CN1961068A - Compositions and methods to culturing neural stem cells with bone marrow stromal cells - Google Patents

Abstract

The present invention encompasses methods and compositions for enhancing the growth of neural stem cells. Methods for modulating MHC molecule expression on a neural stem cell (NSC) are also included in the invention. Figure 1 is a graph depicting the proliferation of BMSCs in BMSC medium (DMEM-low glucose, 10% lot tested fetal bovine serum) or NSC medium (DMEM-F12, N2-Supplement, EGF 20 ng/ml, bFGF 10 ng/ml, heparin 8 ug/ml, penicillin-streptomycin).

Description

The method and composition of culturing neural stem cells with bone marrow stromal cells

Background technology

Marrow comprises at least two kinds of stem cells: hemopoietic stem cell and non-hemopoietic tissue stem cell, the latter has various formulation: mescenchymal stem cell, spinal cord stroma cell (MSCs) or marrow stromal cell (BMSC), these terms are synonym in this article.The spinal cord stroma cell causes that people's interest is because they can separate easily from ossiculum marrow aspirate, and generates the unicellular colony of deriving easily.The unicellular colony of deriving can have 50 times population doublings in 10 weeks, and is divided into scleroblast, adipocyte, chondrocyte (people .1970 Cell Tissue Kinet.3:393-403 such as A.J.Friedenstein; People .1980Blood 56:289-301 such as H.Castro-Malaspina; People .1992 J.Cell Sci.102:341-351 such as N.N.Beresford; D.J.Prockop 1997 Science 276:71-74), myocyte (people .1995 Muscle Nerve 18:1417-1426 such as S.Wakitani), astroglia cell, oligodendrocyte and neurone (people .1998 Proc.Natl.Acad.Sci.USA 95:3908-3913 such as S.A.Azizi; People .1999 Proc.Natl.Acad.Sci.USA 96:10711-10716 such as G.C.Kopen; People .2000 Neuroreport 11 such as M.Chopp, 3001-3005; People .2000 Neuroscience Res.61:364-370 such as D.Woodbury).

And spinal cord stroma cell (MSCs) generates cell (Kopen, the people .1999 Proc.Natl.Acad.Sci.96:10711-10716 such as G C. of all three germinal layers; Liechty, people .2000 Nature Med.6:1282-1286 such as K.W.); Kotton, people .200lDevelopment 128:5181-5188 such as D.N.; Toma, people .20002 Circulation 105:93-98 such as C.; Jiang, people .2002 Nature 418:41-49 such as Y.).Evidence shows the same epithelial cell that all generates of the pure cell mass of undifferentiated bone marrow-derived cells and MSCs in the body, comprises that (Krause waits people .2001 Cell 105:369-377 for the epithelial cell of lung; Petersen waits people .1999Science 284:1168-1170).And several the nearest transplanting that studies show that MSCs are increased (Ferrari, people .1998 Science 279:1528-1530 such as G. owing to tissue injury; Okamoto, people .2002 Nature Med.8:1101-1017 such as R.).For these reasons, investigating at present the spinal cord stroma cell the cell therapy of multiple human diseases and the application in the gene therapy (people such as Horwitz., 1999 Nat.Med.5:309-313; Caplan waits people .2000 Clin.Orthoped.379:567-570).

The spinal cord stroma cell has constituted a selection source of multipotential stem cell.Under physiological condition, the spinal cord stroma cell is kept generation (Clark, the B.R.﹠amp of myeloid tissue structure and regulation and control hematopoietic cell respectively under the help of different cell adhesion molecules and cytokine; Keating, A.1995 Ann NY Acad Sci 770:70-78).By selective adsorption in tissue culturing plastic and in that the spinal cord stroma cell of marrow outgrowth can (Azizi, S.A. wait people .1998 Proc Natl Acad Sci USA, 95:3908-3913 by efficient amplification; Colter, D.C. waits people .2000Proc Natl Acad Sci USA 97:3213-218), and can carry out genetic manipulation (Schwarz, E.J. wait people .1999 Hum Gene Ther 10:2539-2549) to it.

The spinal cord stroma cell is also referred to as mescenchymal stem cell, because they can be divided into multiple mesoderm tissue, comprise bone (Beresford, J.N., wait people .1992 J Cell Sci 102:341-351), cartilage (Lennon, D.P, Deng people .1995 Exp Cell Res 219:211-222), fat (Beresford, J.N., wait people .1992 J Cell Sci 102:341-351) and muscle (Wakitani waits people .1995 Muscle Nerve 18:1417-1426).In addition, reported that (Woodbury, D. wait people .2000J Neurosci Res 61:364-370 to the class neuronal cell that is divided into expression neurone mark; Sanchez-Ramos, J. waits people .2000 Exp Neurol164:247-256; Deng, W. waits people .2001 Biochem Biophys Res Commun282:148-152), show that perhaps the spinal cord stroma cell can overcome the germinal layer typing.

Stem cell is the multi-functional progenitor cell of self, in the particular organization of specific period, have the most widely developmental potentiality people .1997 Cell 88:287-298 such as () Morrison. recently, the research of neural system stem cell has caused extensive interest, because they are to understanding neurodevelopmental importance and the treatment potentiality in the treatment neurodegenerative disease.

Separate and keep the existing method of embryo and other stem cell lines to depend on the use rat embryo fibroblast cell as trophoderm.Have been found that in nutrient solution and to use the serum alternative and make embryonic stem cell clone's frequency improve several times people .2000 Dev Biol 227:271-278 such as () Amit.The clone embryonic stem cell continues and the breeding of not having a differentiation needs the existence of basic fibroblast growth factor.Be used at present to separate, the method for cultivation and amplifying human embryonic stem cell all is restricted because of depending on the rat embryo fibroblast cell trophoderm.What still need to prove is: when lacking nurse cell, stem cell can a kind of undifferentiated state of unconfined maintenance.

For improving the growth velocity of human foetus's abr cell, many different research teams have used several diverse ways and somatomedin in the past during the decade.The amplification and the maintenance that have shown human foetus's neurocyte dried (hNSC) need basic fibroblast growth factor (bFGF) and Urogastron (EGF).These human nerve's stem cell culture are with the form normal growth of free unmanaged flexibility cell cluster (neural ball), but when only having bFGF and EGF to exist, neural ball can not infinite multiplication.Leukaemia inhibitory factor (LIF) show improve growth velocity and prolong FGF and the survival time of EGF responsiveness neural stem cell (people such as Carpenter., people such as 1999 and Wright., 2003).Except regulating growth velocity, leukaemia inhibitory factor is also actively regulated and control several genes, comprise the neural stem cell surface main histocompatibility complex molecule (people such as Wright., 2003).

The human fetal abr cell is considered to be used for the attractive candidate of damaged tissue regenerated stem cell transplantation.Need homotransplantation from embryo or the fetus donor stem cell that comes that derives.Transplanted cells in the heredity between the different individuality is usually with the danger of host's rejection.Nearly all cell is all expressed the major histocompatibility complex product, I class MHC molecule.And many kinds of cells can be expressed II class MHC molecule under the inducing of struvite cytokine.The rejection of allotransplantation is mainly cell-mediated by the T of CD4 and CD8 subclass.(people .1992 Annu.Rev.Immunol.10:333 such as Rosenberg).The cd4 t cell of alloreactivity generates some cytokines, and the CD8 that has aggravated cytolysis is to alloantigenic reaction.In these subclass, competitive cell subsets is formed at after the antigenic stimulation, and described antigenic stimulation has the feature that cytokine that described subclass generates is given.The Th1 cell that generates interleukin II (IL-2) and IFN-γ (IFN-γ) mainly with allograft rejection react relevant (people such as Mossmann., 1989 Annu.Rev.Immunol.7:145).The Th1 that the Th2 cell that generates interleukin 4 (IL-4) and generate interleukin 10 (IL-10) can be reduced the IL-10 mediation reacts people .1989J.Exp.Med.170:2081 such as () Fiorentino.In fact, carried out much making great efforts to make not wishing that the Th1 reaction that takes place turns to the Th2 approach.For the bad allosome rejection of T cell in the patient body to graft, typical treatment is to adopt immune suppressant drug, as Bo Nisong, azathioprine and cyclosporine A.Unfortunately, for patient's life need use these medicines usually, and they have the side effect of many danger, comprise the general immunosuppression.

Neural stem cell is expressed the I class MHC of low (maybe can ignore) level and/or II class MHC antigen people .2001J.Neuroimmunol.112:35 such as () McLaren, but these cells are repelled after being implanted into the allogeneic receptor usually, unless use immunosuppressive drug.After the surface of cell membrane MHC molecular level that is exposed to the struvite cytokine of this class of IFN is raised, may cause rejection.Therefore, be starved of the stdn of culture condition, make the cell proliferation of nerve cord and multiple with the largest potentialityization of therepic use.In addition, it is believed that at present: the neural stem cells transplantation of a success is by preventing and/or reduce the neural stem cell immune response of undesired immune effector cell mediation, and the host who transforms neural stem cell repels.

Therefore, undesired immunoreactive method in the permanent nerve cells transplantation process of thirsting for suppressing or to prevent between the hereditary different individuality, the present invention has then satisfied this demand.

Summary of the invention

The present invention includes the method and composition of culture of neural stem cells neural.The present invention comprises the cell that utilizes above-mentioned composition and method to generate simultaneously.

The present invention includes a kind of composition, said composition comprises a kind of isolating marrow stromal cell (BMSC) and the definite developing medium of a kind of chemical ingredients, and described developing medium comprises neural stem cell (NSC) growth medium and the described BMSC excretory factor.

An aspect, described developing medium do not contain exogenous leukaemia inhibitory factor (LIF).

Another aspect, the BMSC excretory factor is selected from somatomedin, nutritional factor and cytokine.

Another aspect, the described factor is selected from LIF, Brain Derived Neurotrophic Factor (BDNF), EGF-R ELISA (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), pHGF (HGF), IFN-γ, trypsin-like growth factor bindin (IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, MCP (MCP-1), mononuclear phagocyte G CFS (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinase (TIMP-1), TIMP-2, tumour necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, UPA acceptor (uPAR), Delicious peptide (BMP4), IL1-a, IL-3, Leptin (leptin), STEM CELL FACTOR (SCF), mesenchymal cell derivative factor-1 (SDF-1), Thr6 PDGF BB-BB (PDGFBB), transforming growth factor-beta (TGF β-1) and TGF β-3.

The present invention also is included in the developing medium that a kind of chemistry that comprises the neural stem cell growth medium determines and cultivates BMSC and NSC altogether.

An aspect, BMSC cultivates in a kind of mode that contacts that relies on NSC, and wherein BMSC is that physics contacts with NSC.

Another aspect, BMSC cultivates in the mode that a kind of non-dependence contacts with NSC, and wherein BMSC is not that physics contacts with NSC.

Another aspect, NSC derives from people's central nervous system.

Another aspect, BMSC derives from the people.

Another aspect is incorporated into exogenous genetic material in the cell of the present invention.

The present invention also comprises a kind of marrow stromal cell condition developing medium (BMSC-CM), comprises the developing medium that a kind of chemistry is determined, wherein comprises the neural stem cell growth medium and the isolating BMSC excretory factor.

An aspect, BMSC-CM does not comprise exogenous leukaemia inhibitory factor (LIF).

Another aspect, BMSC-CM does not contain BMSC substantially.

Another aspect of the present invention, the factor that BMSC-CM comprises is selected from somatomedin, nutritional factor and cytokine.

Another aspect, the described factor are selected from leukaemia inhibitory factor (LIF), Brain Derived Neurotrophic Factor (BDNF), EGF-R ELISA (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), pHGF (HGF), IFN-γ, trypsin-like growth factor bindin (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, MCP (MCP-1), mononuclear phagocyte G CFS (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinase (TIMP-1), TIMP-2, tumour necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, UPA acceptor (uPAR), Delicious peptide (BMP4), IL1-a, IL-3, Leptin (leptin), STEM CELL FACTOR (SCF), mesenchymal cell derivative factor-1 (SDF-1), Thr6 PDGF BB-BB (PDGFBB), transforming growth factor-beta (TGF β-1) and TGF β-3.

The present invention also comprises the regulate and control method of a kind of main histocompatibility complex molecule at isolating NSC surface expression.

An aspect, the expression of MHC molecule on the NSC surface regulated in the common cultivation of isolating BMSC and isolating NSC.

Another aspect, the expression of MHC molecule on NSC can be regulated by cultivating NSC with BMSC-CM.

The present invention includes a kind of isolating NSC, prepare through cultivating BMSC and NSC altogether.

One aspect of the present invention, the minimizing expression that the NSC that generates by the inventive method shows I class MHC molecule.

Another aspect, the NSC that generates by the inventive method shows the baseline values of II class MHC molecule.

The present invention has also comprised a kind of neuronal cell cultures equipment, wherein contains separative NSC, isolating BMSC, NSC growth medium and a device that prevents physics contact between NSC and the BMSC.

Another aspect, aforesaid device also comprise a strainer or film, make and do not produce the physics contact between NSC and the BMSC.

Another aspect, above-mentioned strainer or film have the hole, make the described BMSC excretory factor by described strainer or film.

Description of drawings

For explanation the present invention, some the specific embodiment of the present invention has been described in the accompanying drawing.But, the arrangement of the embodiment that the invention is not restricted to describe in the accompanying drawing and means.

Fig. 1: described the propagation of BMSC in BMSC developing medium (foetal calf serum of DMEM-low dextrose, 10% sampling test (FBS)) or NSC substratum (DMEM-F12, N2-additive, Urogastron (EGF) 20ng/ml, basic fibroblast growth factor (bFGF) 10ng/ml, heparin 8 μ g/ml, penicillin-Streptomycin sulphate (P/S)).

Fig. 2: comprising Fig. 2 A-2C, is the image that a series of NSC of describing grow into neural ball.Fig. 2 B and 2C have described outside, and there is the NSC of growth down in derived leukocythemia supressor (LIF).Fig. 2 A has described to lack the LIF neural ball of growth down.

Fig. 3: comprise Fig. 3 A-3D, be a series of when describing to exist exogenous LIF (Fig. 3 C and 3D) and when not having exogenous LIF (Fig. 3 A and 3B) cultivate the image of BMSC and NSC altogether.

Fig. 4: comprising Fig. 4 A-4F, is a series of NSC of describing and BMSC nidogen (nestin) and glial fibrillary acidic protein (GFAP) is expressed, the image (Fig. 4 A-4F) of pre-differentiation when cultivating altogether.Fig. 4 G and 4H have described to lack when independent BMSC cultivates the expression of nidogen and GFAP.

Fig. 5: comprising Fig. 5 A-5D, is that the NSC that a series of proofs grow on the BMSC remains with the image that is divided into neurone and Astrocytic potentiality.Fig. 5 A-5D has described the MAP2-GFAP-DAPI dyeing of the coculture of differentiation.MAP2 is a kind of neurocyte skelemin.DAPI be used for 4 of nucleus dyeing ', 6 '-diamidino-2-benzene indole hydrochloride.

Fig. 6: the painted image of describing to break up of coculture nestin-GFAP has shown the trace expression of nestin after the differentiation.

Fig. 7: the painted image of the GFAP-DAPI of the NSC that describes to break up.

Fig. 8: comprising Fig. 8 A and 8B, is the painted image of MAP2-GFAP-DAPI of a series of BMSC that describe to break up, has shown the trace expression of neurone and stellate cell mark.

Fig. 9: comprising 9A and 9B, is that a series of fluorescence activated cell sorter are analyzed collection of illustrative plates (FACS), has described the phenotype of the NSC after BMSC lacks.It is the CD-105 positive (mark of BMSC) that Fig. 9 A explanation is less than 2% cell.Fig. 9 B explanation is the CD-133 positive more than 90%.

Figure 10: comprising Figure 10 A and Figure 10 B, is that a series of fluorescence activated cell sorter are analyzed collection of illustrative plates, described the nestin expression deletion (Figure 10 A) of BMSC and from the BMSC coculture nestin of isolating NSC express (Figure 10 B).

Figure 11: illustrate that the NSC that grows on the BMSC has kept it and has been divided into neurone and Astrocytic versatility.

Figure 12: described to exist or lack under the BMSC condition, NSC is at Transwell ^TMIn growth.

Figure 13: comprise Figure 13 A and Figure 13 B, described in Transwell, to exist under BMSC (Figure 13 A) or shortage BMSC (Figure 13 B) condition that NSC be not coated with by the image of the growth on the flat board.

Figure 14: comprise Figure 14 A-14D, described the NSC (Figure 14 A and 14B) that cultivates with marrow stromal cell conditioned medium (BMSC-CM) and containing exogenous LIF (Figure 14 C) or lacking the NSC that cultivates in the NSC-substratum of exogenous LIF (Figure 14 D).

Figure 15: compared BMSC-CM and the effect of the standard NSC substratum that contains or do not contain exogenous LIF to the NSC growth.The BMSC-CM1 substratum derives from the BMSC that is incubated in the NSC substratum that contains EGF and FGF.The BMSC-CM2 substratum comes from the BMSC that is incubated in the NSC substratum that does not contain EGF and FGF.

Figure 16: comprising Figure 16 A-16D, is a series of facs analysis collection of illustrative plates, has described the phenotype curve (Figure 16 A and 16B) of the NSC that separates from the BMSC donor different with two kinds cultivated altogether.Figure 16 C and Figure 16 D have described respectively at the NSC substratum with contain the phenotype curve of the NSC that does not add BMSC in the NSC substratum of exogenous LIF and cultivate out.

Figure 17: comprising Figure 17 A-17D, is a series of facs analysis collection of illustrative plates, has described containing and do not containing the phenotype of the NSC that cultivates in the NSC substratum of exogenous LIF and containing and the phenotype that does not contain the NSC that cultivates in the BMSC-CM substratum of somatomedin.Figure 17 A-17D has described the curve of CD56, CD133, II class MHC molecule and I class MHC molecule respectively.

Figure 18: comprise Figure 18 A-18D, be a series of facs analysis collection of illustrative plates, described containing (Figure 18 B) and do not containing the phenotype of the NSC that cultivates in the NSC substratum of (Figure 18 A) exogenous LIF and the phenotype of the NSC that in complete NSC substratum (Figure 18 C and 18D), cultivates altogether.Figure 18 has described the I class MHC molecule of the NSC that cultivates under various conditions and the expression of II class MHC molecule, and (black=abnormal shape controlled; Grey=I class or II class).

Embodiment

The present invention includes and be used to induce and/or improve composition and the method that neural stem cell (NSC) is bred, kept its versatility simultaneously.The present invention also comprises the composition and the method for the MHC developed by molecule that is used to regulate NSC.

The present invention relates to a discovery, promptly marrow stromal cell (BMSC) can be supported the propagation of NSC as trophoderm.Like this, the present invention includes composition and the method that is used to induce and/or improves NSC propagation, keep its versatility simultaneously, cultivate NSC with BMSC as trophoderm.

In addition, the present invention openly illustrates: with compare at other identical NSC that do not have BMSC Culture of neural stem cells medium (NSC substratum) trophoblastic, that added the exogenous LIF that is used to improve amplification to cultivate, on the BMSC trophoderm, cultivate altogether NSC reduced NSC the MHC developed by molecule rise and/or induce.Therefore, the present invention includes and use the BMSC NSC that cultivates and increase as trophoderm, be used to reduce and/or prevent the composition and the method for the MHC developed by molecule of NSC.

Data disclosed herein have illustrated that also the BMSC secretion to the useful somatomedin of NSC, nutritional factor and/or cytokine, has kept their versatility simultaneously.The BMSC excretory factor can be collected in the following way: cultivation BMSC for some time and collection condition substratum are standby in a kind of substratum.Therefore, the present invention also comprises a kind of marrow stromal cell conditioned medium (BMSC-CM), to the propagation of NSC and to keep versatility be useful.

The invention still further relates to following discovery: by the BMSC excretory, be present in the factor among the BMSC-CM reduced NSC the MHC developed by molecule rise and/or induce.Therefore, the present invention includes, do not exist and/or the NSC that reduces surface MHC developed by molecule on timing, use BMSC-CM increase composition and the method for NSC.

Accordingly, the present invention includes the method and composition of the NSC that is used to generate medical use.Therefore, the present invention includes the composition and the method that are used to generate NSC, described NSC helps to treat the patient who is subjected to central nervous system disease, imbalance or state image.Described method comprises cultivation, amplification NSC and is the step of patient's administration NSC.

Definition

Following term used herein has the described implication of this section.

" one " is meant that in the use of this paper the target compound of its qualification is one or more than one (promptly being at least " one ").For example, " element " is meant one or more elements.

Term " approximately " can be understood by those of ordinary skills, and with changing to some extent according to its context to a certain extent.

Term used herein " autologous " meaning is meant any material that comes and introduce again this individuality from same syntaxy.

Term used herein " allochthonous " refers to from any material of deriving out a kind of different animals body.

Term used herein " marrow stromal cell ", " stroma cell ", " mescenchymal stem cell " or " MSCs " can be replaced use each other, refer to the small portion cell in the marrow, can be as the stem-like cell precursor of osteocyte, chondrocyte and adipocyte, and utilize its ability that adheres to the plastics plate and separated from marrow.The spinal cord stroma cell can derive from any animal.In some embodiments, stroma cell preferably derives from primates.

" differentiation " is used to refer to a kind of cell that has obtained sophisticated final state here, and such cell development fully and show biospecificity and/or to certain specific environment and/or adaptability of function.Typically, a kind of feature of noble cells is to express the relevant proteic gene of coding differentiation in specific cells.For example, the formation of proteic expression of myelin and myelin is the exemplary of the ultimate differentiation of neurogliocyte in neurogliocyte.Will " break up " when speaking of a kind of cell, as the use here of this term, then described cell is in an atomization.

" division culture medium " is used to refer to a kind of cell growth medium here, comprise or do not comprise a kind of additive, as: stem cell, embryonic stem cell, similar embryonic stem cell, neural ball, NSC or other similar precursor cells, not differentiation fully develops into a kind of cell with some or all noble cells characteristics when cultivating in developing medium.

" amplification ability " is used to refer to the fecundity of cell here, for example, and the situation of number amplification or cell mass experience population doublings.

" trophoderm " is to represent such cell: generate somatomedin, cytokine, other cell-derived products, and provide physical support by necessary contact in cultivating altogether, with the propagation that improves stem cell and keep the undifferentiated versatility of stem cell.

" trophoderm " is used for describing a kind of cell of first types of organization here, the co-culture of cells of these cells and second types of organization, the environment that provides a kind of second types of organization's cell to grow.

Term used herein " growth medium " means a kind of developing medium that promotes the cell growth.Growth medium generally can contain animal serum.In some cases, growth medium can not contain animal serum.

Term used herein " NSC substratum " means the developing medium that a kind of NSC of being used for cultivates and increases.Typical NSC substratum comprises DMEM/F12, N2 additive, EGF, bFGF and liver phosphorus matter.In some cases, the NSC substratum can not contain somatomedin (as EGF and bFGF).

" marrow stromal cell conditioned medium " (BMSC-CM) is used to refer to the developing medium of utilize cultivating BMSC and being defined condition here.Based on the disclosure, BMSC-CM obtains by cultivate BMSC in the NSC substratum, therefore, secrete the mode that is present in somatomedin, nutritional factor and cytokine in other compounds by making BMSC in the NSC substratum, the BMSC in the condition cultured object of BMSC-CM limits.Can be enough BMSC-CM cultivate NSC, the breeding that improves NSC keeps the versatility of NSC simultaneously.In addition, can cultivate NSC in a kind of mode of the MHC of adjusting developed by molecule by enough BMSC-CM.

" leukaemia inhibitory factor " (LIF) is used to refer to the protein of a kind of 22KDa in the interleukin-6 family here, and it has the various biological function.Proved that LIF has following ability: the ultimate differentiation in the inducing leukemia cell, induce in normal and the myelocytic leukemia cell the synthetic of acute phase protein in the hematopoietic differentiation and cell cultured supernatant.Here, LIF has also demonstrated the breeding that improves undifferentiated state NSC, keeps the versatility of NSC simultaneously.

" exogenous LIF " refers to from a kind of organism, cell or system introduces or by the LIF of its generation and discharge.

A kind of differentiation of stem cells that term used herein " versatility " or " versatility " mean central nervous system becomes the ability of broad variety cell.For example, a kind of central nervous system stem cell of versatility can be divided into, but is not limited to, neurone, stellate cell and oligodendroglia.

" neural ball " is used to refer to a kind of neural stem cell/progenitor cell here, wherein can detect the expression of nidogen, comprises, especially detects nidogen in the cell by immunostaining.The aggregation of neural ball reproduction neural stem cell, and form the characteristics that neural ball is the neural stem cell culturing in vivo.

" neural stem cell " is used to refer to a kind of neurocyte of undifferentiated, polyenergic, self here.Neural stem cell is the former multipotential stem cell of a kind of clone, can break up and has the self ability under optimum conditions, and can finally be divided into neurone, stellate cell and oligodendroglia.Therefore, neural stem cell is " polyenergic ", because the filial generation of stem cell has multiple differentiation pathway.Neural stem cell can the oneself keep, and means that the daughter cell that each cell fission obtains generally also can be a stem cell.

" neurocyte " is used to refer to the cell that a kind of and neurogliocyte that derives from central nervous system and/or peripheral nerve system and neurone have similar form, function and phenotypic characteristic here.

" neuron cell " is used to refer to a kind of cell that has similar form to neurone here, the expression neuronal specificity mark of its detectability, for example (but being not limited to) MAP2, neural fibril 200kDa, neural fibril-L, neural fibril-M, synaptophysin, β-tubulin (TUJI), taurine (Tau), N-n acetylneuraminic acid n (NeuN), neurofilament protein and joint conference's albumen (synaptic protein).

" star like cell " is used to refer to a kind of cell that has similar phenotype to stellate cell that has here, and it expresses stellate cell specific marker, for example (but being not limited to) GFAP.

" oligodendroglia like cell " is used to refer to a kind of oligodendroglia specific marker similar to oligodendroglia that have here, for example, but is not limited to O-4.

" process of cell cycle " is used to refer to a cell here and prepares and/or enter mitotic division and/or maiotic process.The process of cell cycle comprises G1 phase, S phase, G2 phase and M-phase.

" breeding " is used to refer to duplicating of similar type (particularly cell) here or doubles.I.e. breeding comprises the generation of greater amount cell, can be by the simple computation cell number, detect the 3H-thymidine and be integrated into similar approach such as cell and obtain measuring.

Any material that the term that this paper is suitable for " exogenous " refers to introduce from a kind of organism, cell or system or that discharge by their.

" coding " refers to the inherent nature of specific nucleotide sequence in a kind of polynucleotide, for example gene, cDNA or mRNA have the polymkeric substance and the macromole of definite nucleotide sequence (being rRNA, tRNA and mRNA) or aminoacid sequence and consequent biological property as template synthetic other in bioprocess.Therefore, if the corresponding mRNA of a gene in cell or the other biological system is transcribed and is translated and generate a kind of albumen, this gene this albumen of just encoding so.Coding strand (its nucleotide sequence is identical with the mRNA sequence, and the form with sequence list provides usually) and noncoding strand (as the template of transcribing of gene or cDNA) can be regarded as protein or other products of described gene of coding or cDNA.

Unless miscellaneous stipulations are arranged, a kind of " nucleotide sequence of a kind of aminoacid sequence of encoding " comprises that all are the nucleotide sequence of denatured form each other, their same seed amino acids of encoding.The nucleotide sequence of coded protein and RNA can comprise intron.

" isolating nucleic acid " refers to the nucleic acid fragment of separating from the naturally occurring sequence that is arranged in its both sides, and for example a kind of dna segment is from removing with its normal adjacent sequence, for example natural separation on the sequence adjacent with this segment from genome.This term also refers to the nucleic acid of large-scale purification from other natural compositions of following, the composition followed natural with it such as RNA, DNA or protein in the cell.Therefore this term comprises; for example; be integrated into the plasmid of carrier, self-replacation or the recombinant DNA in virus or protokaryon or the eukaryotic genomic dna, or have, be independent of other sequence recombinant DNAs (for example cDNA that produces by PCR or restriction enzyme digestion, genomic fragment or cDNA segment) as independent molecule.This term also comprises a kind of recombinant DNA, as the part of heterozygous genes and the extra peptide sequence of encoding.

In the context of the present invention, be suitable for the abbreviation of the ubiquitous nucleic acid base of following representative." A " refers to VITAMIN B4, and " C " refers to cytosine(Cyt), and " G " refers to guanine, and " T " refers to thymus pyrimidine, and " U " refers to uridylic.

" carrier " is a kind of composition of matter, comprises that an isolating nucleic acid also can be used for this isolating nucleic acid is sent into cell interior.A large amount of carrier well known in the prior art include, but not limited to linear polynucleotide, with ionic compound or amphoteric substance bonded polynucleotide, plasmid and virus.Therefore, term " carrier " comprises a kind of plasmid or virus of self-replacation.This term also should be interpreted as comprising non-plasmid and the non-virus compound that promotes nucleic acid to change cell over to, as poly-lysine compound, liposome and analogue.The example of virus vector includes, but are not limited to adenovirus carrier, adeno-associated virus vector, retroviral vector and analogue.

" expression vector " refers to a kind of carrier that comprises the polynucleotide of recombinating, and described reorganization polynucleotide contains regulating and controlling sequence, and this regulating and controlling sequence is connected to wants the nucleotide sequence of expressing.An expression vector comprises enough cis-acting elements that is used to express, and other elements that are used to express can be provided by host or vivoexpression system.Expression vector comprises the known carrier of all that prior art, Cos plasmid for example, plasmid (for example exposed or be included in the liposome) and integrate the virus of the polynucleotide of recombinating.

Describe

The present invention includes the breeding of a kind of NSC of raising, keep the method for its versatility simultaneously.This method comprises the known method separation of employing prior art NSC, and NSC and BMSC are cultivated the versatility that the breeding that improves NSC also keeps NSC simultaneously altogether.The mode that the contact that the cultivation of two kinds of cell types can take NSC and BMSC to have physics to contact relies on, perhaps NSC relies on mode with the noncontact that BMSC does not have physics to contact.

The amplification ability (NSC self-replacation ability repeatedly) that the present invention relates to such discovery: NSC can be improved by the common cultivation with BMSC.That is, an embodiment of the invention relate to such discovery: BMSC can serve NSC as sustenticular cell in co-culture system amplification.The personnel that are familiar with prior art can recognize: according to content disclosed by the invention, BMSC can be used as the trophoderm of NSC and provides and comprises-but be not limited to-factor of somatomedin, nutritional factor and cytokine, support the cultivation of NSC, keep the versatility of NSC simultaneously.The BMSC trophoderm also can be used as an individual layer, and NSC can grow in the above.

The personnel that are familiar with prior art will recognize that based on content disclosed by the invention, BMSC and NSC also can cultivate the breeding that improves NSC altogether under the condition that the known factor of other prior aries exists.

Among the present invention, BMSC and NSC can cultivate altogether lacking under the condition of exogenous LIF, and the breeding that improves NSC keeps the versatility of NSC simultaneously.Content disclosed by the invention shows the breeding of described NSC and the level of amplification that level of amplification is significantly higher than the NSC of single culture on the spread plate that contains exogenous LIF (being polyornithine/fibronectin spread plate).Therefore, the invention provides a kind of method that does not need to use the cultivation NSC of spread plate and/or exogenous LIF.

Another one embodiment of the present invention comprises a kind of method of getting rid of or separating BMSC from BMSC and NSC coculture.The present invention relates to such discovery, BMSC can separate from coculture by the following method: cultivate a kind of and BMSC bonded antibody in coculture, additional subsequently separating step is including, but not limited to magnetic resolution.An example in conjunction with the antibody of BMSC is anti-CD13 antibody.The magnetic resolution process is followed the use magnetic bead, including, but not limited to Dynabead (Dynal Biotech, Brown Deer, WI).About the use of Dynabead, (Miltenyi Biotec, Auburn CA) removes BMSC from coculture can to use MACs to separate solvent.A result of separating step has obtained pure NSC.FACS also can be with removing BMSC, and perhaps, forward is selected NSC.

In NSC cultivation/amplification method of the present invention, NSC has kept its versatility (being divided into the ability of different cell types, as neurone, stellate cell, oligodendroglia and other).In this another anti-bright embodiment,, adopt the NSC of method amplification of the present invention to keep the differentiation capability of (promptly bigger ratio) more with respect to the NSC that adopts the art methods amplification or cultivate.

NSC cultivation/amplification method described herein has solved a major issue of using in the NSC treatment human diseases, that is, before the disclosure, be difficult to separate from culture and amplification NSC (promptly being difficult to induce their breeding liberal quantities).Description of contents NSC disclosed herein can be cultivated and be separated in a large number, satisfies therepic use.

The MHC developed by molecule that the present invention also relates to such discovery: NSC can be by cultivating BMSC and NSC regulates altogether.Description of contents disclosed herein except the breeding that improves NSC and keep its versatility, is compared with the NSC that adopts method known in the state of the art to cultivate, cultivate altogether BMSC and NSC also reduced NSC the MHC developed by molecule rise and/or induce.In other words, the invention provides the method for a kind of NSC of cultivation, this method provides than the more benefit of standard method that improves NSC breeding in the culture.

Co-culture system provides the method for a kind of NSC of cultivation, and its benefit obviously is better than at spread plate or contains single culture NSC on the spread plate of exogenous LIF.Discuss as this paper elsewhere, co-culture system provides a kind of for the first time not to be needed to use spread plate or exogenous LIF and generates the method for a large amount of NSC.In addition, the method for co-culture system is compared with the method for use standard NSC substratum single culture NSC in the prior art, has extra benefit and/or bigger benefit.These benefits include, but is not limited to: improve the breeding of NSC and keep the versatility of NSC and the MHC developed by molecule of adjusting NSC.

In another one embodiment of the present invention, NSC can cultivate with BMSC altogether lacking under the condition of exogenous LIF, reduce NSC the MHC developed by molecule rise and/or induce.That is, the invention provides a kind of method that does not need to use the cultivation NSC of exogenous LIF.BMSC in the description of contents co-culture system disclosed herein can come to provide the factor to the NSC that cultivates altogether as trophoderm, includes, but is not limited to somatomedin, nutritional factor and cytokine.Believe benefit that the factor that BMSC provides provides for the NSC that cultivates altogether, will exceed single culture NSC on the spread plate of NSC substratum at the beneficial effect aspect amplification and the MHC developed by molecule.

Use method disclosed herein can regulate such discovery of MHC developed by molecule, the method for a kind of NSC of breeding is provided, NSC is useful at aspects such as treatment, diagnosis, experimental uses.For example, compare, utilize method disclosed herein and the MHC developed by molecule of the NSC that reduced provides a kind of reduction NSC immunogenic method with method known in the state of the art.Preferably, the reduction of NSC the MHC developed by molecule provide a kind of NSC of increasing to be implanted into the method for acceptor success ratio.

Cultivate NSC and BMSC altogether and provide a kind of with contacting of two kinds of cells relies on or non-dependence mode is regulated the MHC developed by molecule of NSC method.In an embodiment of the invention, NSC can cultivate altogether with contact dependence mode and BMSC.Do not wish to be subjected to the constraint of any particular theory, the physics of BMSC and NSC contacts to the breeding of NSC and amplification and has brought beneficial effect.The adjusting of the MHC developed by molecule of physics contact also the having facilitated NSC of two kinds of cells.Preferably, the same NSC that use to add the standard NSC substratum of exogenous LIF with other and do not have the spread plate of BMSC to cultivate compares, the mode that relies on two kinds of cells contacting cultivate altogether NSC and BMSC reduced NSC the MHC developed by molecule rise and/or induce.

In another one embodiment of the present invention, NSC can cultivate altogether with the mode and the BMSC of non-dependence contact, regulates the MHC developed by molecule of NSC.The present invention relates to such discovery: NSC and BMSC can be at Costar Transwell ^TMIn cultivate altogether, by a kind of permeable film filter separate two kinds of cells and prevent NSC and BMSC between the physics contact.The phenotype that described permeable film filter allows the BMSC excretory factor to see through film and therefore effectively facilitate NSC for example improves the breeding of NSC and keeps its versatility and the MHC developed by molecule of regulating NSC.

Use Transwell ^TMExperimental results show that, content disclosed by the invention shows: by BMSC being added (the BMSC excretory factor also enters developing medium) in the developing medium, BMSC can lack growth and the amplification of supporting NSC in the co-culture system under the condition of direct contact between two kinds of cells.Do not wish to be subjected to the constraint of any particular theory, the phenotype that the BMSC excretory factor has been facilitated NSC.Therefore, the invention provides a kind of method of cultivating BMSC and NSC altogether, do not need BMSC as NSC direct growth trophoderm thereon.The invention provides a kind ofly to contact the method that non-dependence mode is cultivated BMSC and NSC altogether, wherein, therefore NSC obtains benefit from BMSC by absorbing BMSC excretory in the co-culture system.

Those skilled in the art will recognize that based on content disclosed by the invention the method that any NSC of preventing and BMSC formation physics contact can both be used for co-culture system.For example, can use and be different from Transwell ^TMAny system/device, to contact the mode co-cultured cell of non-dependence.Such system/device includes, but is not limited to strainer and film, and its aperture that has will prevent the direct contact of two kinds of cells, but allows some factors by strainer/film.The benefit of using such system/device to cultivate BMSC and NSC altogether is that a kind of method of cultivating NSC in an individual system can be provided, and described system has successive, is used for the factor source of NSC propagation.Like this, the present invention includes a kind of molectron, comprise: a kind of Culture of neural stem cells equipment wherein contains separative NSC, isolating BMSC, NSC growth medium and by the described isolating BMSC excretory factor; The device that a kind of NSC of preventing and BMSC are in contact with one another.

Based on such discovery, be that BMSC can enter developing medium by excreted factor and supports in the co-culture system NSC growth to contact non-dependence mode, estimate the growth whether the BMSC conditioned medium can support NSC in the mode that improves the NSC breeding and keep its versatility and regulate the MHC developed by molecule of NSC.Description of contents BMSC conditioned medium disclosed by the invention can be supported the growth of NSC, and has significant beneficial effect, although it is more effective not contact the common cultivation of dependence mode.Therefore, the invention provides a kind of use marrow stromal cell conditioned medium (BMSC-CM) and cultivate the NSC method, and do not use co-culture system.Use BMSC-CM to cultivate NSC, the method for another breeding NSC also is provided, NSC that this method generates and the NSC that adopts co-culture system to cultivate have the character that is equal to.

In addition, present disclosure illustrates that BMSC-CM can substitute the NSC substratum of having added exogenous LIF and be used to cultivate NSC.The cell number that the NSC substratum that the present disclosure explanation has been added exogenous LIF according to method founder cell number and the use of cultivation NSC in BMSC-CM is generated is suitable.Therefore, the invention provides a kind of BMSC-CM of use, the reproduction speed of NSC is equivalent to or has added the speed that the NSC substratum of exogenous LIF is bred NSC greater than use as some methods that is of value to the factor source of inducing NSC breeding.

The present invention illustrates that also BMSC-CM can substitute spread plate, and polyornithine/fibronectin spread plate for example is used to adopt the NSC substratum NSC that cultivates and increase.Description of contents NSC disclosed herein can utilize BMSC-CM to increase on uncoated flat board.Can observe, NSC utilizes BMSC-CM being expanded on uncoated flat board to be equivalent to less or greater than at the NSC substratum, or even contains the amplification of the NSC of single culture on the spread plate of NSC substratum of exogenous LIF.Therefore, the present invention includes and a kind ofly do not need spread plate and exogenous LIF and utilize BMSC-CM to cultivate the method for NSC.

The use of BMSC-CM also provides the method for a kind of NSC of cultivation, and its mode is not use BMSC and provide benefit as NSC in co-culture system.Present disclosure explanation BMSC-CM can support the cultivation of NSC and be equivalent to use the viewed benefit of the co-culture system that comprises BMSC and NSC for it provides.Like this, the present invention includes a kind of BMSC-CM of use and cultivate NSC with breeding that improves NSC and the method that keeps its versatility and regulate the MHC developed by molecule of NSC.As proving that here BMSC can be used for producing marrow stromal cell conditioned medium (BMSC-CM).BMSC-CM is a kind of developing medium by the BMSC conditioning in the culture, by cultivating BMSC and make BMSC secrete somatomedin, nutritional factor and/or cytokine in the NSC substratum in the NSC substratum.BMSC-CM comprises by BMSC excretory somatomedin, nutritional factor and/or cytokine also include, but is not limited to: LIF, Brain Derived Neurotrophic Factor (BDNF), basic fibroblast growth factor (bFGF), FGF-6, glial cell derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), pHGF (HGF), IFN-γ, trypsin-like growth factor bindin (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, MCP (MCP-1), mononuclear phagocyte G CFS (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinase (TIMP-1), TIMP-2, tumour necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, UPA acceptor (uPAR), Delicious peptide (BMP4), IL1-a, IL-3, Leptin (leptin), STEM CELL FACTOR (SCF), mesenchymal cell derivative factor-1 (SDF-1), Thr6 PDGF BB-BB (PDGFBB), transforming growth factor-beta (TGF β-1) and TGF β-3.

BMSC-CM is to breeding and the amplification of NSC and to keep its versatility be useful.The use of BMSC-CM provides a kind of will introduce NSC by BMSC excretory somatomedin, nutritional factor and/or cytokine etc., be used for breeding and the amplification of NSC.

Speed breeding when the use of BMSC-CM provides the NSC culture medium culturing of a kind of NSC of inducing to be equivalent to or to have added exogenous LIF greater than use, or even on uncoated flat board.Therefore, the invention provides molectron and method, be used to use BMSC-CM to generate NSC a large amount of, that be used for the treatment of use.

Except BMSC-CM is used to support NSC breeding and keeps its versatility, present disclosure also illustrates: cultivate the method that NSC provides the MHC developed by molecule of a kind of NSC of adjusting in BMSC-CM.Preferably, compare, in BMSC-CM, cultivate the MHC developed by molecule that NSC has reduced NSC with other identical NSC that in containing the NSC substratum of exogenous LIF, cultivate.The MHC developed by molecule that uses BMSC-CM to regulate NSC is based on such discovery: be used to detect under the condition of II class MHC molecule, compare with other same NSC that are incubated in the NSC substratum that contains exogenous LIF, the NSC that grows in BMSC-CM does not express II class MHC molecule and shows the I class MHC molecule of lower level.This discovery is with consistent with the discovery that NSC co-culture method (relying on or contact non-dependence mode to contact) reduces the MHC developed by molecule of NSC according to BMSC.No matter relying on contact still is the mode that noncontact relies on, and is to adopt BMSC to cultivate NSC as trophoderm or in BMSC-CM, and the method for the MHC developed by molecule of a kind of NSC of minimizing all is provided.The discovery of using method disclosed by the invention can regulate the MHC developed by molecule provides a kind of NSC propagation method that helps therepic use.The HMC developed by molecule of the NSC that uses method disclosed by the invention and reduce also provides a kind of NSC of increasing to be implanted into the method for acceptor success ratio.

Based on present disclosure, the present invention includes and use BMSC-CM cultivate and increase NSC and reduce the MHC developed by molecule of NSC.That is, the present invention is based on such discovery: compare with other identical NSC that are incubated at the NSC substratum that contains exogenous LIF, use BMSC-CM amplification NSC to reduce and/or prevented the rise of the surperficial MHC developed by molecule of NSC.Like this, the present invention includes a kind of method that helps the propagated cell of therepic use.

Extensively determine: the danger of transplant rejection is usually followed in the Transplanted cells in the heredity between the different individuality (allogeneic).All expression groups of nearly all cell are wanted the I of histocompatibility complex (MHC) quasi-molecule.In addition, many cell types can both be by abduction delivering II class MHC molecule when being exposed to struvite cytokine.The rejection of allotransplantation is mainly mediated by the T cell CD4 and the CD8 subclass of identification I class and II class MHC molecule.Major objective in the transplanting is the permanent implantation of donor graft, does not induce the transplant rejection immune response that results from acceptor simultaneously.Like this, the present invention includes minimizing and/or eliminate the immunoreactive method of recipient cell at the intravital NSC of acceptor that is implanted into, cultivate NSC by use method disclosed herein before transplanting, purpose is to reduce the MHC developed by molecule of NSC.Do not wish to be subjected to the constraint of any particular theory, use method disclosed herein, the MHC developed by molecule of NSC is reduced, can reduce the amount of the MHC molecule that is presented on the NSC cytolemma, thereby reduce the immunogenicity of NSC in the acceptor body.

The present invention also can be used for obtaining the NSC of expression alien gene, so NSC can be used in cell therapy or gene therapy.That is, the present invention has considered the mass production of the NSC of expression alien gene.Foreign gene can be the external source version (that is, the wild-type version of homologous genes can be used for replacing the defective type allelotrope that contains sudden change) of a native gene.Allogenic gene can comprise the cytotoxic group that is used for central nervous system (CNS) regenerated nutritional factor or target on cancer because of.Allogenic gene is (but nonessential) and one or more other genes covalently bound (that is, " merging (fused with) ") usually." other " gene of example comprises: be applied to " positive " and choose the gene of the cell of having integrated foreign gene and be applied to " feminine gender " and choose the gene of exogenous origin gene integrator being gone into the cell of the same chromosomal foci of native gene, perhaps the both has.

The NSC that the inventive method obtains can be induced and is divided into neurone, stellate cell, oligodendrocyte etc., carries out selectivity by the known culture condition of prior art and cultivates the cell that makes NSC be divided into a kind of selection type.

The described NSC through cultivating or increasing of present disclosure before or after being divided into selected cell type, can be used to treat the illness that the known available NSC of multiple prior art treats.Useful NSC can comprise the NSC of the foreign gene that contains insertion and not contain the NSC of the foreign gene of insertion in these methods of treatment.Described illness includes, but is not limited to cerebral trauma, Huntington Chorea (Huntington ' disease), degenerative brain disorder (Alzheimer ' s disease), Parkinson's disease (Parkinson ' s disease), Spinal injury, apoplexy, multiple sclerosis, cancer, CNS LSD and head trauma.

NSC and BMSC separate

NSC can be preferably the mankind from Mammals, central nervous system in obtain.These cells can obtain from multiple tissue, include, but is not limited to forebrain, hindbrain, whole brain and spinal cord.NSC can use the currently known methods of method that this paper elsewhere describes in detail or prior art and separated and cultivate, and for example uses disclosed method in the United States Patent (USP) 5958767, therefore incorporates its full content into this paper in the mode of quoting as proof here.The method that other are commonly known in the art to be used to separate NSC can be easy to use by the technician, comprise the method that develops in the future.The present invention be limited to absolutely not these or any other obtain the method for relevant cell.

Can from many dissimilar tissues, separate NSC, for example: donor tissue, separate individual cells by connection extracellular matrix from this tissue; Perhaps from the commercial source of NSC.Among the embodiment, adopt aseptic technique to peel off brain source tissue, adopt the known any method of prior art to come dissociated cells then, comprise method of enzymatically treating, as trypsinase, collagenase or the like, perhaps adopt the physics dissociation methods, as adopting blunt to grind or handling.The disassociation of neurocyte and other multipotential stem cells can be carried out in the sterile tissue nutrient solution.Dissociated cells is drawn supernatant liquor through low-speed centrifugal (between 200rpm and 2000rpm), uses the nutrient solution suspension cell then.

Prior art has been described the source of BMSC and obtained the method for BMSC from these source.BMSC can obtain from any marrow in a large number, comprises the marrow that for example suction obtains from the crista iliaca of human donor.The method that obtains marrow from donor is that prior art is known, and is illustrated in as United States Patent (USP) 6653134 and WO96/30031, so the full content of incorporating them here into as a reference.Human mescenchymal stem cell can from Cambrex company (Walkersville MD.) buys,

The application of isolating neural stem cell

Isolating neural stem cell all has application in many aspects.These cells can be used to rebuild the intravital cell of Mammals, and described mammiferous cell loses because of i or I.Genopathy can obtain medical treatment, and comes corrective gene defective or defence disease by the genetic modification from body homology or allosome homology neural stem cell.The disease relevant with lacking specific secretory product (as hormone, enzyme, somatomedin etc.) also can use NSC to treat.Central nervous system disorder comprises a lot of diseases, as neurodegenerative disease (for example degenerative brain disorder and Parkinson's disease), acute cerebral insult (for example apoplexy, brain injury, cerebral paralysis, tumor resection, chemotherapy and radiation supportive treatment) and many central nervous system dysfunctions (for example depression, epilepsy and neural division).It is all relevant with the neurocyte degeneration of neural system specific position at interior some diseases to include, but is not limited to degenerative brain disorder, multiple sclerosis (MS), Huntington Chorea (Huntington ' s Chorea), amyotrophic lateral sclerosis (ALS) and Parkinson's disease, and cell degradation causes these cells or brain not to have ability to carry out the function of its expection.NSC through separation and cultivation described here can be used as the progenitor cell source, is used for specific cells and treats these diseases.NSC can be used as the nutritional factor source, comes stimulation of endogenous stem cell and neural regeneration.NSC through cultivating described here can freezing and long-term preservation under liquid nitrogen temperature, can reuse after thawing.Described cell is stored in the medium of 10%DMSO and 90%NSC usually.Once thawing, the method that can use this paper elsewhere the to describe described cell that increases.

Genetic modification

Cell of the present invention can by genetic modification, generate nutritional factor, somatomedin, cytokine, neurotrophic factor and other similar factors by the importing of exogenous genetic material, and this cultivation to NSC is benefited.For example, compare with BMSC without genetic modification, BMSC can express the high-caliber EGF of justacrine by genetic modification.Do not wish to be subjected to the constraint of any particular theory, express through genetic modification with the BMSC that secretes EGF will be with the same factor of secretion on the level that increases than identical BMSC without genetic modification.

The effect of using engineering BMSC in co-culture system is to make the BMSC after the modification continue to provide exogenous factor in co-culture system.Exogenous factor can be the BMSC of cultivation or NSC or both and brings benefit.The exogenous genetic material that is imported into BMSC can also help the secretion of other endogenous factors among the engineering BMSC.In addition, the BMSC of genetic modification can help the secretion of endogenous factors in the flanking cell.Therefore, the present invention includes the BMSC that uses genetic modification and come to be that co-culture system continues the supply endogenous factors, in some instances, the exogenous genetic material that is imported into BMSC helps the secretion of endogenous factors in the BMSC of genetic modification and/or the flanking cell.In any case BMSC excretory exogenous factor and/or endogenous factors provide the useful factor for cultivation and the propagation of NSC.

And, express through genetic modification and the BMSC that secretes a kind of factor (as EGF) can also be used to generate the BMSC-CM of the EGF level with increase.Except providing for BMSC-CM the exogenous factor that increases level, the BMSC of genetic modification also helps the secretion of endogenous factors in engineering BMSC and/or the flanking cell.The described factor includes, but is not limited to: LIF, Brain Derived Neurotrophic Factor (BDNF), basic fibroblast growth factor (bFGF), FGF-6, glial cell derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), pHGF (HGF), IFN-γ, trypsin-like growth factor bindin (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, MCP (MCP-1), mononuclear phagocyte G CFS (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinase (TIMP-1), TIMP-2, tumour necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, UPA acceptor (uPAR), Delicious peptide (BMP4), IL1-a, IL-3, Leptin (leptin), STEM CELL FACTOR (SCF), mesenchymal cell derivative factor-1 (SDF-1), Thr6 PDGF BB-BB (PDGFBB), transforming growth factor-beta (TGF β-1) and TGF β-3.

The benefit of using the BMSC of genetic modification to generate BMSC-CM is to improve the level of exogenous factor, and for example EGF makes EGF be secreted into substratum from engineering BMSC, rather than identical from other but secrete the BMSC without genetic modification.Along with the raising from genetically modified cell excretory EGF level, more EGF comes across BMSC-CM.In addition, the EGF level of engineering BMSC excretory raising has the secretion that helps other endogenous factors in engineering BMSC and/or the flanking cell.BMSC-CM with the EGF of raising and/or other factor levels can be used in cultivation and the amplification of NSC.

In aspect another one, BMSC can be expressed HSV-thymidine kinase or the such albumen of green fluorescent protein (GFP) by genetic modification, these albumen can be used in the separation after the NSC amplification in the BMSC coculture, handle by Gancyclovir respectively or separate on flow cytometer.

Except genetic modification BMSC, the present invention also comprises the NSC of genetic modification.The NSC of genetic modification can be used in and replaces defective cell in the individuality.The present invention can also be used to express the protein of being wanted by excretory.In other words, NSC can be separated and be imported a kind of gene of target protein, is imported into individuality then, and target protein will generate and bring into play or produce result of treatment in this individuality.This aspect of the present invention relates to gene therapy, wherein, introduces individuality by the NSC with genetic modification and comes to inject therapeutic protein for this individuality.The NSC of genetic modification through method disclosed herein cultivate, separate also implant individual, this individuality will described albumen by body in NSC express and secretion back and being benefited.

According to the present invention, the structure gene that contains the nucleotide sequence of the heterologous protein of encoding is imported among the NSC.Be that the NSC cell is introduced a kind of gene by gene alteration, this expression of gene has therapeutic action in individuality.According to certain aspects of the invention, the NSC that derives from body one by one or another one individuality or non-human animal can be come the substitutional defect gene by gene alteration and/or introduce a kind of its to be expressed in the gene that has therapeutic action in the individuality.

Under all situations, make up gene and all be transfected into cell, foreign gene is connected to the required regulating and controlling sequence of genetic expression in the cell by controlled.Such regulating and controlling sequence comprises promotor and polyadenylation signal.

Described structure gene preferably is provided as expression vector, comprises and the encoding sequence of the controlled foreign protein that is connected of basic regulating and controlling sequence that after this carrier is transfected into cell, encoding sequence will be expressed by cell.Encoding sequence with link to each other at the necessary controlling element of this sequence of cell inner expression.The nucleotide sequence of encoding said proteins can be cDNA, genomic dna, synthetic DNA or its heterozygote or such as the RNA molecule of mRNA.

Make up gene and comprise coding useful proteic and the controlled nucleotide sequence that is connected of controlling element, it can be used as functional cell matter molecule, functional free molecule is present in the cell, also can be integrated among the cell homologous chromosomes DNA.Exogenous genetic material can be imported into cell, with independently genetic material form (as plasmid) existence.Perhaps, can be with the linear DNA transfered cell that can be integrated in the homologous chromosomes.In the time of in the DNA transfered cell, can add the reagent that some promote that DNA and homologous chromosomes are integrated.Can also comprise in the dna molecular and be used to promote the dna sequence dna integrated.Perhaps, can be with in the RNA transfered cell.

The controlling element that is used for genetic expression comprises: promotor, initiator codon, terminator codon and polyadenylation signal.Preferably, these elements are exercisable in cell of the present invention.In addition, preferred, these elements are connected with the nucleotide sequence of coded protein is controlled, and nucleotide sequence can be expressed in cell like this, thereby generate protein.Initiator codon and terminator codon are generally considered to be the part of the nucleotide sequence of proteins encoded.But preferred, these elements are functional in cell.Equally, the promotor of use and polyadenylation signal must be functional in cell of the present invention.Being used for implementing promotor of the present invention includes, but is not limited in all effective promotor of many cells, as cytomegalovirus promotor, SV40 promotor and retrovirus promotor.Other are used to implement promotor of the present invention and include, but is not limited to: promptly there is function in tissue-specific promoter and does not have the promotor of function in its hetero-organization in some tissue; The promotor of normal expression in cell has or do not have special or common enhancer sequence.In some embodiments, used the promotor that has or do not have enhancer sequence, continuous expression gene in cell.When suitably or when needing, in these embodiments, provide enhancer sequence.

Cell of the present invention can be transfected by the known technology that those of ordinary skills obtain easily.Standard method that can be by will making up the gene transfered cell is with in the foreign gene transfered cell, and described cell is with expressing said gene encoded protein matter.In some implementations, cell is transfected by the following method: calcium phosphate precipitation transfection, the transfection of diethyllaminoethyl dextran, electroporation, microinjection, liposome-mediated transfer, chemistry mediation are shifted, the aglucon mediation is shifted or recombinant viral vector shifts.

In some embodiments, recombinant adenoviral vector is used for wanting having the DNA transfered cell of sequence.In some embodiments, recombinant retroviral vector is used for wanting having the DNA transfered cell of sequence.In some embodiments, use standard C aPO4, diethyllaminoethyl dextran or lipid carrier mediation rotaring dyeing technology that want DNA is integrated into isolated cell.Can use the antibiotics resistance triage techniques of standard to discern and choose transfected cell.In some embodiments, DNA is by the direct transfered cell of microinjection quilt.Similarly, known electroporation or particle bombardment technology also can be used in the foreign DNA transfered cell.A kind of second gene usually and therapeutic genes cotransfection or be connected in therapeutic genes.Described second gene often is a kind of selectable antibiotics resistance gene.Transfected cell can be cultivated by microbiotic and be selected, and described microbiotic will be killed and not receive the described cell of selecting gene.In most cases, these two kinds of genes are isolating and by cotransfection, and the cell of surviving after antibiotic treatment has these two kinds of genes simultaneously and expresses them.

Following embodiment is for more complete description preferred implementation of the present invention.These embodiment should never be interpreted as the restriction of the scope of the invention that claim is limited.

Embodiment

Embodiment 1:BMSC is used for cultivation and the amplification of NSC as the trophocyte

The cultivation of NSC is challenging, because they need somatomedin and special matrix to improve its growth velocity and amplification.For somatomedin, in the uncertain substratum of the serum that comprises FGF and/or EGF, add exogenous LIF, can significantly improve the amplification of NSC.Discuss as other parts of this paper, the combination of exogenous LIF and to the be further improved level of amplification of NSC of the bag of culture dish has kept its versatility simultaneously.

Marrow stromal cell (BMSC) can obtain and be expanded to essence homologous cell mass easily in cultivation.In addition, BMSC also secretes several nutritional factor that can promote the NSC growth.Therefore, present embodiment explanation BMSC can be used for the amplification of NSC as sustenticular cell in co-culture system.

Now, employed material of experiment and method in the present embodiment are described as follows:

The foundation of human fetal neural stem cell (NSC), keep and identify

(Alameda CA) provides human brain (from big fetus of 11-14 week) by Advanced Bioscience Resources company.Cerebral tissue grinds in cold PBS.Cell is suspended in 10mlNSC growth medium (DMEM/F12,8mM glucose, L-Glutamine deaminase, 20mM sodium bicarbonate, 15mM HEPES, 8 μ g/ml heparin, N2 additive, 10ng/ml bFGF, 20ng/ml EGF) after centrifugation.Above-mentioned cell is placed T-25cm through polyornithine and Zeta protein bag quilt ²On the flask, in 5%CO ₂37 ℃ of cultivations in the incubator.Nutrient solution used fresh culture to change 50% in per 2 days, went down to posterity by tryptic digestion in per 14 days.Cell is liquid nitrogen storage in the NSC substratum that contains 10%DMSO.Cell is through thawing and in having initial incubation 1-2 generation under bFGF and the EGF, place afterwards the complete growth medium of having added 10ng/ml LIF.

Other NSC growth mediums can be used in method disclosed herein.For example, cell can be cultivated in the Neurobasal substratum, has wherein added L-glutaminate, bFGF, EGF and B27, but do not contain vitamin A acid (Invitrogen, Carlsbad, CA).Another NSC substratum is NeuroCult, wherein added somatomedin (StemCell Technologies, Vancouver BC, Canada).Do not wish to be subjected to the constraint of any particular theory, any substratum can both be used to cultivate NSC.But the suitable culture base can be grown and increase cell, keeps them to be divided into the ability of various kinds of cell type simultaneously.

In order to identify, the NSC that is in different generations is placed on the cell cultures slide (chamber slide) of bag quilt, uses 4% Paraformaldehyde 96 to fix and nidogen (nestin) and glial fibrillary acidic protein (GFAP) are dyeed.By recalling bFGF, EGF and LIF, and use Neurobasal substratum, B27 additive and BDNF to handle, NSC was broken up about 14 days.It is particular lineage that other differentiation condition can be used for making cytodifferentiation.Cell is fixed and to microtubule-associated protein dyeing (MAP2; Neuronic generation person) and GFAP (Astrocytic generation person).Be identification neurone hypotype, cell dyes with anti-gamma amino butyric acid (GABA) and anti-tyrosine hydroxylase (TH).The main antibody that uses is the human specific nidogen, 1: 10 (R﹠amp; D Systems); MAP2,1: 500 (Sigma); GFAP, 1: 1000 (DAKO); O4,1: 100; NG-2 (1: 200) (Chemicon).The second antibody of using in these experiments is the anti-mouse of Alexa Fluor488 chicken 1: 500 (Molecular Probes) and anti-rabbit of Alexa Fluor 594 chickens, 1: 500 (Molecular Probes).

Several marker expression with flow cytometry analysis NSC comprise hematopoietic cell mark CD45 and CD14, stem cell markers CD34, CD133 and CD56 and immunogenicity/stimulation mark CD80, CD86, I class MHC and II class MHC.

The quantitative analysis of nidogen express cell is also measured by flow cytometer.Carry out this analysis and used anti-nidogen (R﹠amp; D) and with the crosslinked mountain sheep anti mouse Ig of Alexa-fluor488.Use the Cytofix/Cytoperm test kit and the instrument of Becton-Dickinson company, the improvement through too small fixes NSC.

The foundation of marrow stromal cell (BMSC), keep and identify

BMSC produces by the prior art currently known methods.For example, by syringe needle collected at suction people marrow.Karyocyte counting to marrow.Marrow mixes with hydroxyethylamyle after the PBS dilution.Hydroxyethylamyle/cell suspension left standstill 45 minutes-1 hour.During this period, the red corpuscle deposition makes karyocyte be present in supernatant layer.After removing red corpuscle, washing karyocyte and counting.

In tissue culture treated container (as polystyrene), cultivate karyocyte, DMEM-low dextrose, 10%FBS.Former being commissioned to train supported lasting 12-17 days, per 3 or 4 days replacing substratum.When the adhesin with enough numbers, cambiform cell, use the trypsinase subculture to remove adherent cell.Cell is by coated plate again and cultivate about 1 week, and follow-up per generation is changed a subculture.

Flow cytometer uses particular marker to detect cell purity.CD45 negative and 90% above CD90 and CD13 male the 1st or 2 generations (P1 or P2) BMSC in co-culture experiments, have been used.

In the NSC growth medium, cultivate BMSC

BMSC places 6 orifice plates with different densities, makes it to adhere in BMSC substratum (DMEM-low dextrose, the FBS of 10% sampling test) to spend the night.Second day, the substratum of a plate replaced with NSC substratum (DMEM-F12, N2-additive, EGF, 20ng/mL, bFGF, 10ng/mL, 8 μ g/mL heparin, P/S), and another plate is accepted fresh BMSC substratum.Be additional fresh BMSC or the NSC substratum of cell in per three days.One group of culture is through trypsin treatment, counting cells after 7 days.Another group cell is collected and counting after 12 days.As table 1 and Fig. 1, how a lot of BMSC breeding ratio in the BMSC substratum is in the NSC substratum.In the NSC substratum, the initial increase of cell number is with respect to fewer in the BMSC substratum.But, in the NSC substratum, to cultivate after 7 days, the cell number of BMSC does not significantly increase.In the BMSC substratum, cell continued propagation and highly converges in this latter stage in 12 days.In the NSC substratum, BMSC does not form any vortex that converges cell, and display form is normal, does not have the visible necrocytosis; The cellular form of observing in the BMSC substratum is just in time opposite.

Table 1

Substratum	Initial cell	7 days cell numbers	12 days cell numbers
				The NSC substratum	50,000	210,000	240,000
The NSC substratum	100,000	300,000	460,000
				The NSC substratum	200,000	615,000	650,000
The BMSC substratum	50,000	310,000	1,300,000
				The BMSC substratum	100,000	683,000	1,200,000
The BMSC substratum	200,000	855,000	2,200,000

These presentation of results, when NSC and BMSC cultivated in the NSC substratum altogether, BMSC was not and NSC competition and hypertrophy.

The common cultivation of BMSC and NSC; NSC wraps by survival on the flat board and amplification at BMSC

(hBM-03-016 P1) is placed on 12 orifice plates, 75,000 cells/well to people BMSC through thawing, wash, counting.Walk abreast, another one 12 orifice plates are spent the night through 15 μ g/ml polyornithines and 10 μ g/ml people Zeta protein bags subsequently.Second day, collect the hNSC that grows in the substratum and also be suspended in once more in the NSC substratum that contains EGF and FGF.The NSC that uses is hHB-007, and P7 cultivates in containing the NSC substratum of EGF, FGF and LIF.The major part of these cells is grown to neural ball.When these cells being placed BMSC or bag by plate hole, neural ball is broken into unicellular as much as possible.3 parts of NSC place BMSC or bag by on the flat board under the following conditions.

1.25000NSC+NSC substratum, no LIF

2.25000NSC+NSC substratum+LIF

3.50000NSC+NSC substratum, no LIF

4.50000NSC+NSC substratum+LIF

Every other day culture is changed 50% fresh culture.Taken the picture that differs of cell at different time.Cultivate after 12-13 days, use the trypsinase collecting cell.That is, used 0.05% trypsinase culturing cell 5 minutes or break away from culture dish until all cells.Use in the Trypsin inhibitor SBTI (SBTI) then and trypsinase.Cell uses trypan blue dyeing to count in Hematocyte Counter after phosphate buffered saline buffer (PBS) washing.Microscopically is observed the cell of two kinds of different sizes, bigger BMSC and less NSC.Count this two kinds of cell masses respectively.Counting is at the 12nd day, by two mixing with 3 copy type cultures for the first time.

Place NSC on the BMSC to adhere to accumulation area and sprawl and be monolayer.It is not difficult to change substratum on these cells.On the other hand, NSC particularly is grown to the NSC (Fig. 2) of neural ball in the presence of exogenous LIF, be not that adhesivity is arranged very much, therefore wants extreme care when changing substratum.Sometimes need the aerated culture base is carried out the centrifugal loss that prevents neural ball.Simultaneously, when NSC density is low, can not survive, unless they and BMSC cultivate altogether.

On the form, the NSC in cultivating has altogether formed roundlet to oval-shaped network on the big BMSC that sprawls.NSC has formed isolating NSC colony, and the BMSC that is become fiber sometimes is around (Fig. 3).In the colony of each increase, it is many that the NSC number of NSC colony becomes.The NSC of single culture only exists and has under the higher inoculum density and could survive and breed at exogenous LIF; Yet in cultivating altogether, even without LIF, remarkable amplification also takes place in NSC.Therefore, as if BMSC can be as trophoderm and is provided nutritional support for NSC.With compare having added the NSC that grows on the bag of exogenous LIF (the being polyornithine/Fiberonectin) culture dish, the NSC in cultivating has altogether obtained remarkable amplification.Table 2 is described the NSC that collects in the culture after 12 days and the number of BMSC.

Table 2

The amplification of the NSC that cultivates altogether with BMSC

Initial #NSC	LIF	+BMSC	There is not BMSC
				2.5e4	-	8.8e4	1.3e4
2.5e4	+	10e4	7.5e4
				5.0e4	-	25e4	11e4
5.0e4	+	24e4	33e4

The characterized of the NSC that on BMSC, cultivates: the expression of nidogen

Express for the nidogen of estimating the NSC that cultivates altogether with BMSC by immunostaining, culture is grown on cover glass.As described in this paper elsewhere, the cover glass (10mm) that places 24 hole culture dish is by polyornithine and people's Fiberonectin bag quilt.One group of cover glass is spent the night cell adhesion by BMSC bedding (35,000/ cover glass).Second day, collect culture (THD-015+LIF, P12) NSC of middle growth and counting.The NSC in 25000/ hole be bedded in that BMSC goes up or directly in bag by cover glass on.Some have the hole of BMSC not having single culture under the NSC condition.All different cultures are grown in NSC substratum or NSC substratum+LIF.Change 50% fresh culture for cell in each one day, and continued 10 days.After 10 days, some cultures are fixed and prepare nidogen dyeing.Other cultures were allowed to break up 2 weeks.

For fixed culture, this culture through the PBS washing once.Add 4% Paraformaldehyde 96 then, incubated at room temperature 15 minutes, PBS washing 3 times.At this moment, the fixed culture is stored among 4 ℃ of PBS or dyeing immediately.

The nidogen dyeing of follow-up culture realizes by the dyeing of anti-nidogen antibody+anti-GFAP antibody.Culture comprises BMSC+NSC, BMSC+NSC+LIF, independent BMSC, BMSC+LIF, independent NSC and NSC+LIF.A replicate sample (does not have initial antibodies) in contrast

The NSC of single culture has expressed nidogen in the NSC substratum that contains or do not contain exogenous LIF.In the time of in being incubated at NSC substratum+LIF, except express nestin, NSC is coexpression GFAP also.

All cocultures show a large amount of nidogen positive cells, sprawl on BMSC (Fig. 4 A-4F).The only fuzzy background dyeing (Fig. 4 G-4H) of performance of BMSC itself.The form of nidogen positive cell is allogenic.Many nidogen positive cells also are positive to GFAP, and other are the nidogen positive and GFAP feminine gender.Some nidogen positive cells are little circular or oval, have or do not have one or both thread stretching, extensions.The two positive cells of some nidogen-GFAP are big, flat similar stellate cells.Between the common cultivation of adding and do not add exogenous LIF, there is not significant difference.

The differentiation of coculture

Cultivate after 10 days in the NSC substratum, coculture carries out the differentiation in 2 weeks.Differentiation is arranged to comprise liquid feeding 2 times, is 1 at every turn) added N2 but do not have EGF or the DMEM/F1 of FGF, 2) DMEM/F12+B27 additive and 3) a kind of among the DMEM/F12+B27+BDNF.The culture of differentiation is fixed in the Paraformaldehyde 96, uses the combination of following antibody to dye then: for example nidogen/GFAP, MAP2, GFAP and O4/GFAP.Under all conditions, GFAP detects by red fluorescence (Alexa 594), and nidogen, MAP2 or O4 detect by green fluorescence (Alexa 488).Use DAPI that culture is redyed all cells nuclear is carried out the green fluorescence mark.

The NSC that is grown on the BMSC has kept it to be divided into neurone and Astrocytic ability (Fig. 5 A-5D).Through the cultivation of division culture medium, several cell expressings neural mark MAP-2.These cells are to have oval cell nuclear and minicell bipolar or the multilevel cell body.These projections be extendability and forming complicated neural network on the BMSC or on the GFAP staining cell.GFAP male stellate cell also is present in the whole culture.It is big, polygonal, flat that these cells are compared with neurone.According to approximation, neuron number and stellate cell quite or more, it is impossible measuring accurately, because neuronic vast network has formed a three-dimensional cell web.Under used dilution, the O4 antibody staining shows does not have the cytodifferentiation oligodendroblast.May need to produce oligodendrocyte between longer differentiation phase.But, when using NG2 (a kind of chondroitin sulfate proteoglycan that is sent on the oligodendroglia precursor surface) to estimate existing of oligodendrocyte, find that cell seldom is positive to NG2 as the mark of oligodendrocyte differentiation.The nidogen dyeing of the coculture of differentiation has shown that the positive stellate cells of some GFAP are the weak positive to nidogen, but these cells show with break up before the little filiform cell that sees at modal different (Fig. 6).The differentiation in coculture does not bring difference for NSC for the existence of exogenous LIF or shortage in the process of growth.When the NSC of single culture as mentioned above by differentiation phase, observe them and be divided into neurone (the MAP2 positive) and stellate cell (the GFAP positive) (Fig. 7).

When BMSC accepted identical differentiation condition alone, they showed nidogen weak, disperse and GFAP dyeing, and the GFAP dyeing in additional exogenous LIF cultured cells to a certain extent is denseer.It is identical with BMSC that cellular form keeps, and be different from stellate cell.Seldom cell has the nidogen or the MAP2 positive (Fig. 8) that shows the height that is divided into NSC or neurone pedigree in the culture.

Embodiment 2: remove BMSC from BMSC and NSC coculture

BMSC can cultivate coculture by use mouse-anti people's CD13 antibody (being negative being positive on the BMSC) with the cell in the NSC coculture and be separated on NSC.The magnetic bead that utilization links to each other with pan mouse IgG, BMSC can separate from NSC.Represent NSC not in conjunction with cell by facs analysis or put back in the nutrient solution.For facs analysis, use different BMSC traget antibody (mouse-anti people CD105) to detect the BMSC that is contaminted, and use mouse-anti people CD133 to detect NSC.

BMSC is placed in the 6 hole culture dish, 150,000 cells/well, and make it in the BMSC substratum, to adhere to and spend the night.Collection grows in the NSC (THD-hWB-015+LIFP17) in the culture and suspends once more, smashes neural ball.From the BMSC culture, remove the BMSC substratum and place the BMSC in the NSC substratum (contain EGF and FGE, contain or do not contain LIF) to go up (75,000 cells/well) NSC that collects.Coculture was cultivated 13 days in the NSC substratum.Every other day or at weekend is that cell is changed fresh culture.

At the 13rd day, use trypsinase to collect coculture, use Trypsin inhibitor SBTI subsequently.Cell mixture is suspended among the PBS that contains 0.1%BSA again.With anti-CD13 culturing cell mixture 30 minutes on ice.Use PBS (0.1%BSA) pair cell to carry out centrifuge washing.Simultaneously, with PBS (0.1%BSA) Dynal pearl-Pan mouse IgG is washed 3 times, at every turn by magnetic-particle concentrator (Dynal-MPC). they are separated.Cultivated 30 minutes for last 4 ℃ at inclination/swivel arrangement (Dynal sample mix device) among cell through washing and the washed magnetic bead PBS (0.1%BSA).The test tube that contains cell-pearl mixture was placed in MPC 2-3 minute.Magnetic bead with adhere to cell adhesion on it near test tube one side of magnet.Collect supernatant liquor and place an independent test tube.

A part of cell in the supernatant liquor is used to facs analysis, and a part of cell has been placed in bag by in the cell cultures slide of polyornithine and Fiberonectin.Use anti-CD105 and anti-CD133 (identification NSC) to carry out facs analysis.Place the cell on the cell cultures slide after the NSC substratum is cultivated 1-2 days, to be fixed, be used for the immunostaining of nidogen/GFAP.

In the time in 2 weeks, NSC increases on the BMSC layer.Observe some big colonies on it, infer that it results from little neurone.Other NSC unicellular or several cells form are grown to several less NSC colonies in 2 weeks.

After magnetic was got rid of BMSC, isolating NSC was through facs analysis.Cell above 90% is the CD133 positive (Fig. 9 B).

Being lower than 2% is the CD105 positive (Fig. 9 A).Cell adhesion on the cell cultures slide is in culture dish, and is similar to NSC on the form.The nidogen immunostaining is used to confirm the existence of NSC.Similar to BMSC on the BMSC-pearl blend morphology in the culture, it combines a large amount of magnetic beads and is attached to tissue culture flasks.The result shows, BMSC can stay spissated NSC cell mass by cultivating and subsequently magnetic resolution and being separated with the antibody that is incorporated into BMSC is simple from coculture.

Embodiment 3

Cultivate altogether and amplify and use the NSC amplification that minimum quantity BMSC improves as trophoderm

In this experiment, coculture is expanded in the T75 tissue culture flasks.(P1) two different inoculum densities with about 2.5e5 or 5.0e5/ bottle place the BMSC substratum to BMSC for donor BM-022, P1 and BM-024.Two days later, cell is through NSC substratum washing, and (THD-015 P14) is placed in the complete NSC substratum of 15ml on the BMSC 5.0e5NSC.Co-culture of cells 15 days, every other day or changed 50% fresh culture in two days for cell.After 15 days, come collecting cell by tryptic digestion.Big BMSC and less NSC count on Hematocyte Counter.As described in this paper elsewhere,, remove BMSC as embodiment 2.Suspension cell to 5 * 10e7/ml was cultivated on ice 15 minutes with anti-people CD13 antibody.Washed cell was once cultivated 30 minutes with washed Pan mouse IgG Dyna-pearl then.Pearl is separated in the magnetic-particle concentrator in conjunction with cell.The supernatant liquor that comprises NSC is counted the NSC number that reclaims with the washing of NSC substratum.Some cells are through facs analysis, and other cells are through differentiation and immunocytochemical assay.

Cultivate NSC on the fewer purpose BMSC trophoderm of description of contents disclosed herein and generate more NSC amplification than on more number BMSC trophoderm, cultivating.By changing the ratio of BMSC and NSC inoculum density, find to work as BMSC in about 30% low converging down, produce higher NSC multiplication, with respect to 50-60% or higher converging.For example, as shown in table 3, can see when the BMSC inoculum density in the T75 bottle under about 2.5e5 and the NSC inoculum density in 5.0e5 following time, 82 times of NSC multiplications, and the NSC of similar number is when placing the BMSC of double number to go up, 47 times of NSC multiplications.Compare with it, in other experiments when NSC be incubated at lack BMSC bag by the interpolation of culture dish in the standard NSC substratum of external source LIF the time, the maximum multiplication of the NSC of acquisition is approximately 20-25 times.After the NSC amplification in the presence of BMSC, use anti-BMSC antibody and immune magnetic pearl effectively to remove the BMSC in the coculture, the rate of recovery is 83-93% (table 3).

Table 3

The amplification of hNSC on the BMSC nurse cell

BMSC donor (inoculum density)	Initial # NSC	#NSC is in the time of the 15th day	The NSC multiplication	Rate of recovery % after BMSC removes	The NSC (multiplication) that # reclaims
						BM-03-022 (approximately 2.5e5)	5.0e5	41.1e6	82.2 doubly	93％	(38.3e6 76.5 times)
BM-04-024 (approximately 5.0e5)	5.0e5	23.5e6	47 times	83％	(19.4e6 39 times)

Cultivate amplification and isolating NSC altogether and be observed the mark that continues to express progenitor cell, include, but is not limited to nidogen (Figure 10).Isolating NSC has also kept it to be divided into neurone and Astrocytic ability (Figure 11).Prior art personnel can recognize that based on the disclosure of invention present method of the NSC that is used to increase can further be amplified and be suitable for being verified the production process of the closed system that is used to produce BMSC.

Embodiment 4:BMSC and NSC are at Transwell ^TMIn common cultivation (not relying on the common cultivation of contact)

This paper is verified, and BMSC can be used for the breeding and the amplification of human nerve stem cell (hNSC) as outstanding nourishing/supporting layer.In the present embodiment, experiment is designed to illustrate whether need two kinds of physics contacts or the BMSC mode that whether can contact by the non-dependence that provides soluble nutritional factor to support NSC breeding and amplification breeding and the propagation that support NSC between the cell.

BMSC is appointed as hBM-03-016-P1 and hBM-012-P2, through thaw, BMSC substratum washing, place Transwell ^TMIn.Transwell ^TMIn experiment in 6 plate hole Costar culture dish, carry out.BMSC is placed in Transwell ^TMIn, and NSC is placed in the plate hole bottom.Two kinds of cells all adopt identical NSC culture medium culturing.Transwell ^TMApplication can be implemented in and cultivate dissimilar cells, for example BMSC and NSC under the condition that does not have physics contact between two types of cells.

About 30,000 BMSC place Transwell ^TMMovably porous filter on.Make cell in the BMSC substratum to Transwell ^TMAfter surface adhesion was spent the night, cell was incubated at complete NSC substratum (in the DMEM/F12+N2 additive+EGF+FGF) after the flushing of the free substratum of blood plasma.NSC (being appointed as THD-hWB-015-P13) is through thawing, wash and placing on the 6 hole culture dish of polyornithine/Fiberonectin bag quilt with the density of 40,000 cells/well.The Transwell that will contain BMSC then ^TMPlace on 6 orifice plates that contain NSC.Contrast is Transwell ^TMComprise NSC and do not have BMSC.Transwell ^TMAdd the capacity substratum with the bottom, hole.Every other day change about 50% substratum.Cultivate after two weeks, from each plate hole, collect NSC and counting.Cell is analyzed the expression of NSC mark through flow cytometer.

Presentation of results is at Transwell ^TMIn with the BMSC co-cultivation grow in bag by the breeding of the NSC on the hole be better than cultivation at the bag that does not have BMSC by the NSC on the orifice plate (Figure 12).Find that BMSC has supported growth and the propagation of NSC, even under the situation that does not have two kinds of cells directly to contact.Do not wish to be subjected to the constraint of any particular theory, the phase believer in a certain religion BMSC excretory factor can be used for replenishing the NSC substratum, thereby helps to support breeding and the propagation of NSC.To growing in the Transwell that contains BMSC or do not contain BMSC ^TMIn NSC carry out facs analysis, illustrate that the phenotype of NSC is similar.For example, the NSC that grows under two kinds of conditions has about 100% the CD133 positive.

Repeat above-mentioned experiment, except 6 hole culture dish do not wrap by polyornithine/Fiberonectin.Nearly 50,000 BMSC are placed in Transwell ^TMIn.NSC (being appointed as THD-hWB-015-P12) is placed in after thawing among the Falcon or Costar plate in 6 holes.Be grown in Transwell ^TMOn BMSC be placed in 6 orifice plates, cultivated for 2 weeks.In contrast, the NSC single culture is in the NSC substratum that contains or do not contain exogenous LIF.Transwell ^TMIn the growth pattern of the NSC that cultivates altogether to BMSC similar to the NSC of single culture in the NSC that contains exogenous LIF cultivates, and its propagation significantly is better than at the NSC that does not contain single culture under the exogenous LIF (Figure 13).Find that also the Costar culture dish quantitatively provides than the better result of Falcon culture dish at cell proliferation.

Embodiment 5:NSC grows in the BMSC conditioned medium

The experimental result explanation BMSC of embodiment has generated the soluble factor of supporting the NSC growth.Next the conditioned medium that derives from the BMSC through cultivating has been used in one group of experiment carrying out, further specifies the effect of BMSC excretory factor pair NSC growth.Description of test disclosed herein BMSC excretory growth and/or other factors whether can support and improve NSC growth and propagation.This experimental design is to estimate 1) whether the substratum that limited by BMSC in the culture can substitute the NSC substratum that is added with exogenous LIF and the NSC in the culture brought the effect 2 that promotes growth) whether the conditioned medium that limited by BMSC in the culture can bring the beneficial effect that is similar to cultivation NSC on the substratum of polyornithine/Fiberonectin bag quilt for the cultivation of NSC.

For generating BMSC-CM, at first BMSC is placed the T-80 bottle, in the BMSC substratum, cultivate.After cultivating for some time, replace the BMSC substratum with the NSC substratum.Obtain BMSC-CM thus, that obtain with the complete NSC culture medium culturing BMSC that contains bFGF and EGF is BMSC-CM1, and that obtain with the NSC culture medium culturing that does not contain bFGF and EGF is BMSC-CM2.Changed fresh NSC substratum for BMSC in per 48 hours.At this moment, substratum is moved out of and centrifugal removal particulate matter, is used for cultivating NSC or in-80 ℃ of freezing evaluations that are used for cytokine.For BMSC-CM experiment, NSC uses the NSC growth medium that contains about 25-50%BMSC-CM to cultivate, and described BMSC-CM is new that collect or stored for 2 weeks in 4 ℃ from BMSC.BMSC-CM is through the existence of Cytokine Arrays (RayBiotech Inc.) or the various cytokines of elisa assay.

Whether can in BMSC-CM, breed for estimating NSC, NSC is placed on the culture dish of polyornithine/Fiberonectin bag quilt, every other day change 50% substratum, the replacement substratum that uses is a) BMSC-CM1, b) BMSC-CM2, c) contain the complete NSC substratum of exogenous LIF, or d) do not contain the complete NSC substratum of exogenous LIF.Twice BMSC-CM1 of beginning and the replacing of BMSC-CM2 added fresh EGF and FGF among every kind of BMSC-CM, and the interpolation of this EGF and FGF stop after a while.

The growth of experimental result explanation NSC in BMSC-CM or complete NSC substratum is good equally.Figure 14 has described the representative area of the NSC that cultivates under the different condition.In the presence of BMSC-CM1, can see that NSC has formed the neural ball of big viscosity, and have cell to grow neural ball.Can also see that BMSC-CM1 exists some NSC that cultivate down to form monolayer.In BMSC-CM2, cultivate under the situation of NSC, can see that also NSC has formed neural ball, still compare, seldom have big neural ball to form, and monolayer seldom occurs with the NSC that BMSC-CM1 cultivates down.In the presence of complete NSC substratum, NSC grows up to several interconnective neural balls, and shows the cell of hypertrophy from neural ball.In any case, the cell in each cultivation group all is collected and the counting cells number after cultivating 10 days, comes the influence of more different culture condition to the NSC breeding.The cell of gathering in the crops from BMSC-CM1 (approximately 3.4e6 cell) is suitable with the cell of gathering in the crops from the complete NSC substratum that contains exogenous LIF (approximately 3.3e6 cell) number.The cell number that obtains in BMSC-CM2 is approximately 2.85e6.

Next group experiment test NSC whether can in BMSC-CM, breed, or even do not wrapping on the culture dish of quilt.4 groups of NSC are as follows: 1) be incubated at the NSC among the BMSC-CM1; 2) be incubated at NSC among the BMSC-CM2; 3) be incubated at NSC in the complete NSC substratum that contains exogenous LIF; With 4) be incubated at the NSC in the complete NSC substratum that does not contain exogenous LIF.In 2 weeks of cell cultures, go down to posterity and handle time in another 2 week under the same conditions by tryptic digestion.Discovery is in first 2 week, and NSC has maximum propagation in BMSC-CM1, secondly be BMSC-CM2, is that (bar graph is shown P1 to NSC substratum+LIF, Figure 15) then.After going down to posterity, the NSC of BMSC-CM1 and NSC substratum+LIF propagation is similar (to be expressed as P2, Figure 15).The cell of handling through BMSC-CM2 continues breeding, good than the cell performance of handling with the NSC substratum that does not contain LIF.Therefore, available data shows that BMSC-CM is a kind of good somatomedin source to hNSC, the breeding that can induce NSC, and reproduction speed is equal or higher with the NSC substratum that has added exogenous LIF.In addition, the NSC that uses BMSC-CM to cultivate can go down to posterity by tryptic digestion, and further increases cell quantity.

The cytokine chip analysis of BMSC-CM

BMSC is incubated in the complete NSC substratum.Collect substratum at 48 hours, use the cytokine chip to measure the cytokine curve.1000 (combinations of chip VI and VIT) of RayBio human cell factor antibody chip C series buy from RayBiotech company (Norcross, GA).Use the sample of describing in manufacturer's the specification sheets to come the process chip film.(Amersham, Piscataway NJ) carry out the final detection of cytokine to use the ECL-Plus system.Signal be revealed on the X-ray film (Amersham, Piscataway, NJ).

The various cytokines of the presentation of results of cytokine curve/other factors are present among the BMSC-CM, and its strength of signal is higher than the negative control on the same chip.Cytokine/other factors include, but is not limited to LIF, brain derived neurotrophic factor (BDNF), EGF-R ELISA (EGF), basic fibroblast growth factor (bFGF), FGF-6, glial cell derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (GCSF), pHGF (HGF), IFN-γ, trypsin-like growth factor bindin (IGFBP-2), IGFBP-6, IL-lra, IL-6, IL-8, MCP (MCP-1), mononuclear phagocyte G CFS (M-CSF), neurotrophic factor (NT3), tissue inhibitor of metalloproteinase (TIMP-1), TIMP-2, tumour necrosis factor (TNF-β), vascular endothelial growth factor (VEGF), VEGF-D, UPA acceptor (uPAR), Delicious peptide (BMP4), IL1-a, IL-3, Leptin (leptin), STEM CELL FACTOR (SCF), mesenchymal cell derivative factor-1 (SDF-1), Thr6 PDGF BB-BB (PDGFBB), transforming growth factor-beta (TGF β-1) and TGF β-3.

Do not wish to be subjected to the constraint of any particular theory, BMSC-CM comprises several factors, comprises the factor that is present in the substratum that contains EGF and bFGF, except by the BMSC excretory factor or by the substratum inductive factor.The factor of listing above is those factors that signal evaluation is higher than negative control on the chip in 120 cytokines on the detection chip.

The cytokine that is higher than background level after testing includes, but is not limited to EGF and bFGF.Other clear cytokines of expressing are HGF (pHGF), M-CSF (mononuclear phagocyte G CFS) and TIMP-1, TIMP-2 (being respectively the tissue depressant of metalloprotease 1 and 2).Neurotrophic factor includes, but is not limited to BDNF, NT3, GDNF and GNTF.IGFBPs and FGF-6 are demonstrated.Many chemokineses, proinflammatory cytokine and angiogenesis factor as VEGF, also can generate.ELISA has confirmed that also BMSC also generates LIF.In the ELISA test, deduct the actual quantity that the control medium level has been assessed each detection factor of BMSC excretory by observed level from BMSC-CM.

Can draw from content disclosed herein: BMSC produces the soluble factor that promotes the NSC growth.NSC contacts not necessarily with the direct of BMSC, because the BMSC condition is cultivated growth and the propagation that also can support NSC.The present disclosure demonstration relies on the maximum propagation that the common cultivation that contacts has produced NSC, has shown that the soluble factor that BMSC or BMSC product and BMSC generate contacts the synergy that NSC is bred with the physics of NSC.

Embodiment 6: the NSC that cultivates in BMSC or BMSC-CM shows the MHC developed by molecule level of reduction, but has kept and the place an order identical phenotype of NSC of only length of standard conditions.

The various BMSC of the NSC of propagation and the expression of NSC mark among the flow cytometry analysis embodiment 3.Keeping the phenotypic characteristic identical (for example to the CD56 and the CD133 positive with NSC that BMSC cultivates altogether with do not contain the NSC that cultivates in the NSC substratum of BMSC having added exogenous LIF, to the CD105 feminine gender), except the NSC that cultivates altogether with BMSC shows can not detected II class MHC developed by molecule (Figure 16).When exogenous LIF adds in the NSC growth medium, can observe II class MHC molecule and be induced and result from the NSC.On the contrary, do not observe the NSC that cultivates altogether with BMSC and express the II class MHC molecule that is higher than baseline, this quasi-molecule may be useful to transplanting.In addition, isolating NSC does not have to express the BMSC mark CD105 that is higher than baseline from coculture, illustrates that BMSC effectively removes from coculture.

Similarly, the NSC that cultivates with BMSC-CM has also kept the identical phenotypic characteristic of NSC cultivated with standard NSC substratum+LIF, except they show the II class MHC of baseline values and the I class MHC (Figure 17) of minimizing.4 groups of NSC have accepted the analysis (Figure 17) that FACS expresses CD133, II class MHC molecule, I class MHC molecule and CD56.Observation before the result has confirmed, promptly the NSC of growth has expressed II class MHC molecule and has shown the average fluorescent strength that I class MHC molecule increases in the presence of exogenous LIF.But the NSC that grows in BMSC-CM does not observe it and expresses II class MHC molecule, and has lower I class MHC developed by molecule level.In all 4 groups of NSC, the expression of CD133 is similar.All have detected cell expressings CD56 is although cultured cells shows the average fluorescent strength of CD56 reduction in the presence of exogenous LIF.

Embodiment 7: by breeding the I class of NSC and the regulation and control of II class MHC developed by molecule on BMSC

Carry out other experiment and estimated the I class of NSC of common cultivation and the expression of II class MHC molecule.NSC from fetal brain, separate and in the NSC substratum, cultivated for 13 generations (THD-WB-015, P13).Under following 4 conditions, NSC was cultivated 12 days then, every other day change substratum.

1.62,500 NSC the bag of complete NSC substratum by the T25 bottle in single culture

2.62,500 NSC the bag of complete NSC substratum+LIF by the T25 bottle in single culture

3.62,500 NSC in complete NSC substratum 250, cultivate on 000BMSC (donor-012) trophoderm

4.NSC cultivate on 250,000 BMSC (donor-016) trophoderm in complete NSC substratum

Cell was collected by tryptic digestion after 12 days.Counting cells is also used the expression of flow cytometry analysis CD133, CD105, I class MHC and II class MHC molecule.In cultivating altogether, need all cells and express gate NSC based on scattering of light and CD133.Figure 18 has shown I class MHC and the II class MHC developed by molecule (black=isotype contrast of the NSC that cultivates under various conditions; Grey=I or II class).The result shows that all NSC have expressed I class MHC.But, when in the presence of exogenous LIF, cultivating NSC, I class MHC developed by molecule significantly increases on the cell, as being reflected in and the increase that does not contain NSC that exogenous LIF cultivates down Log average fluorescent strength (Log Median FluorescenceIntensity) relatively the time.Compare with the NSC that increases under LIF, the NSC that cultivates has expressed obvious low-level I class MHC molecule (table 4) altogether.

Table 4

Culture condition	The contrast of Log MF I class MHC isotype	The cell multiplication
			NSC is independent, does not contain LIF	209	8.16X
NSC separately+LIF	469	13.5X
			NSC+BMSC-012	323	16X
NSC+BMSC-016	366	26.7X

NSC does not have constructive expression II class MHC molecule.But LIF has induced the II class MHC developed by molecule of 38% cell.The NSC that cultivates with BMSC in that not contain the NSC that cultivates under the LIF similar, can not express the II class MHC molecule that is higher than baseline.

The The above results explanation is minimizing and/or prevents immunoregulatory I and the expression of II class MHC molecule on NSC in the advantage of propagation NSC on the BMSC, they are more suitable in clinical transplantation.

Here each patent, patent application and announcement and the disclosed content thereof quoted are all incorporated this paper in the mode of reference.

Obviously, concerning the prior art personnel, do not departing under design of the present invention or the scope, can make various adjustment and variation the inventive method and composition.Therefore, adjustment and the variation that is provided in claim and equivalency range has been provided in the present invention.

Claims (43)

1. a composition comprises: (a) a kind of isolating marrow stromal cell; (b) the definite developing medium of a kind of chemistry, described developing medium comprises the neural stem cell growth medium and the described marrow stromal cell excretory factor.

2. the described composition of claim 1, wherein said developing medium does not contain exogenous leukaemia inhibitory factor.

3. the described composition of claim 1, the wherein said factor is selected from: somatomedin, nutritional factor and cytokine.

4. the described composition of claim 1, the wherein said factor is selected from: leukaemia inhibitory factor, brain derived neurotrophic factor, EGF-R ELISA, basic fibroblast growth factor, FGF-6, glial cell derived neurotrophic factor, granulocyte colony-stimulating factor, pHGF, IFN-γ, the trypsin-like growth factor bindin, IGFBP-6, IL-1ra, IL-6, IL-8, MCP, the mononuclear phagocyte G CFS, neurotrophic factor, TIMP-1, TIMP-2, tumour necrosis factor, vascular endothelial growth factor, VEGF-D, the UPA acceptor, Delicious peptide, IL1-a, IL-3, Leptin, STEM CELL FACTOR, mesenchymal cell derivative factor-1, PDGFBB, TGF β-1 and TGF β-3.

5. the described composition of claim 1 also comprises a kind of isolating neural stem cell.

6. the described composition of claim 5, wherein said neural stem cell is that physics contacts with described marrow stromal cell.

7. the described composition of claim 5, wherein said neural stem cell is not that physics contacts with described marrow stromal cell.

8. the described composition of claim 5, wherein said neural stem cell derives from people's central nervous system.

9. the described composition of claim 1, wherein said bone marrow substrate cell source is in the people.

10. the described composition of claim 5, wherein exogenous genetic material is imported into described neural stem cell.

11. the described composition of claim 1, wherein exogenous genetic material is imported into described marrow stromal cell

12. a marrow stromal cell conditioned medium comprises the developing medium that a kind of chemistry is determined, described developing medium comprises the neural stem cell growth medium and by the isolating marrow stromal cell excretory factor.

13. the described marrow stromal cell conditioned medium of claim 12, wherein said bone marrow matrix conditioned medium does not comprise exogenous leukaemia inhibitory factor.

14. the described marrow stromal cell conditioned medium of claim 12, the wherein said factor is selected from: somatomedin, nutritional factor and cytokine.

15. the described marrow stromal cell conditioned medium of claim 12, the wherein said factor is selected from: leukaemia inhibitory factor, brain derived neurotrophic factor, EGF-R ELISA, basic fibroblast growth factor, FGF-6, glial cell derived neurotrophic factor, granulocyte colony-stimulating factor, pHGF, IFN-γ, the trypsin-like growth factor bindin, IGFBP-6, IL-1ra, IL-6, IL-8, MCP, the mononuclear phagocyte G CFS, neurotrophic factor, TIMP-1, TIMP-2, tumour necrosis factor, vascular endothelial growth factor, VEGF-D, the UPA acceptor, Delicious peptide, IL1-a, IL-3, Leptin, STEM CELL FACTOR, mesenchymal cell derivative factor-1, PDGFBB, TGF β-1 and TGF β-3.

16. regulate the method that main histocompatibility complex molecule is expressed for one kind on isolating neural stem cell, described method comprises co-cultured cell, described cell comprises isolating marrow stromal cell and isolating neural stem cell.

17. the described method of claim 16, wherein said cell is lacking cultivation altogether under the exogenous leukaemia inhibitory factor condition.

18. the described method of claim 16, wherein said neural stem cell is that physics contacts with described marrow stromal cell.

19. the described method of claim 16, wherein said neural stem cell is not that physics contacts with described marrow stromal cell.

20. the described method of claim 16, wherein said neural stem cell derive from people's nervous center system.

21. the described method of claim 16, wherein said bone marrow substrate cell source is in the people.

22. the described method of claim 16, wherein exogenous genetic material is imported into described neural stem cell.

23. the described method of claim 16, wherein exogenous genetic material is imported into described marrow stromal cell.

24. regulate the method that main histocompatibility complex molecule is expressed for one kind on isolating neural stem cell, described method comprises with the marrow stromal cell conditioned medium cultivates described neural stem cell, and wherein said marrow stromal cell conditioned medium comprises nerve stem cell culture medium and by the described marrow stromal cell excretory factor.

25. the described method of claim 24, wherein said marrow stromal cell conditioned medium does not comprise exogenous leukaemia inhibitory factor.

26. the described method of claim 24, wherein said marrow stromal cell conditioned medium does not contain marrow stromal cell substantially.

27. the described method of claim 24, the wherein said factor is selected from: somatomedin, nutritional factor and cytokine.

28. the described method of claim 24, the wherein said factor is selected from: leukaemia inhibitory factor, brain derived neurotrophic factor, EGF-R ELISA, basic fibroblast growth factor, FGF-6, glial cell derived neurotrophic factor, granulocyte colony-stimulating factor, pHGF, IFN-γ, the trypsin-like growth factor bindin, IGFBP-6, IL-1ra, IL-6, IL-8, MCP, the mononuclear phagocyte G CFS, neurotrophic factor, TIMP-1, TIMP-2, tumour necrosis factor, vascular endothelial growth factor, VEGF-D, the UPA acceptor, Delicious peptide, IL1-a, IL-3, Leptin, STEM CELL FACTOR, mesenchymal cell derivative factor-1, PDGFBB, TGF β-1 and TGF β-3.

29. the described method of claim 24, wherein said neural stem cell derive from people's nervous center system.

30. the described method of claim 24, wherein exogenous genetic material is imported into described neural stem cell.

31. an isolating neural stem cell prepares through the method for the marrow stromal cell of culture of isolated altogether and isolating neural stem cell.

32. the described isolating neural stem cell of claim 31, wherein said neural stem cell show the minimizing of I class MHC molecule and express.

33. the described isolating neural stem cell of claim 31, wherein said neural stem cell shows the baseline values of II class MHC molecule.

34. the described isolating neural stem cell of claim 31, wherein said neural stem cell derives from people's nervous center system.

35. the described isolating neural stem cell of claim 31, wherein exogenous genetic material is imported into described neural stem cell.

36. isolating neural stem cell, through in marrow stromal cell condition developing medium culture of isolated neural stem cell and prepare, wherein said marrow stromal cell condition developing medium comprises the developing medium that a kind of chemistry is determined, comprises the neural stem cell growth medium and the isolating BMSC excretory factor.

37. the described isolating neural stem cell of claim 36, wherein said neural stem cell show the minimizing of I class MHC molecule and express.

38. the described isolating neural stem cell of claim 36, wherein said neural stem cell shows the baseline values of II class MHC molecule.

39. the described isolating neural stem cell of claim 36, wherein said neural stem cell derives from people's nervous center system.

40. the described isolating neural stem cell of claim 36, wherein exogenous genetic material is imported into described neural stem cell.

41. a neuronal cell cultures equipment contains

(a). isolating neural stem cell;

(b). isolating marrow stromal cell;

(c). neural stem cell growth medium, wherein said neural stem cell growth medium comprise from the described isolating marrow stromal cell excretory factor; With

(d). prevent the device of physics contact between described neural stem cell and the described marrow stromal cell.

42. the described equipment of claim 41 also comprises a strainer or film, makes and does not produce the physics contact between described neural stem cell and the described marrow stromal cell.

43. having the hole, the described equipment of claim 42, described strainer or film make the described marrow stromal cell excretory factor by described strainer or film.

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