CN101914493B - Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells - Google Patents
Inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, in particular to an inducing method for differentiating umbilical cord mesenchymal stem cells into nerve cells, which comprises the following steps of: cultivating the separated umbilical cord mesenchymal stem cells in an MSC culture medium to spread a whole culture dish; cultivating the stably-subculturing mesenchymal stem cells in the culture dish which is spread with the extracellular matrix by means of the MSC culture medium to be 70% confluence; and leading the 70% confluent mesenchymal stem cells to sequentially pass through four inducing culture mediums to cultivate, wherein the four inducing culture mediums comprise the following factors of 5-azacytidine+Pam3CSK4, bFGF+Noggin, bFGF+RA+FGF8+Wnt3a, and Bmp4+Shh+RA+NGF+BDNF. The inducing method has the benefit effects of being free of chemical substances with cytotoxicity, transgenic technology and nerve cell co-cultivation, and being high in differentiating efficiency and shorter in time consumption.
Description
Technical field
The present invention relates to biological technical field, particularly umbilical cord mesenchymal stem cells is divided into the induction method of neurocyte.
Background technology
(Mesenchymal stem cell is the mesoderm origin MSC) to mescenchymal stem cell, can carry out self, and possesses to skeletonization, cartilage and three kinds of a kind of adult stem cells that mesoderm is a cytodifferentiation potential of fat.Recent study finds that MSC outside mesoderm, can also stride germinal layer and change differentiation, as is divided into the neurocyte of ectoderm system and the liver cell and the pancreatic cell of epithelial cell and entoderm system.Because the MSC wide material sources are easy to separation and Culture, immunogenicity is lower, and the ethnics Problem that does not have embryonic stem cell and faced, and this makes its application aspect regenerative medicine possess the unexistent advantage of other stem cells.Therefore the human problem of long-term puzzlement such as the nerve injury that the nerve degenerative diseases of cns such as parkinson and exterior trauma cause also will have more real feasible solution.
In past 10 years, domestic and international many research groups have reported the external different methods that the MSC directional induction is divided into neurone, spongiocyte.Summarize, these methods roughly are divided into four kinds: chemical small molecules is induced, growth factor and neurotrophic factor mix induce, the transfection of specific gene and cultivate altogether with neurocyte.To summarize the present Research in this field below.
1. micromolecular compound is induced
Existing up to now a lot of micromolecular compounds are used for that MSC is neurophilic to induce differentiation.For example people such as the Deng dbcAMP that has a membrane permeability through use improves intracellular cAMP concentration and unites and use a kind of phosphodiesterase inhibitor IBMX to handle MSC; After six days; Have 25% cell to change typical neurocyte form into approximately, and neuronspecific enolase (NSE) and vimentin (vimentin) expression amount increase [Deng, Biochem.Biophys.Res.Commun.282; 148-152, (2001)].And people such as Woodbury have started a kind of three one-step inducing methods; Promptly induce in advance containing in the substratum of beta-mercaptoethanol earlier; Re-use the serum free medium that contains inhibitor, butyl hydroxyanisole (BHA), DMSO and induce, on the second step medium base, add valproic acid (valproic acid), forskolin (forskolin) and N2 assistant agent at last and keep cultivation.The cell expressing neuronal specificity nucleoprotein (NeuN) that these two kinds of methods obtain; In latter's method, also follow the sign nestin that neural precursor occurs after inducing 5h, to express phenomenon [Woodbury et al.J.Neurosci.Res, 61 of rising; 364-370, (2000)].The most attractive characteristic of Woodbury method is the quick startup of cellular form change and NeuN up-regulated---only occurs in and induces within the several hrs of back.Compare with the former, this method has also caused tangible necrocytosis.Can keep survival yet inducing cell is kept in the substratum at Woodbury, find that cell has lost the expression of nestin, the expression of neurotrophic factor acceptor TrkA occurred though detect after 6 days.
Yet nearest some research beginning let us are queried these small molecules, particularly as in the Woodbury method employed those.Induce the phenomenon in the process to be likely or at least in part because intracellular structural changes rather than cytodifferentiation cause.At first the change of cellular form is not accompanied by the appearance of neurite outgrowth awl, and has caused very high cell mortality.Secondly, the more important thing is that this commentaries on classics differentiation occurs in the very short time period (generally being in several hours).Is to compare the required time of cytodifferentiation with MSC to mesoderm, and this makes us a bit inconceivable.The experimental result of three research groups shows that cell changes the neurocyte form into and is actually actin filament owing to cytoskeleton and is destroyed and causes cellular contraction and cell bottom to stick together spot to lose [the Lu et al that causes; J.Neurosci.Res.77; 174-191, (2004); Neuhuber et al, J.Neurosci.Res.77,192-204, (2004); Bertani et al, J.Cell Sci.118,3925-3936, (2005)].Cellular stress such as the environmental stress of low pH, high salt or EDTA, CD, stain remover are former can to cause that equally similar cellular form changes.And this change obtains other cells such as inoblast being carried out can observe equally when BHA/DMSO handles.Though the expression of typical neural labelled protein NeuN and 200-kDa neurofilament (NF-200) has rising from the immunofluorescence result, it seems to seem unusual in intracellular location.And the lifting of this protein level can not be proved conclusively from reverse transcription RT-PCR and western blot experiment.And the cycloheximide arrestin can not stop cell after chemically induced, to be transformed into the neurocyte form after synthetic.Another point it should be noted that MSC itself just expresses the neurone of certain level and the labelled protein of spongiocyte.Human microarray technologies such as Bertani have been analyzed MSC 21000 gene transcription groups after BHA/DMSO handles 6h and 48h and have been changed, and find that the overall spectrum of transcribing of cell does not produce evident difference.This processing had not both significantly changed the neural labelled protein that MSC just expresses originally, did not change the expression situation of other neural marks yet.All these discoveries show that the appearance of neural like cell form is the acute reaction of answering that cell is faced the quick startup of poisonous chemical substance, are not by causing based on the differentiation of transcribing the group variation.Therefore this shortage is transcribed or the what is called of translation basis changes that differentiation very is worth querying.
2. growth factor/induced by neurotrophin
In the growth course of mammalian nervous system, from start to finish accurately regulated and control by different growth factors always.They are controlling heterogeneic expression or reticent through activating some important signal paths, accurately sophisticated neurone, astroglia cell or the oligodendrocyte with difference in functionality of cell guiding differentiation becoming step by step.
People such as Kondo find the member that MSC can constructive expression Shh signal path and the nuclear receptor of vitamin A acid (RA); And show that with experiment Shh and RA can pass through bFGF, forskolin and the pretreated BMSC of IBMX by co-induction; Make it to be divided into Sensory neurone [the Kondo et al. of L-glutamic acid ability; Proc.Natl.Acad.Sci.USA 102,4789-4794. (2005)].People such as Locatelli cultivate the marrow MSC of mouse in having added the neurone substratum of bFGF and EGF, obtain cerebral nerve ball appearance tissue after 5-7 days, and then induce with RA and Shh; Observe and have the neurocyte form; And the cell of expressing neurone sign tubulin III (TuJ-1) and neurofilament (NF) produces [Locatelli et al., J.Hematother.Stem Cell Res, 12; 727-734, (2003)].People such as Jiang are with multipotent adult progenitor cells (the multipotent adult progenitor cells of derived from bone marrow; MAPCs) containing bFGF respectively; FGF8, Shh, and respectively cultivate successively in the inducing culture of BDNF obtained after 7 days more sophisticated expression dopaminergic, serotonin can and the cell of GABAergic neuron sign [Jiang et al., Proc.Natl.Acad.Sci.USA 100; 11854-11860, (2003)].People's such as Tohill research shows that glial growth factor (GGF) can induce MSC to form astroglia cell appearance, expresses the cell [Tohill et al., Neurosci.Lett, 362,200-203, (2004)] of labelled proteins such as GFA and S100.
The reasonably combined use of growth factor helps MSC to break up to the accurate pointing of specific neurocyte kind, the emphasis of studying in the future beyond doubt, but still there are deficiencies such as lower, the consuming time length of differentiation efficiency in this mode at present.
3. the transfection of specific gene impels the differentiation of MSC neuralward
Noggin can antagonism bomeplasty albumen (BMP), in the progenesis process of cns, plays crucial effects.When people such as Kohyama when MSC clone KUSA/A1 changes the Noggin gene over to; Cell detachment ware wall forms the aggregate of neural ball appearance; After it is inoculated into the petridish that is covered with ornithine/FTN; These cells present the neuron form, have 50% to express the neurone mark MAP-2 that mitotic division stops attitude approximately.And when damping fluid that receives high K+ and glutaminate stimulation, cell performance Ca
2+Inflow [Kohyama et al., Differentiation, 68,235-244, (2001)].The Notch signal path has extensive and important effect at embryo development procedure, particularly aspect the decision of cell fate.Existing research shows that Notch can promote embryo's the nervous system development and the differentiation of embryonic stem cell neuralward direction.Reports such as Dezawa have changed the MSC of Notch albuminous cell intracellular domain gene over to, after under Prostatropin (bFGF), cilium neurotrophic factor (CNTF) and the forskolin effect 5 days, demonstrate the neuron cell form.These cells have-50 to-60mV tranquillization electromotive force and outward rectification potassium current.It should be noted that most that it is to be in mitotic division to stop attitude that these cells look, and express back mitotic division neurone mark MAP-2.Continuation is handled these cells with the neurotrophic factor (GDNF) in spongiocyte source, can produce the tyrosine oxidase positive cell, secretion Dopamine HCL [Dezawa et al., J.Clin.Invest, 113,1701-1710, (2004)].
Though the transfection of specific gene can improve the efficient and the ripening degree of cytodifferentiation,, make the security of its clinical use become an outstanding issue because the use of foreign vector and possible genome insert.Certainly the research of this respect is still very useful for the understanding that relates to concrete mechanism in the neurocyte growth course to us.
4. with the common cultivation of neurocyte
Neurocyte grow with process of growth in understand through modes such as autocrine or some growth factors of paracrine and iuntercellular contact and carry out self-control.Therefore, cultivate altogether with neurocyte or the conditioned medium that uses neurocyte preparation analog neuron cell microenvironment in vivo to a certain extent, promote the commentaries on classics differentiation of MSC.After people such as Sanchez-Ramos cultivate the marrow MSC of rat and rat embryo midbrain cell in the N5 substratum that adds RA, BDNF altogether; Most MSC of two weeks back discovery changes the neurocyte form into; And the expression amount of NeuN obviously improves [Sanchez-Ramos et al., Exp.Neurol, 164; 247-256, (2000)].People's such as Wislet-Gendebien research be illustrated in serum-free NSC (NSC) screening of medium to the positive rat MSC of nestin after cultivating altogether, can be divided into GFAP male astroglia cell [Wislet-Gendebien et al. with rat NSC; BMC Neurosci.5; 33, (2004)].
Yet an inconvenience of this kind method is that the cell that obtains at last can not effectively separate with co-cultured cell, and possibly have the morbific risk of potential foreign pathogens.
Summary of the invention
In order to address the above problem, the invention provides a kind of do not use have Cytotoxic chemical substance, do not use transgenic technology, do not use that neurocyte is cultivated altogether, induction method that high, the consuming time short umbilical cord mesenchymal stem cells of differentiation efficiency is divided into neurocyte.
The present invention realizes in the following manner.
Umbilical cord mesenchymal stem cells is divided into the induction method of neurocyte, it is characterized in that adopting following steps:
(1) isolating umbilical cord mesenchymal stem cells is cultured to the confluent culture ware in the MSC substratum;
(2) getting the stable mescenchymal stem cell that goes down to posterity converges with MSC culture medium culturing to 70% in being covered with the vessel of extracellular matrix;
(3) 70% mescenchymal stem cells that converge are cultivated through four kinds of inducing cultures that are added with the following factor successively,
First kind of inducing culture: add U-18496 and three acylazide lipoprotein (Pam
3CSK
4),
Second kind of inducing culture: add Prostatropin (bFGF) and Noggin,
The third inducing culture: add Prostatropin (bFGF), vitamin A acid (RA), fibroblast growth factor 8 (FGF8) and Wnt3a albumen,
The 4th kind of inducing culture: add bomeplasty albumen 4 (Bmp4), Shh albumen, vitamin A acid (RA), NGFF (NGF) and BDNF (BDNF).
Described induction method is characterized in that: the amount of adding the factor in four kinds of inducing cultures is respectively:
First kind: U-18496 1-50uM and Pam
3CSK
40.1-1ug/ml,
Second kind: bFGF 1-200ng/ml and Noggin 1-500ng/ml,
The third: bFGF 1-200ng/ml, RA 0.5-50uM, FGF8 1-100ng/ml and Wnt3a1-500ng/ml,
The 4th kind: Bmp4 1-200ng/ml, Shh 1-500ng/ml, RA 0.5-50uM, NGF1-500ng/ml and BDNF 1-500ng/ml.
Described induction method is characterized in that: the amount of adding the factor in four kinds of inducing cultures is preferably
First kind: U-18496 10uM and Pam
3CSK
40.5ug/ml,
Second kind: bFGF 25ng/ml and Noggin 100ng/ml,
The third: bFGF 25ng/ml, RA 10uM, FGF8 10ng/ml and Wnt3a 25ng/ml,
The 4th kind: Bmp4 20ng/ml, Shh 10ng/ml, RA 10uM, NGF 10ng/ml and BDNF10ng/ml.
Described induction method is characterized in that: the basic medium of four kinds of inducing cultures is the DMEM/F12 that is added with the Neurobasal of B27 assistant agent or is added with the BIT9500 assistant agent.
Described induction method is characterized in that: the MSC substratum consists of and adds foetal calf serum (FBS) 10%, Stimulina 2mM, penicillium mould and strepto-1%, Prostatropin (bFGF) and Urogastron (EGF) 2ng/ml among the low sugar DMEM.
Described induction method is characterized in that: said extracellular matrix is for containing the aqueous solution of ln 1-100ug/ml or matrigel (matrigel) 1-100ug/ml.
Described induction method is characterized in that: in four kinds of inducing cultures, respectively cultivated 3 days.
Described induction method is characterized in that: get the stable mescenchymal stem cell that goes down to posterity in being covered with the vessel of extracellular matrix with 5 * 10
3Individual cell/cm
2Density cultivate.
Described induction method is characterized in that: get the stable mescenchymal stem cell that goes down to posterity in the step (2) and in being covered with the vessel of extracellular matrix, converge with MSC culture medium culturing 24-48h to 70%.
The alleged neurocyte of the present invention comprises neurone, astroglia cell and oligodendrocyte.
Though cytodifferentiation is not at present very clear with the mechanism of changeing differentiation, say that in essence the two all relates to unlatching and silence that the different genes crowd expresses.That is to say difference in functionality or be in that to be in the active assortment of genes of expressing in its genome of cell of different states be special.So so change differentiation (a kind of cell cross germinal layer of preliminary differentiation is divided into the cell of another germinal layer again),, must find to promote this different genes crowd to express the external stimulus material of accomplishing conversion if realize smoothly.We know that expression of gene normally realizes through means such as chromatin histone modification, dna methylations in cell with silence.Wherein dna methylation is very important control methods, if there be methylating of higher degree in the promoter region of gene, will stop the combination of RNA polymerase, and gene transcription can't be carried out, thereby causes the silence of gene.Therefore, demethylation or hypomethylated gene usually can enliven expression.The U-18496 that uses in first kind of inducing culture of the present invention promptly is a kind of compound (Nikolas Zagri et al that can demethylation; Int.J.Dev.Biol.38:741-749 (1994)); The gene that it can make some originally be in silence state reopens, thereby possibly enlarge the differentiation potential of cell.Pam that simultaneously should the stage
3CSK
4There are some researches show that (Pevsner-Fischer M et al, Blood.109 (4): 1422-1432, (2007)) can promote MSC propagation and suppress its mesoderm differentiation capability to skeletonization, cartilage and fat.So just make that outwards germinal layer or entoderm carry out for the differentiation of MSC.Also receive the forward of various growth factor conduction and the accuracy controlling of negative going signal in the atomization of cell.They can activate some signal of interest paths usually, and the passing to of stimulation one-level one-level in the external world examined certain interior transcription factor, and transcription factor is then perhaps reticent according to the startup of signal deciding specific gene.Among the present invention second and third, what use in four kinds of inducing cultures all is the factor that in the neurodevelopment process, plays an important role.In Neural Differentiation and transition process, extracellular matrix also is indispensable influence factor in addition, and the extracellular matrix that the present invention selects for use is the ln or the matrigel aqueous solution.
Beneficial effect of the present invention: do not use to have Cytotoxic chemical substance, can not cause false positive reaction and apoptosis; Do not use transgenic technology, therefore can not produce and insert sudden change; Therefore do not use neurocyte to cultivate altogether yet, do not exist target cell to be difficult to separate the problem with potential foreign pathogens.And employed demethylation compound U-18496 and MSC are to the suppressor factor Pam of mesoderm differentiation
3CSK
4, make MSC open to the outside world more, more easily accept the token stimulus of its neuralward ectoderm differentiation of guiding and make correct reaction.Simultaneously, the present invention is no matter from the selection of extracellular matrix, still second and third, the use of inducible factor in four kinds of inducing cultures, all at utmost simulation neurocyte residing microenvironment in growth course.Therefore, the present invention can promote the differentiation of MSC neuralward cell more effectively.The patent No. is that 02808091.1 patent also is to use the inducible factor inducing mesenchymal stem cell to be divided into neurocyte, but its induction duration is 4-8 week, and induction duration of the present invention was merely about three weeks, had improved inductive efficient greatly, had saved the time.
Description of drawings
Fig. 1 is after umbilical cord mesenchymal stem cells is cultivated in the MSC substratum, the form under the opticmicroscope
Fig. 2 umbilical cord mesenchymal stem cells is through inducing, and a is behind the immunocytochemical stain of nerve-specific nucleoprotein (NeuN), the form under the opticmicroscope; B is behind the immunocytochemical stain of neuronspecific enolase (NSE), the form under the opticmicroscope; C is behind the immunocytochemical stain of microtubule associating albumen-2 (MAP-2), the form under the opticmicroscope; D is behind the immunocytochemical stain of astroglia cell mark GFAP (GFAP), the form under the opticmicroscope
Embodiment
Below in conjunction with specific embodiment the present invention is described further.
Embodiment 1
(1) extraction of umbilical cord mesenchymal stem cells
This embodiment uses neonatal umbilical cord that the pluripara agrees the to authorize source as mescenchymal stem cell.
Neonatal umbilical cord is washed three times in containing 1% pair of anti-saline water, remove the blood stains on surface, be cut into the segment of about 1 cm long then.With eye scissors umbilical cord is vertically cut open along the blood vessel parallel direction, 2 Umbilical artery and 1 umbilical vein blood vessel are peeled off from umbilical cord totally.Peel surperficial amnion off, the logical glue of China shreds to about 1mm partly with containing 1% pair of anti-saline water thorough washing 3 times
3Size.The tissue block that shreds is tiled in 75cm uniformly
2In the culturing bottle, room temperature is placed 5-10min, and tissue block is adjacent to.Add the 5mlMSC substratum.Be placed on 37 ℃, 5%CO
2Incubator in cultivate.The MSC medium component is: add 10%FBS (GIBCO), 2mM Stimulina (GIBCO), 1% penicillium mould and Streptomycin sulphate (GIBCO), 2ng/mlbFGF and EGF (Biovison) among the low sugar DMEM (GIBCO).Every at a distance from three days replacing one subcultures, cell grows to the cultivation of going down to posterity behind the confluent culture ware.
Referring to accompanying drawing 1 is umbilical cord mesenchymal stem cells after through the MSC culture medium culturing, the form under the opticmicroscope.
The preparation of (2) four kinds of inducing cultures
The used basic medium of inducing culture is for being added with the Neurobasal (Invitrogen) of 2%B27 (Invitrogen) assistant agent.
The factor of adding in four kinds of inducing cultures is following.
First kind: U-18496 (10uM, Sigma) and Pam
3CSK
4(0.5ug/ml, Invivogen)
Second kind: bFGF (25ng/ml, Biovison) and Noggin (100ng/ml, Biovison)
The third: bFGF (25ng/ml, Biovison), RA (10uM, Sigma), FGF8 (10ng/ml, R&D System) and Wnt3a (25ng/ml, R&D System)
The 4th kind: Bmp4 (20ng/ml, R&D System), Shh (10ng/ml, R&D System), RA (10uM, Sigma), NGF (10ng/ml, R&D System) and BDNF (10ng/ml, R&D System)
(3) umbilical cord mesenchymal stem cells induces differentiation
In advance with 24 porocyte culture plates with the aqueous solution of 5ug/ml ln (Invitrogen) shop by good, then with the stable mescenchymal stem cell that goes down to posterity in the step (1) with 5 * 10
3/ cm
2Density inoculation, in the MSC substratum, cultivate and converged to 70% in 48 hours, then the MSC substratum is replaced with first kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, then first kind of inducing culture replaced with second kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, then second kind of inducing culture replaced with the third inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, at last the third inducing culture is replaced with the 4th kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, induce end of processing, obtain the neurocyte that mescenchymal stem cell is induced to differentiate into.
(4) neurocyte of inducing differentiation to obtain being carried out immunocytochemistry detects
With the 24 orifice plate cells that obtain in the step (3), to inhale and abandon substratum, every hole adds 250ulPBS (phosphate buffered saline buffer), and room temperature leaves standstill 3min with washed cell, and repeated washing washs three times altogether; Every then hole adds the solution that 250ul contains the 4V% Paraformaldehyde 96, and room temperature is 25min fixedly; Repeat as above PBS washing 3 times; Carry out sealing treatment with serum that contains the 10% 2 anti-animal of originating and the PBS confining liquid pair cell that contains 0.3%TritonX-100 (DOWANOL), incubated at room was inhaled deblocking liquid after 30 minutes, directly dripped with one of corresponding confining liquid dilution to resist, and hatched 2h for 37 ℃.The one anti-specificity olefinic alcohol enzyme (NSE of anti human nerve unit that selects for use; Santa Cruz), anti human nerve specific nucleoprotein (NeuN; Santa Cruz), anti-people's microtubule associating albumen (MAP-2; Santa Cruz), anti-people's GFAP (GFAP, Santa Cruz) and anti-people OX-42 (Santa Cruz) albumen.Repeat PBS washing 8 times then; Drip fluorescently-labeled two and resist, NSE and NeuN are TRITC (Santa Cruz) mark, and Map-2, GFAP and OX-42 are FITC (Santa Cruz) mark, and the room temperature dark is hatched 40min, repeat PBS washing three times then; DAPI redyes nucleus 5min; The PBS flushing twice then, each 3min.Add PBS and preserve, the fluorescence inverted microscope is observed down.
Coloration result, observe the neurone mark for example nerve-specific nucleoprotein (NeuN) dyeing be positive, shown in Fig. 2 a; Neuronspecific enolase (NSE) dyeing is positive, and shown in Fig. 2 b, microtubule associating albumen-2 (MAP-2) dyeing is positive; Shown in Fig. 2 c, astroglia cell mark GFAP (GFAP) dyeing is positive, shown in Fig. 2 d; Oligodendrocyte mark OX-42 dyeing is negative, shows that the cell of acquisition does not have oligodendrocyte.Coloration result shows that the neurocyte of inducing acquisition is made up of neurone and astroglia cell.
Nearly 80% mescenchymal stem cell is divided into neurocyte, and wherein about 85% neurocyte is a neurone, and about 15% neurocyte is an astroglia cell.
Embodiment 2
(1) extraction of umbilical cord mesenchymal stem cells
This embodiment uses neonatal umbilical cord that the pluripara agrees the to authorize source as mescenchymal stem cell.
Neonatal umbilical cord is washed three times in containing 1% pair of anti-saline water, remove the blood stains on surface, be cut into the segment of about 1 cm long then.With eye scissors umbilical cord is vertically cut open along the blood vessel parallel direction, 2 Umbilical artery and 1 umbilical vein blood vessel are peeled off from umbilical cord totally.Peel surperficial amnion off, the logical glue of China shreds to about 1mm partly with containing 1% pair of anti-saline water thorough washing 3 times
3Size.The tissue block that shreds is tiled in 75cm uniformly
2In the culturing bottle, room temperature is placed 5-10min, and tissue block is adjacent to.Add the 5mlMSC substratum.Be placed on 37 ℃, 5%CO
2Incubator in cultivate.The MSC medium component is: add 10%FBS (GIBCO), 2mM Stimulina (GIBCO), 1% penicillium mould and Streptomycin sulphate (GIBCO), 2ng/mlbFGF and EGF (Biovison) among the low sugar DMEM (GIBCO).Every at a distance from three days replacing one subcultures, cell covers with the cultivation of going down to posterity behind the petridish.
The preparation of (2) four kinds of inducing cultures
The used basic medium of inducing culture is for being added with the DMEM/F12 (GIBCO) of 20%BIT 9500 (STEMCELL).
The factor of adding in four kinds of inducing cultures is following.
First kind: U-18496 (1uM, Sigma) and Pam
3CSK
4(1ug/ml, Invivogen)
Second kind: bFGF (1ng/ml, Biovison) and Noggin (500ng/ml, Biovison)
The third: bFGF (1ng/ml, Biovison), RA (0.5uM, Sigma), FGF8 (100ng/ml, R&D System) and Wnt3a (500ng/ml, R&D System)
The 4th kind: Bmp4 (1ng/ml, R&D System), Shh (1ng/ml, R&D System), RA (0.5uM, Sigma), NGF (500ng/ml, R&D System) and BDNF (500ng/ml, R&D System)
(3) umbilical cord mesenchymal stem cells induces differentiation
In advance with 24 porocyte culture plates with the aqueous solution of 1ug/ml matrigel (BD Bioscience) shop by good, then with the stable mescenchymal stem cell that goes down to posterity in the step (1) with 5 * 10
3/ cm
2Density inoculation, in the MSC substratum, cultivate and converged to 70% in 24 hours, then the MSC substratum is replaced with first kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, then first kind of inducing culture replaced with step
(2) second kind of inducing culture in, at 37 ℃, 5%CO
2Incubator in cultivated 3 days, then second kind of inducing culture replaced with the third inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, at last the third inducing culture is replaced with the 4th kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, induce end of processing, obtain the neurocyte that umbilical cord mesenchymal stem cells is divided into.
(4) neurocyte of inducing differentiation to obtain being carried out immunocytochemistry detects
With the 24 orifice plate cells that obtain in the step (3), to inhale and abandon substratum, every hole adds 250ulPBS (PBS), and room temperature leaves standstill 3min with washed cell, and repeated washing washs three times altogether; Every then hole adds the solution that 250ul contains the 4V% Paraformaldehyde 96, and room temperature is 25min fixedly; Repeat as above PBS washing 3 times; Carry out sealing treatment with serum that contains the 10% 2 anti-animal of originating and the PBS confining liquid pair cell that contains 0.3%TritonX-100 (DOWANOL), incubated at room was inhaled deblocking liquid after 30 minutes, directly dripped with one of corresponding confining liquid dilution to resist, and hatched 2h for 37 ℃.The one anti-specificity olefinic alcohol enzyme (NSE of anti human nerve unit that selects for use; Santa Cruz), anti human nerve specific nucleoprotein (NeuN; Santa Cruz), anti-people's microtubule associating albumen (MAP-2; Santa Cruz), anti-people's GFAP (GFAP, Santa Cruz) and anti-people OX-42 (Santa Cruz) albumen.Repeat PBS washing 8 times then; Drip fluorescently-labeled two and resist, NSE and NeuN are TRITC (Santa Cruz) mark, and Map-2, GFAP and OX-42 are FITC (Santa Cruz) mark, and the room temperature dark is hatched 40min, repeat PBS washing three times then; DAPI redyes nucleus 5min; The PBS flushing twice then, each 3min.Add PBS and preserve, the fluorescence inverted microscope is observed down
Coloration result; For example nerve-specific nucleoprotein (NeuN), neuronspecific enolase (NSE) and microtubule are united albumen-2 (MAP-2) and astroglia cell mark GFAP (GFAP) dyeing all is positive to observe the neurone mark; Oligodendrocyte mark OX-42 dyeing is negative, shows that the cell of acquisition does not have oligodendrocyte.Coloration result shows that the neurocyte of inducing acquisition is made up of neurone and astroglia cell.
Nearly 78% mescenchymal stem cell is divided into neurocyte, and wherein about 85% neurocyte is a neurone, and about 15% neurocyte is an astroglia cell.
Embodiment 3
(1) extraction of umbilical cord mesenchymal stem cells
Extraction step is with identical among the embodiment 1.
The preparation of (2) four kinds of inducing cultures
The used basic medium of inducing culture is for containing the Neurobasal (Invitrogen) of 2%B27 (Invitrogen) assistant agent.
The factor of adding in four kinds of inducing cultures is following.
First kind: U-18496 (50uM, Sigma) and Pam
3CSK
4(0.1ug/ml, Invivogen)
Second kind: bFGF (200ng/ml, Biovison) and Noggin (1ng/ml, Biovison)
The third: bFGF (200ng/ml, Biovison), RA (50uM, Sigma), FGF8 (1ng/ml, R&D System) and Wnt3a (1ng/ml, R&D System)
The 4th kind: Bmp4 (200ng/ml, R&D System), Shh (500ng/ml, R&D System), RA (50uM, Sigma), NGF (1ng/ml, R&D System) and BDNF (1ng/ml, R&D System)
(3) umbilical cord mesenchymal stem cells induces differentiation
In advance with 24 porocyte culture plates with the aqueous solution of 100ug/ml ln (Invitrogen) shop by good, then with the stable mescenchymal stem cell that goes down to posterity in the step (1) with 5 * 10
3/ cm
2Density inoculation, in the MSC substratum, cultivate and converged to 70% in 48 hours, then the MSC substratum is replaced with first kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, then first kind of inducing culture replaced with second kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, then second kind of inducing culture replaced with the third inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, at last the third inducing culture is replaced with the 4th kind of inducing culture in the step (2), at 37 ℃, 5%CO
2Incubator in cultivated 3 days, induce end of processing, obtain the neurocyte that umbilical cord mesenchymal stem cells is divided into.
(4) neurocyte of inducing differentiation to obtain being carried out immunocytochemistry detects
With the 24 orifice plate cells that obtain in the step (3), to inhale and abandon substratum, every hole adds 250ulPBS (PBS), and room temperature leaves standstill 3min with washed cell, and repeated washing washs three times altogether; Every then hole adds the solution that 250ul contains the 4V% Paraformaldehyde 96, and room temperature is 25min fixedly; Repeat as above PBS washing 3 times; Serum with containing 10% 2 anti-source animal carries out sealing treatment with the PBS confining liquid pair cell that contains 0.3%TritonX-100 (DOWANOL); After the incubated at room 30 minutes; Inhale deblocking liquid, directly dropping resists with one of corresponding confining liquid dilution, hatches 2h for 37 ℃.The one anti-specificity olefinic alcohol enzyme (NSE of anti human nerve unit that selects for use; Santa Cruz), anti human nerve specific nucleoprotein (NeuN; Santa Cruz), anti-people's microtubule associating albumen (MAP-2; Santa Cruz), anti-people's GFAP (GFAP, Santa Cruz) and anti-people OX-42 (Santa Cruz) albumen.Repeat PBS washing 8 times then; Drip fluorescently-labeled two and resist, NSE and NeuN are TRITC (Santa Cruz) mark, and Map-2, GFAP and OX-42 are FITC (Santa Cruz) mark, and the room temperature dark is hatched 40min, repeat PBS washing three times then; DAPI redyes nucleus 5min; The PBS flushing twice then, each 3min.Add PBS and preserve, the fluorescence inverted microscope is observed down
Coloration result; For example nerve-specific nucleoprotein (NeuN), neuronspecific enolase (NSE) and microtubule are united albumen-2 (MAP-2) and astroglia cell mark GFAP (GFAP) dyeing all is positive to observe the neurone mark; Oligodendrocyte mark OX-42 dyeing is negative, shows that the cell of acquisition does not have oligodendrocyte.Coloration result shows that the neurocyte of inducing acquisition is made up of neurone and astroglia cell.
Nearly 78% mescenchymal stem cell is divided into neurocyte, and wherein about 85% neurocyte is a neurone, and about 15% neurocyte is an astroglia cell.
Claims (8)
1. a umbilical cord mesenchymal stem cells is divided into the induction method of neurocyte, it is characterized in that adopting following steps:
(1) umbilical cord mesenchymal stem cells is cultured to the confluent culture ware in the MSC substratum;
(2) getting the stable mescenchymal stem cell that goes down to posterity converges with MSC culture medium culturing to 70% in being covered with the petridish of extracellular matrix;
(3) 70% mescenchymal stem cells that converge are cultivated through four kinds of inducing cultures that are added with the following factor successively,
First kind of inducing culture: add U-18496 and three acylazide lipoprotein,
Second kind of inducing culture: add Prostatropin and Noggin albumen,
The third inducing culture: add Prostatropin, vitamin A acid, fibroblast growth factor 8 and Wnt3a albumen,
The 4th kind of inducing culture: add bomeplasty albumen 4, Shh albumen, vitamin A acid, NGFF and BDNF;
The amount of adding the factor in four kinds of inducing cultures is respectively:
First kind: U-18496 1-50uM and three acylazide lipoprotein 0.1-1ug/ml,
Second kind: Prostatropin 1-200ng/ml and Noggin 1-500ng/ml,
The third: Prostatropin 1-200ng/ml, vitamin A acid 0.5-50uM, fibroblast growth factor 81-100ng/ml and Wnt3a albumen 1-500ng/ml,
The 4th kind: bomeplasty albumen 4 1-200ng/ml, Shh albumen 1-500ng/ml, vitamin A acid 0.5-50uM, NGFF 1-500ng/ml and BDNF 1-500ng/ml.
2. induction method according to claim 1 is characterized in that: the amount of adding the factor in four kinds of inducing cultures is preferably
First kind: U-18496 10uM and three acylazide lipoprotein 0.5ug/ml,
Second kind: Prostatropin 25ng/ml and Noggin 100ng/ml,
The third: Prostatropin 25ng/ml, vitamin A acid 10uM, fibroblast growth factor 810ng/ml and Wnt3a 25ng/ml,
The 4th kind: bomeplasty protein 42 0ng/ml, Shh protein 10 ng/ml, vitamin A acid 10uM, NGFF 10ng/ml and BDNF 10ng/ml.
3. induction method according to claim 1 is characterized in that: the basic medium of four kinds of inducing cultures is the DMEM/F12 that is added with the Neurobasal of B27 assistant agent or is added with BIT9500.
4. induction method according to claim 1 is characterized in that: the MSC substratum consists of and adds foetal calf serum 10%, Stimulina 2mM, penicillium mould and Streptomycin sulphate 1%, Prostatropin and Urogastron 2ng/ml among the DMEM.
5. induction method according to claim 1 is characterized in that: said extracellular matrix is the aqueous solution of 1-100ug/ml ln or 1-100ug/ml matrigel.
6. induction method according to claim 1 is characterized in that: in four kinds of inducing cultures, respectively cultivated 3 days.
7. induction method according to claim 1 is characterized in that: get the stable mescenchymal stem cell that goes down to posterity in being covered with the petridish of extracellular matrix with 5 * 10
3Individual cell/cm
2Density cultivate.
8. induction method according to claim 1 is characterized in that: get in the step (2) the stable mescenchymal stem cell that goes down to posterity in being covered with the petridish of extracellular matrix with MSC culture medium culturing 24-48h.
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| CN102220338B (en) * | 2011-05-06 | 2013-08-21 | 武汉北度生物科技有限公司 | Method for expressing human 2.5 S beta-nerve growth factor in human umbilical cord mesenchymal stem cell, and separating and purifying the human 2.5 S beta-nerve growth factor |
| 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 |
| TWI493034B (en) * | 2011-12-14 | 2015-07-21 | Nat Univ Chung Hsing | Neuronal epithelial cells differentiated by universal stem cells and the medium used and their differentiation methods |
| KR101542847B1 (en) * | 2013-11-01 | 2015-08-10 | 주식회사 비비에이치씨 | Method for Differentiating Pluripotency Stem Cell Induced from Mesenchymal Stem Cell into Neuron |
| CN104004713A (en) * | 2014-05-14 | 2014-08-27 | 安沂华 | Method for inducing and culturing umbilical cord mesenchymal stem cells into nerve cells |
| CN104388384B (en) * | 2014-10-29 | 2018-07-20 | 新乡医学院 | The application that oxidized low-density lipoprotein breaks up in inducing bone mesenchymal stem cell Cardiocytes |
| CN104789531B (en) * | 2015-03-27 | 2017-10-27 | 安沂华 | A kind of method that umbilical cord mesenchymal stem cells are induced differentiation into dopaminergic neuron |
| CN105176929B (en) * | 2015-10-30 | 2018-03-20 | 山东省齐鲁干细胞工程有限公司 | A kind of method of inducing neural blastoma cell Differentiating Into Neurons |
| CN106566810A (en) * | 2016-10-19 | 2017-04-19 | 广州赛莱拉干细胞科技股份有限公司 | Composition, inducer containing composition and induction method |
| CN107254441A (en) * | 2017-07-24 | 2017-10-17 | 陈强 | A kind of induced fibroblast is the method for nerve cell like cell |
| WO2020212996A1 (en) * | 2019-04-18 | 2020-10-22 | Exostem Biotec Ltd. | Differentiated and nondifferentiated msc compositions and use thereof |
| CN112795541B (en) * | 2020-11-18 | 2023-02-10 | 天津市康婷生物工程集团有限公司 | Method for inducing differentiation of neural stem cells into neurons and astrocytes by umbilical cord mesenchymal stem cells |
| CN112522181B (en) * | 2020-12-29 | 2023-02-17 | 苏州方舟生物科技有限公司 | Culture medium system and method for inducing and generating human islet beta cells in vitro |
| CN116426478B (en) * | 2023-06-14 | 2023-11-24 | 中科聚研干细胞有限公司 | Brain-derived neural precursor cell culture method |
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