CN105925528A - Schwarm progenitor cell derived from marrow neural crest cell and application of Schwarm progenitor cell to promotion of nerve regeneration - Google Patents

Schwarm progenitor cell derived from marrow neural crest cell and application of Schwarm progenitor cell to promotion of nerve regeneration Download PDF

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CN105925528A
CN105925528A CN201610338493.9A CN201610338493A CN105925528A CN 105925528 A CN105925528 A CN 105925528A CN 201610338493 A CN201610338493 A CN 201610338493A CN 105925528 A CN105925528 A CN 105925528A
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neural crest
ncc
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differentiation
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施海燕
丁斐
强亮
余英奇
孙晓婷
胡静静
孙晓欢
何欢
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Nantong University
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Abstract

The invention relates to the technical field of tissue engineering, in particular to a Schwarm progenitor cell derived from a marrow neural crest cell and application of the Schwarm progenitor cell to promotion of nerve regeneration. The rat marrow-derived neural crest cell N1, namely, BM-NCC clone N1, is provided, the preservation number is CCTCC NO:C201647, and the rat marrow-derived neural crest cell N1 has the performance of the Schwarm progenitor cell (SCP). The invention further provides a subculture method of the BM-NCC clone N1 and a method for induced directional differentiation to the Schwarm cell (SC). The BM-NCC clone N1 is expected to be applied to nerve regeneration promotion therapy based on cells.

Description

Come from bone marrow neural crest cell executes Wang Shi CFU-GM and the application in promoting neuranagenesis thereof
Technical field
The present invention relates to tissue engineering technique field, be specifically related to that a kind of to come from executing of bone marrow neural crest cell prosperous Family name's CFU-GM and the application in promoting neuranagenesis thereof.
Background technology
In nervous system, schwann's cell (Schwann cell, SC) is the colloid of peripheral nervous system Cell, becomes the systematism of myelin, peripheral nerve unit, the maintenance of neuron normal function, synapse in aixs cylinder Form and the aspect such as nerve injury and neuralgic reaction played the effect of key.Neural damage around Wallerian denaturation stage after wound, SC dedifferentes as immaturity SC or precursor sample SC, participation marrow phosphorus Fat and the removing of aixs cylinder fragment, produce multiple somatomedin and cytokine, breeds and migrates formation beneficially The Bungner band of axon regeneration length, the one-tenth myelin SC being divided into maturation the most again supports aixs cylinder again Form myelin.At present, neural tissue engineering based on SC has a lot of application target, including peripheral nerve The treatment of i or I, the treatment potential of central nervous system (CNS) disease, and SC are correlated with disease The treatment in sick body external model stage.But the source of SC and purity still limit it in terms of cell therapy Application.
Execute Wang Shi CFU-GM (Schwann cell precursor, SCP) and represent the early stage of schwann's cell Stage.In vertebrate growth course, the neural crest that SCP and immature SC originates from migration is thin Born of the same parents (neural crest cell, NCC), just disappear to adulthood neural crest tissue.Will be around embryo After the SCP and ripe SC of neural enrichment is transplanted to CNS, the former than the latter have preferably existence and Transfer ability.SCP can also be divided into into the maincenter glial cell of myelin and repair cerebral lesion.Additionally, After the birth of enteric nervous system, neural generation has also been observed that the neuronotropic differentiation of SCP.
At present, there is multiple different exogenous stem cells type to be used to derivative SC, done including embryo Cell and induced multi-potent stem cell [Ma MS, Boddeke E, Copray S.Pluripotent stem cells for Schwann cell engineering.Stem Cell Rev.2015；11:205-18.][Li W,Huang L,Lin W, Ke Q,Chen R,Lai X,et al.Engraftable neural crest stem cells derived from cynomolgus monkey embryonic stem cells.Biomaterials.2015；39:75-84.][Liu Q, Spusta SC,Mi R,Lassiter RN,Stark MR,Hoke A,et al.Human neural crest stem cells derived from human ESCs and induced pluripotent stem cells:induction, maintenance,and differentiation into functional schwann cells.Stem Cells Transl Med.2012；1:266-78.][Ren YJ,Zhang S,Mi R,Liu Q,Zeng X,Rao M,et al. Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix.Acta Biomater.2013；9:7727-36.][Wang A,Tang Z,Park IH,Zhu Y,Patel S,Daley GQ,et al.Induced pluripotent stem cells for neural tissue engineering.Biomaterials.2011；32:5023-32.], all demonstrate that they are to being subject to Damage the repair of neuron.It is true that adult stem cell has polyphyly differentiation potential in vitro, and They can obtain from autologous, can avoid the puzzlement of immunologic rejection effect and ethical issues, therefore becomes soma thin Born of the same parents it appear that more prospect alternative SC source cell type, including mescenchymal stem cell (mesenchymal stem cell, MSC) and migrate after NCC [Neirinckx V, Coste C, Rogister B, Wislet-Gendebien S.Concise review:adult mesenchymal stem cells,adult neural crest stem cells,and therapy of neurological pathologies:a state of play.Stem Cells Transl Med.2013；2:284-96.].
It has been reported that NCC and derivative SC can obtain [Nagoshi from dorsal root ganglion (DRG) after Qian Yiing N,Shibata S,Kubota Y,Nakamura M,Nagai Y,Satoh E,et al.Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow,dorsal root ganglia,and whisker pad.Cell Stem Cell.2008；2:392-403][Li HY,Say EH,Zhou XF.Isolation and characterization of neural crest progenitors from adult dorsal root ganglia.Stem Cells.2007；25:2053-65.][Vidal M,Maniglier M,Deboux C, Bachelin C,Zujovic V,Baron-Van Evercooren A.Adult DRG Stem/Progenitor Cells Generate Pericytes in the Presence of Central Nervous System(CNS) Developmental Cues,and Schwann Cells in Response to CNS Demyelination. Stem Cells.2015；33:2011-24.][Zujovic V,Thibaud J,Bachelin C,Vidal M, Coulpier F,Charnay P,et al.Boundary cap cells are highly competitive for CNS remyelination:fast migration and efficient differentiation in PNS and CNS myelin-forming cells.Stem Cells.2010；28:470-9.], also can from impaired or intact around Neural enrichment culture goes out Schwann cell ball [Takagi T, Ishii K, the Shibata S, Yasuda of pluripotency A,Sato M,Nagoshi N,et al.Schwann-spheres derived from injured peripheral nerves in adult mice--their in vitro characterization and therapeutic potential.PLoS One.2011；6:e21497.][Martin I,Nguyen TD,Krell V,Greiner JF,Muller J,Hauser S,et al.Generation of Schwann cell-derived multipotent neurospheres isolated from intact sciatic nerve.Stem Cell Rev.2012；8:1178-87.], there is the identity of NCC With the potential to SC directed differentiation, these Schwann cell balls can be that neuranagenesis provides autogenous cell Source, but the damage that donor nerve is caused by corresponding biopsy means, in fact for translational medicine It is unpractical.So, scholars are intended to search out the non-nerve that can obtain adult NCC and derivative SC Tissue-derived.Have latest report [Mendez-Ferrer S, Michurina TV, Ferraro F, Mazloom AR, Macarthur BD,Lira SA,et al.Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.Nature.2010；466:829-34.][Isern J,Martin-Antonio B, Ghazanfari R,Martin AM,Lopez JA,del Toro R,et al.Self-renewing human bone marrow mesenspheres promote hematopoietic stem cell expansion.Cell Rep. 2013；3:1714-24.][Isern J,Garcia-Garcia A,Martin AM,Arranz L,Martin-Perez D, Torroja C,et al.The neural crest is a source of mesenchymal stem cells with specialized hematopoietic stem cell niche function.Elife.2014；3:e03696.], find The marrow stromal cell (bone marrow stromal cell, BMSC) of the Nestin positive and neural crest origin SCP there is common nerve-epithelial development origin, and both cells may reside in long bone simultaneously Bone marrow, establishes the microenvironment of hematopoietic stem cell jointly；During In vitro culture, these Nestin are positive, blood The BMSC of platelet source growth factor receptors (PDGFR) feminine gender can be formed has self-renewal capacity with many The cell ball of differentiation potential, and it is dry thin to include neural crest in these mesenchyme balls (Mesensphere) Born of the same parents (neural crest stem cell, NCSC) and neural crest CFU-GM (neural crest precursor, NCP), And thus further infer that in bone marrow the residence having SCP cell mass.
Although BMSC is well-known high heterogeneous population, seminar's early stage at the present inventor place Research work has shown that [Shi H, Zhang T, Qiang L, Man L, Shen Y, Ding F.Mesenspheres of neural crest-derived cells enriched from bone marrow stromal cell subpopulation.Neurosci Lett.2013；532:70-5.], by CMC model, can answer from bone marrow Answer epidermal growth factor/fibroblast growth factor BMSC (EGF/FGF2-responsive BMSC, E/Fr-BMSC) subgroup raises the mesenchymal cell ball (Mesensphere) of suspension growth, i.e. reside in NCC after the migration of bone marrow, it is critical that bone marrow neural crest cell (bone marrow derived neural Crest cell, BM-NCC) there is self-renewal capacity and directed differentiation is the latent of neuron and glial cell Energy.
Summary of the invention
It is an object of the invention to provide a kind of BM-NCC and clone N1, and BM-NCC clones N1 Secondary Culture method, to SC induction differentiation method, and BM-NCC clone N1 preparation promote Application in neuranagenesis medicine.
How SCP is derived from BM-NCC it is contemplated that probe into.Then, we are obtaining rat After BM-NCC, isolate multiple can frozen and recovery cell clone, be then shown experimentally that BM-NCC clones and can be induced to differentiate into the positive SC of S100 β, and the cell institute of N1 therein clone Derivative SC purity is higher than other cell clone, and without gene regulation or be further purified, N1 cell spreads out Raw SC also has external one-tenth myelin potential.
Testing result is it is also shown that in protein level and mRNA level in-site, NCC indicates at N1 cell height table Reach, and at derivative SC down-regulated expression；At derivative SC, the SC mark relevant with SC differentiation state Then raise.We are further with the conditioned medium (N1-CM) of N1 cell, i.e. cell culture supernatant Processing the DRG neuron after (OGD) damage deprived by oxygen sugar, the survival of neuron and the growth of aixs cylinder are equal Improved；It addition, N1 cell infusion is entered the rat sciatic nerve after crush injury by us, result shows Show that N1 cell SC more cell-derived than N1 (N1derived Schwann cell, N1-d-SC) has more Good rush neuranagenesis and function repair, show that N1 cell the most all can promote injured neurons Reparation.Present invention demonstrates that N1 cell can be identified as SCP identity (or undifferentiated SCP sample state), The SCP coming from BM-NCC is expected to the rush treatment of nerve regeneration based on being applied to cell future.
A first aspect of the present invention, it is provided that a kind of rat marrow source neural crest CFU-GM N1, preserving number is CCTCC NO:C201647.
Rat marrow source of the present invention neural crest CFU-GM N1, i.e. BM-NCC clones N1, possesses Execute the performance of Wang Shi CFU-GM (SCP).
The present invention test result indicate that: N1 clone compared to obtain simultaneously other BM-NCC clone (as N3, N4 and N13), there is the strongest potential to SC differentiation, after Induction of committed differentiation, S100 β is positive SC ratio reach more than 95%, it is not necessary to follow-up cell sorting step；Meanwhile, to have colloid thin in display The NCC of born of the same parents' differentiation potential indicates CD44, HES1 and SOX10, and its mrna expression level is at N1 gram The grand BM-NCC higher than other clones (such as N4)；And show the NCC mark with neuron differentiation potential Will WNT1, ID2, p75NTR and CXCR4, its mrna expression level is then less than other N1 clone BM-NCC clone (such as N4).
A second aspect of the present invention, additionally provides the Secondary Culture method of BM-NCC clone N1.
Described Secondary Culture method, is by after cell recovery that preserving number is CCTCC NO:C201647, Cultivate to most cells ball increase and ball center's cell start to shade time, with accutase enzyme by cell ball digest For single cell suspension, then cell presses≤104The density of individual/ml is inoculated into Tissue Culture Plate, carries out balling-up and passes on Cultivating, half amount changes liquid 2 times weekly；Every 7～14d pass on 1 time, or pass on as required.
A third aspect of the present invention, additionally provides BM-NCC clone N1 fixed to schwann's cell (SC) Method to induction differentiation.
Described Induction of committed differentiation method, is to be answered by the cell that preserving number is CCTCC NO:C201647 Su Hou, cultivates to exponential phase, is separated into individual cells, and is inoculated into Laminin lens/poly-D-lysine (laminin/PLL) on coated culture plate, cultivation is hatched with SC Induction of committed differentiation liquid, i.e. containing 5% tire Ox blood serum (FBS), 35ng/ml all-trans retinoic acid (tRA), 5 μMs of forskolin, 200ng/ml HRG-1 β, DMEM/F12 (v/v, the 1:1) culture fluid of 10ng/ml FGF2,10ng/ml PDGF-AA and 1%N2, Every 2～3d change liquid 1 time.
The invention provides a kind of simple efficient method, this induction differentiation scheme is cloned in 2d to BM-NCC The most i.e. start onset, no longer than 1 week.
A fourth aspect of the present invention, additionally provides BM-NCC clone N1 and promotes drug for nerve regeneration in preparation Application in thing.
Described application is specially the treatment based on cell, and the BM-NCC clone N1 of the present invention is for promoting The research entering injured nerve regeneration provides unlimited cell derived.
Treatment based on described cell uses mode can be that local injection enters damaged tissues, intravenous injection Or be used for preparing tissue engineering nerve graft row implant region etc..
Described medicine can be cell suspension, the conditioned medium of cell, the factor of emiocytosis, cell The vesicle (secreting outward body etc.) of secretion or vesicle (secreting the outward body etc.) extract etc. of emiocytosis.
The concrete technical scheme of the present invention is as follows:
We have cultivated BM-NCC and separation obtains BM-NCC single cell clone, have detected theirs The expression of NCC mark and self-renewal capacity.According to each BM-NCC clone to the efficiency of SC differentiation, We have selected the N1 clone cell subsets as research further.N1 cell is with embryo's DRG neuron altogether Cultivating, to detect the one-tenth myelin potential of N1 cell, result shows that N1 cell can derive SC and promote in vitro Enter the one-tenth myelin of aixs cylinder.N1-CM is used for processing the DRG neuron of OGD damage, and result shows to improve The survival of DRG neuron and axon growth situation.Additionally, N1 cell or N1-d-SC are implanted into respectively The rat sciatic nerve crushed, result shows to promote the recovery of sciatic regeneration and function.
Existing researcher analyzes the similar and difference of MSC with NCC deriving from adult mice bone marrow.Also There is researcher to pass through the effect of Wnt1 and BMP2, be enriched to NCC single cell clone from adult BMSC, Although experiment shows the medullary cell that Nestin/SOX10/p75NTR is positive, it may be possible to have neurad unit and The NCC of glial cell differentiation potential, but from the cell of different clone's differentiation, GFAP or Tuj1 is positive The percentage ratio of cell is different, and some clones tend to derivative glial cell (GFAP positive cell), and Other clones tend to derivative neuronal cell (Tuj1 positive cell).Similar, in the present invention we Separation obtains multiple BM-NCC single cell clone, and it was found that the cell of 4 clones is divided into SC's Ratio is different.Result shows that N1 clone has the strongest ability to SC differentiation, display SC differentiation The mrna expression level of NCC mark CD44, HES1 and SOX10 of potential is higher than other at N1 BM-NCC clones (such as N4), and shows that the NCC of neuron differentiation potential indicates WNT1, ID2, p75NTR Then (such as N4) is cloned less than other BM-NCC at N1 with the mrna expression level of CXCR4.
Break up to SC for induction N1 cell, used laminin/PLL stromal surface and clear and definite one group thin Intracellular cytokine.In atomization, we do not observe the of short duration squamous epithelium presented having document to report before Like cell form [Lobsiger CS, Smith PM, Buchstaller J, Schweitzer B, Franklin RJ, Suter U,et al.SpL201:a conditionally immortalized Schwann cell precursor line that generates myelin.Glia.2001；36:31-47.][Xu Y,Xiong F,Liu L,Zhang C.Rat bone marrow stromal cells could be induced into Schwann cell precursor-like cells in vitro. Neurosci Lett.2011；488:229-33.].The difference of this form be likely due to different inductive conditions and Different cell deriveds.By contrast, the N1 cell in the present invention is being induced in atomization to SC, 0h, The gene expression pattern that 12h, 24h and 48h detect respectively is basically identical with existing document report [Ziegler L,Grigoryan S,Yang IH,Thakor NV,Goldstein RS.Efficient generation of schwann cells from human embryonic stem cell-derived neurospheres.Stem Cell Rev. 2011；7:394-403.][Liu Z,Jin YQ,Chen L,Wang Y,Yang X,Cheng J,et al.Specific marker expression and cell state of Schwann cells during culture in vitro.PLoS One. 2015；10:e0123278.][Finzsch M,Schreiner S,Kichko T,Reeh P,Tamm ER,Bosl MR,et al.Sox10is required for Schwann cell identity and progression beyond the immature Schwann cell stage.J Cell Biol.2010；189:701-12.].Absorbing, calcium sticks Even protein 19 (Cadherin 19) is as the distinctive cell sign of SCP, along with N1 cell is to the differentiation of SC Present inverted U-shaped change, at induction 12h apparently higher than 0h, but at induction 24h and 48h significantly lower than 0h. These time dependent changes can be compared to simulate dynamic process from NCC to ripe SC differentiation [Jessen KR, Mirsky R,Lloyd AC.Schwann Cells:Development and Role in Nerve Repair.Cold Spring Harb Perspect Biol.2015；7:a020487.][Woodhoo A,Sommer L.Development of the Schwann cell lineage:from the neural crest to the myelinated nerve.Glia. 2008；56:1481-90.][Takahashi M,Osumi N.Identification of a novel type II classical cadherin:rat cadherin19is expressed in the cranial ganglia and Schwann cell precursors during development.Dev Dyn.2005；232:200-8.], result shows N1 cell SCP feature may progressively weaken, and N1-d-SC such as the prolongation of induction time progressively by immaturity shape State is to maturity state transition.
Existing research report, application Laminin/PLL substrate and HRG1 beta induced skin progenitor cell (skin Precursor, SKP) after SC breaks up, use different means purification SC the most again, can be with one circle Column casing (cylinder) selects SC colony, carry out digesting and collect Secondary Culture after cell [Biernaskie JA, McKenzie IA,Toma JG,Miller FD.Isolation of skin-derived precursors(SKPs)and differentiation and enrichment of their Schwann cell progeny.Nat Protoc. 2006；1:2803-12.], it is also possible to carry out the streaming living cells sorting based on p75NTR immunofluorescence dyeing [Khuong HT,Kumar R,Senjaya F,Grochmal J,Ivanovic A,Shakhbazau A,et al. Skin derived precursor Schwann cells improve behavioral recovery for acute and delayed nerve repair.Exp Neurol.2014；254:168-79.].By contrast, the invention provides one Planting simple efficient method, use the method, 4 clone's subgroups in the present invention can have 40% to 95% Cell is induced to differentiate into SC, and wherein N1 clone has 95% more than, and induction N1 cell is divided into after SC not Need follow-up cell sorting step.Many existing research report adult stem cells can be induced to differentiate into SC, Such as from bone marrow [Caddick J, Kingham PJ, Gardiner NJ, Wiberg M, Terenghi G. Phenotypic and functional characteristics of mesenchymal stem cells differentiated along a Schwann cell lineage.Glia.2006；54:840-9.][Park HW,Lim MJ,Jung H,Lee SP,Paik KS,Chang MS.Human mesenchymal stem cell-derived Schwann cell-like cells exhibit neurotrophic effects,via distinct growth factor production,in a model of spinal cord injury.Glia.2010；58:1118-32.] or Cord blood [Peng J, Wang Y, Zhang L, Zhao B,Zhao Z,Chen J,et al.Human umbilical cord Wharton's jelly-derived mesenchymal stem cells differentiate into a Schwann-cell phenotype and promote neurite outgrowth in vitro.Brain Res Bull.2011；84:235-43.][Lee JH,Chung WH, Kang EH,Chung DJ,Choi CB,Chang HS,et al.Schwann cell-like remyelination following transplantation of human umbilical cord blood(hUCB)-derived mesenchymal stem cells in dogs with acute spinal cord injury.J Neurol Sci. 2011；300:86-96.] MSC [Pan Y, the Cai S.Current state of the development of that originates mesenchymal stem cells into clinically applicable Schwann cell transplants.Mol Cell Biochem.2012；368:127-35.], adipose-derived stem cell [Kingham PJ, Kalbermatten DF, Mahay D,Armstrong SJ,Wiberg M,Terenghi G.Adipose-derived stem cells differentiate into a Schwann cell phenotype and promote neurite outgrowth in vitro. Exp Neurol.2007；207:267-74.][Kaewkhaw R,Scutt AM,Haycock JW.Anatomical site influences the differentiation of adipose-derived stem cells for Schwann-cell phenotype and function.Glia.2011；59:734-49.], the NCSC of epidermis [Sieber-Blum M, Grim M,Hu YF,Szeder V.Pluripotent neural crest stem cells in the adult hair follicle. Dev Dyn.2004；231:258-69.] and dental pulp NCSC [Al-Zer H, Apel C, Heiland M, Friedrich RE,Jung O,Kroeger N,et al.Enrichment and Schwann Cell Differentiation of Neural Crest-derived Dental Pulp Stem Cells.In Vivo.2015；29:319-26.] etc., but this The ratio being divided into SC in a little researchs is only below 40% or does not reports differentiation rate.This programme not only can obtain more Many SC, differentiation speed is also fast than (>=1 week) that existing document is reported, it is thus achieved that so break up possibility efficiently It is derived from NCP clone rather than NCSC or MSC owing to derivative SC.Special one is mentioned that have The feature N1 clone of SCP is beneficial to set up an external SC differentiated system, and, this system includes Immaturity SC is to the differentiation becoming myelin SC.
Becoming myelin is the mark that SC function is ripe, becomes the SC of myelin to be different from other SC hypotype, the most not Ripe SC with non-become myelin SC.In the present invention, N1 cell and DRG neuron co-culture system are formed One-tenth myelin SC, and, sheath structure positive for MBP and MAG confirms that it has typical SC function. Result shows, the one-tenth myelin SC that SCP directed differentiation is, also needs to solvable in addition to needs paracrine signal Sex factor, and also it is helpful to [Joseph NM, Mukouyama YS, Mosher with the intimate contact of aixs cylinder JT,Jaegle M,Crone SA,Dormand EL,et al.Neural crest stem cells undergo multilineage differentiation in developing peripheral nerves to generate endoneurial fibroblasts in addition to Schwann cells.Development.2004；131:5599-612.].
The BM-NCC of the immortality selected in the present invention clones N1, has the identity characteristic of SCP, it is possible to The most frozen and recovery, therefore can be cell therapy provide a unlimited cell derived, especially can promote god The regeneration of warp.Additionally, these clone cells also can avoid in primary cell preparation cell between different batches poor Different harmful effect.Existing scholar's report takes out SC from rat embryo sciatic nerve, then uses reverse transcription disease The EGFR/neu fusion protein of poison coding establishes the SCP cell line of the immortality that a condition relies on (SpL201)[Lobsiger CS,Smith PM,Buchstaller J,Schweitzer B,Franklin RJ,Suter U,et al.SpL201:a conditionally immortalized Schwann cell precursor line that generates myelin.Glia.2001；36:31-47.].Present invention BM-NCC after adult rat migrates separates Obtain BM-NCC cloned cell line, overcome ethical issues, and avoid nerve injury and genetic modification, Also it is not required to as SpL201, rely on the EGF in environment just can be survived.It addition, also scholar's application Different regulatory factors, from mouse skin establish immortality birth after NCSC cell line [Sviderskaya EV, Easty DJ,Lawrence MA,Sanchez DP,Negulyaev YA,Patel RH,et al.Functional neurons and melanocytes induced from immortal lines of postnatal neural crest-like stem cells.FASEB J.2009；23:3179-92.], however it is necessary that regulation and control Wnt, shh and TGF signal beta leads to Road, epidermis NCSC can be as a kind of cell derived [Sakaue M, Sieber-Blum M.Human of SC epidermal neural crest stem cells as a source of Schwann cells.Development. 2015；142:3188-97.].By contrast, present invention report for the first time can obtain and build from adult rat bone marrow The NCC system of vertical immortality, and this NCC system can as biology relevant cell derived offer SCP with The most derivative SC.
In the present invention, N1-CM can repair the DRG neuron of OGD damage, internal transplanting N1 in vitro Sciatic nerve crush can be repaired, show N1 can play cell based on therapeutical effect.Contained by N1-CM Some neurotrophic factors promote survival and the aixs cylinder of the DRG neuron of OGD damage in cell is cultivated Growth, and after N1 cell infusion enters the rat sciatic nerve crushed, the repair risen is better than N1-d-SC.For preferably application cell transplantation treatment nerve injury, the cell of injection survival feelings in vivo Condition is the importance needing to consider, is additionally also required to consider to inject precellular differentiation state, thus Regeneration microenvironment [Walsh S, the Midha R.Practical considerations building together good with surrounding tissue concerning the use of stem cells for peripheral nerve repair.Neurosurg Focus. 2009；26:E2.].
The BM-NCC clone N1 of the present invention is expected to be applied to the rush treatment of nerve regeneration based on cell.
Accompanying drawing explanation
Fig. 1 is aspect graph (A) and the immunocytochemical stain figure (B) of BMSC and derivative Mesensphere.
Fig. 2 is E/Fr-BMSC and flow cytometer (FCM) analysis chart of derivative Mesensphere.
Fig. 3 is BM-NCC single cell clone aspect graph (A) and growth curve (B) and growth curve chart (C).
Fig. 4 is MSC and the NCC mark mrna expression level at N1, N4 and E/Fr-BMSC: express water Putting down and indicate (A) at N1 and N4 higher than the NCC of E/Fr-BMSC, expression is less than at N1 and N4 MSC mark (B) of E/Fr-BMSC, expression is at N1, N4 and E/Fr-BMSC no significant difference Mark (C).
Fig. 5 is that BM-NCC clone clones N1 to SC differentiation 12h and 48h to the differentiation of SC: BM-NCC Aspect graph (A and B), the ratio of each BM-NCC clonal derivation SC (S100 β positive) compares (C), The immunocytochemical stain figure (D) of the SC that BM-NCC clone N1 is derivative.
Fig. 6 is that SC and NCC mark derives the mRNA of SC induction differentiation 0h, 12h, 24h and 48h at N1 Expression change (A) of expression: SC mark, expression change (B) of NCC mark, SCP mark Expressing change (C), SC and NCC has expression change (D) of mark.
Fig. 7 is the detection figure that N1 cell and DRG neuron co culture system in vitro become myelin: co-culture 0d, 1d, 14d With aspect graph and the immunocytochemical stain figure (A) of 21d, co-culture the myelin aspect graph (B) of 28d, Co-culture myelin immunocytochemical stain figure (C) of 28d, become myelin mark co-culturing 7d's Yu 28d Mrna expression level (D).
Fig. 8 is the DRG neuron β Tubulin III immunocytochemistry dye of N1-CM external reparation OGD damage Chromatic graph.
Fig. 9 is the rat sciatic nerve form and function detection figure that cell infusion reparation crushes: after cell infusion 3 weeks Each experimental group sciatic nerve function index (SFI) of interior different time points is compared (A), and cell infusion is after 3 weeks Each experimental group compound muscle action potential (CMAP) wave amplitude ratio relatively (B), respectively tested after 3 weeks by cell infusion The immunohistochemical staining figure (C) of group regenerating nerve.
Preservation information: the cell line of the present invention, named rat marrow source neural crest CFU-GM N1, preservation In China typical culture collection center (being called for short CCTCC), address: Wuhan, China university, preservation day April 16 2016 phase, deposit number CCTCC NO:C201647.
Rat marrow source neural crest CFU-GM N1 in CCTCC preservation is the 50th and 51 generation cells.
Detailed description of the invention
Describe the present invention below in conjunction with embodiment and accompanying drawing.But it is right that the following example should not regarded as The restriction of the scope of the invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
The cultivation of embodiment 1. bone marrow neural crest cell (BM-NCC) and qualification
The cultivation of BM-NCC, applies the following two step CMC model programs repeated to cultivate and obtains BM-NCC:
The first step, after adult Wistar rat (8 week old) is condemned to death, flushes out bone from tibia and femur immediately Marrow, by medullary cell inoculated and cultured in the IMDM culture fluid containing 10%FBS, utilizes BMSC to attach and moulds The characteristic of material culture dish growth, repeatedly changes liquid and removes floating hematopoietic stem cell, and separation and Culture goes out Primary rat BMSC；
Second step, with containing 20ng/ml EGF, 20ng/ml FGF2,1%N2 (v/v), 2%B27 (v/v)), 2mM glutamine, 100U/ml penicillin, the DMEM/F12 (v/v, 1:1) of 0.1mg/ml streptomycin Balling-up culture fluid, carries out balling-up cultivation to primary BMSC, and inoculating cell density is 2～3 × 105Individual/ml, shape Become primary cell ball (Sphere).Balling-up culture fluid i.e. the culture fluid of NCC.
Then, repeat the first step, i.e. collect the primary cell ball inoculated and cultured again of suspension in containing 10%FBS IMDM culture fluid, utilize BMSC attach plastic culture dish growth characteristic, it is thus achieved that E/Fr-BMSC；
Repeat second step, with DMEM/F12 balling-up culture fluid, E/Fr-BMSC subgroup carried out balling-up cultivation, The cell ball formed is also referred to as mesenchymal cell ball (Mesensphere).
Cell or cell ball phase contrast microscope in above incubation are observed and gather picture；Thin with immunity Born of the same parents' chemistry (ICC) colouring method detection MSC and NCC marker protein；With flow cytometer (FCM) Analyze the expression of cell sign albumen.
The fibroblast-like cells form of primary BMSC and E/Fr-BMSC display adherent growth；BMSC derives Mesensphere suspension balling-up growth (Figure 1A) derivative for Sphere and E/Fr-BMSC.
Separate the process obtaining Mesensphere: (1) separates primary BMSC cell from primary BMSC (a1) Ball (a2)；(2) from the derivative E/Fr-BMSC (a3) of primary BMSC cell ball (a2)；(3) from E/Fr-BMSC (a3) Separate Mesensphere (a4).ICC coloration result display E/Fr-BMSC expresses MSC marker protein CD90, And Mesensphere expresses NCC marker protein CD29, CD133 and p75NTR.E/Fr-BMSC and Mesensphere all expresses stem-cell marker albumen Vimentin and Nestin (Figure 1B).
FCM analysis result shows, E/Fr-BMSC high expressed MSC marker protein CD90 and CD73, and Mesensphere high expressed NCC marker protein CD133.The highest table of E/Fr-BMSC and Mesensphere Reach CD44, CD29 and Nestin (Fig. 2).
The separation of embodiment 2.BM-NCC clone and propagation and cell sign detection
Application limiting dilution assay cultivation acquisition BM-NCC clone [Glejzer A, Laudet E, Leprince P, Hennuy B,Poulet C,Shakhova O,et al.Wnt1and BMP2:two factors recruiting multipotent neural crest progenitors isolated from adult bone marrow.Cell Mol Life Sci.2011；68:2101-14.].Cell ball Secondary Culture is to after more than 4th generation, with accutase enzyme by cell Ball digestion is individual cells suspension, by the density in 0.7/hole, cell is inoculated into 96 porocyte culture plates and carries out (every porocyte number is 0,1 or 2 etc. at random) is cultivated in unicellular balling-up, and half amount changes liquid 2 times weekly. Microscope observes every porocyte, when the cell number contained by the individual cells ball that individual cells propagation is formed reaches When more than 100, just being digested by this cell ball with accutase enzyme is single cell suspension, is further continued for passing The balling-up in generation is cultivated.Single cell suspension is with≤5 × 104The cell density inoculation of individual/ml, every 7～14d pass on 1 time, Or pass on as required.
Obtained cell clone subgroup routine passage, cell clone N1 therein, N3, N4 and N13 length Phase breeds, and for drawing the Long-term Proliferation curve of above-mentioned 4 cell clonies, each cell clone is in every generation (P) accumulative total cellular score below equation calculates and obtains: Cn=Cn-1×Tn/Sn, CnIn=the n-th generation, is accumulative thin Born of the same parents' sum, Cn-1In=the (n-1)th generation, added up total cellular score, TnIn=the n-th generation, added up total cellular score, Sn=the n-th pickup kind Cell number.
For detection vitro growth rates, N1 and the N4 clone cell being passaged to P31, P32 and P33 is divided Not being seeded in the 2ml culture fluid of 6 well culture plates, inoculum density is 1 × 104Individual/ml.Incubation time every 24h, collects cell 1 time and also counts total cellular score, co-culture 10d, with 10 time points countings average the most carefully Born of the same parents' number draws out growth curve.
Result shows, it is thus achieved that BM-NCC single cell clone subgroup N1 of 4 Long-term Proliferations, N3, N4 and N13 (Fig. 3 A), and continuous passage at least 8 generation in vitro, N1 and N4 passes on and reaches 16 months more than 37 generations Above (Fig. 3 B).Additionally, the 10d growth curve of N1 and the N4 cell in P31, P32 and P33 generation (figure 3C) show that single cell clone N1 and N4 also remains in that good self renewal passing on 30 generations more than Ability.
PCR (qRT-PCR) result shows significant difference: NCC mark at N1 and N4 the most in real time Mrna expression level obviously higher than the mrna expression level at E/Fr-BMSCs, including Nestin, Musashi1、CD133、TFAP2α、HNK1、CD49d、WNT1、ID2、p75NTR、CD44、 HES1 and SOX10 (Fig. 4 A)；But, MSC mark is equal in the mrna expression level of N1 and N4 Significantly lower than in the mrna expression level of E/Fr-BMSC, including CD90, CD73, Vimentin, N-Cadherin and CXCR4 (Fig. 4 B).Additionally, CD29, SOX2 and Snail are N1's or N4 Mrna expression level is similar to the mrna expression level (Fig. 4 C) at E/Fr-BMSCs.Experiment knot Fruit indicates N1 and N4 cell clone and maintains BM-NCC identity.
Embodiment 3.BM-NCC is to the Induction of committed differentiation of schwann's cell (SC)
Apply specific differentiation liquid induction BM-NCC monoclonal cell (N1, N3, N4 and N13) to SC Differentiation.
Separate each cell clone of obtaining P5～P7 for time be separated into individual cells, and be inoculated into On PLL/laminin coated roundlet slide, with containing 5%FBS, 35ng/ml tRA, 5 μMs of forskolin, 200ng/ml HRG-1 β, 10ng/ml FGF2, the DMEM/F12 of 10ng/ml PDGF-AA and 1%N2 (v/v, 1:1) culture fluid (i.e. SC Induction of committed differentiation liquid) hatches cultivation, and every 3d changes liquid 1 time.
N1 cell is after induction differentiation 12h, and adherent cell starts elongation, and cellular morphology becomes spinning from circle Capitate (Fig. 5 A)；After 48h, fusiform cell presents sample arrangement (Fig. 5 B) shoulder to shoulder；After 60h, cell The oldest and the most feeble and dead.The cell selecting induction 48h carries out the ICC of S100 β and dyes to confirm BM-NCC Monoclonal cell is to the differentiation of SC, and counts the cell percentages of S100 β positive expression, compare N1, N3, These 4 monoclonal differentiation efficiency of BM-NCC of N4 and N13, result shows significant difference, N1 gram Grand directed differentiation is that the ratio of SC positive for S100 β is apparently higher than other 3 BM-NCC clone (Fig. 5 C). Additionally, ICC coloration result also show N1-d-SC coexpression GFAP, CNPase positive for S100 β and P75NTR (Fig. 5 D).
QRT-PCR analyzes not isogeneous induction divergaence time point (0h, 12h, 24h and 48h) in 48h, SC Change in the mrna expression level of N1-d-SC with NCC mark.Result shows significant difference: SC Mark, expresses water including S100 β, GFAP, CNPase, O4 and ErbB3 along with the prolongation of induction time Put down and be gradually increased (Fig. 6 A)；And NCC mark, including CD44, CD29, CD49d, CXCR4, WNT1, Nestin, Musashi1, CD133, HNK1, SOX2, ID2 and HES1, then along with induction The prolongation mrna expression level of time is gradually lowered (Fig. 6 B)；Additionally, SCP mark Cadherin19 exists The mrna expression level of induction 12h significantly increases than at 0h, but brighter than at 0h at induction 24h and 48h Aobvious reduction, it is notable that be significantly higher than in induction 24h and 48h (Fig. 6 C) at induction 12h；It addition, SOX10 and p75NTR is as the total mark of SC and NCC, and p75NTR is along with the prolongation of induction time Mrna expression level is gradually increased, and SOX10 is then that mrna expression level is the most notable after induction 12h to be increased High and remain that high level is to 48h (Fig. 6 D).
The cultivation of embodiment 4.DRG neuron and DRG neuron form myelin with N1 co-culture of cells
(1) original cuiture of DRG neuron [Shea GK, Tsui AY, Chan YS, Shum as described in existing document DK.Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.Exp Neurol.2010；224:448-58.][Gu Y,Wang J,Ding F,Hu N,Wang Y,Gu X.Neurotrophic actions of bone marrow stromal cells on primary culture of dorsal root ganglion tissues and neurons.J Mol Neurosci. 2010；40:332-41.].
DRG is got at fetal rat (embryo 14.5～15.5) or both sides, neonate rat spinal column back vertebral foramen, After application 3mg/ml collagenase and 0.25% pancreatin digest 30 minutes respectively successively, inhaling and abandon enzyme liquid, addition contains The DMEM culture fluid of 5%FBS, machinery piping and druming is to obtain single cell suspension gently, then application differential patch Wall approach removes adherent neurogliocyte faster and fibroblast, reapplies containing anti-mitosis medicine The neuronal cultured solution of 10 μMs of fluorodeoxyuridines and 10 μMs of uridnine processes, the growth of suppression non-neuronal cell To be further purified neuron.Cell is with 5 × 104Individual/cm2Density be inoculated in the coated culture dish of PLL, The 24 every holes of orifice plate about 1 × 105Individual neuron, neuronal cultured solution is containing 1%B27 (v/v), 50ng/ml NGF With the Neurobasal culture fluid of the glutamine of 2mM, every 2～3d change liquid 1 time, at least cultivate 7d.
When cell forms the neurite network of densification, fetal rat DRG neuron is used for into myelin experiment. Postnatal rat DRG neuron is used for neuronal damage model experiment, sees embodiment 5.
(2) DRG neuron forms myelin with N1 co-culture of cells
The experiment of external one-tenth myelin carries out [Shea GK, Tsui AY, Chan YS, Shum with reference to existing document report DK.Bone marrow-derived Schwann cells achieve fate commitment--a prerequisite for remyelination therapy.Exp Neurol.2010；224:448-58.].
After N1 cell being inoculated in the coated culture dish of PLL/laminin cultivation 6h with NCC culture fluid, Being changed to SC directed differentiation liquid and hatch 12h, the N1 cell after induction is divided into the short shuttle of stretching, extension by round cell Shape cell, is inoculated into the DRG neuron neurite formation in the above-mentioned 24 every holes of orifice plate after being collected by N1 cell In network, every hole inoculation about 5 × 104Individual N1 cell.The condition of co-culturing is: without glutamine DMEM/F12 and Neurobasal culture medium (1:1, v/v), add whole SC DIFs and Whole DRG Neuronal Growth Factor (seeing above), every 2～3d half amounts change liquid 1 time.
After co-culturing 1 week, N1 be can be observed cell-derived for spindle shape SC, and along the aixs cylinder of neuron Arrangement, then starts to add L-AA (50 μ g/ml)) and glucose (4mg/ml), remove tRA, PDGF-AA, FGF2 and HRG-1 β, to stimulate the formation of aixs cylinder myelin.Co-culture and continue to 3 weeks, Every 2～3d change liquid 1 time.
DRG neuron co-cultures with N1 cell step as described above.N1-d-SC Yu DRG is neural Unit keeps contact, gradually presents the bipolar morphology of elongation, and after co-culturing 14d and 21d, a lot of cells are opened Begin to be formed the myelin of DRG neuron, more significantly during 21d (Fig. 7 A)；After co-culturing 28d, DRG god The myelin formed through unit's aixs cylinder presents the morphosis (Fig. 7 B) of ring sample；ICC dyeing display myelin ring sample Structure coexpression becomes myelin marker protein MBP and MAG (Fig. 7 C)；QRT-PCR analyzes display further Having significant difference, MBP, MAG, P0, PMP22, PLP and Krox20 are co-culturing the mRNA of 28d Expression is apparently higher than when co-culturing 7d (Fig. 7 D).
The DRG neuron of embodiment 5.N1-CM external reparation OGD damage
With the N1 cell of Neurobasal culture medium culturing exponential phase, without somatomedin, cell is close Degree is 2 × 105Individual/ml, collects culture medium, i.e. N1-CM, is stored in-20 DEG C after 24h.
With the neonate rat DRG neuron of sugar-free Neurobasal culture medium culturing purification, condition of culture is 37 DEG C, 5%CO2Concentration and 0.1%O2Concentration, i.e. OGD damages.Change liquid with N1-CM after 8h to process, Condition of culture is 37 DEG C, 5%CO2Concentration and 21%O2Concentration, after cultivating 24h, i.e. OGD damage N1-CM repair process.
The table of the OGD damage DRG neuron β Tubulin III before and after ICC dyeing detection N1-CM reparation Reach.Result shows, compared with the DRG neuron that conventional medium processes, and the DRG god that N1-CM processes (Fig. 8) is all made moderate progress in survival and neurite-outgrowth these two aspects through unit.
Transplant N1 cell after embodiment 6. sciatic nerve crush or N1-d-SC carries out cell therapy
(1) cell transplantation operation after sciatic nerve crush
Lumbar injection combined anesthesia medicine (10mg/kg xylazine, 95mg/kg ketamine, 0.7mg/kg acetyl Promazine) with anesthetized rat；In the middle part of hind leg thigh, perform an operation otch in side, exposes left sciatic；? Sciatic nerve separates the position near-end of tibial nerve and common peroneal nerve, crushes sciatic nerve 3 times with mosquito forceps, often Secondary lasting 10 seconds, every minor tick 10 seconds, it is careful not to block the blood circulation of surrounding tissue；With 1 not Absorbable No. 9/0 silk thread is through level and carries out labelling on the muscle crush position；Crushing portion immediately after The far-end of position is given along neural parallel inserting needle and is crushed neural injection 1 × 106Individual N1 cell or N1-d-SC (induction N1 breaks up 48h)；Finally sew up operative incision.
Rat is divided into saline injection (NS) group, N1 cell transplantation group, N1-d-SC cell transplantation group, Often group 10.
(2) the functional rehabilitation situation of foot printing test test rat
Rat foot printing test, method reference is carried out the most before surgery with Post operation 8d, 12d, 16d and 20d Existing document [Yuan Y, Shen H, Yao J, Hu N, Ding F, Gu X.The protective effects of Achyranthes bidentata polypeptides in an experimental model of mouse sciatic nerve crush injury.Brain Res Bull.2010；81:25-32.].It is summarized as follows literary composition: the rat biped rear solid end palm dips in red Color stamp-pad ink, passes by and is covered with the gallery (wide 15cm, high 15cm, long 80cm) of blank sheet of paper rice paper, stay red The footprint of color.Check and choose footprint clearly, and measure normal and art parapodum toe width (normal toe Spread, NTS；Experimental toe spread, ETS), footmark length (normal print length, NPL；Experimental print length, EPL), middle toes width (normal intermediary toe Spread, NIT；Experimental intermediary toe spread, EIT), calculate every finally according to formula The sciatic nerve function index (sciatic function index, SFI) of rat, SFI represents nerve close to 0 Function is normal, and SFI close-100 represents that function of nervous system damages completely.The formula that Bain etc. are given is as follows:
SFI=-38.3 [(EPL-NPL)/NPL]+109.5 [(ETS-NTS)/NTS]+13.3 [(EIT-NIT)/NIT]-8.8
In foot printing test, SFI value representative is moved one's steps activity, is used to refer to crush rat hindlimb sense after sciatic nerve Feel and the recovery situation of motion.Operation consent SFI value is normal value 0, and Post operation SFI value drastically drops to-100, Gradually rise then as sciatic regeneration.Relatively NS process group, N1-d-SC process group and N1 is thin The result of born of the same parents' process group shows significant difference: the SFI value 20d after surgery of N1-d-SC process group is obvious Higher than NS process group；SFI value 12d, 16d and 20d after surgery of N1 cell process group apparently higher than NS process group；Additionally, the SFI value 20d after surgery of N1 cell process group is apparently higher than at N1-d-SC Reason group (Fig. 9 A).
(3) electro physiology detection compound muscle action potential (CMAP)
Within 3 weeks, carry out the electro physiology detection of CMAP after surgery.Ischium god on the left of again exposing after anesthetized rat Warp, after sciatic nerve-trunk proximal part gives electricity irritation, at homonymy gastrocnemius abdominal part record CMAP, with right The CMAP of the non-operated hind limb in side is as normal control, to inject NS group as negative control.Testing result Showing significant difference, the CMAP amplitude peak of N1 cell process group is apparently higher than N1-d-SC process Group；Meanwhile, the recovery of two kinds of cell process group electrophysiological functions is substantially better than NS process group (Fig. 9 B).
(4) sciatic immunohistochemistry (IHC) dyeing detection
Taking out the sciatic nerve of each group of regeneration after performing the operation 3 weeks, the non-surgical denervation of offside is as normal control.God Fix after specimen is carried out, preparation nerves transected face frozen section, the table of IHC detection S100 β and NF200 Reach.Result shows, rat sciatic nerve processes and regenerated nervous fibers through cell infusion after crushing, positive table The aixs cylinder reaching NF200 is surrounded by the myelin of positive expression S100 β；Further, N1 cell processes composition marrow The axon diameter of sheath and Myelin thickness are all higher than N1-d-SC process group；Meanwhile, two kinds of cell process groups are again Raw neural axon diameter and Myelin thickness are all higher than NS process group (Fig. 9 C).
Conclusion:
After migration, BM-NCC is a kind of cell derived easily obtained from myeloid tissue.Height due to BMSC Heterogeneity, separable acquisition multiple BM-NCC monoclonal cell subgroup, wherein N1 cell clone can be The most high-purity derivative SC effectively, and need not genetic modification or be further purified.Mainly entering of this research Exhibition is can to derive, from BM-NCC, the SC offspring that homogeneity is high, and according to Cadherin19 at N1 cell High expressed, N1 cell is to the differentiation capability of SC, the one-tenth myelin ability of N1 cell, and N1 cell is to being subject to Damage neural repair, it was demonstrated that N1 cell clone has SCP (or undifferentiated SCP sample state) body The inference of part.In sum, the SCP (BM-NCC clones N1) coming from BM-NCC is expected in the future should Rush treatment of nerve regeneration based on cell.
In the present invention, " BM-NCC " is from mammal, including but be not limited to rat, mice, rabbit, cat, Canis familiaris L., monkey, people.
The ultimate principle of the present invention, principal character and advantages of the present invention have more than been shown and described.The industry Skilled person will appreciate that, the present invention is not restricted to the described embodiments, in above-described embodiment and description The principle that the present invention is simply described described, the present invention is also without departing from the spirit and scope of the present invention Having various changes and modifications, these changes and improvements both fall within scope of the claimed invention.This Bright claimed scope is defined by appending claims and equivalent thereof.

Claims (8)

1. a rat marrow source neural crest CFU-GM N1, preserving number is CCTCC NO:C201647.
Rat marrow source the most according to claim 1 neural crest CFU-GM N1, it is characterised in that its tool The standby performance executing Wang Shi CFU-GM.
3. the Secondary Culture method of rat marrow source as claimed in claim 1 neural crest CFU-GM N1, it is special Levying and be, described Secondary Culture method is as follows:
By after cell recovery that preserving number is CCTCC NO:C201647, cultivate to most cells ball and increase Big and time ball center's cell starts to shade, be single cell suspension with accutase enzyme by cell ball digestion, the most carefully Born of the same parents press≤104The density of individual/ml is inoculated into Tissue Culture Plate, carries out balling-up Secondary Culture, and half amount changes liquid 2 weekly Secondary；Pass on as required.
4. rat marrow source as claimed in claim 1 neural crest CFU-GM N1 is to schwann's cell directional induction The method of differentiation, it is characterised in that described Induction of committed differentiation method is as follows:
By after cell recovery that preserving number is CCTCC NO:C201647, cultivate to exponential phase, point From for individual cells, and it is inoculated on the coated culture plate of Laminin lens/poly-D-lysine, thin with executing Wang Shi Born of the same parents' Induction of committed differentiation liquid hatches cultivation, and every 2～3d change liquid 1 time；
Described schwann's cell Induction of committed differentiation liquid is: regard containing 5% hyclone, 35ng/ml alltrans Yellow acid, 5 μMs of forskolin, 200ng/ml HRG-1 β, 10ng/ml FGF2,10ng/ml PDGF-AA and DMEM/F12 (v/v, the 1:1) culture fluid of 1%N2.
5. rat marrow source as claimed in claim 1 neural crest CFU-GM N1 promotes drug for nerve regeneration in preparation Application in thing.
Rat marrow source the most according to claim 5 neural crest CFU-GM N1 promotes neuranagenesis in preparation Application in medicine, it is characterised in that described application is rat marrow source neural crest CFU-GM N1 to be made For promoting the cell derived of neuranagenesis.
Rat marrow source the most according to claim 5 neural crest CFU-GM N1 promotes neuranagenesis in preparation Application in medicine, it is characterised in that described application is the treatment based on cell, the mode of employing Damaged tissues, intravenous injection or for preparing tissue engineering nerve graft row implant region is entered for local injection.
Rat marrow source the most according to claim 5 neural crest CFU-GM N1 promotes neuranagenesis in preparation Application in medicine, it is characterised in that described medicine is cell suspension, the conditioned medium of cell, thin The vesicle extract of the factor of intracrine, the vesicle of emiocytosis or emiocytosis.
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Cited By (2)

* Cited by examiner, † Cited by third party
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CN108624560A (en) * 2018-06-01 2018-10-09 南京艾尔普再生医学科技有限公司 A kind of preparation method of differential medium and oligodendrocyte precursor cells
CN117448267A (en) * 2023-12-22 2024-01-26 上海元戊医学技术有限公司 Mesenchymal stem cell construction method and application for osteoarthritis medicine

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108624560A (en) * 2018-06-01 2018-10-09 南京艾尔普再生医学科技有限公司 A kind of preparation method of differential medium and oligodendrocyte precursor cells
CN108624560B (en) * 2018-06-01 2022-04-08 南京艾尔普再生医学科技有限公司 Differentiation culture medium and preparation method of oligodendrocyte precursor cells
CN117448267A (en) * 2023-12-22 2024-01-26 上海元戊医学技术有限公司 Mesenchymal stem cell construction method and application for osteoarthritis medicine

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