CN112746057A - Culture system, method and application for inducing human pluripotent stem cells into neuromesodermal progenitor cells in vitro and maintaining self-renewal - Google Patents

Abstract

The invention relates to the technical field of biomedical engineering, and provides a culture system for inducing human pluripotent stem cells into neuromesodermal progenitor cells in vitro and maintaining self-renewal, which comprises a basic culture medium and differentiation-inducing factors added in the basic culture medium, wherein the differentiation-inducing factors comprise 0.01-500 mu MTGF beta inhibitor, 0.01-500 mu MBMP inhibitor, 0.01-50 mu MGSK3 inhibitor, 0.01-500ng/mLFGF family growth factor and 0.01-500ng/mLEGF family growth factor. Wherein the basic culture medium comprises liquid basic culture medium, 0-5 XB 27 additive, 0-5 XN 2 additive, 1% penicillin/streptomycin, and 0-500 μ g/mL 2-phosphoric acid-vitamin C. The induction method comprises the following steps: A. adopting an hPSCs culture medium to culture the hPSCs until the cell density is 50% -70%; B. and (3) carrying out cell induction by using the culture system, changing the liquid half a day, carrying out cell passage according to the ratio of 1:6 when the cell density reaches 90%, adding Blebbistatin into the culture system during subculture, and continuously inducing for more than 10 days.

Description

Culture system, method and application for inducing human pluripotent stem cells into neuromesodermal progenitor cells in vitro and maintaining self-renewal

Technical Field

The invention relates to the technical field of biomedical engineering, in particular to a system and a method for inducing human pluripotent stem cells to differentiate into neuromesodermal progenitor cells in vitro and maintaining long-term culture of the neuromesodermal progenitor cells.

Background

Human pluripotent stem cells (hPSCs) can be classified into human embryonic stem cells and human induced pluripotent stem cells according to their sources. Human embryonic stem cells (hESCs) are isolated from the inner cell mass of early blastocysts and cell lines that can be stably cultured in vitro for long periods of time by optimizing the culture conditions. hESCs can maintain stable self-renewal and multipotent differentiation potential in vitro with the potential to differentiate to produce all types of adult cells. The acquisition of hESCs and the establishment of cell lines provide potential cell sources for cell replacement therapy, have great development prospects in the aspect of regenerative medicine application, and particularly have great development prospects in recent years in research on Induced Pluripotent Stem Cells (iPSCs), so that individual pluripotent stem cells can be obtained without depending on embryos for the first time, and the regenerative medicine is in a new period of vigorous development.

The spinal cord, as the central nervous system, has a plurality of functions of conducting nerve signals up and down, integrating nerve signals, coordinating movement and the like. Spinal cord degenerative changes such as amyotrophic lateral sclerosis and neuromuscular atrophy and exogenous trauma cause spinal cord injury, however, the treatment effect of the existing surgical and pharmaceutical treatment modes on spinal cord injury is limited, and cell transplantation is expected to become a new means for treating spinal cord injury. Neuromesodermal progenitors (NMPs) can differentiate into nerve cells in the spinal cord, and can also generate mesodermal tissues around the spinal cord, and have unique application value in cell therapy of spinal cord injury.

At present, the literature reports that the pluripotent stem cells are induced to generate the neuromesodermal progenitor cells, but how to realize the self-renewal of the neuromesodermal progenitor cells still remains to be solved. There is no efficient induction method for inducing hPSCs into neuromesodermal progenitors (srNMPs) that are stable in self-renewal, especially under chemically defined culture conditions.

Disclosure of Invention

The present invention has been made to solve the above-mentioned problems, and provides a culture method for inducing pluripotent stem cells (hPSCs) into neuromesodermal progenitor cells (srNMPs) capable of stably self-renewal, aiming at the defect that neuromesodermal progenitor cells induced and generated in the prior art are difficult to maintain stable self-renewal.

In a first aspect of the invention, there is provided a culture system for inducing hPSCs into srNMPs in vitro and maintaining self-renewal, the system comprising a chemically defined neuromesodermal progenitor medium. By using the culture system, the hPSCs can be rapidly induced into srNMPs after 10 days, and meanwhile, the cells can be stably passaged in vitro for more than 30 generations and the cell phenotype is maintained to be stable.

The culture system comprises a basic culture medium and an induced differentiation factor added in the basic culture medium: 0.01-500 μ M TGF beta inhibitor, 0.01-500 μ M BMP inhibitor, 0.01-50 μ M GSK3 inhibitor, 0.01-500ng/mL FGF family growth factor, 0.01-500ng/mL EGF family growth factor. The above differentiation inducing factors are key factors capable of inducing and maintaining molecular phenotypes of srNMPs and stabilizing self-renewal.

Wherein the basic culture medium (marked as N2B27) comprises a liquid basic culture medium, 0-5 XB 27 additive, 0-5 XN 2 additive, 1% penicillin/streptomycin and 0-500 mug/mL 2-phosphoric acid-vitamin C.

Preferably, the liquid basal medium is DMEM/F12, 0.5 XN 2, 0.5 XB 27, 60. mu.g/mL 2-phospho-vitamin C, 1% penicillin/streptomycin;

TGF beta inhibitors include, but are not limited to, one or more of SB431542, LY2109761, A-83-01, and other small molecule TGF beta inhibitors;

BMP inhibitors include but are not limited to one or more of small molecular BMP inhibitors such as DMH1, K02288, LDN-193189 and the like, and BMP inhibitors such as Noggin and the like;

the GSK3 inhibitor includes but is not limited to one or more of small molecule GSK3 inhibitors such as CHIR99021, BIO and LY 2090314;

EGF family growth factors include, but are not limited to, one or more of EGF, TGF alpha, and HB-EGF;

the FGF family growth factors include, but are not limited to, any one or more of FGF 1 to FGF 23.

Further preferably, the components and the optimal concentration of the differentiation inducing factor are as follows: TGF beta inhibitor is 2 mu M SB431542, BMP inhibitor is 2 mu M DMH1, GSK3 inhibitor is 3 mu M CHIR99021, FGF of 10ng/mL FGF2, and EGF of 10 ng/mL.

In the embodiment of the present invention, the experiment was carried out using the above components, but each component is not limited to the above specific components. For example, 2 μ M SB431542 was selected as the TGF inhibitor, but LY2109761 and A-83-01 also performed the corresponding functions. The component choices of the other components are also possible.

Preferably, the hPSCs are human embryonic stem cell lines derived from inner cell masses of human embryos or induced pluripotent stem cells obtained by somatic reprogramming.

In a second aspect of the present invention, there is provided a method for inducing hPSCs into srNMPs and maintaining self-renewal thereof using the above culture system, comprising the steps of:

A. adopting an hPSCs culture medium to culture the hPSCs until the cell density is 50% -70%;

B. when the density of hPSCs reaches 50% -70%, the culture system is used for cell induction, liquid is changed half a day, cell passage is carried out according to the ratio of 1:6 when the cell density reaches 90%, non-muscle myosin IIATPase inhibitor is added into the culture system during subculture, and continuous induction is carried out for more than 10 days.

Preferably, in step A, the composition of the hPSCs medium is as follows: basal medium DMEM/F12, 0.5 XN 2, 0.5 XB 27, 60. mu.g/mL 2-phospho-vitamin C, 1% penicillin/streptomycin, 40ng/mL basic fibroblast growth factor, 2ng/mL transforming growth factor-beta. In step B, the non-muscle myosin II ATPase inhibitor is 5. mu.M Blebbistatin.

The inventor carries out unicellular clone spontaneous differentiation on the srNMPs obtained by the culture system, proves the bidirectional differentiation potential of the nerve and mesoderm of the srNMPs from the level of unicellular, and cell balls generated by the suspension culture differentiation of the srNMPs can generate a structure similar to a spine after long-term in vitro culture. The srNMPs are directionally differentiated towards the nerve and mesoderm to generate dorsal spinal cord interneurons and ventral spinal cord motor neurons and skeletal muscle cells respectively, and further prove that the srNMPs have the potential of bidirectional differentiation of the nerve and mesoderm.

Accordingly, the third aspect of the present invention provides the application of srNMPs obtained by the culture method of the present invention, such as the application potential in the mechanism of generation of neuromesodermal progenitor cells, the mechanism of self-renewal of neuromesodermal progenitor cells, the induction of spinal neurons, the induction of paraaxial mesoderm, the cell model of spinal development, in vitro drug screening of spinal cord injury diseases, cell therapy of spinal cord injury diseases, and tissue engineering of spinal cord injury repair.

The invention has the following beneficial guarantee and effects:

through experiments, hPSCs can be rapidly induced into srNMPs after 10 days by using the culture system with definite chemical components, the cell co-expresses the neural stem cell molecular marker SOX2 and the mesoderm molecular marker T, and meanwhile, the cell can be stably passaged in vitro for more than 30 generations and the cell phenotype is maintained to be stable. The single cell clone spontaneous differentiation experiment and the suspension culture differentiation experiment of the srNMPs prove the neural and mesoderm bidirectional differentiation potential of the srNMPs. The srNMPs are directionally differentiated towards the nerve and mesoderm to generate dorsal spinal cord interneurons, ventral spinal cord motor neurons and skeletal muscle cells respectively, and further prove that the srNMPs have the potential of bidirectional differentiation of the nerve and mesoderm.

Further, srNMPs were subjected to spheronization experiments, allowing them to spontaneously differentiate, giving rise to a tissue mass similar to the spinal structure. The cell can be differentiated to generate nerve cells and mesoderm cells simultaneously, and the important application potential and advantage of the cell in the aspect of spinal injury are suggested.

Drawings

FIG. 1 shows the induction process of srNMPs and the results of molecular phenotype identification. Wherein (A) the hPSCs grow in a clonal-like manner under a light microscope at low density, typically in an epithelial-like manner (shot under a 200-fold visual field), and the expression levels of the pluripotency marker genes OCT4, SSEA3 (shot under a 200-fold visual field) and SOX2 and SSEA4 (shot under a 100-fold visual field) are detected by immunofluorescence staining; (B) induction protocols for srNMPs; (C) under light microscopy, srNMPs appear as clonal forms at low density and srNMPs appear as paving stone-like typical epithelial cell forms at high density (images taken in 200-fold visual field). (D-H) detecting the expression of the molecular marker of the neuromesodermal progenitor cell by immunofluorescence staining and flow cytometry, and detecting the expression of the molecular marker of mesodermal, endodermal and epidermal ectodermal by RT-PCR; (I) and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is used for detecting the gene change condition in the induction process of srNMPs.

FIG. 2 shows the results of the assays of srNMPs having bidirectional differentiation potential in the neural and mesoderm. Wherein, (A-B) is the result of the spontaneous differentiation experiment of single-cell clone of srNMPs; (C-E) the neural tissue, the cartilage tissue and the skeletal muscle tissue can be detected by the cell mass of srNMPs cultured in suspension for four months; (F-H) is the result of detecting the cell mass of srNMPs cultured in suspension for eight months. Note: the right panel in panel F is a photograph of the Spinal Cord in a vertebrate embryo (pictures from the network, Vertebrand Spinal Cord Development is aphograph by Steve Gschmeissner white roads loaded on June 27th, 2016.).

FIG. 3 shows the experimental results of spontaneous differentiation of srNMPs into nerves. Wherein (A-B) is spinal cord neuron and precursor cell classification and marker gene thereof; (C) experimental protocol for spontaneous neural differentiation of srNMPs; (D-F) analyzing the spinal cord location of the obtained neuron by immunofluorescence staining and RT-PCR detection of related molecular marker results.

FIG. 4 shows the results of experiments of directional differentiation of srNMPs into spinal ventral motor neurons. (A) An experimental scheme for directed differentiation of srNMPs into spinal ventral motor neurons; (B and C) qRT-PCR and immunofluorescence staining detection ventral motor neuron molecular marker results.

Fig. 5 shows the results of experiments directed differentiation of srNMPs into spinal cord dorsal interneurons. (A) An experimental protocol for directed differentiation of srNMPs into spinal cord dorsal interneurons; (B and C) qRT-PCR and immunofluorescence staining detection of dorsal central neuron molecular marker results.

FIG. 6 shows the results of experiments directed differentiation of srNMPs into skeletal muscle cells. (A) qRT-PCR detects the differentiation condition of srNMPs to mesoderm; (B) and (3) detecting a skeletal muscle cell molecular marker by immunofluorescence staining.

Detailed Description

The present invention will now be described in detail with reference to examples, but the practice of the present invention is not limited thereto.

The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless otherwise indicated, percentages and parts are by weight.

Example 1: induction of srNMPs and molecular phenotype identification

1. Resuscitation, culture and identification of hPSCs

The dish surface of the cells was pre-coated with DMEM/F12 medium containing 1% matrigel (Corning, Cat #356231) and stored at 4 ℃ overnight. The cryopreserved hPSCs in this laboratory were H1(passage 70-85) and HUES9 (passage 20-35). Taking out from a refrigerator at minus 80 ℃, and rapidly shaking in a water bath at 37 ℃ until a little ice residue is dissolved. Quickly placing into a low-temperature horizontal centrifuge for centrifuging cells for 5min at 1000rpm, sucking away frozen stock solution, resuspending with hPSCs culture medium, inoculating into a culture plate coated with matrigel, and changing the culture solution every day. After digestion of the cells with TrypLE digestive enzyme (Gibco, Cat # 12605-010) and passage at 1:10, 5. mu.M Blebbistatin (Selleck, S7099) was added to prevent apoptosis due to spontaneous blebbing of the cells.

The optimum composition of the hPSCs medium is as follows: basal media DMEM/F12(Thermo Fisher Scientific), 0.5 XN 2(Thermo Fisher Scientific, Cat #17502048), 0.5 XB 27(Thermo Fisher Scientific, Cat #17504044), 60. mu.g/mL 2-phospho-vitamin C (Sigma-Aldrich, Cat # A8960), 1% penicillin/streptomycin, 40ng/mL basic fibroblast growth factor (PeproTech, AF-100-18B), 2ng/mL transforming growth factor-beta (PeproTech, 100-21).

Observed under an inverted microscope, the hPSCs grow in a clone shape, are in a typical paving stone-like epithelial cell shape, and have obvious nucleolus and large nucleoplasmic ratio. The undifferentiated state of the cells was evaluated by immunofluorescence staining periodically, and 99% or more of the hPSCs remained positive for the pluripotency marker genes OCT4, SOX2, SSEA3, and SSEA4 (FIG. 1A).

2. Induction process and molecular phenotype identification of srNMPs

The culture system for inducing hPSCs into srNMPs comprises the following components: liquid basal medium, 0.5 XB 27 additive, 0.5 XN 2 additive, 1% penicillin/streptomycin, 60. mu.g/mL 2-phospho-vitamin C, 2. mu.M small molecule TGF beta inhibitor SB431542(Selleck, S1067), 2. mu.M small molecule BMP inhibitor SB431542(Selleck, S7146), 3. mu.M small molecule GSK3 inhibitor CHIR99021(Selleck, S2924), 10ng/mL FGF family growth factor FGF2(PeproTech, AF-100-18B), 10ng/mL EGF family growth factor EGF (PeproTech, AF-100-15), and the medium is named as srNMPs medium.

When the hPSCs density reached 50% -70%, induction with srNMPs medium was started and continued for more than 10 days with half of the medium change every day (FIG. 1B). The cells were passaged at 90% cell density and the cell plates were pre-coated with 1% matrigel in DMEM/F12 and stored overnight at 4 ℃.

The experimental results shown were achieved in both H1 and HUES9 cell lines, indicating that the induction of srNMPs is well reproducible for different hPSCs. The display results are all exemplified by srNMPs derived from H1 cell line.

srNMPs can be cultured in vitro for a long period of time, maintain stable self-renewal, and maintain typical epithelial-like cell morphology. Observed under a light microscope, the nucleolus is obvious, the nucleoplasm is high, and when the cells are subjected to low-density passage, the cells are converged into clone, the form is uniform, and the cloning edge is clear. The cells have contact inhibition phenomenon, and the cells stop proliferating and rapidly undergo apoptosis after the culture dish is full of the cells, so that the phenomenon of overlapping growth cannot occur. At high density passage, the cells were evenly distributed and uniform in morphology, still maintaining an epithelial-like morphology (fig. 1C).

Further, the results of immunofluorescence staining and flow cytometry detection show that srNMPs positively express the neural stem cell molecular marker SOX2, NESTIN, NCAD, CD133, early mesoderm molecular marker T and cell proliferation molecular marker MKI67, SOX2 and T can be co-expressed, the proportion of double positive cells reaches 75.5%, and the srNMPs can maintain long-term stable self-renewal in vitro (FIG. 1D, FIG. 1E, FIG. 1F and FIG. 1G).

The results of reverse transcription-PCR detection show that srNMPs do not express molecular markers of other germ layers, such as mesoderm molecular marker EOMES, endoderm molecular marker SOX17 and ectoderm epidermal molecular marker K14, indicating that the cells are not differentiated in mesoderm, endoderm and ectoderm during induction (FIG. 1H).

3. Early induction process of srNMPs

The quantitative PCR results showed that the expression of the pluripotency molecular markers OCT4, NANOG decreased rapidly during the induction of srNMPs, indicating that all hPSCs had exited the pluripotency state by three days of induction. The other pluripotency molecular marker, namely the molecular marker SOX2 of the neural stem cells is basically unchanged in the induction process and is always in a high expression state. The mesoderm molecular marker T gradually increases in expression during induction, and the expression is gradually stabilized after 10 days of induction. This indicates that hPSCs can be induced into srNMPs over a 10 day induction period. In addition, during induction, the expression of the genes of the spinal cord molecular marker CDX2 and HOX family is quickly up-regulated and always kept in a high expression state. Among them, HOXB9 was the highest in expression level, and HOX gene was not expressed further in the posterior (results not shown), indicating that this method induces srNMPs in the anterior-posterior axis corresponding to the location of the thoracic spinal cord (fig. 1I).

The above results show that hPSCs can be rapidly induced into srNMPs after 10 days by using the culture system with definite chemical components, and the cells can be stably passaged in vitro for more than 30 generations and maintain the cell phenotype to be stable.

Example 2: srNMPs have bidirectional differentiation potential of nerve and mesoderm

1. single cell differentiation experiments with srNMPs:

after digesting srNMPs into single cells, inoculating the single cells into a matrigel-coated six-well plate at a very low density (about 50 cells per well), growing the srNMPs under the culture condition for five days, forming clones by most of the single cells (figure 2A), then changing the culture condition into a N2B27 basal medium to make the single cells spontaneously differentiate, continuing to culture for two to three weeks, detecting the clones of the srNMPs from the single cells by immunofluorescence staining, and simultaneously expressing a neural marker MAP2 and a mesoderm molecular marker alpha SMA (figure 2B), which shows that the clones formed by the single srNMPs can differentiate to generate neurons and mesoderm cells, and the single cell level proves that the srNMPs have the bidirectional differentiation potential of the neurons and the mesoderm.

2. suspension culture differentiation experiments of srNMPs:

the srNMPs are digested to form single cells, and then the single cells are subcultured in a low adsorption culture plate under the condition of N2B27 basic culture medium. The srNMPs are cultured in suspension to form cell balls which can be cultured in vitro for a long time.

Cell pellets cultured in suspension for four months were sectioned by paraffin embedding and H & E stained. It was observed that there were dense nuclei, fibrous prominent nerve tissue with a crotch-like distribution, cartilage tissue with a deep blue stained cytoplasmic portion with large gaps between the cartilage capsule and the cells, and skeletal muscle tissue with bright red cytoplasm and nuclear translocation, as well as fibroblasts and fibroblasts rich in wavy collagen fibers with light red color (FIG. 2C). The presence of nerve and cartilage tissue in the cell sphere was further confirmed by the dark brown color of nerve fibers with glycine-silver staining, light red-fast green color of cartilage tissue with light red color and light blue color of collagen fibers with MASSON staining (fig. 2D). The molecular marker NEUN of mature neurons in the nerve tissue is detected to be positive by immunohistochemical staining, the molecular marker S-100 of astrocytes is detected to be positive in the periphery of the nerve tissue, and the skeletal muscle transcription factor MYOD is detected to be positive in the skeletal muscle tissue. The above results indicate that the cell balls cultured in suspension for four months contained astrocytes and skeletal muscle cells in addition to neurons (FIG. 2E).

Each tissue in the cell pellet cultured in suspension for eight months developed more mature, appearing with more complex tissue structures, similar to the spine in early embryos (fig. 2F). The cell pellet cultured in suspension for eight months was frozen and sectioned, and the expression of mature neuron molecular marker NEUN, skeletal muscle cell molecular marker DESMIN, and cartilage molecular marker COL2A1 in the corresponding region was detected to be positive by immunofluorescence staining, and the nerve tissue, skeletal muscle tissue, and cartilage tissue in the cell pellet were verified from the protein level (FIG. 2G, FIG. 2H).

Example 3: neural directed differentiation of srNMPs

1. spontaneous differentiation of srNMPs into the neural direction

srNMPs were subjected to neural differentiation induction in neural differentiation medium without any morphogen for three consecutive weeks, under the condition of B27 medium as a basis, to which 0.2. mu.M of gamma-secretase inhibitor RO4929097(EMD Chemicals), 10ng/mL BDNF (PeproTech, 450-02), and 10ng/mL GDNF (PeproTech, 450-10) were added, and this neuronal differentiation condition was designated as BGRO (FIG. 3C).

The neurons differentiated under BGRO conditions from srNMPs expressed a broad spectrum of neuronal markers TUJ1, MAP2, and HOXB8, HOXB9 of the HOX family (fig. 3D, fig. 3E), indicating inductionThe posterior neuron still maintains its localization at the posterior spinal cord. The V0 molecular markers DBX1 and PAX2 were expressed positively and the glutamate transporter gene SLC1A1 was expressed positively by RT-PCR (FIG. 3E), which indicates that srNMPs can spontaneously generate excitatory glutamatergic neurons. Further immunofluorescence staining results show that neurons generated by srNMPs differentiated under BGRO conditions can differentiate into NEUN positive mature neurons, and V0 is detected_GPositive expression of the cellular molecular markers TACR1 (tachykinin receptor 1, also known as NK1R), SST (somatostatin), glutamatergic neuronal molecular marker vGLUT2 (vesicular glutamate transporter 2) (fig. 3F).

In conclusion, after spontaneous neural differentiation of srNMPs, the neurons produced were mainly glutamatergic and were associated with V0 in the spinal cord_GThe genetic phenotype of the cells is consistent.

2. srNMPs can be directionally differentiated into spinal ventral motor neurons

srNMPs were treated for one week ventrally using SHH protein, SAG (SHH agonist), and Trans retinoic acid RA in combination, and the culture conditions were N2B27 medium as the basis, and 10ng/ml of SHH (R & D, 8908-SH), 0.2. mu.M SAG (Selleck, S7779), and 1ng/ml of Trans retinoic acid RA (Sigma, PHR1187) were added, and this condition was designated as SSR. Further culturing the ventral srNMPs for about 3 weeks using a neuron differentiation medium to form differentiated mature neurons, and detecting changes in the relevant molecular markers. The neuron differentiation medium was based on B27 medium, supplemented with 5. mu.M Forskolin (Selleck, S2449), 10ng/mL BDNF, 10ng/mL GDNF, and the neuron differentiation conditions were designated as BGF (FIG. 4A).

The quantitative PCR results showed that after one week of ventral induction, the mesodermal molecular marker T was no longer expressed, while the motor neuron precursor cell molecular marker OLIG2 increased significantly, indicating that srNMPs were induced into motor neuron progenitor cells after ventral induction. Continued differentiation resulted in motor neurons that highly expressed the motor neuron critical transcription factor ISL1, as well as acetylcholine transferase ChAT and the vesicular acetylcholine transporter vAChT (fig. 4B). Immunofluorescence staining results show that the motor neurons produced by srNMPs differentiation express the general nerve molecule markers TUJ1 and MAP2, and the motor neuron molecule marker ISL1 is also detected and can be co-expressed with ChAT and vAChT (FIG. 4C).

The above results indicate that srNMPs can be transformed into OLIG2 positive motor precursor cells in response to induction of a ventral signaling and further cultured in vitro to produce mature motor neurons.

3. srNMPs can be directionally differentiated into spinal cord dorsal interneurons

WNT and BMP signal pathways are activated simultaneously, and srNMPs are subjected to dorsal induction treatment for one week. The culture conditions were based on N2B27 medium, supplemented with 3. mu.M CHIR99021, 20ng/ml BMP4(Gibco, PHC9531), and recorded as B20C3, and finally differentiated for 2-3 weeks using BGF neuronal differentiation medium (FIG. 5A).

Quantitative PCR results and immunofluorescent staining results showed that srNMPs before induction, i.e. highly expressed transcription factors PAX3, PAX7, indicated that untreated srNMPs were localized on the dorsal side. One week after induction of srNMPs in dorsal culture conditions, expression of the dorsal transcription factors PAX3, PAX7 was elevated. The differentiated mature neurons highly express the dorsal molecular marker PAX3, PAX7, LHX5 and the neuronal molecular marker ATOH1 (also called MATH1) of pd1 region. This indicates that the neurons induced by dorsal chemo-treatment were mainly pd1 neurons (fig. 5B, fig. 5C).

The above experimental results demonstrate that srNMPs can be directionally differentiated into neurons and can respond to signals of dorsoventral axis to further differentiate to generate neurons of dorsal and ventral spinal cords.

Example 4: skeletal muscle differentiation of srNMPs

And WNT and FGF signal channels are activated simultaneously, so that srNMPs are differentiated to mesoderm. The culture conditions were C10F40, which was obtained by adding 10. mu.M CHIR99021 and 40ng/ml FGF2 to N2B27 medium. The results of quantitative PCR showed that simultaneous activation of WNT and FGF signaling pathways could differentiate srNMPs into mesoderm, down-regulate expression of neural stem cell markers SOX2 and PAX6, and up-regulate expression of both mesoderm markers T, TBX6 and MSGN1 (fig. 6A).

The mesoderm differentiated srNMPs were further differentiated into skeletal muscle using muscle differentiation medium for three weeks. The culture conditions were based on N2B27 medium supplemented with 10ng/ml IGF1(Gibco, PHG0071) and 10ng/ml HGF (Gibco, PHG 0254). Immunofluorescent staining results showed that the molecular markers of muscle cells, DESMIN, MF20, TTN, MYOG and PAX7, were all expressed positively, indicating that srNMPs differentiated in the mesoderm direction can be further differentiated to generate skeletal muscle cells (FIG. 6B).

The above experimental results demonstrate that srNMPs can differentiate towards the mesoderm and further differentiate to give skeletal muscle cells.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Claims (9)

1. A culture system for inducing human pluripotent stem cells into neuromesodermal progenitors in vitro and maintaining self-renewal, comprising a basal medium and 0.01-500 μ M TGF beta inhibitor, 0.01-500 μ M BMP inhibitor, 0.01-50 μ M GSK3 inhibitor, 0.01-500ng/mL FGF family growth factor, 0.01-500ng/mL EGF family growth factor added in the basal medium.

2. The culture system for inducing human pluripotent stem cells into neuromesodermal progenitors and maintaining self-renewal in vitro according to claim 1, wherein the culture system comprises:

wherein the basic culture medium comprises DMEM/F12 liquid basic culture medium, 0-5 xB 27 additive, 0-5 xN 2 additive, 1% penicillin/streptomycin, and 0.01-500 mug/mL 2-phosphoric acid-vitamin C;

the GSK3 inhibitor comprises one or more of CHIR99021, BIO and LY 2090314;

TGF beta inhibitors include one or more of SB431542, LY2109761 and A-83-01;

BMP inhibitors include one or more of DMH1, K02288, LDN-193189, Noggin;

the EGF family growth factor comprises one or more of EGF, TGF alpha and HB-EGF;

the FGF family growth factor comprises any one or more of FGF 1 to FGF 23.

3. The culture system for inducing human pluripotent stem cells into neuromesodermal progenitors and maintaining self-renewal in vitro according to claim 2, wherein the culture system comprises:

wherein the amount of B27 additive is 0.5 XB 27 additive, the amount of N2 additive is 0.5 XN 2 additive, and the concentration of 2-phospho-vitamin C is 60 μ g/mL.

4. The culture system for inducing human pluripotent stem cells into neuromesodermal progenitors and maintaining self-renewal in vitro according to claim 2, wherein the culture system comprises:

wherein the TGF beta inhibitor is 2 mu M SB431542, the BMP inhibitor is 2 mu M DMH1, the GSK3 inhibitor is 3 mu M CHIR99021, the EGF family growth factor is 10ng/mL EGF, and the FGF family growth factor is 10ng/mL FGF 2.

5. The culture system for inducing human pluripotent stem cells into neuromesodermal progenitors and maintaining self-renewal in vitro according to claim 1, wherein the culture system comprises:

wherein, the human pluripotent stem cell is an embryonic stem cell line derived from a human embryonic inner cell mass or an induced pluripotent stem cell obtained by reprogramming a somatic cell.

6. A method of inducing the production of neuromesodermal progenitor cells and maintaining self-renewal thereof using the culture system of any one of claims 1 to 5, comprising the steps of:

A. culturing human pluripotent stem cells by adopting a human pluripotent stem cell culture medium until the cell density is 50-70%;

B. when the density of human pluripotent stem cells reaches 50% -70%, the culture system is used for cell induction, liquid is changed half a day, cell passage is carried out according to the ratio of 1:6 when the cell density reaches 90%, a non-muscle myosin IIATPase inhibitor is added into the culture system during subculture, and continuous induction is carried out for more than 10 days.

7. The method of inducing the production and maintenance of self-renewal of neuromesodermal progenitor cells as claimed in claim 6, wherein:

wherein, in the step A, the components of the culture medium of the human pluripotent stem cells are as follows: basal medium DMEM/F12, 0.5 XN 2, 0.5 XB 27, 60. mu.g/mL 2-phospho-vitamin C, 1% penicillin/streptomycin, 40ng/mL basic fibroblast growth factor, 2ng/mL transforming growth factor-beta.

8. The method of inducing the production and maintenance of self-renewal of neuromesodermal progenitor cells as claimed in claim 6, wherein:

wherein, in step B, the non-muscle myosin IIATPase inhibitor is 5 μ M Blebbistatin.

9. Use of the neuromesodermal progenitor cells obtained by the method according to claim 6 in the study of the mechanism of generation and self-renewal of neuromesodermal progenitor cells, in vitro drug screening for spinal cord injury diseases, cells for treating spinal cord injury diseases, and tissue engineering for repairing spinal cord injury.

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