, as well as cells that stain α-fetoprotein, which is a marker of the endoderm lineage. The ability of undifferentiated cells to form teratomas by intramuscular injection into SCID mice was also tested. The resulting tumor was removed 78-84 days later. Cell types from all three germ layers were identified by histological analysis.

Example 2: Establishment of differentiated cell line

[0144] Embryo-like bodies are produced as follows. The confluent hES cell monolayer culture was collected by culturing in 1 mg/mL collagenase for 5-20 minutes, and the cells were scraped from the plate. The cells are then separated into pieces and plated on non-adherent cell culture plates (Costar). The medium includes 80% K0 ("knock out") DMEM (Gibco) and 20% non-heat-inactivated FBS (Hyclone), supplemented with 1% Non-essential amino acids, ImM L-glutamine, and 0.1MMB-seryl ethanol. Cells were seeded in 2mL medium per well (6-well plate) at a ratio of 1:1 or 1:2. Ebs was fed every other day by adding 2 mL of medium/well. When the volume of the medium exceeded 4 mL/well, EBs were collected and resuspended in fresh medium. After 4-8 days of suspension, EBs were plated on the substrate.

[0145] Differentiated cell lines are established by collecting embryonic body-derived cells and allowing them to differentiate further. The cells were collected by incubating in 2mg/mL type II collagenase in PBS for 30 minutes at 37°C. The cells were separated, centrifuged, resuspended in differentiation medium and plated in 6-well plates. The proliferating cells were passaged in hEF medium (90% DMEM, 10% heat-inactivated FBS, 0.1 mM non-essential amino acids and 2 mM L-glutamine) and fed every 2-3 days. After two passages, the morphological characteristics of the cell population are similar to that of fibroblasts. This cell line was named HEF1.

[0146] A subpopulation of cells was transduced for the expression of human telomerase reverse transcriptase (hTERT). This was accompanied by infection with the retroviral construct pBABE puro hTERT, which contained the hTERT coding sequence driven by MoLV LTR and SV40 early promoter driven terrin-resistance gene. The growth medium contains 5mL retrovirus stock solution (lX10⁶pfu/mL) and 4pg/mL polybrene (l,5-monomethyl-1,5-mononitrogen H^-methylene polymethionide) and incubate at 37°C. After 8 hours, another 5 mL of retrovirus/polybrene mixture was added, and the cells were incubated at 37°C. The next day, the retrovirus/polybrene mixture was removed and replaced with fresh growth medium. The next day, the medium was replaced with growth medium supplemented with 0.5 μg/mL terrinin. The cells were divided in a ratio of 1:4 in the pterinomycin medium once a week for 8 weeks, and then the cells were tested for telomerase activity.

[0147] Figure 2 (Panel A) shows the morphology of the telomerized HEF1 cell line. Panel B (below) shows the telomerase activity measured in the TRAP test. The cells transduced with the hTERT expression cassette showed positive telomerase activity 20 or 65 days after transduction. Untransduced cell lines or cells transduced with the control vector did not show telomerase activity. HTERT-transduced HEF1 cells and cells transduced with the control vector doubled approximately every 2 days until the control cells stopped dividing on the 38th day. HTERT-transfected cells continuously proliferate at a constant growth rate for more than 60 days (30 doublings).

[0148] ES cell growth medium used the conditions in Example 8, using 6000 rad irradiated HEF1 cells and seeded at ~4.1 to 5.5×10, cells cm-. The test medium supports the growth of the H9hES cell line cultured on Matrigel® substrates. The HEF1 conditioned medium was used to maintain hES cells for more than 4 passages, showing the morphology of undifferentiated ES cells, and maintaining the expression of HTERT and Oct-4.

Example 3: Further differentiation of osteoblast-like cells

[0150] As described above, human ES cells (H1 cell line, passaged 30 times) were maintained in feeder-free conditions. For this experiment, hES cells were seeded on Matrige in mEF-conditioned medium at ~1X. Telomerization HEF1

CN 101696397 Β

The cells were cultured in a 3.lXl()3cm-2 plate in DMEM containing 10% FBS, 1% non-essential amino acids and 2 mM L-glutamine. Normal human mesenchymal stem cells (hMSC) were obtained from BioWhittaker Inc., MD (a subsidiary of Cambrex). They are maintained in MSC growth medium (BioWhittaker Part#PT-3001) according to the manufacturer's instructions. The BJ5ta fibroblast cell line (Bodnar et al., Science279:349, 1998) is maintained in a standard medium made from 10% FBS in 1:3M199/DMEM.

[0151] Two days after the last passaging, each medium was replaced with osteoblast induction medium (01M) to induce differentiation. 0ΙM is based on MSC growth medium (ClonTech catalog number #3238) (U.S. Patent No. 5,486,359), and the medium is supplemented with 0.1PM dexamethasone, 5pM ascorbic acid-2-phosphate, 10mM B-glycerophosphate and 100ng/mL BMP-4_oCells are fed with fresh OIMo every 2-3 days

[0152] After 11 days in 0IM, all cells showed changes in cell morphology. HEF1 cells, hMSC and BJ cells changed from a spindle shape to a square shape, and some cells became flatter. hES cells show different morphologies of mixed differentiation groups.

[0153] The cells were fixed in PBS containing 2% paraformaldehyde for 20 minutes, washed with PBS and analyzed for osteoblast markers. Alkaline phosphatase (AP) was detected with Vector substrate (Vector Laboratories, Inc., Burlingame, CA). The expression of AP was clearly localized in the cell clusters of differentiated H1 cells and HEFKBJ and hMSC cells.

[0154] Matrix protein is produced by osteoblasts, collagen and osteocalcin, and detected by immunostaining. Treatment with 100% ethanol for 2 minutes increased the permeability of the culture. After washing with PBS, the culture was incubated with PBS containing 5% normal goat serum for 2 hours, and then treated with the first rabbit antibody anti-collagen-1 (1:10, Monosa catalog number #P5041) or osteocalcin (1:50, Biomedical Technologies Inc. catalog number #13T593). The staining was visualized with FITC-labeled second goat anti-rabbit immunoglobulin (1:100, Southern Biotechnology Associate Inc. catalog number #4050-02).

[0155] Figure 3 shows the results. Groups A and B show immunocytochemistry labeled osteocalcin and collagen T. Group C shows alkaline phosphatase activity staining. These characteristics are characteristic of cells of the osteoblast lineage.

[0156] These data are consistent with the hypothesis that hES cells and HEF1 cells have the ability to produce osteoblasts when subjected to appropriate differentiation operations in vitro.

[0157] It should be understood that some changes of the invention described in the present disclosure are routine optimization matters for those skilled in the art, and can be completed without departing from the spirit of the invention or the scope of the appended claims.

CN 101696397 Β