CN101451123A - Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte - Google Patents
Method for inducing human mesenchymal stem cells differentiation to oligoden drocyte Download PDFInfo
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- CN101451123A CN101451123A CNA2007101958446A CN200710195844A CN101451123A CN 101451123 A CN101451123 A CN 101451123A CN A2007101958446 A CNA2007101958446 A CN A2007101958446A CN 200710195844 A CN200710195844 A CN 200710195844A CN 101451123 A CN101451123 A CN 101451123A
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Abstract
The invention discloses a method for inducing differentiation from human mesenchymal stem cells to oligodendrocyte. The invention aims to provide a method for obtaining a large quantity of oligodendrocyte and using the oligodendrocyte to treat diseases such as myelin sheath damage or demyelination. The method is to obtain a large quantity of the autogenous human mesenchymal stem cells by a simple platy adhesion method, and to use adhesion molecules F3/Contactin to induce differentiation from the human mesenchymal stem cells to the oligodendrocyte. The method adopts an easy, simple and convenient means to perform in vitro regulation on differentiation from the human mesenchymal stem cells to the oligodendrocyte, and achieves the aims of acquisition of the autogenous oligodendrocyte, preparation of a large quantity of the autogenous oligodendrocyte, and low cost. The method realizes the aim of applying the oligodendrocyte from the autogenous human mesenchymal stem cells to treat the demyelination disease, and has better economic benefit and social benefit.
Description
Technical field
The present invention relates to a kind of method of regulating the cell induction differentiation, relate in particular to a kind of novel method of regulating the human marrow mesenchymal stem cell of vitro culture to the oligodendrocyte differentiation.
Background technology
Oligodendrocyte is that the myelin of nervus centralis forms cell, it holds the nerve fiber aixs cylinder and has formed insulating myelin sheath, thereby the formation of vertebrates central nervous system myelin plays crucial effect for the transmission of the growth of neuron axon, growth, nerve signal.When the myelin damage, the myelin that no matter is structural integrity is damaged-demyelination (demyelination), as wound, multiple sclerosis (multiplesclerosis, MS), still the myelin due to the oligodendrocyte heteroplasia forms bad (dysmyelination), as congenital cerebellum disease etc., all can cause serious central nervous system pathological change.The oligodendrocyte or the progenitor cell shortage that form cell as myelin are one of reasons of Remyelination failure.Thereby oligodendrocyte is transplanted to the damage of nervus centralis such as the treatment of multiple sclerosis and demyelination new approaches is provided.
The method that obtains oligodendrocyte or oligodendroglia precursor cell at present has: 1) can still be difficult to obtain enough donorcellses, and have the allosome rejection from embryo or separation of adult cerebral tissue and vitro culture acquisition.2) also can have stem cell such as embryonic stem cell, the neural stem cell etc. of versatility from broad variety, induce differentiation to obtain required cell type by vitro culture, but subject matter is the oligodendrocyte in embryonic stem cell or neural stem cell source, can not autotransplantation, there is the allosome rejection; 3) from the oligodendrocyte in body MSC source, it is to separate with paramagnetic particle method that traditional method is separated the MSC cell, costs an arm and a leg, and the sepn process complexity is easy to pollute, and growth factor-induced differentiation reaction has tumorigenicity; Still there is not at present simple and reliable method optionally to be divided into less prominent precursor cell or oligodendrocyte from human marrow mesenchymal stem cell (hBMSCs), therefore explore a kind of effective means, directional induction hBMSC breaks up to oligodendrocyte, is the prerequisite of cellular transplantation therapy nerve injury such as multiple sclerosis and demyelination.
Mesenchymal stem cells MSCs has characteristics such as the separation of being easy to, amplification, manipulation in vitro be easy, at the ideal seed cell of can yet be regarded as aspect the cell replacement treatment.Animal data shows that the stem cell of derived from bone marrow after peripheral vein input or intracerebroventricular transplanting, has the potential of treatment diffusivity central nervous system pathological change.Experiment in vitro proves that also the stem cell of derived from bone marrow can be expressed neurone, astroglia cell and schwann cell mark by inducing, but less expression oligodendrocyte mark.F3/contactin contactin member, F3/contactin contactin member, its film outskirt all has 6 Ig structural domains and 4 fibronectin III to repeat segment, is anchored on the cytolemma through the glycosyl-phosphatidyl inositol combination.F3 extensively distributes in brain, and is closely related with the growth of oligodendrocyte.The present invention obtains hMSC with simple method, and breaks up to oligodendrocyte with adhesion molecule F3/Contactin inducing human mesenchymal stem cells, thereby has obtained a large amount of oligodendrocytes.
Summary of the invention
The purpose of this invention is to provide a kind of method that obtains a large amount of oligodendrocytes, be used for the treatment of diseases such as myelin damage or demyelination.Adopt following technical scheme:
1) utilize simple method to obtain human marrow mesenchymal stem cell, the present invention by flat board stick method can obtain the higher CD90 (+) of purity from the body human marrow mesenchymal stem cell.
2) invented the method for a kind of human marrow mesenchymal stem cell to the oligodendrocyte differentiation.Human marrow mesenchymal stem cell is through after inducing (β-ME and RA) and adhesion molecule F3/Contactin in advance and inducing, and the per-cent of NG2 (oligodendrocyte precursor cells mark) and O4 (oligodendrocyte mark) positive cell reaches 41.5%.
Content of the present invention is unexposed to be delivered, and those skilled in the art can not obtain method of inducing differentiation of the present invention according to existing technology deduction as not spending creative work at all.
Description of drawings:
The flow cytometry figure of Fig. 1 hBMSCs CD90 positive cell
The flow cytometry figure of Fig. 2 MFS group O4 positive cell
The flow cytometry figure of Fig. 3 F3 group O4 positive cell
Fig. 4 NG2 and O4 positive cell statistic analysis result histogram
Embodiment
Embodiment 1 human marrow mesenchymal stem cell vitro culture and amplification
HBMSCs (〉=70%) derives from Cambrex company, and collection of specimens was from 38 years old young donor of healthy male.Former generation hBMSCs is inoculated in and contains 10% foetal calf serum DMEM nutrient solution, and 37 ℃, 5% CO2 incubator are cultivated.With PBS flushing twice, remove not adherent cell, and more renew nutrient solution behind the 48h.Treat that attached cell 90% merges, use 0.25% tryptic digestion, by the 1:3 continuation amplification cultivation that goes down to posterity.
Cultivate the morphological character of the hBMSCs that obtains with this method: when cell had just been cultivated, a large amount of round cells of culture dish inner suspension can be removed these not adherent hemocytes by changing liquid, and attached cell then is hBMSCs.Just the hBMSCs of inoculation is rounded, and volume is bigger, and cell space is bright, and refractivity is strong; Cell begins adherent stretching, extension behind the inoculation 24h, stretches out 2-3 long projections, and cell space is broad, form is various, and fusiformis, fusiform are arranged, polygon, and nucleus is circular or oval, and 1-3 kernels are arranged; Attached cell quantity increases gradually behind the 3d, is the growth of clone's property, and the cellular form great majority change spindle shape into; Cell is bred rapidly behind the 5d, and merges mutually in flakes, and arranging closely has certain directivity, is the inoblast sample and distributes.
The characteristic of embodiment 2 usefulness flow cytometry analysis mesenchymal stem cells MSCs
When cell grew to 90% fusion, collecting cell carried out flow cytometer and detects, with 0.25% pancreatin that contains EDTA for unicellular, is given a baby a bath on the third day after its birth hMSC digestion adherent in the plastic ware time with PBS, counts and is adjusted to suitable concentration (1 * 106/ml), alcohol fixation is given a baby a bath on the third day after its birth time with PBS; Handle with TritonX-100 and hydrogen peroxide; Add normal sheep serum room temperature sealing 30 minutes; Add CD34 respectively, CD45, or CD90 antibody room temperature is after 2 hours; Add the FITC fluorescence antibody and hatch jointly, use the positive cell number of facs analysis CD90 then.
The result shows: adherent cell carries out flow cytometry when merging near 90%.The result shows that CD90 (+) cell reaches 83.8% to 97.7%, and average out to 94.78+4.52% (accompanying drawing 1) also has 4.7% cell expressing hemopoietic stem cell surface sign CD34 approximately, and 5.8% cell expressing leukocyte surface sign CD45 is arranged in addition.Prove absolutely that institute's cultured cells really is hBMSCs, and can obtain higher CD90 (+) hBMSCs of purity through adherent partition method.
Embodiment 3 human marrow mesenchymal stem cells external evoked to oligodendrocyte differentiation and phenotypic evaluation with reference to (Dezawa M such as De zawa, Takahashi I, Esaki M, et al.Sciatic nerve regenerationin rats induced by transplantation of in vitro differentiated bone-marrow stromalcells.Eur J Neurosci, 2001, the 14:1771-1776) method of chemical induction hBMSCs: (β-ME) DMEM induces 24h in advance with 1mM β-mercaptoethanol near the cell that merges; Induce 3d with the 10% foetal calf serum DMEM induced liquid that contains 35ng/ml vitamin A acid (RA) again; Subsequently cell is divided into MFS group (positive controls) and F3 group and continues to cultivate 3d, MFS group cell cultures is in containing 10% foetal calf serum, 5mM forskolin, 10ng/ml Basic Fibroblast Growth Factor (bFGF), 5ng/ml PDGF (PDGF), 200ng/mLheregulin (HRG) DMEM substratum; F3 group cell cultures is in the DMEM substratum that contains 10% foetal calf serum, 20nM F3.
Induce characteristic with the flow cytometry analysis mesenchymal stem cells MSCs into oligodendrocyte.With 0.25% pancreatin that contains EDTA with hMSC digestion adherent in the plastic ware for unicellular, give a baby a bath on the third day after its birth time with PBS, count and be adjusted to suitable concentration (1 * 106/ml), alcohol fixation is given a baby a bath on the third day after its birth time with PBS; Handle with TritonX-100 and hydrogen peroxide; Add normal sheep serum room temperature sealing 30 minutes; Add respectively multi-clone rabbit NG2 antibody (1:200, Chemicon, Temecula, CA) and monoclonal mouse O4 antibody (1:200, Chemicon, Temecula, CA) room temperature is after 2 hours; Add two of the IgG of the anti-rabbit of PE bonded respectively and the anti-mouse of FITC bonded and anti-ly hatch jointly, PBS washing 2 times, each centrifugal 5min of 1000rpm abandons supernatant.Detect oligodendroglia or precursor cell with flow cytometry analysis then.
The flow cytometry result shows: after process β-ME and RA induce step by step, again with after using MFS (positive controls) and F3 group to continue to cultivate 3d respectively, find that F3 group and MFS group O4 positive cell significantly increase (F3 group 31.55 ± 5.8%, MFS group 32.61 ± 6.8%; Fig. 2 and 3), and the NG2 positive cell (F3 group 9.94 ± 3.3%, MFS organizes 7.75 ± 4.3%; Fig. 4), F3 group compare with the MFS group O4 and NG2 expression level no difference of science of statistics (p〉0.05; Fig. 4).After F3 and MFS processing, the tiling of cell cell space launches to form flat-section, stretches out 1-2 short and small projections, and expresses ripe oligodendrocyte mark MBP.Above result shows similar to the MFS effect, and F3 can induce and produce oligodendrocyte and get positive rate and reach 41.5%.
Claims (3)
1. an inducing human mesenchymal stem cells is characterized in that utilizing the characteristics of mesenchymal stem cells MSCs neuralward cytodifferentiation to the method that oligodendrocyte breaks up, and adopting stage by stage, the inductive method obtains oligodendrocyte.
2. according to the induction scheme of claim 1, adopting a kind of nerve adhesion molecule inducing bone mesenchymal differentiation of stem cells is O4 and NG2 male oligodendrocyte.
3. according to the induction scheme of claim 2, it is characterized in that the oligodendrocyte that obtains is used for the treatment of diseases such as myelin damage or demyelination, has avoided variant cell to transplant the immunological rejection that causes.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337246A (en) * | 2011-10-12 | 2012-02-01 | 北京弘润天源生物技术有限公司 | Development of oligodendrocyte progenitor cells prepared by differentiating mesenchymal stem cells through eleutheroside and kit, and medicine for nerve system disease |
| CN104046676A (en) * | 2013-03-13 | 2014-09-17 | 中国科学院上海生命科学研究院 | Cell for removing amyloid polypeptide and use thereof |
| 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 |
| CN110431149A (en) * | 2016-12-05 | 2019-11-08 | 埃克斯欧力提斯药物公司 | Peptide compounds are used to promote the purposes of survival, growth and cell differentiation |
-
2007
- 2007-11-30 CN CNA2007101958446A patent/CN101451123A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102337246A (en) * | 2011-10-12 | 2012-02-01 | 北京弘润天源生物技术有限公司 | Development of oligodendrocyte progenitor cells prepared by differentiating mesenchymal stem cells through eleutheroside and kit, and medicine for nerve system disease |
| CN104046676A (en) * | 2013-03-13 | 2014-09-17 | 中国科学院上海生命科学研究院 | Cell for removing amyloid polypeptide and use thereof |
| CN110431149A (en) * | 2016-12-05 | 2019-11-08 | 埃克斯欧力提斯药物公司 | Peptide compounds are used to promote the purposes of survival, growth and cell differentiation |
| CN110431149B (en) * | 2016-12-05 | 2023-08-08 | 埃克斯欧力提斯药物公司 | Use of peptide compounds for promoting survival, growth and cell differentiation |
| 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 |
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