Process for producing nerve stem cells, motor neurons and gabaergic neurons from embryonic stem cells

The neural stem cell, motor neuron, and G A B A operation neuron from detailed embryonic stem cells

Technical field

the present invention -- from embryonic stem cells (E S cell) -- alternative -- a neural stem cell -- or still more nearly alternative One -- the method of manufacturing a motor neuron and a GA B A operation neuron efficiently -- To involve. Background art

in a mammalian central nervous system, a neural stem cell continues existing through the lifetime of an individual -- many -- producing Neuron Greer [ like ] -- generating and maintenance of a central nervous system It is thought that it has contributed. the brain of the mammal which includes humans recently -- nerve Staircase separation and the art to cultivate are developed in A cow -- Cell migration to various neurodegenerating diseases and damages The application to Medical treatment is expected. however, it sets in a generating process -- various -- inner -- -like -- and various types which are produced from a stem cell by control by an external factor, and specialize by it Neuron, The trial which produces the motor neuron etc. which are specifically produced especially in early stages of an embryonic growth from the neural stem cell which carried out cultivation amplification by in vi tro should be achieving sure success. It is and is ..

From a E S cell with the present invention and the capability to specialize in all the cells of an individual to therefore, generating From there to the efficient specialization guidance means to the neural stem cell holding early character, and movement It is a subject about providing the art of producing a neuron with a specific neuron etc. alternatively. It carries out. the indication of an invention -- there -- this artificer -- the embryo from a E S cell -- formation of a state, the specialization guidance to a neural stem cell, and 2 Result of having examined many things about the conditions of the specialization guidance to Yuron, E the embryo from S cell -- state formation -- N o g g i n (noggin) Especially proteinic addition is excellent. an embryo -- a state -- coming out to inside use of the culture medium which added the Sonic hedgehog protein for amplification of the neural stem cell which carries out present in addition to fibroblast growth factor (f ibroblas t growth fac tor: FGF) -- very -- efficiency -like -- it is furthermore -- if the neural stem cell guided by doing in this way is made to specialize -- movement New See 1 Ron and a GA B A operation neuron produced alternatively and efficiently. it takes out -- it came to complete the present invention.

namely, the thing which the present invention does for the suspended cell culture of the E S cell under noggin protein existence -- Special the embryo considered as the mark -- the formation method of a state is provided.

moreover -- the present invention carries out the suspended cell culture of the E S cell under existence of noggin protein or nonexistence Embryo -- a state is made to form -- ranking next -- this -- a fibroblast growth factor and Sonique Hedgehog the manufacturing process of the neural stem cell carrying out a suspended cell culture under existence of A protein -- To provide It is To.

furthermore -- the present invention carries out the suspended cell culture of the E S cell under existence of noggin protein or nonexistence Embryo -- making a state form this -- a fibroblast growth factor and Sonique -- Djihog protein it floats under existence of quality and guides to a neural stem cell -- ranking next and making this specialize -- the feature It is what provides the manufacturing process of the motor neuron to carry out and a G A B A operation neuron. It is. Brief explanation of the drawings

Drawing 1 -- an embryo -- it is a figure showing the relation between the cultivation days of a state, and New Ross fair formation. Drawing 2 is a figure showing the result of having carried out immunity dyeing of the New Ross fair. dyeing parts --]3 -- I I I -- tubulin is revealed -- it is shown that it is a neuron.

Drawing 3, It is a figure showing the result of having carried out immunity dyeing of the New Ross fair.

Drawing 4, It is a figure showing the result of having carried out immunity dyeing of the New Ross fair. Drawing 5, It is a figure showing the immunity dyeing result after New Ross fair Succession cost cultivation.

Drawing 6, The neuron after the New Ross Fare cost cultivation and the ratio of a Dalya cell are shown. It is a figure.

Drawing 7, It is a figure showing the addition effect of noggin protein.

Drawing 8, It is a figure showing the addition effect of Djihog protein to Sonique. The best form for inventing

It is used for the present invention. As an embryonic stem cell, it is already established as a cultured cell. An embryonic stem cell can be used. For example, a mouse, a hamster, a pig, humans, etc. An embryonic stem cell stock can be used. as an example -- 129 / 01 a system -- Mau The A origin An embryonic stem cell, EB3, and E 14 t g 2 grade are mentioned. being concerned -- As for an embryonic stem cell, it is preferred to carry out Culture inheritance cost in the GMEM culture medium containing serum.

E the embryo from S cell -- carry out a suspended cell culture by the culture medium which added noggin protein for formation of the state The specialization guidance efficiency from an embryonic stem cell to a neural stem cell improves. noggin protein -- example although For example, American noggin can be used -- American cane full length cDNA of noggin is introduced into COS 7 cell -- transient -- noggin protein -- revelation -- A Did culture supernatant may be used as it is. the concentration in the culture medium of noggin protein -- this -- A grade is preferred 1*50% (v/v) at culture supernatant conversion. the suspended cell culture of an embryonic stem cell -- for example -- an embryonic stem cell -- serum content alpha -- a MEM culture medium --X[ 1 ] 10⁵It may carry out for 4* eight days at the concentration about eel ls_mL. here -- as serum -- Sushi serum, pig serum, etc. -- mentioning -- Re and 8*12% of especially its concentration are desirable 5*15%. In a ￡* 1 MEM culture medium, it is 2. -- They are 0. 01*0. 5mM, especially 0. 05*0. 2mM about mercaptoethanol. Adding like is preferred. Cultivation is 5%CO.₂bottom of condition, and 35*40 degreeC -- line That which is obtained is preferred.

addition of noggin protein -- an embryo -- a state -- adding to the time of formation (i.e., cultivation 1*6 day) Especially that of Keep it is preferred. the embryo formed as mentioned above -- neural stem cell obtained from the E S cell via the state In order to amplify, a suspended cell culture is carried out by the neural stem cell multiplication culture medium which contains the Sonic hedgehog protein in addition to a fibroblast growth factor. addition of the Sonic hedgehog protein the specialization guidance efficiency and multiplication efficiency to a motor neuron precursor cell of a neural stem cell -- Toward it stages -- subsequent specialization cultivation -- a motor neuron and G A B A operation neuron It actually specializes.

As fibroblast growth factor (F G F), it is F G F. -- 2 is preferred. The content of F G F in a culture medium is 5*5. 0 ngZmL, especially 1 0*4 0 ngZmL is preferred. As Djihog protein, it is Djihog protein for example, to mouse Sonic in Sonique. It is desirable. The content of the Sonic hedgehog protein in a culture medium is 1*2. 0 nM, especially 1*1 O nM are preferred.

As a culture medium, they are glucose, glutamine, an insulin, and tolan besides the above-mentioned ingredient. Palin etc. are added to Spheerin, projet Steron, Putrescine, and chlorination selenium. It is preferred to use a It was DM EM culture medium. DM E M: It is especially to use F-1 2 culture medium. It is desirable. Cultivation is 5 % CO.₂The bottom of a condition, and 3 5*4 It is preferred to carry out by 0 °C. At the time of cultivation 7* nine days are preferred in between.

it is called New Ross fair (neurosphere) by the above-mentioned suspended cell culture -- single -- thin -- The cell condensation lump of Cell derivation forms.

Obtained New Ross fair, It originates only in a neural stem cell. Above-mentioned Culture. It turns out that specialization guidance efficiency by the Nursing method is very high.

Thus, it will be movement 2 if the obtained neural stem cell is cultured by the usual specialization culture medium. Specialization guidance only of Yuron and the G A B A operation New mouthful A is carried out. It is specialization guidance here. As a culture medium, they are glucose, glutamine, and an insulin, transformer Ferrin -- pro -- DM E M containing Jesty Ron, Putrescine, and chlorination selenium: F-1 2 culture medium (culture medium excluding Palin to In other words and F G F from the culture medium for neural stem cell multiplication), using -- good -- better -- There. This time, the Sonic hedgehog protein may exist or there may not be. [ any ] Cultivation is 5%CO.₂It is preferred the bottom of a condition and to carry out for 5*7 days by 35*40.

the former Neural cell by which specialization guidance was carried out from an embryonic stem cell, not only a neuron -- many -- A neuroglia of quantity, etc. are included. The utility value was restricted extremely. this it receives -- the neuron obtained by the present invention -- they are almost only a motor neuron and a GAB A operation neuron. EXAMPLE

Next, although an example is given and the present invention is explained still in detail, the present invention is Limit in any way to this. Constant is not carried out.

A. Material and a method

(1) mouse the Culture inheritance cost of an embryonic stem cell, and an embryo -- formation of a state

129/01 a system mouse origin A blast Scidin resistance gene is inserted in an embryonic stem cell, E 14 t g 2 a, and its Oc t 3/4 gene loci, and there is undifferentiated. EB3 which can choose an embryonic stem cell, 10% calf embryo serum, nonessential amino acid, ImM sodium pyruvate, 0. ImM 2 -- Mercaptoethanol and 1 0 0O UZmL leukemia inhibitory factor

(Leukemia inhibitory factor, LIF) (GMEM) the included Glasgow minimum essential medium culture medium -- a law -- method (5%C0)₂if it is only called "cultivation" 37 and the following it comes -- Culture inheritance cost was carried out according to this condition.

the embryo from an embryonic stem cell -- a state -- performing as follows formation of (Embryoid body: EB) -- Went. They are after washing and 0. 25% trypsin ImM EDTA Processing at PBS about an embryonic stem cell. A cow is stopped, About the cell distributed by pipetting, he is 10% calf embryo. Serum and 0. 1 niM 2 -- Ba filled with the a_M E M culture medium containing mercaptoethanol It isX[ 1 ] 10 in the culture dish for Kateria.⁵sowing is carried out at the concentration of cellsZmL -- noggin protein (full length cDNA of Afrikatsumegael Noggin is introduced into COS 7 cell transient revelation culture supernatant carried out) Carry out a suspended cell culture for 4*8 days under existence and nonexistence, and make EB form. It was. (2) Separation by alternative cultivation of the neural stem cell from EB

moving EB formed as mentioned above to a centrifugal tube with culture solution -- 10 -- by settling between parts, it brings together in a tube bottom, top Fresh is removed, and it is re-suspended in PBS -- it settles for 10 minutes again. ￡ after removing top Fresh is re-suspended in 0. 25% trypsin 1 lmM EDT A solution, incubates for 5 minutes by 37 °C, and is 10 % calf embryo blood. It is a part about a cell with the pipette after stopping a protein decomposition reaction by the Facial 1 MEM culture medium containing Fresh. It was made to scatter. Distributed cell alpha -- Centrifugal operation washes twice in a MEM culture medium, Glucose (0. 6%), glutamine (2mM), and insulin (25 gZmL) Transformer Ferrin (l O O gZmL) and projet Steron (20nM) Putrescine (60 M), chlorination selenium (30nM), and FGF -- 2 (20 ng/mL), Dulbecco's modified Eagle's medium which passed and acted as Attach Callot of Palin (2 n g/mL) (DMEM) : F-12 (1 : 1) -- inside of a culture medium (neural stem cell multiplication culture medium) Into Or he is further Mau there. It isX[ 5 ] 10 to the culture medium which added Ssonic hedgehog protein mouse sonic hedgehogl (5nM).⁴It is New by carrying out sowing and carrying out a suspended cell culture for 7* nine days at the concentration of cells/mL. -- The cell condensation lump of the single cell origin called loss fair (neurosphere) is made to form. It was (the neurosphere method). These New Ross fairs are the above-mentioned neural stem cell multiplication culture media. After carrying out centrifugal washing by the specialization culture medium except Palin to Ri F G F_2, then -- or -- Polly L1 Orr 2 who filled with the specialization culture medium the cell distributed by pipetting Carry out sowing to the culture dish which carried out the coat by Ching (poly-L- ornithin). Sonic hedgehog protein [ (5nM) ] -- cultivating for 5*7 days under existence or nonexistence -- specialization It carried out. the New Ross fair obtained as mentioned above is again distributed into a single cell -- it Succession-cost[ during seven days ]-cultivates by a neural stem cell multiplication culture medium, and makes secondary New Ross fair form -- this , -- it is made to specialize like the above

(3) Identification of the specialization neuron by immunity dyeing, and a neuroglia

immunity using a fluorescent antibody in the Neuron neuroglia made to specialize as mentioned above the law of dyeing -- it identified by the method. A motor neuron is mouse anti-I s 1. -- One monochrome 1 Null antibody, A Jack anti-ChAT Pori Cros 1 Null antibody and mouse anti-j3_III tubulin A Using a Nookuro 1 Null antibody, GAB A operation neurons are 67 points of Usagi anti-GAD. It identified using the Recreal antibody. The oligodendrocyte was identified for the neuroglia and the Astro site by the Usagi anti-GFAP Pori Cros 1 Null antibody using the Null [ Mouse anti-04 monochrome - ] antibody.

B. Experimental result

(1) Separation refining by the alternative cultivating method of the neural stem cell from EB

it sets to initial specialization of the E S cell by formation of EB first -- a neural stem cell -- cultivation throat It was examined whether it would appear at time. it will be single in being concrete and EB on cultivation 4* the 8th -- thin -- it distributes to A cow -- it cultivates for seven days in a neural stem cell culture medium -- making the New Ross fair form It was. these New Ross fairs are made to move and specialize in a specialization culture medium -- the specialization ability is authorized Self-duplicate ability was also authorized by carrying out Succession cost.

It is into a single cell about the 6 after the formation start of EB according to suspended cell culture in Drawing 1, and eight-day back, and EB. It distributed and the result of having performed alternative cultivation of the neural stem cell (the neurosphere method) was shown. The number of the obtained New Ross fairs was made into the number of the neural stem cells which appeared in EB. As a result, it is identified by this method. (the New Ross fair can be formed) It turned out that a neural stem cell will almost increase the 4th day of cultivation of E B gradually with 1. 1 % by detection in all the 0in cell. 25%, and the 8th day a crack and on the 6th day of cultivation.

the New Ross fair obtained from EB on the 6th as shown in Drawing 1 -- specialization conditions -- 7 -- it cultivates during a day -- the specialization ability was authorized by immunity dyeing. the result -- a figure -- 2*4 -- To indicate. it is marker 1 of a neuron -- 3 -- marker 1 of III_tubulin and a neuroglia -- Ah Place which performed To GFAP and 3-fold immunity dyeing by the antibody of 04, all the neuro Spare -- almost -- i3 it comprises only a neuron which reveals -III- tubulin -- (figure 2) from which the neuroglia was not detected. and the neuron of them -- few -- Both -- I s 1 -- GABA operation neuron (a part for fibrous part [ Figure ] of 4) which reveals motor neuron (figure 3 round portions and Fiber-like portions) which reveals 1 and ChAT, and GAD 67 It contained.

what the obtained New Ross fair is Succession-cost-cultivated for -- secondary neuro Spare is obtained -- they are cultivated for seven days on specialization conditions -- specialization ability -- immunity dyeing -- Inspection It carried out constant. As a result, all the New Ross fair contains the neuroglia. (figure)

5) 84. 2% contained both the neuron and the neuroglia before long. (figure)

6) . Figure [ It is a result. ] 5, 3 -- III -- tubulin (thin clear fiber type), GFAP (surrounding portion of /3 -III- tubulin), and 04 (further outside portion [ GFAP ]) 3-fold immunity dyeing

As a result, into a single cell, it distributes, these neurosphere(s) are Succession-cost-cultivated, and it is the new new. If loss fair is made to form and it is made to specialize, Many of those clones are a neuron and A. It is [ in / that both Lear cells come to be included / generating of an actual central nervous system ] a neuroglia. Many [ so that it may appear in the second half / the neural stem cell separated from E B / cultivation / Succession cost ] It turned out that-izing ability comes to be shown.

(2) Increase in efficiency of specialization guidance of the neural stem cell by noggin protein

It is a part of a neural stem cell by adding noggin protein to (for six days) at the time of EB formation. The increase in efficiency of-izing guidance was tried. full length cDNA of noggin and Xenopus laevis noggin -- pEF -- it includes in a BOS expression vector and introduces into COS 7 cell -- transient -- revelation let the culture supernatant carried out be noggin solution -- COS 7 cell which introduced only the expression vector The culture supernatant was considered as contrast. Figure Like and the number of neural stem cells which forms the New Ross fair by which specialization guidance is carried out in EB depending on the quantity of a noggin culture supernatant which are shown in 7 increases, and is 1. The peak was reached in Hiroshi 10 times.

(3) Increase in efficiency of the motor neuron specialization by the Sonic hedgehog protein

E aiming at the increase in efficiency of the motor neuron production from B origin neural stem cell, and specialization -- Sonique -- Djihog protein -- primary culture 2 of the time of neural stem cell multiplication, i.e., the E B origin, it adds at the time of formation of you loss A fair -- the effect was examined. a motor neuron -- 2 you loss A fair is distributed into a single cell -- a specialization culture medium -- after [ the cultivation during five days ], and I s 1 -- 1 -- and -- i3 -- I I I -- it identifies by performing double immunity dyeing of tubul in -- turning a fixed quantity of the number It was. As shown in Drawing 8 as a result, it is movement 2 by Sonic hedgehog of 5 nM. Production of Yuron was doubled. It is the Sonique Hedgehog to a specialization culture medium at the time of nerve specialization. The effect was not accepted even if it added A. Industrial applicability

According to the present invention method, they are a motor neuron and G A B A operation nature at least than a E S cell. It was which that a neuron can produce systematically and efficiently divides. Are alternative. If a neuron is obtained, they will be amyotrophic lateralsclerosis, Huntington's chorea, and Al. It may be applicable to transplant medical treatment, such as a Haiheimer disease.