CN102827812B - Preparation method and application of induction type neural stem cells - Google Patents
Preparation method and application of induction type neural stem cells Download PDFInfo
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Abstract
The invention provides a preparation method and application of induction type neural stem cells. The preparation method of the induction type neural stem cells comprises the steps of: carrying out overexpression of exogenous factors Ascl1, Neurog2, Pax6, Hes1, Id1, Brn2, c-Myc and Klf4 in sustentacular cells of testis, and culturing in a base culture fluid containing cell growth factors to obtain the induction type neural stem cells. According to the preparation method of the induction type neural stem cells, sustentacular cells of testis derived from the mesoblast are reprogrammed to be neural stem cells derived from the ectoderm, the neural stem cells obtained in the reprogramming manner are proved to have physiological functions in vivo or in vitro, and the cells are safer than pluripotent stem cells for transplantation.
Description
Technical field
The present invention relates to preparation method and the application of induction type neural stem cell, particularly relate to and make cell without pluripotent state, directly across germinal layer transdifferentiated cells, the preparation method of induction type neural stem cell rapidly and efficiently and application.
Background technology
People rise year by year to the mechanism of nervous system disorders and treatment means attention rate.At a tremendous pace in society scientific-technical progress, new drug development emerges in an endless stream, but undeniable, and in global range, neural system class Disease quantity grows with each passing day.Such as Parkinson's disease, stages alzheimer's disease etc. nerve degenerative diseases has seriously reduced the quality of life of sufferers themselves, and adds the living burden of patient home.So how fundamentally treating nervous system disorders, particularly nerve degenerative diseases has become the focus that scholars contends, and China also attaches great importance to the research and probe in this field.The traditional treatment means mainly pharmacological agent of nerve degenerative diseases.But, actual clinical drug treatment most likely causes serious complication (Willis, G.L., The therapeutic effects of dopamine replacement therapy and its psychiatric side effects are mediated by pineal function.Behav Brain Res, 2005.160 (1): p.148-60.).Mostly nerve degenerative diseases Producing reason is a large amount of apoptosis of certain neurone of specific region in brain.Research finds that Mammals comprises the mankind, and neural regenerative power is very poor, so following effectively treatment nerve degenerative diseases depends on neural stem cell or neuronic Transplanted cells.Because operation needs the neurocyte meeting clinical criteria in a large number, so exploitation is a kind of, cellular resources is particularly important reliably.Pluripotent embryonic stem cells can be divided into various types of neurocyte, but induced efficiency is very low, has tumorigenesis risk after transplanting.Consider postoperative clinical complication and Social And Ethical Issues, carry out Transplanted cells by the nervous tissue of autologous patient and have more using value (Chen, Z.and T.D.Palmer, Cellular repair of CNS disorders:an immunological perspective.Hum Mol Genet, 2008.17 (R1): p.R84-92.).Research before shows, some important gene of process LAN in somatocyte, inducing pluripotent stem cells (the iPSCs) (Takahashi consistent with original donorcells genetic background can be obtained, K.and S.Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell, p.663-76.) and induction type neurone (iNs) (Vierbuchen 2006.126 (4):, T., et al., Direct conversion of fibroblasts tofunctional neurons by defined factors.Nature.463 (7284): p.1035-41).Although iPSCs cell can produce neural precursor endlessly, but the tumorigenicity problem produced after transplanting constrains clinical application (the Wer nig of this kind of multipotent stem cells, M., et al., Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of ratswith Parkinson ' s disease.Proc Natl Acad Sci U S A, 2008.105 (15): p.5856-61.).On the contrary, iNs cell can not carry out cell fission completely, so be difficult to survival in body after transplanting go back to sufferer place.In addition, studying the iNs cell obtained can only be a class peripheral neurons before, obtains other types of neurons temporarily have no report by means of directly inducing.
In order to reach clinical effectiveness more safely and effectively, the induction method developing a kind of reliable and stable acquisition neural stem cell is very urgent.
Summary of the invention
Therefore, the object of this invention is to provide one can make cell without pluripotent state, directly across germinal layer transdifferentiation sustentacular cell of testis, the preparation method of induction type neural stem cell (iNSCs) rapidly and efficiently, and prove that this neural stem cell obtained by reprogrammed mode in vivo or externally all have physiologic function, this kind of cell is safer than the multipotent stem cells before for transplanting.
The method preparing induction type neural stem cell of the present invention, comprise exogenous factor Ascl1, Neurog2, Pax6, Hes1, Id1, Brn2, c-Myc and Klf4 process LAN in sustentacular cell of testis, then cultivate in containing the basic culture solution of cell growth factor, obtain induction type neural stem cell.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, described cell growth factor is EGF and bFGF.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, described EGF and described bFGF is 20 nanograms/milliliter.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, described basic culture solution is N2B27 nutrient solution.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, described cultivation D type poly-lysine (PDL) and ln (Laminin) wrap quilt culture dish in carry out.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, the method of described exogenous factor Ascl1, Neurog2, Pax6, Hesl, Id1, Brn2, c-Myc and Klf4 process LAN in sustentacular cell of testis is: be cloned into respectively on retroviral vector by the cDNA of exogenous factor Ascl1, Neurog2, Pax6, Hesl, Id1, Brn2, c-Myc and Klf4, obtain recombinant vectors, described recombinant vectors is packaged into virus respectively, utilizes described virus infection sustentacular cell of testis.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, described virus infection sustentacular cell of testis carries out in accordance with the following steps:
200, in 000 described virus infection sustentacular cell of testis, add the concentrating virus of often kind of exogenous factor, add the sustentacular cell of testis nutrient solution of the polybrene containing 4 mcg/ml simultaneously, infect 24 hours, then change normal sustentacular cell of testis nutrient solution into, renewal cultivation 24 hours, superinfection, renewal cultivation are once.
Preferably, of the present inventionly prepare in the method for induction type neural stem cell, described sustentacular cell of testis is prepared in accordance with the following steps:
Remove the tunica albuginea of mouse testis, then divide three steps to digest: (one) 0.1% collagenase IV 37 DEG C digestion 20 minutes; (2) digest 10 minutes with 0.1% collagenase IV and 0.1% Unidasa 37 DEG C simultaneously, clean with PBS after digestion; (3), the mixture of 0.1% collagenase IV, 0.1% Unidasa, 0.25% pancreatin, 0.04%DNA enzyme I, 37 DEG C digest 20 minutes, stop digestion with foetal calf serum, 200 object cells sieve filtration cells, again with DMEM/F12 nutrient solution cleaning twice, cultivate in sustentacular cell of testis nutrient solution, the composition of sustentacular cell of testis nutrient solution is: DMEM/F12,10%FBS, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates.
Another object of the present invention is to provide induction type neural stem cell prepared by above-mentioned method.
Induction type neural stem cell of the present invention can be applied in the medicine of preparation treatment nerve degenerative diseases.
Classical developmental biology theory is pointed out, sustentacular cell of testis be a class from mesoblastic specialization somatocyte, after ripe, cell just no longer divides, and do not have versatility, physiological function mainly provides spermatogenetic microenvironment.
The present invention is separated and obtains highly purified sustentacular cell of testis in body, under determining not exist the prerequisite that neurocyte pollutes, by process LAN only 8 external source transcription factors, in suitable culture system, without pluripotent state, be successfully the neural stem cell of ectodermal origin by direct for the sustentacular cell of testis of mesoderma origin reprogrammed.Through the systems axiol-ogy from molecular level to physiological level, the neural stem cell (iNSCs) that these inductions obtain can be stablized in vitro and goes down to posterity, identical cellular form and proliferating cycle is had compared with normal neuronal stem cell, Immunofluorescence test finds that iNSC expresses the molecular marked compound the same with NSC, as: Nestin, Olig2, Pax6 and Sox2, gene chip results confirms that the whole genomic expression of iNSC is also similar to positive control height; Functionally, iNSCs can maintain self and can be become the various neurone and the spongiocyte that have electrophysiological function, as dopaminergic neuron, γ-aminobutyric acid neurone and ACh neuron by efficiently differentiation-inducing.Importantly, after inducing the Transplanted cells that obtains to return the dentate gyrus of mouse brain hippocampus these, iNSCs can survive form Synaptic junction with peripheral nerve unit.The above results proves that iNSCs will become a kind of cellular resources screened for clinical treatment nerve degenerative diseases and novel drugs.
Accompanying drawing explanation
Below, describe embodiment of the present invention in detail by reference to the accompanying drawings, wherein:
Specific proteins-the Nestin of Figure 1A: iNSC expression neural stem cell;
Specific proteins-the Olig2 of Figure 1B: iNSC expression neural stem cell;
Fig. 1 C: iNSC is closer to normal neural stem cell for the display of full-length genome expression pattern analysis, and being different from its donorcells-sustentacular cell of testis, three groups of cells are followed successively by sustentacular cell of testis, normal type neural stem cell, induction type neural stem cell from left to right.
Fig. 2 A:iNSC can be divided into the two positive neurone of MAP2 and GABA;
Fig. 2 B:iNSC can be divided into the two positive neurone of MAP2 and TH;
Fig. 2 C:iNSC, in differentiation 2 weeks to 3 weeks processes, demonstrates normal bioelectrical activity.
The iNSC that Fig. 3 GFP marks, can survive behind implantation mouse brain hippocampal dentate region.
Embodiment
Ascl1：
Gene?ID：17172
Neurog2：
Gene?ID：11924
Pax6：
Gene?ID：18508
Hes1：
Gene?ID：15205
Id1：
Gene?ID：15901
Brn2：
Gene?ID：18992
c-Myc：
Gene?ID：17869
Klf4：
Gene?ID：16600
embodiment 1
The separation of sustentacular cell of testis:
Utilize a kind of method (Richardson of improvement, L.L., H.K.Kleinman, and M.Dym, Basement membrane gene expressionby Sertoli and peritubular myoid cells in vitro in the rat.Biol Reprod, 1995.52 (2): p.320-30.) highly purified sustentacular cell of testis can be separated to.Concrete steps are as follows:
Take out the testis of the mouse of raw latter 5 days.The tunica albuginea of testis is removed.Then divide three steps to digest: (one) 0.1% (mass percent) collagenase IV, 37 DEG C digest 20 minutes, (2) use 0.1% (mass percent) collagenase IV and 0.1% (mass percent) Unidasa, 37 DEG C digest 10 minutes, should clean after digestion with PBS simultaneously, (3) 0.1% (mass percent) collagenase IV, 0.1% (mass percent) Unidasa, 0.25% (mass percent) pancreatin, the mixture of 0.04% (mass percent) DNA enzymatic I, 37 DEG C digest 20 minutes, use foetal calf serum (FBS, Invitrogen) digestion is stopped, use 200 object cell sieve filtration cells, again with DMEM/F12 nutrient solution cleaning twice, cultivate in sustentacular cell of testis nutrient solution, the composition of nutrient solution is: DMEM/F12, 10%FBS, 100U/ml penicillin, 100 μ g/ml Streptomycin sulphates, at 37 DEG C, 5%CO
2environment in cultivate, carry out after cell covers with about 80% going down to posterity or frozen.
The induction of induction type neural stem cell (iNSC):
By exogenous factor Ascl1, Neurog2, Pax6, Hes1, Id1, Brn2, c-Myc and Klf4) and the cDNA of EGFP be cloned into (Takahashi on retroviral vector respectively, K.and S.Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell, 2006.126 (4): p.663-76), obtain recombinant vectors, recombinant vectors is proceeded to respectively in gag/pol-293T cell again and carry out virus packaging, and utilize ultrafiltration/ultracentrifugal method to carry out the concentrated of virus, virus after concentrated be stored in-80 ° stand-by.Virus infection 293T cell after concentrated, and utilize coubling dilution to detect the titre of virus.
Utilize EGFP virus infection sustentacular cell of testis, find that efficiency of infection only has about 30%, this may be relevant slowly with sustentacular cell of testis rate of propagation.
About 200, in 000 donorcells, the concentrating virus of often kind of factor adds 10 microlitres, add polybrene (final concentration is 4 mcg/ml) simultaneously, infect about 24 hours, then change normal sustentacular cell of testis nutrient solution into, renewal cultivation 24 hours, superinfection/renewal cultivation once, to improve the efficiency of infection of donorcells.After this, seed cells in the culture dish of PDL and Laminin bag quilt, utilize N
2b
27nutrient solution is cultivated, add EGF (20 nanograms/milliliter) and bFGF (20 nanograms/milliliter) two kinds of cell growth factor (Ying simultaneously, Q.L., et al., Conversion of embryonic stem cellsinto neuroectodermal precursors in adherent monoculture.Nat Biotechnol, 2003.21 (2): p.183-6.), in 37 °, 5%CO
2in cultivate, the cell colony of iNSC can be formed within 3 days after inoculation.In Induction Process, within every two days, carry out changing liquid.INSC clone can about set up within one month.
The vitro differentiation of induction type neural stem cell (iNSC) detects
Neuron differentiation: about 5,000-10,000 iNSC cell is inoculated in 24 orifice plates of PDL and Laminin bag quilt, adds the N containing BDNF (10 nanograms/milliliter) and NT-3 (10 nanograms/milliliter)
2b
27nutrient solution, carries out half amount in every three days and changes liquid.Differentiation culture is after 2 weeks in vitro, by N
2b
27in nutrient solution, the concentration of BDNF and NT-3 all brings up to 20 nanograms/milliliter.
When differentiation culture 2 weeks, part cell can express MAP2 and NeuN two kinds of neuronic labelled proteins, has the labelled protein O4 of part cell expressing oligodendrocyte simultaneously, when differentiation culture 3 weeks, part cell can express TH, the neuronic labelled protein of the specific types such as GABA, ChAT.
Differentiation (the Conti of astroglia cell, L., et al., Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.PLoS Biol, 2005.3 (9): p.e283.): iNSC is incubated at factor-containing BMP4 (10 nanograms/milliliter) N
2b
27in nutrient solution.The astroglia cell of expressing GFAP can occur within these few days.
The external electro physiology of induction type neural stem cell (iNSC) detects
INSCs after differentiation 2 to 3 week time, carry out the record of electrophysiological function.The sheet glass of developing approach be positioned over (ASCF) in the artificial cerebrospinal fluid constantly passing into oxygen and carbonic acid gas, the formula of ASCF is: NaCl 119mM, NaHCO
326.2mM, glucose 11mM, KCl 2.5mM, K
2hPO
41.0mM, CaCl
22.5mM, MgCl
21.3mM.Under full cell currents pincers pattern, record spontaneity and pungency action potential, under whole-cell voltage-clamp pattern, the dependent electric current of record ionic channel.In electrode, the composition of liquid is: potassium gluconate 120mM, NaCl 5mM, KCl 10mM, MgCl
21mM, EGTA 1mM, Hepes 10mM, ATP 2mM, GTP 0.5mM, utilizes 1MKOH that pH value is adjusted to 7.2.
Result shows (see Fig. 2), and iNSCs can maintain self and differentiation becomes the various neurone having electrophysiological function, as dopaminergic neuron, γ-aminobutyric acid neurone and ACh neuron.
Transplantation experiments in the body of induction type neural stem cell (iNSC)
Utilize the virus infection iNSC of expressing green fluorescent protein (GFP), obtain the iNSC cell of continuous expression GFP, for transplanting.The mouse of first treating transplanting is anaesthetized, and anesthetic formulations is 80mg/kg Ketamine and 10mg/kg xylazine.Utilize mouse brain stereotaxic instrument to carry out Transplanted cells, in the dentate gyrus of every side hippocampus, be implanted into about 10
5individual iNSC.The 2nd week after transplanting, 3 weeks, Immunofluorescence test was carried out to the hippocampal dentate of the mouse transplanted in 4 weeks.Collective's step is: first utilize 0.9% physiological saline and 4% paraformaldehyde to carry out perfusion, then entirely go out full brain fixedly to spend the night in 4% paraformaldehyde, recycle 30% sucrose solution to dewater, dewater and carry out cutting into slices and identified by immunofluorescence after 2-3 days, transplant latter 2 weeks, can observe the iNSC expressing GFP is distributed in hippocampal dentate, 3-4 week after transplanting, the neuronic labelled protein of the maturations such as NeuN and Synapsin can be detected, prove that iNSC can be survived in vivo, differentiation, and set up functional connection in other neurones.
Result shows (see Fig. 3), and after these induce the Transplanted cells that obtains to return the dentate gyrus of mouse brain hippocampus, iNSCs can survive form Synaptic junction with peripheral nerve unit.Prove that iNSCs will become a kind of cellular resources screened for clinical treatment nerve degenerative diseases and novel drugs.
Full-length genome expression pattern analysis
Utilize Illumina company MouseWG-6v2.0 Expression BeadChips type chip, we compare analysis in cell transcription level.From NSC cell iNSC cell and sustentacular cell of testis, extract total serum IgE respectively, each clone has three technology to repeat respectively.Process chip raw data in R environment, linearizing data empirical Bayes methods analyst, the differential gene of selection must ensure more than 2 times change multiples and P value is less than 0.05.Data clusters analysis uses Euclidean distance matrix and complete linkage clustering method.
Result shows (see Fig. 1), and the neural stem cell (iNSCs) that these inductions obtain expresses the molecular marked compound the same with normal neuronal stem cell, and whole genomic expression is also similar to positive control height.
Claims (4)
1. prepare the method for induction type neural stem cell for one kind, comprise exogenous factor Ascl1, Neuro g2, Pax6, Hes1, Id1, Brn2, c-Myc and Klf4 process LAN in sustentacular cell of testis, then cultivate in containing the basic culture solution of cell growth factor, obtain induction type neural stem cell;
Wherein, described cell growth factor is EGF and bFGF;
Described cultivation is carried out in the culture dish of D type poly-lysine and ln bag quilt; And
The method of described exogenous factor Ascl1, Neurog2, Pax6, Hes1, Id1, Brn2, c-Myc and Klf4 process LAN in sustentacular cell of testis is: be cloned into respectively on retroviral vector by the cDNA of exogenous factor Ascl1, Neurog2, P ax6, Hes1, Id1, Brn2, c-Myc and Klf4, obtain recombinant vectors, described recombinant vectors is packaged into virus, utilizes described virus infection sustentacular cell of testis.
2. method according to claim 1, is characterized in that, described EGF and described bFGF is 20 nanograms/milliliter.
3. method according to claim 1 and 2, is characterized in that, described basic culture solution is N2B27 nutrient solution.
4. method according to claim 1 and 2, is characterized in that, described virus infection sustentacular cell of testis carries out in accordance with the following steps:
200, in 000 described sustentacular cell of testis, add the concentrating virus of often kind of exogenous factor, add the sustentacular cell of testis nutrient solution of the polybrene containing 4 mcg/ml simultaneously, infect 24 hours, then change normal sustentacular cell of testis nutrient solution into, renewal cultivation 24 hours, superinfection, renewal cultivation are once.
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