CN107496457B

CN107496457B - Method for growing OPCs (OpCs) trophoblastic sensory neuron axons from MSCs (mesenchymal stem cells)

Info

Publication number: CN107496457B
Application number: CN201710654622.XA
Authority: CN
Inventors: 张洪钿; 苑春慧
Original assignee: Beijing Regeneration Biology Technology Research Institute Co ltd
Current assignee: Beijing Regeneration Biology Technology Research Institute Co ltd
Priority date: 2017-08-03
Filing date: 2017-08-03
Publication date: 2020-08-21
Anticipated expiration: 2037-08-03
Also published as: CN107496457A

Abstract

The invention discloses a method for growing axons of trophoblastic sensory neurons by OPCs (optical fiber) from MSCs (mesenchymal stem cells), which comprises the following steps of: s1, culturing neonatal umbilical cord Wharton jelly in a MSCs culture medium to obtain P2 umbilical cord MSCs; s2, suspending the P2 umbilical cord MSCs by using an MSCs culture medium, inoculating the MSCs into a cell culture plate coated by the recombinant human laminin and the recombinant human vitronectin, culturing to 60-70% confluence, and replacing a neural differentiation culture medium to prepare neural epithelial pre-induced cells; s3, culturing the neural epithelial pre-induced cells in an OPCs culture medium to obtain P0 generation OPCs, and subculturing to P2 generation OPCs; s4, preparing OPCs trophoblasts by using P2 generation OPCs; s5, preparing DRG sensory neuron cells, and co-culturing OPCs trophoblasts and the DRG sensory neuron cells. The method for nourishing and sensing neuron axon growth by OPCs from MSCs provides a new idea for cell therapy of central nervous system nerve injury repair.

Description

Method for growing OPCs (OpCs) trophoblastic sensory neuron axons from MSCs (mesenchymal stem cells)

Technical Field

The invention relates to the technical field of cell culture, in particular to a method for growing OPCs (optical fiber Cs) trophoblastic neuron axons from MSCs.

Background

The degenerative change of the nervous system and the regeneration repair and functional reconstruction after injury are the major problems to be explored and solved by neuroscience researchers and the most difficult and serious challenges to be overcome. Because of the structural fragility and functional complexity of central nervous tissue, its damage often smells a huge, irreversible destruction, seriously affecting patient life safety and quality of life. Repair of damage to the nervous system involves both structural repair and functional recovery. Structural repair encompasses neuronal regeneration and neogenesis, axonal growth, and synaptic reconnection. The current medical technology can not solve the difficult problems of neuron regeneration and new growth, and the axon growth is a controllable aspect. Previous researches show that 5% of residual nerve fibers of paraplegic rats can restore partial or all joint movement of paralyzed hind limbs; when the residue reaches 10%, the paraplegic animal can even recover part of the walking function; when the residual proportion exceeds 40%, the hindlimb motor function of the rat can reach normal. It is reasonable to speculate that the degenerative change or loss of the human nervous system (brain and spinal cord) can keep nearly 85% -90% of functions as long as 10% -15% of nerve structures are complete, and how to promote nerve fiber regeneration is important content for recovering the nerve functions and improving the life quality.

At present, most of the drugs applied in clinical treatment play a role in neuroprotection and/or possible neurorestoration, including: neurotrophic drugs such as ganglioside, cerebroprotein hydrolysate, cytidine disodium triphosphate and oxiracetam, drugs for weakening and inhibiting nerve growth such as chondroitin sulfate and chondroitinase ABC, metabolic drugs such as selegiline, creatine and idebenone, anti-excitotoxic drugs such as lamotrigine and riluzole, and neurotransmitter drugs such as memantine, reboxetine, ritalin and tacrine, but the effects of these drugs on axonal regeneration and neogenesis and nerve function recovery are extremely limited. The study of new nerve repair preparations or drugs is an important issue in nerve repair medicine.

An effective solution to the problems in the related art has not been proposed yet.

Disclosure of Invention

Aiming at the technical problems in the related art, the invention provides a method for growing the axons of the trophoblastic neuron by using the OPCs from the MSCs, and provides a new idea for cell treatment of repairing the nerve injury of the central nervous system.

In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:

a method for MSCs-derived OPCs to nourish the growth of sensory neuron axons, comprising the steps of:

s1, culturing neonatal umbilical cord Wharton jelly in a MSCs culture medium to obtain P2 umbilical cord MSCs;

s2, suspending the P2 umbilical cord MSCs by using an MSCs culture medium, inoculating the MSCs into a cell culture plate coated by the recombinant human laminin and the recombinant human vitronectin, culturing to 60-70% confluence, and replacing a neural differentiation culture medium to prepare neural epithelial pre-induced cells;

s3, culturing the neural epithelial pre-induced cells in an OPCs culture medium to obtain P0 generation OPCs, and subculturing to P2 generation OPCs;

s4, preparing OPCs trophoblasts by using P2 generation OPCs;

s5, preparing DRG sensory neuron cells, and co-culturing OPCs trophoblasts and the DRG sensory neuron cells.

Further, the MSCs medium is a serum-free medium containing 2% serum replacement.

Further, the neural differentiation medium was DMEM/F12 containing the following components:

1×B27，

1×N2，

1uM 1, 25-dihydroxyvitamin D3,

the concentration of triiodothyronine is 40ng/mL,

0.5ug/mL of human melatonin,

20ng/mL recombinant human basic fibroblast growth factor,

20ng/mL recombinant human epidermal growth factor.

Further, the OPCs culture medium is DMEM/F12 containing the following components:

1×B27，

1×N2，

2mM of L-glutamine (Glutamine),

1uM 1, 25-dihydroxyvitamin D3,

the concentration of triiodothyronine is 40ng/mL,

0.5ug/mL of human melatonin,

20ng/mL recombinant human basic fibroblast growth factor,

20ng/mL of recombinant human epidermal growth factor,

20ng/mL of recombinant human platelet-derived growth factor AA,

4ng/mL recombinant human neurotrophic factor 3.

Further, the step S1 further includes the following steps: washing umbilical cord of healthy newborn in term, dissecting aseptically to peel off Wharton's jelly, shearing, suspending with MSCs culture medium, transferring to culture bottle, and placing in CO₂Culturing in an incubator, adding a digestive juice when culturing is carried out till the 14 th day, digesting for 5 minutes at room temperature, then terminating, centrifuging, discarding supernatant, collecting precipitate, filtering by using a nylon cell sieve, collecting filtrate, centrifuging, discarding supernatant, and collecting precipitate, wherein the precipitate is marked as P0 generation umbilical cord MSCs; suspending the cells by the culture medium of the MSCs, adjusting the cell density, inoculating the cells into a new tissue culture flask to culture the cells to the 4 th day, harvesting the cells, marking as the umbilical cord MSCs of the P1 generation, and continuously culturing the P1 generation to the P2 generation.

Further, the step S2 further includes the following steps: suspending P2 umbilical cord MSCs in MSCs culture medium, inoculating to culture plate coated with recombinant human laminin and recombinant human vitronectin, culturing to 60-70% confluence, changing neural differentiation culture medium, 37 deg.C, and 5% CO₂Culturing for 4 days under the saturated humidity condition, sucking and removing culture supernatant, washing the culture surface for 1 time by PBS, adding trypsin solution, digesting for 5 minutes at room temperature, terminating, centrifuging and removing the supernatant, and harvesting precipitated cells, namely the neuroepithelial pre-induced cells.

Further, the step S3 further includes the following steps: suspending neuroepithelial pre-induced cells by using OPCs culture medium, inoculating the cells into a culture plate coated by recombinant human laminin and recombinant human vitronectin, changing the culture solution every other day, sucking and removing culture supernatant when 60-80% of cells are combined, washing the culture surface by PBS, adding Accutase^TMDigesting the digestion solution at room temperature for 5 minutes, then terminating, centrifuging and removing the supernatant, and harvesting the precipitated cells which are marked as P0 generation OPCs, and subculturing the P0 generation OPCs to P2 generation.

Further, the culture system for subculturing the P0 generation OPCs is as follows: suspending the suspension by an OPCs culture medium, and inoculating the suspension to a tissue culture bottle coated by recombinant human laminin and recombinant human vitronectin.

Further, the step S4 further includes the following steps: and (3) resuspending P2 generation OPCs by using an OPCs culture medium, inoculating the OPCs into a culture plate coated by recombinant human laminin and recombinant human vitronectin, culturing for 24 hours, and culturing for 48 hours by using DMEM/F12 containing 3% FBS, wherein the OPCs are used as an OPCs feeder layer.

Further, the step 5 further comprises the following steps: adult male Wistar rats are sacrificed by cervical dislocation, DRG is surgically removed, digested with DMEM/F12 containing 0.1mg/mLNB4, centrifuged at 600g for 10 minutes, and the supernatant is discarded; resuspending the precipitate with trypsin solution, digesting at 37 deg.C for 15 min, terminating, centrifuging, and discarding the supernatant; washing the precipitate with normal saline, sucking and blowing by a pipette, centrifuging and discarding the supernatant to obtain a precipitate as DRG sensory neuron cells; resuspending DRG sensory neurons in DMEM/F12 containing 3% FBS, and adjusting cell density; removing OPCs trophoblast medium, inoculating DRG sensory neuron cell suspension, 37 deg.C, and 5% CO₂The cells were co-cultured under the conditions for 48 hours.

The invention has the beneficial effects that: the method for growing the axons of the OPCs (mesenchymal stem cells) nourishing sensory neurons from the MSCs, disclosed by the invention, adopts the co-culture of the OPCs from the MSCs and the sensory neurons of Dorsal Root Ganglia (DRG) of adult rats for 48 hours, so that 68.19 +/-24.09% of the axons of the DRG sensory neurons grow, the average growth length is 190.22 +/-20.51 um, and a new idea is provided for cell treatment of repairing of central nervous system nerve injury.

Drawings

FIG. 1 is a morphology chart of a tissue mass of Wharton's jelly at the time of inoculation;

FIG. 2 is a morphology chart of Wharton's jelly tissue pieces at day 14 of culture;

FIG. 3 is a morphogram of P2 generation umbilical cord MSCs;

FIG. 4 is a graphical representation of growth of cord P2 generation MSCs on recombinant human laminin, recombinant human vitronectin coated 96-well plates;

FIG. 5 is a diagram of the alteration of the morphology of the neuroepithelial pre-induced cells;

FIG. 6 is a morphogram of high nestin expression by neuroepithelial pre-induced cells;

FIG. 7 is a morphological diagram of high expression A2B5 of OPCs derived from umbilical cord MSCs;

FIG. 8 is a graph comparing the concentration of various bioactive molecules secreted by umbilical cord MSCs and MSCs-derived OPCs over 48 hours of culture;

FIG. 9 is a control DRG sensory neuron morphology map;

FIG. 10 is a DRG sensory neuron morphology map of a co-cultured group of umbilical cord MSCs;

FIG. 11 is a DRG sensory neuron morphology map of OPCs co-cultured group.

Detailed Description

The technical solutions of the present invention will be described more clearly and completely with reference to the following specific examples, in which various reagents and experimental instruments are commercially available without specific reference.

First, reagents, appearing English and abbreviations referred to in the embodiments of the present invention will be briefly described.

Ultra CULTURE: serum-free medium, LONZA manufactured by manufacturers, with a product number of 12-725F;

ultroser G serum substitete: the serum substitute is PALL as a manufacturer, and has the product number of 15950-017;

0.25% recombinant human trypsin: the manufacturer is Shanghai Yaxin biotechnology limited, and the product number is RHT 0301;

0.08% EDTA: the manufacturer is SIGMA-ALDRICH, and the product number is 1233508;

aprotinin solution: the manufacturer is Sigma, and the product number is A1250000;

recombinant human laminin: the manufacturer is BioLamina, and the product number is Lamin-221;

recombinant human vitronectin: the manufacturer is Enzo, and the product number is ENZ-PRT 179-0500;

FBS: fetal bovine serum, manufacturer BI, cat 04-400-1A;

DMEM/F12: is a cell culture medium, the manufacturer is invitrogen, the product number is 12800017;

b27: human leukocyte antigen, the manufacturer is GIBCO, the product number is 17504-;

n2: is a cell culture additive, the manufacturer is GIBCO, the product number is 17502-;

1, 25-dihydroxyvitamin D3: the manufacturer is Sigma, the product number is 740543;

triiodothyronine: the manufacturer is Sigma, and the product number is T2877;

human melatonin: the manufacturer is Sigma, and the product number is M5250;

recombinant human basic fibroblast growth factor: the manufacturer is Sigma, the product number is SRP2092, Sigma;

recombinant human epidermal growth factor: the manufacturer is Sigma, and the product number is E9644;

Accutase^TMdigestion solution: manufacturer Innovative Cell Technologies, Inc, cat # 40506ES 60;

recombinant human platelet-derived growth factor AA: the manufacturer is PeproTech, and the product number is 100-13A;

recombinant human neurotrophic factor 3: the manufacturer is Sigma, and the product number is SPR 3267;

NB 4: collagenase NB4, made by Serva under the trademark of Cathaka, with a product number of 17454;

0.05% trypsin solution: the manufacturer is Biological industries, and the product number is 03-053-1A;

mouse anti-integrin beta 1: mouse anti-integrin beta 1, Abcam by manufacturer, cat No. ab 183666;

coat anti-rabbitIgG: the goat anti-rabbit IgG is Abcam by a manufacturer, Alexa Fluor 488 and ab150077 by the product number;

NGF: a nerve growth factor;

BDNF: brain-derived neurotrophic factor;

NT-3: NT-3 neurotrophic factor;

GDNF: glial cell line-derived neurotrophic factor;

VEGF, vascular endothelial growth factor;

TGF-. beta.1: recombinant human transforming growth factor-beta 1;

TGF-. beta.2: recombinant human transforming growth factor-beta 2;

bFGF, fibroblast growth factor;

PDGF-AA platelet derived factor-AA;

angiogenin;

HGF: hepatocyte growth factor;

CD73 differentiation antigen cluster 73, a glycoprotein in which extracellular-5' -nucleotidase is anchored to the plasma membrane via Glycosylphosphatidylinositol (GPI). (ii) a

CD90 differentiation antigen cluster 90, also known as Thy-1, a 25-37kDa highly conserved N-glycosylated Glycosylphosphatidylinositol (GPI) -anchored membrane protein that is a member of the immunoglobulin family of adhesion molecules;

CD105, a differentiation antigen cluster 105, also called endothelin, is a marker of endothelial cell proliferation, and is one of the components of transforming growth factor beta receptor complex;

nestin, a member of the intermediate filament protein family.

The first embodiment is as follows: culturing of MSCs and establishment of MSCs trophoblasts

Washing umbilical cord of healthy newborn in term with normal saline for 3 times, removing residual blood, dissecting off Wharton's jelly under sterile condition, and cutting into pieces of 2-3mm³The pieces of (4) are suspended in 10 mM MSCs medium, which is Ultra CURTURE containing 2% Ultra G serum substitee.

Transfer to 25cm²In a tissue culture flask (model: easy flash, manufacturer: NUNC), the flask was placed at 37 ℃ with 5% CO₂Culturing in an incubator.

When the culture reached day 14, the culture supernatant was aspirated and removed, the surface was washed with physiological saline 2 times, 5mL of trypsin digestion solution containing 0.25% recombinant human trypsin and 0.08% EDTA was added, digestion was carried out at room temperature for 5 minutes, digestion was stopped with 1mL of aprotinin solution, centrifugation was carried out at 400g, and the precipitate was harvested after 5 min.

Washing the precipitate with normal saline for 2 times, filtering with 100um nylon cell sieve, collecting filtrate, centrifuging at 400g for 5min, discarding supernatant, and collecting precipitate to obtain P0-generation umbilical cord MSCs.

Suspending cells with MSCs culture medium, adjusting cell density to 6000/cm²Inoculating to new 25cm²Culturing in a tissue culture bottle to the 4 th day, and harvesting to obtain the P1 umbilical cord MSCs.

The culture was continued until P2 generation. Suspending P2 umbilical cord MSCs in MSCs culture medium at 6000/cm²The cells were inoculated into recombinant human laminin-recombinant human vitronectin-coated 96-well plates (model: 167008, manufacturer:NUNC), and culturing for 24 hours, and replacing DMEM/F12 containing 3% FBS with DMEM/F12 for 48 hours for later use, so that the MSCs are trophoblasts.

Example two: preparation of neuroepithelial pre-induced cells

Suspending P2 umbilical cord MSCs in MSCs culture medium at 6000/cm²Inoculating into recombinant human laminin and recombinant human vitronectin-coated 6-well plate (model: 174901, manufacturer: NUNC), culturing to 60-70% confluence, changing neural differentiation medium, 37 deg.C, and 5% CO₂And culturing for 4 days under the saturated humidity condition.

The culture supernatant was aspirated, the culture surface was washed 1 time with PBS, 1mL of 0.25% trypsin solution was added, digestion was carried out at room temperature for 5 minutes, and 100uL of aprotinin solution was added to terminate the digestion.

Centrifuging at 400g for 5min, discarding the supernatant, and harvesting the precipitated cells, namely the neural epithelial pre-induced cells.

Wherein the neural differentiation medium is DMEM/F12 containing the following components:

1×B27，

1×N2，

1uM 1, 25-dihydroxyvitamin D3,

the concentration of triiodothyronine is 40ng/mL,

0.5ug/mL of human melatonin,

20ng/mL recombinant human basic fibroblast growth factor,

20ng/mL recombinant human epidermal growth factor.

Example three: culturing of OPCs and establishment of OPCs trophoblasts

Resuspending neuroepithelial pre-induced cells in OPCs culture medium at 1.5 × 10⁴/cm²Inoculating into 6-well plate coated with recombinant human laminin and recombinant human vitronectin, changing liquid every other day, removing culture supernatant when 60-80% of the liquid is combined, washing culture surface with PBS for 1 time, adding Accutase^TMDigesting 1mL of digestive juice at room temperature for 5 minutes, and adding 200uL of aprotinin solution to terminate digestion; centrifuging at 400g for 5min, discarding the supernatant, and harvesting the precipitated cells, which are marked as P0 generation OPCs.

And (3) subculturing the OPCs of the P0 generation, wherein the culture system is an OPCs culture medium, and the OPCs are obtained by subculturing to the P2 generation through a tissue culture bottle coated by the recombinant human laminin and the recombinant human vitronectin.

Resuspending P2 OPCs in OPCs medium at 1.5 × 10⁴/cm²Inoculating into 96-well plate coated with recombinant human laminin and recombinant human vitronectin, culturing for 24 hr, and culturing with DMEM/F12 containing 3% FBS for 48 hr to obtain OPCs trophoblast.

Wherein the OPCs culture medium is DMEM/F12 containing the following components:

1×B27，

1×N2，

2mM of L-glutamine (Glutamine),

1uM 1, 25-dihydroxyvitamin D3,

the concentration of triiodothyronine is 40ng/mL,

0.5ug/mL of human melatonin,

20ng/mL recombinant human basic fibroblast growth factor,

20ng/mL of recombinant human epidermal growth factor,

20ng/mL of recombinant human platelet-derived growth factor AA,

4ng/mL recombinant human neurotrophic factor 3.

Example four: DRG sensory neuron isolation and co-culture

Adult male Wistar rats (model: SPF, manufacturer: vindolite) were sacrificed by cervical dislocation and DRG was surgically removed. DRG was digested with DMEM/F12 containing 0.1mg/mLNB4, centrifuged at 37 ℃ for 90 minutes at 600g for 10 minutes, and the supernatant was discarded.

Resuspend the pellet in 0.05% trypsin solution, digest for 15 minutes at 37 deg.C, stop digestion by adding 10% volume aprotinin solution, centrifuge for 10 minutes at 400g, and discard the supernatant.

The pellet was washed 2 times with physiological saline, aspirated 15 times with a 5mL pipette, centrifuged at 400g for 10 minutes, and the supernatant was discarded to obtain a DRG sensory neuron cell pellet.

Resuspending DRG sensory neurons in DMEM/F12 containing 3% FBS, adjusting cell density to 3 × 10⁴/mL。

The control group was inoculated directly with DRG sensory neuron cell suspension in recombinant human laminin, recombinant human vitronectin-coated 96-well plates.

The MSCs CO-culture group and the OPCs CO-culture group are prepared by firstly absorbing and removing MSCs trophoblast and OPCs trophoblast culture medium, then inoculating DRG sensory neuron cell suspension, 100uL per well, 5% CO at 37 DEG C₂And co-cultured for 48 hours.

Example five: expression detection of multiple bioactive molecules and detection of DRG neuron axons

5.1 expression assay for multiple bioactive molecules

Detecting the expression of the following bioactive molecules in culture supernatants of the P2 generation umbilical cord MSCs cultured for 48 hours and the P2 generation OPCs derived from the MSCs cultured for 48 hours by an ELISA method: NGF, BDNF, NT-3, GDNF, VEGF, TGF-beta 1, TGF-beta 2, HGF, bFGF, PDGF-AA, Angiogenin.

5.2 DRG neuronal axon detection

Co-culturing for 48 hours, sucking out the culture medium, fixing the cells with 4% paraformaldehyde for 10 minutes, rinsing with PBS for 2 times, dropwise adding mouse anti-integrin beta 1 primary antibody, and standing overnight at 4 ℃; rinsing with PBS for 2 times, dripping the goat anti-rabbit IgG, incubating for 1 hour at room temperature, and rinsing with PBS for 2 times; the number of neurons with axons and the length of axons were measured by a cell imaging system (manufacturer: GE Healthcare).

5.3 statistical analysis

Statistical analysis statistical processing was performed using SPSS 17.0. Data to

S represents, the mean value comparison adopts independent sample t-test, the frequency comparison adopts chi-square detection, P represents<0.05 is statistically significant.

Example six: results of the experiment

6.1 trophoblast preparation and analysis

The different stages of culturing of umbilical cord MSCs-derived OPCs are shown in FIGS. 1-7.

The umbilical cord tissue mass was cultured in a serum-free culture system, see fig. 1, with a portion of the mass having a large amount of cells creeping out by day 7 and a typical MSCs culture pattern of polyclonal cells formed around about 45% of the mass by day 14 harvest, see fig. 2.

The cell morphology was gradually normalized with increasing passage number, see fig. 3.

P2 generation umbilical cord MSCs highly express CD73, CD90 and CD105, and low or no hematopoietic immune system targeted phenotype.

The MSCs of the P2 generation are easier to adhere to the 6-well plate coated by the recombinant human laminin and the recombinant human vitronectin, have better migration characteristics, and have typical MSCs morphological characteristics after being cultured for 72 hours, see figure 4.

The differentiation of the MSCs into nerves is induced by a nerve induction culture medium, the cell bodies of the MSCs are gradually elongated and grow in parallel, see figure 5, and the high expression nestin is seen in figure 6.

The differentiation of MSCs-derived neuroepithelial pre-induced cells into OPCs is induced by an OPCs culture medium, after 6 days, the cell morphology is obviously changed in a bipolar and tripolar manner, and 92.09+/-11.43 percent of cells express A2B5 when subcultured to the 2 nd generation, as shown in figure 7.

When the umbilical cord-derived MSCs and MSCs-derived OPCs were cultured for 48 hours, there was a large difference in secretion of the secreted bioactive factors involved in nerve repair. The results are shown in fig. 8, the left side shows the results of expressing various bioactive molecules by MSCs, and the right side shows the results of expressing various bioactive molecules by OPCs, and the results show that after the MSCs are differentiated into OPCs, the secretion amounts of NGF, BDNF, NT-3 and GDNF are remarkably increased to 2.98, 7.30, 13.11 and 112.80 times compared with the MSCs; VEGF, HGF, TGF-beta 1, TGF-beta 2, bFGF, PDGF-AA and Angiogenin secretion are reduced to different degrees and are respectively reduced to 2.72%, 74.88%, 47.76%, 46.58%, 28.39%, 1.83% and 2.44% of the secretion amount of the MSCs.

6.2 trophoblast cells promote neurite outgrowth and growth in DRG sensory neurons

The DRG sensory neuron cells were inoculated onto recombinant human laminin and recombinant human vitronectin-coated 96-well plates, MSCs trophoblasts or OPCs trophoblasts for 48 hours of co-culture, and the neuronal axons of the control group, MSCs co-culture group and OPCs co-culture group all increased and grew to different degrees, as shown in fig. 9, fig. 10 and fig. 11, respectively.

Wherein, the neuron with nerve fiber in the control group is 22.52 +/-9.11%, the MSCs co-culture group is 51.85 +/-20.06%, and the OPCs co-culture group is 68.19 +/-24.09%. The neuronal axons in the OPCs co-cultured group grew longest (190.22 +/-20.51 um), significantly longer than in the MSCs co-cultured group (99.46 +/-3.87 um) and the control group (25.12 +/-3.28 um).

Example seven: analysis of results

In the process of nerve tissue repair, neuron regeneration is very difficult, but residual nerve fiber growth and synaptic reconnection can partially or even completely restore nerve function.

Although some neurotrophic factors have been found to have some promoting effects on neurite outgrowth, they are very limited. With the development of stem cell regeneration medicine, many basic and clinical researches find that the in vitro culture of MSCs, OPCs and the like and neuronal cells can promote the growth of axons, and the transplantation of the MSCs, OPCs and the like in vivo can rebuild nerve transmission channels to a certain extent. But MSCs act more to locally modulate immune activity and mobilize the restored activity in somatic stem/progenitor cells via paracrine action, thereby promoting neural tissue self-repair, whereas OPCs do not. OPCs are myelin structural cells that have a natural role in axon growth and repair, and in vitro and in vivo experiments indicate that OPCs are functional in axon growth and nerve function reconstruction, and are the most recent advances and promising therapeutics for maintaining and repairing damaged axons, reconstructing axon electrical conduction functions.

At present, adult OPCs are difficult to obtain, and although embryonic stem cells and induced pluripotent stem cells can differentiate in vitro to generate OPCs, the risk of tumorigenicity in vivo transplantation exists. Therefore, the induction of adult pluripotent stem cells, particularly MSCs, by some people to generate OPCs in vitro does not cause serious adverse reactions such as ethical obstacles, tumorigenesis applied in vivo and the like.

The MSCs are adult pluripotent stem cells which are most widely and deeply researched and applied except hematopoietic stem cells at present, the sources of samples are wide, in-vitro culture is simple, the cell yield is high, and particularly umbilical cord MSCs are easier to culture. Therefore, the selection of umbilical cord MSCs-derived OPCs as trophoblasts to promote neuronal axonal proliferation and growth is an optimal solution.

MSCs can be differentiated into nestin + neural precursor cells through bFGF, EGF and other growth factors, and then are differentiated into OPCs through an OPCs induction system. The MSCs-neurosphere-OPCs induction system is adopted in the past, the operation is complex, the culture process is optimized, the MSCs-neuroepithelial pre-induced cell-OPCs subculture system is adopted, the operation steps are simplified, 92.09+/-11.43% of cells express A2B5 when the generation of P2 is reached, and the purity is high. And the secretion of bioactive factors such as NGF, BDNF, NT-3, GDNF and the like related to nerve repair is greatly improved compared with that of MSCs.

The co-culture result shows that the number of growth cones of the DRG sensory neurons fed by the P2 generation OPCs from the umbilical cord MSCs is increased by 3.9 times and 1.32 times compared with a control group without a nourishing layer and the MSCs feeding layer, and the length of the nerve fibers is increased by 7.57 times and 1.91 times after the co-culture is carried out for 48 hours. Accordingly, the present invention provides a method for efficiently culturing DRG sensory neuron axon outgrowth and growth via umbilical cord MSCs-derived OPCs.

Claims

1. A method for the trophoblast sensory neuron axon growth by MSCs-derived OPCs, comprising the steps of:

s4, preparing OPCs trophoblasts by using P2 generation OPCs;

s5, preparing DRG sensory neuron cells, and co-culturing OPCs trophoblasts and the DRG sensory neuron cells;

the MSCs culture medium is a serum-free culture medium containing 2% serum substitute;

the neural differentiation medium is DMEM/F12 containing the following components:

1×B27，

1×N2，

1uM 1, 25-dihydroxyvitamin D3,

the concentration of triiodothyronine is 40ng/mL,

0.5ug/mL of human melatonin,

20ng/mL recombinant human basic fibroblast growth factor,

20ng/mL recombinant human epidermal growth factor;

the OPCs culture medium is DMEM/F12 containing the following components:

1×B27，

1×N2，

2mM of L-glutamine (Glutamine),

1uM 1, 25-dihydroxyvitamin D3,

the concentration of triiodothyronine is 40ng/mL,

0.5ug/mL of human melatonin,

20ng/mL recombinant human basic fibroblast growth factor,

20ng/mL of recombinant human epidermal growth factor,

20ng/mL of recombinant human platelet-derived growth factor AA,

4ng/mL recombinant human neurotrophic factor 3.

2. The method of claim 1, wherein said step S1 further comprises the steps of: washing umbilical cord of healthy newborn in term, dissecting aseptically to peel off Wharton's jelly, shearing, suspending with MSCs culture medium, transferring to culture bottle, and placing in CO₂Culturing in an incubator, adding a digestive juice when culturing is carried out till the 14 th day, digesting for 5 minutes at room temperature, then terminating, centrifuging, discarding supernatant, collecting precipitate, filtering by using a nylon cell sieve, collecting filtrate, centrifuging, discarding supernatant, and collecting precipitate, wherein the precipitate is marked as P0 generation umbilical cord MSCs; suspending the cells by the culture medium of the MSCs, adjusting the cell density, inoculating the cells into a new tissue culture flask to culture the cells to the 4 th day, harvesting the cells, marking as the umbilical cord MSCs of the P1 generation, and continuously culturing the P1 generation to the P2 generation.

3. The source of MSCs according to claim 1The method for OPCs trophoblast sensory neuron axon growth according to (1), wherein said step S2 further comprises the steps of: suspending P2 umbilical cord MSCs in MSCs culture medium, inoculating to culture plate coated with recombinant human laminin and recombinant human vitronectin, culturing to 60-70% confluence, changing neural differentiation culture medium, 37 deg.C, and 5% CO₂Culturing for 4 days under the saturated humidity condition, sucking and removing culture supernatant, washing the culture surface for 1 time by PBS, adding trypsin solution, digesting for 5 minutes at room temperature, terminating, centrifuging and removing the supernatant, and harvesting precipitated cells, namely the neuroepithelial pre-induced cells.

4. The method of claim 1, wherein said step S3 further comprises the steps of: suspending neuroepithelial pre-induced cells by using OPCs culture medium, inoculating the cells into a culture plate coated by recombinant human laminin and recombinant human vitronectin, changing the culture solution every other day, sucking and removing culture supernatant when 60-80% of cells are combined, washing the culture surface by PBS, adding Accutase^TMDigesting the digestion solution at room temperature for 5 minutes, then terminating, centrifuging and removing the supernatant, and harvesting the precipitated cells which are marked as P0 generation OPCs, and subculturing the P0 generation OPCs to P2 generation.

5. The method of claim 4, wherein the OPCs of the P0 generation are subcultured in a culture system comprising: suspending the suspension by an OPCs culture medium, and inoculating the suspension to a tissue culture bottle coated by recombinant human laminin and recombinant human vitronectin.

6. The method of claim 1, wherein said step S4 further comprises the steps of: and (3) resuspending P2 generation OPCs by using an OPCs culture medium, inoculating the OPCs into a culture plate coated by recombinant human laminin and recombinant human vitronectin, culturing for 24 hours, and culturing for 48 hours by using DMEM/F12 containing 3% FBS, wherein the OPCs are used as an OPCs feeder layer.

7. The method of claim 1, wherein said step 5 further comprises the steps of: adult male Wistar rats are sacrificed by cervical dislocation, DRG is surgically removed, digested with DMEM/F12 containing 0.1mg/mLNB4, centrifuged at 600g for 10 minutes, and the supernatant is discarded; resuspending the precipitate with trypsin solution, digesting at 37 deg.C for 15 min, terminating, centrifuging, and discarding the supernatant; washing the precipitate with normal saline, sucking and blowing by a pipette, centrifuging and discarding the supernatant to obtain a precipitate as DRG sensory neuron cells; resuspending DRG sensory neurons in DMEM/F12 containing 3% FBS, and adjusting cell density; removing OPCs trophoblast medium, inoculating DRG sensory neuron cell suspension, 37 deg.C, and 5% CO₂The cells were co-cultured under the conditions for 48 hours.