Composition for inducing reprogramming of mesenchymal stem cells and application thereof

Abstract

The invention relates to a composition for inducing reprogramming of mesenchymal stem cells and application of the composition, and belongs to the technical field of biological medicine. According to the present invention, it is firstly found that the composition of CHIR99021, Y27632, SB431542 and purmorphamine can induce the reprogramming of mesenchymal stem cells into cholinergic motor neurons and glutamic acid energy interneurons, the cholinergic motor neurons are mainly adopted, and the induction efficiency of the composition is the highest; and inoculating the mesenchymal stem cells onto the silk fibroin scaffold, adding CYSP for induced culture, and finding that the induced neuron-like cells wrap the periphery of silk fibroin fibers to form a transplantable neuron network. When the neural network is transplanted into a spinal cord injury rat model, the deposition of collagen in a spinal cord injury cavity of a rat transplanted with the neural network is reduced, host neurons are saved, nerve fiber regeneration is promoted, and the injured spinal cord is repaired.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a composition for inducing mesenchymal stem cell reprogramming and its application. Background Art

[0002] Spinal cord injury (SCI) often causes patients to experience pathological symptoms such as limited sensory / motor function, sphincter dysfunction, and abnormal muscle tone. Over the past few decades, the global incidence of SCI has gradually increased from 236 cases per million to 1298 cases per million, and the number of SCI patients worldwide remains between 250,000 and 500,000 cases per year. Permanent functional and neurological deficits and limited regenerative capacity after SCI are attributed to many secondary cellular and molecular responses, such as neuronal death, axonal loss, demyelination, inflammation, necrosis, glial scarring, and neurotrophic responses. Currently, clinical treatment options are limited (such as surgical decompression, medication, hyperbaric oxygen, etc.), and the results are often unsatisfactory. The high disability rate and long treatment cycle of SCI increase the life burden of patients, seriously affect the quality of life of patients, and pose a huge challenge to clinical treatment.

[0003] Transplantation of mesenchymal stem cells (MSCs) is a promising therapeutic strategy favored by researchers. Its main mechanisms include neuronal protection, immunomodulation, promotion of axonal regeneration, neurotransmission formation, and remyelination. Although many studies have demonstrated that it can improve spinal cord function to some extent, transplanted MSCs rarely differentiate into neurons at the lesion site, failing to truly replace lost neurons and rebuild the neurotransmission network. In recent years, in vitro neuronal reprogramming of MSCs has been extensively studied, including small molecule induction, transcription factor and / or miRNA transduction, and exosome delivery. In existing studies, the use of seven small molecule compounds to induce dermal fibroblast reprogramming into neurons effectively avoids the high tumor risk, transplant rejection, and ethical issues associated with the introduction of foreign genes. Furthermore, this method offers advantages such as rapidity, high efficiency, reversibility, and high permeability. However, while this induction method promotes spinal cord repair, it does not yield functionally specific neurons, and the mechanism of transdifferentiation remains to be fully elucidated. In another study, four additional small molecules were used to efficiently induce the differentiation of human umbilical cord mesenchymal stem cells into dopaminergic neurons, promoting functional recovery in Parkinson's disease model mice after transplantation. However, there are currently no reports on the use of small molecules to induce neuronal reprogramming of MSCs for the treatment of SCI. Summary of the Invention

[0004] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a method for treating SCI by using a small molecule composition to induce mesenchymal stem cells to perform neuronal reprogramming.

[0005] To solve the above technical problems, the present invention provides a composition for inducing mesenchymal stem cell reprogramming and its application. The present invention first discovered that a composition of CHIR99021, Y27632, SB431542 and purmorphamine (hereinafter referred to as "CYSP") can induce mesenchymal stem cells to reprogram into cholinergic motor neurons and glutamatergic interneurons, with cholinergic motor neurons as the main ones, and the induction efficiency of the composition is the highest. After that, the present invention further constructed a silk fibroin scaffold with a compression modulus of about 45.6kPa, and after mesenchymal stem cells were seeded on the silk fibroin scaffold and CYSP was added for induction culture. It was found that the induced neuron-like cells were wrapped around the silk fibroin fibers to form a transplantable neuronal network. This neuronal network was transplanted into a rat model of spinal cord injury, and it was found that the deposition of collagen in the damaged cavity of the rats transplanted with the neural network was reduced, thereby significantly reducing the area of ​​spinal cord cavitation, and was able to significantly inhibit glial epilepsy scars, save host neurons and promote nerve fiber regeneration to promote the repair of SCI.

[0006] The first object of the present invention is to provide a method for inducing mesenchymal stem cell reprogramming, the method comprising: inoculating mesenchymal stem cells into a culture carrier containing a neuron induction medium, and then adding a composition for induction culture; wherein the composition includes a GSK-3β inhibitor CHIR99021, a ROCK inhibitor Y27632, a TGF-β inhibitor SB431542 and a smooth muscle receptor agonist purmorphamine, and the composition induces the reprogramming of mesenchymal stem cells into cholinergic motor neurons and glutamatergic interneurons.

[0007] Furthermore, the culture carrier comprises a silk fibroin scaffold or a culture dish coated with poly-D-lysine, wherein the silk fibroin scaffold is prepared by physically shearing and freeze-drying tussah silk.

[0008] Furthermore, the silk fibroin scaffold is prepared by degumming tussah silk and then physically shearing it for 30 minutes to obtain a nanofiber slurry, pouring the nanofiber slurry into a polytetrafluoroethylene mold and freeze-drying it to obtain a silk fibroin scaffold; wherein the physical shearing speed is 30,000-40,000 r / min. Because the effects of the physical shearing and freeze-drying processes on the molecular structure of silk fibroin are negligible, the silk fibroin scaffold prepared by the present invention is primarily a β-sheet structure. The silk fibroin scaffold prepared by the present invention has a biomimetic nanofiber structure, a 3D porous structure, and suitable mechanical properties, making it suitable for in vitro cell attachment and in vivo nerve regeneration.

[0009] Furthermore, the concentration ratio of CHIR99021:Y27632:SB431542:purmorphamine is (1-5):(1-5):(1-5):(1-5).

[0010] Furthermore, the neuron induction medium is selected from one or more of insulin, transferrin, sodium selenite, progesterone, putrescine, basic fibroblast growth factor, valproic acid, vitamin C and forskolin.

[0011] The second object of the present invention is to provide a composition for inducing mesenchymal stem cell reprogramming, which comprises a GSK-3β inhibitor CHIR99021, a ROCK inhibitor Y27632, a TGF-β inhibitor SB431542 and a smoothened receptor agonist purmorphamine, and the composition induces mesenchymal stem cells to reprogram into cholinergic motor neurons and glutamatergic interneurons.

[0012] Furthermore, the mesenchymal stem cells are derived from bone marrow.

[0013] The third object of the present invention is to provide a use of the above composition in preparing a spinal cord injury treatment product, wherein the spinal cord injury treatment product further comprises a silk fibroin scaffold.

[0014] Furthermore, after SCI occurs, when stem cells are directly transplanted in situ, most stem cells will die rapidly due to the inflammatory microenvironment formed by local damage and the occupation of a large number of glial scars. Within 1 hour after transplantation, disordered migration of stem cells around the transplanted site can be observed. The use of tissue engineering methods to prepare biomimetic scaffolds can well ensure the long-term survival of cells in the injured area. Cells and scaffolds together construct the microenvironment required for spinal cord repair, which has a better promoting effect on SCI treatment. Silk fibroin (SF) nanofibers are artificial nanofiber scaffolds made by electrospinning technology. As a natural protein, it is a good extracellular matrix with excellent biocompatibility, cell adhesion sites and biodegradability.

[0015] Furthermore, the compression modulus of the silk fibroin scaffold is 40kPa-50kPa. When the compression modulus of the silk fibroin scaffold is 40kPa-50kPa, it is close to the reported compression modulus of the spinal cord. Therefore, the silk fibroin scaffold (BSS) of the present invention has a biomimetic nanofiber structure, a 3D porous structure and appropriate mechanical properties, and is suitable for in vitro cell attachment and in vivo nerve regeneration.

[0016] Furthermore, the spinal cord injury treatment product can inhibit glial scarring and induce mesenchymal stem cells to reprogram into cholinergic motor neurons and glutamatergic interneurons.

[0017] Furthermore, the spinal cord injury treatment product inhibits the precipitation of collagen in the damaged cavity caused by spinal cord injury.

[0018] Beneficial effects of the present invention:

[0019] The present invention first discovered that CYSP can induce mesenchymal stem cells to reprogram into cholinergic motor neurons and glutamatergic interneurons, with cholinergic motor neurons as the main ones, and the induction efficiency of the composition is the highest. After that, the present invention further constructed a silk fibroin scaffold with a compression modulus of about 45.6kPa, and after inoculating mesenchymal stem cells on the silk fibroin scaffold, CYSP was added for induction culture. It was found that the induced neuron-like cells were wrapped around the silk fibroin fibers to form a transplantable neuronal network. This neuronal network was transplanted into a rat model of spinal cord injury, and it was found that the deposition of collagen in the damaged cavity of the rats transplanted with the neural network was reduced, thereby reducing the area of ​​spinal cord cavitation, and was able to significantly inhibit glial epileptic scars, rescue host neurons, promote nerve fiber regeneration, and thus achieve repair of the damaged spinal cord. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0021] Figure 1 Figure 1 is the result of CYSP-induced differentiation of rMSCs into neuron-like cells; (A) Identification of rMSCs; (B) Neuron-specific marker DCX promoter reporter system, red indicates mCherry expression, scale bar = 100 μm; (C) Phase contrast images of rMSCs (left) and iN (induced neuron-like cells) (right) after 7 days of induction, scale bar = 100 μm; (D) Expression of Tubb3 and mature neuron marker Map2 (red) after 7 days of induction; (E) The positive rates of Tubb3 and Map2 were 88.7±1.6% and 84.4±0.8%, respectively, scale bar = 250 μm; (F) Expression of functional neuronal synaptic proteins Synapsin-1 (green), Tubb3 (red), and Hoechst (blue) after 14 days of induction, scale bar = 100 μm; **p < 0.01;

[0022] Figure 2 This is a diagram of the screening results of different small molecule compositions;

[0023] Figure 3Figure 3. Transcriptome sequencing analysis of the mechanism of CYSP-induced rMSCs differentiation into neuron-like cells (7 days); (A) Schematic diagram of CYSP-induced transdifferentiation; (B) Volcano plot of differentially expressed genes; (C) KEGG pathway enrichment analysis of differentially expressed genes; (D) Heat map showing a significant decrease in the expression of genes related to cell proliferation and a significant increase in the expression of genes related to synapse formation and transmission and neuronal development; (EF) GO enrichment analysis of differentially expressed genes; (G) GSEA analysis showing significantly upregulated signaling pathways (HIF1a, JAK / STAT, and PI3K / AKT) and a significantly downregulated signaling pathway (Hippo);

[0024] Figure 4 CYSP induces the differentiation of rMSCs into motor neurons and glutamatergic neurons; (AC) Western blot analysis of HIF1a, JAK / STAT, PI3K / AKT, and Hippo signaling pathway activities in iNs after 7 days of differentiation Blot verification; (D) Transcriptome data enrichment showed a significant increase in the expression of the cholinergic motor neuron-specific neurotransmitter Ache and receptor Chrm4; (E) Immunofluorescence staining showed that differentiated iNs expressed the motor neuron-specific transcription factor Islet1 (green) and the choline acetyltransferase CHAT (green), co-labeled with Tubb3 (red), Hoechst (blue), scale bar = 50 μm; (F) Transcriptome data enrichment showed an increase in the expression of the glutamatergic neuron-specific transporters Slc1a1 and Slc17a7 (also known as vGlut1); (G) Immunofluorescence staining showed that differentiated iNs expressed vGlut1 (green), co-labeled with Tubb3 (red), Hoechst (blue), scale bar = 50 μm; (H) The proportion of Islet1 or vGlut1-positive cells among Tubb3-positive cells; (I) Differentiation-induced iNs did not express the GABARAP-specific marker of GABAergic neurons, *p < 0.05, **p < 0.01;

[0025] Figure 5BSS supports the transdifferentiation of rMSCs into neuron-like cells; (A) Photograph and SEM image of degummed silk fibroin fibers; (B) Photograph and SEM image of BSS; (C) Compressive stress-strain curve; (D) FTIR spectrum of BSS; (E) rMSCs seeded on BSS were double-stained with TRITC-phalloidin (red) and Hoechst (blue), scale bar = 50 μm; (F) Compared with the 2D-PDL-rMSCs group in each state, the rMSCs on the first day of culture were significantly different from those in the 2D-PDL-rMSCs group. Proliferation of rMSCs on BSS on days 7, 2, and 3. Data represent SEM ± mean of at least three independent experiments; (G) SEM images of rMSCs (left) or iNs (right) on BSS, showing that iNs were distributed along the scaffold or spread as long processes; (H) iNs expressed GFP and Tubb3 on day 7, scale bar = 50 μm; (I) Neuron-like cells induced on BSS expressed the mature neuronal marker NeuN and were also positive for the motor neuron marker CHAT, **p < 0.01;

[0026] Figure 6 BSS+iNs transplantation significantly promotes motor recovery and injury repair in SCI rats. (A) Representative images of hindlimb movement in SCI rats under four different treatment strategies. (B) Basso, Beattie, and Bresnahan (BBB) ​​scores showed that hindlimb motor function was significantly improved after neural scaffold transplantation. Compared with the SCI group, *p<0.05, **p<0.01; compared with the BSS group, #p<0.05, ##p<0.01, indicating statistically significant differences. (C) Hindlimb motor evoked potentials (MEPs) were obtained in the Sham, SCI, BSS, and BSS+iNs groups by electrophysiological analysis. (DG) Eight weeks after treatment, H&E and Masson staining showed that the area of ​​spinal cord cavitation and collagen deposition in the injured area was significantly reduced in the BSS+iNs group. Scale bar = 1 mm. **p<0.01, ##p<0.01 indicate statistically significant differences, while ns indicates no statistically significant differences.

[0027] Figure 7BSS+iNs transplantation significantly promoted nerve regeneration. (A, E) Immunofluorescence staining showed that compared with the SCI and BSS groups, the BSS+iNs group showed a large number of NF-H (red)-positive areas with a fibrous distribution near the injury site 8 weeks after treatment. Hoechst (blue), scale bar = 1 mm; (B, F) The BSS+iNs group showed an increase in the number of GAP43-positive (red) cells near the injury site, indicating an increase in the formation of newly formed nerve fibers. Hoechst (blue), scale bar = 1 mm; (C, G) The BSS+iNs group significantly enhanced the survival rate of host neurons, such as NeuN ( (D, H) BSS+iNs group significantly inhibited glial scar formation, as shown by reduced GFAP (red) staining, Hoechst (blue), scale bar = 1 mm; (I) Regenerated nerve fibers were observed in and around the transplanted area as shown by NF-H (red) staining, Hoechst (blue), scale bar = 50 μm; (JK) Transplanted iNs were positively stained for NeuN, Islet1, and CHAT, indicating that they survived, differentiated, and integrated into the host spinal cord tissue, Hoechst (blue), scale bar = 50 μm. DETAILED DESCRIPTION

[0028] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0029] Example 1: Isolation, culture and identification of rMSCs

[0030] rMSCs were obtained from 6-week-old Sprague-Dawley rats (100-150 g) purchased from the Experimental Animal Center of Soochow University. The use of animals was approved by the Veterinary Bureau of Soochow University. Cells were isolated from the total bone marrow of rat tibia and femur using density gradient centrifugation (1.073 g / mL) and incubated at 37°C in an atmosphere of 95% air and 5% CO2. They were supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin-streptomycin, and 2 mM L-glutamine (Gibco). The culture medium was changed every 72 hours. After 7-10 days, when the confluence reached 80%-90%, rMSCs were collected using 0.25% trypsin-EDTA solution (Gibco) and passaged at a ratio of 1:2. All experiments were performed using passages 3 to 10. The expression of CD markers (CD105, CD29, CD34, and CD45) was analyzed using flow cytometry (BD eBioscience) to characterize the expanded rMSCs in culture.

[0031] MSCs-derived neurons are good candidates for neuronal replacement therapy and are used as an important treatment for repairing neurological diseases. First, primary rat rMSCs derived from bone marrow were cultured. Their specific surface markers CD105 and CD29 were positive, while CD34 and CD45 (hematopoietic stem cell markers also derived from bone marrow) were negative ( Figure 1 A). When cultured in vitro, rMSCs exhibit a spindle-shaped or oval appearance similar to fibroblasts ( Figure 1 C).

[0032] Example 2: Screening of small molecule compositions (CYSP)

[0033] In order to find small molecules that can effectively differentiate rMSCs into neuron-like cells, this example uses a DCX promoter reporter system ( Figure 1 B) to enable DCX monitoring (a neuron-specific marker). Various small molecules that play a role in neuronal transdifferentiation were screened using rMSCs-DCX::mCherry, including Y27632, SB431542, CHIR99021, LDN193189, SP600125, DAPT, SU5402, VPA, Forskolin, ISX9, I-BET151, etc. (see Figure 2 ). It was found that the "cocktail" model CYSP (CHIR99021, Y27632, SB431542 and Purmorphamine) could initiate the transformation of rMSCs into neuronal-like cell fate and produced the highest induction efficiency (Tubb3 and Map2 positive rates were 88.7±1.6% and 84.4±0.8%, respectively, Figure 1 CE), as indicated by mCherry expression ( Figure 1 CD), cells that underwent mesenchymal-like to neuronal-like transformation were all red and expressed mCherry. In addition, after 14 days, these cells expressed functional neuronal synapse-related protein Synapsin-1 ( Figure 1 F). The above results demonstrate that the "cocktail" model CYSP can effectively reprogram rMSCs directly into neuron-like cells.

[0034] Example 3: Chemical induction of rMSCs into neuron-like cells

[0035] (1) Chemical induction of rMSCs into neuron-like cells: rMSCs were seeded onto 35 mm culture dishes coated with PDL (poly-D-lysine) or BSS. When the cells reached confluence, the culture medium was replaced with a neuronal induction medium containing N3 medium (F12 + 25 μg / mL insulin, 50 μg / mL transferrin, 30 nM sodium selenite, 20 nM progesterone, 100 nM putrescine, 10 ng / mL bFGF, 0.5 mMVPA, 50 μM Vc, 50 μM forskolin). The small molecule composition of Example 2 was added to the neuronal induction medium to achieve the following final concentrations: 3 μM CHIR99021, 5 μM Y27632, 2 μM SB431542, and 1 μM purmorphamine; the culture medium was replaced every two days. After 7 days, the neuronal induction medium was replaced with maturation medium supplemented with 20 ng / mL BDNF, 20 ng / mL NT3, and 20 ng / mL GDNF to promote neuronal maturation. After 14 days, neurons (iNs) derived from induced mesenchymal stem cells were cultured in neuronal maturation medium without bFGF and small molecules. Half of the neuronal maturation medium was replaced every other day.

[0036] (2) Cell adhesion and morphology were observed using confocal microscopy and scanning electron microscopy (SEM). Confocal microscopy (FV10 inverted microscope, Olympus, Japan) was used to examine cell adhesion. After washing the cells three times with PBS, they were fixed in 4% paraformaldehyde for 1 hour. Subsequently, the cells were permeabilized with 0.1% Triton X-100 and incubated with TRITC-phalloidin (1:2,000, Invitrogen) at room temperature for 2 hours and then stained with DAPI (Sigma) for 15 minutes. Representative fluorescence images of the stained samples were obtained using a confocal laser scanning microscope. For SEM, the scaffolds with seeded cells were washed three times with PBS. Each sample was fixed in 4% paraformaldehyde for 30 minutes, then dehydrated through an ethanol gradient and examined with a 5kV SEM after gold sputtering.

[0037] (3) CCK8 assay was used to evaluate the effect of the scaffold on cell growth. In short, a common culture dish (2D-PDL) was used as a control. A total of 100 μL of reaction solution (10 μL CCK-8 and 90 μL DMEM) was transferred to a 96-well plate. The absorbance at 450 nm was measured using a microplate reader (BioTek) at different time points (1, 2, and 3 days) during the culture process. Each experimental condition was performed in triplicate (n=3). Three independent experiments were performed, and each experiment was repeated twice.

[0038] (4) Immunofluorescence staining of iN. Cells cultured with 2D-PDL or BSS were fixed in 4% paraformaldehyde (PFA) for 30 minutes, washed three times with PBS, and then permeabilized with 0.2% Triton X-100 for 50 minutes. After washing three times with PBS, the cells were blocked with 1% BSA for 1 hour at room temperature. The primary antibody was incubated for 2 hours at room temperature or overnight at 4°C. Subsequently, the cells were washed three times with PBS (10 minutes each time) and then incubated with the secondary antibody. Alexa Fluor 594 goat anti-mouse or rabbit IgG (H+L) (Invitrogen) was diluted 1:200 with PBS / 0.02% NaN3 / 3% BSA and incubated for 1 hour in the dark. The cells were then washed three times with PBS (10 minutes each time) and nuclear visualization was performed with Hoechst33258 (1:100 dilution; Sigma-Aldrich). Fluorescence was examined using CLSM and a Leica DMI 6000B microscope (Germany).

[0039] (5) RNA sequencing. Samples were collected on day 0 and day 7 after differentiation for RNA extraction, library preparation, and quality control. RNA sequencing (RNA-seq) libraries were constructed using the Illumina NovaSeq 6000 system provided by Guangzhou Kidio Biotechnology Co., Ltd. The RNA-Seq data were then used for gene set enrichment analysis (GSEA). For differentially expressed genes, gene ontology (GO) and KEGG analysis were performed using DAVID (https: / / david.ncifcrf.gov).

[0040] The direct transdifferentiation of cells across germ layers (mesenchymal stem cells to neuron-like cells) is a complex and delicate process. To explore the possible mechanism, RNA sequencing was used to study the potential molecular mechanism of the small molecule combination CYSP inducing the transdifferentiation of rMSCs into neuron-like cells for 7 days ( Figure 3 A). Volcano plot depicts the number of differentially expressed genes during transdifferentiation, including 1947 upregulated and 1919 downregulated genes ( Figure 3 B). KEGG enrichment analysis found that signaling pathways closely related to neuron development and differentiation were significantly enriched, including MAPK, Hippo, and P13K / Akt ( Figure 3 C). We further used heat maps and GO analysis to identify up-regulated and down-regulated genes to explain the differentially expressed genes. Notably, we found that genes related to nervous system development, neuronal differentiation, neuronal development, synapse formation and transmission were significantly up-regulated, while genes related to cell proliferation and cell cycle were significantly down-regulated ( Figure 3DF). In addition to specifically targeting their signaling pathways, small molecules can also affect the crosstalk of multiple signaling pathways. Different combinations may lead to changes in neuronal subtypes and conversion efficiency.

[0041] (6) Construction and transduction of lentiviral vectors. The rat DCX promoter (NM_053379) and the fluorescent protein mCherry were linked to the lentiviral vector to evaluate the expression of DCX and the efficiency of differentiation into neuronal cells. The lentiviral vector was packaged using a three-plasmid expression system, including the packaging plasmid pMD2.G (Addgene plasmid 12259) and psPAX2 (Addgene plasmid 12260), and transiently co-transfected into HEK293T cells to produce lentiviral particles. The supernatant containing lentivirus was collected 48 hours and 72 hours after transfection. The viral supernatant was centrifuged at 4500×g for 10 minutes, filtered through a 0.45 μm low protein binding filter membrane (Millipore, Bedford, Massachusetts, USA), and concentrated by ultracentrifugation. rMSCs at approximately 70% confluence were transduced with lentiviral vectors at a multiplicity of infection (MOI) of approximately 10, mixed with polybrene (Sigma-Aldrich) to a final concentration of 4 μg / mL, and incubated for 24 hours. The cells were then incubated overnight in fresh growth medium and cultured at an air-liquid interface 48 hours after transduction for functional studies. After induction with small molecules, the efficiency of promoter activation was assessed using confocal microscopy and flow cytometry.

[0042] To elucidate the neuronal cell types induced by small molecule compounds in rMSCs, transcriptome sequencing results showed that the expression of cholinergic motor neuron-specific neurotransmitter Ache and its receptor Chrm4 increased significantly on the 7th day of differentiation induction ( Figure 4 D). Further immunofluorescence staining also showed that the differentiated neuron-like cells were positive for Islet1 (a motor neuron-specific transcription factor) and choline acetyltransferase (CHAT), and co-labeled with Tubb3 ( Figure 4 E). These results indicate the presence of motor neurons in differentiated neuron-like cells. Furthermore, the expression of glutamatergic neuron-specific transporters Slc1a1 and Slc17a7 (also known as vGlut1) was significantly increased in the induced neuron-like cells ( Figure 4 F). Some cells were positive for vGlut1 in immunofluorescence staining and co-labeled with Tubb3 ( Figure 4 G), indicating the presence of glutamatergic neurons in differentiated neuron-like cells. Notably, statistical analysis of the two types of neurons showed that Islet1 accounted for 68.6±5.9% of Tubb3-positive cells, while vGlut1 accounted for 24.2±6.0% of Tubb3-positive cells ( Figure 4H), and neither expressed GABARAP ( Figure 4 I). In summary, the results indicate that the small molecule mixture CYSP has the potential to induce rMSCs to differentiate into cholinergic motor neurons and glutamatergic interneurons, among which cholinergic motor neurons are the main type.

[0043] (7) Western blot analysis: Cells were lysed using a protein extraction reagent and the lysates were collected. Equal amounts of protein lysates were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, the membranes were probed with primary antibodies overnight at 4°C and then incubated with HRP-conjugated secondary antibodies for 1 hour at room temperature. The primary antibodies used were anti-phospho-YAP, anti-YAP, anti-phospho-TAZ, anti-TAZ, anti-phospho-JAK2, anti-JAK2, anti-phospho-STAT3, anti-STAT3, anti-phospho-AKT, anti-AKT, anti-HIF1α, and β-actin (Cell Signaling Technology).

[0044] GSEA analysis found that the HIF1a, JAK / STAT, and PI3K / AKT signaling pathways were significantly activated, while the Hippo signaling pathway was inhibited after transdifferentiation. To further verify the changes in key signaling pathways, we used Western blot analysis to detect the levels of HIF1a, JAK / STAT, PI3K / AKT, and Hippo signaling pathways in iNs 7 days after differentiation. Compared with the control group (DMSO treatment), we observed several significant changes. The phosphorylation of Yap, a key protein in the Hippo signaling pathway, was significantly reduced, while the phosphorylation of TAZ was also reduced, and the total protein level of Yap increased ( Figure 4 A). In the JAK / STAT pathway, the protein level of the key component JAK2 was significantly increased, and both the phosphorylation and total protein levels of STAT3 were significantly increased ( Figure 4 B). In addition, in the PI3K / AKT pathway, the phosphorylation of AKT was significantly increased. Finally, the expression of HIF1α, a key transcription factor in the HIF1α signaling pathway, was significantly upregulated ( Figure 4 C). These results further confirmed that Hippo signaling was significantly inhibited, while HIF1α, JAK / STAT, and PI3K / AKT signaling pathways were significantly activated. Therefore, combined with transcriptome sequencing results, the small molecule cocktail CYSP effectively inhibited cell proliferation and the phenotype of rMSCs, and by regulating multiple signaling pathways to mediate the expression of genes involved in neuronal differentiation, effectively promoting the reprogramming of rMSCs into neuronal-like cells.

[0045] Example 4: Preparation and characterization of biomimetic silk scaffold (BSS)

[0046] (1) Preparation of BSS: Tussah silk (Haian County Suhao Silk Co., Ltd.) was boiled three times in a 0.5 wt.% Na2CO3 solution and then thoroughly rinsed with deionized water to remove sericin. The degummed silk was cut into 5 mm long fibers. These silk fibers were physically sheared at a speed of 32,000 r / min for 30 min in a high-speed shearing machine (Jiuyang, Shandong, China) to obtain nanofiber slurry. The slurry was poured into a polytetrafluoroethylene (PTFE) mold, placed in liquid nitrogen, and freeze-dried to prepare BSS.

[0047] (2) Characterization of BSS: The morphology of BSS was observed using a scanning electron microscope (Regulus 8100, Hitachi, Japan) at 3.0 kV and 7.0 kV. The secondary structure of the biomimetic silk scaffold was analyzed by Fourier transform infrared spectroscopy (Nicolet 5700, Thermo Scientific, USA), where the wavelength range of 400–4000 cm -1 64 scans were performed over a wavenumber range of 100 nm. The mechanical properties of the BSS were measured using an Instron 5967 universal materials testing machine (Boston, USA). Five scaffolds were compressed in each group to characterize the scaffolds.

[0048] Silk fibroin scaffolds can effectively repair peripheral and central nervous system injuries, making them ideal scaffolds for neural tissue engineering. The natural extracellular matrix (ECM) in neural tissue engineering scaffolds is very important, as it can regulate cell behavior and tissue growth. This example developed a biomimetic scaffold BSS with a 3D silk fibroin nanofiber structure. Microfibers are composed of many nanoscale fibrils, such as Figure 5 A. This example develops a top-down method to obtain silk nanofibrils by physical shearing. These nanofibrils are then aggregated by freeze drying to form a 3D porous scaffold ( Figure 5 B). Since the effects on the molecular structure of silk fibroin during physical shearing and freeze-drying are negligible, the prepared scaffold is mainly a β-sheet structure ( Figure 5 C) To achieve optimal nerve regeneration, the scaffold must mimic the mechanical properties of neural tissue. The BSS showed a compression modulus of 45.6 kPa, similar to the reported spinal cord compression modulus of 40.1 kPa. Therefore, the silk scaffold prepared in this example possesses a biomimetic nanofiber structure, a 3D porous structure, and suitable mechanical properties, making it suitable for in vitro cell attachment and in vivo nerve regeneration.

[0049] Example 5: Biocompatibility of biomimetic silk scaffold (BSS)

[0050] To evaluate the biocompatibility of BSS, rMSCs were seeded in BSS and cultured for 2 days. Cell morphology was analyzed by immunofluorescence staining of cytoskeleton F-actin. The results showed that BSS with nanofibrous structure served as an excellent extracellular matrix, providing a richer spatial structure. The rMSCs indicated by cytoskeleton staining were aligned along the direction of the scaffold and extended protrusions to contact adjacent cells ( Figure 5 E). CCK-8 assay results showed that the three-dimensional culture environment provided by BSS was more conducive to the proliferation of rMSCs than the two-dimensional culture of PDL coating, providing more sufficient space. After 3 days of culture, the number of cells was significantly higher than that of rMSCs in the culture dish coated with PDL ( Figure 5 F).

[0051] Subsequently, we used CYSP to induce neural differentiation of rMSCs seeded on the material. After 7 days of induction, SEM showed that the cells in the CYSP group exhibited neuronal morphology, extending long processes and interweaving into a network on the nanofiber structure compared with undifferentiated rMSCs (DMSO). Figure 5 G). Immunofluorescence staining showed that a large number of rMSCs transformed into neuron-like cells (Tubb3 positive), and the connections between rMSC-derived neuron-like cells were established through long processes or connections between long processes and adjacent cell bodies ( Figure 5 H). In addition, the induced neuron-like cells were wrapped around the silk fibroin fibers, and their processes were distributed along the local filamentous topography of the scaffold. At the same time, these successfully induced neuron-like cells can express the mature neuron marker NeuN and the motor neuron marker CHAT ( Figure 5 These results demonstrate that BSS has excellent biocompatibility and facilitates the adhesion and proliferation of rMSCs. More importantly, BSS can support the transdifferentiation of rMSCs into neuron-like cells and the formation of transplantable neuronal networks.

[0052] Example 6: Spinal Cord Transsection and Transplantation

[0053] (1) Spinal cord transection and transplantation. Female SD rats (220-250 g) were anesthetized with intraperitoneal injection of chloral hydrate. After determining the surgical segment at the T9-T10 level, the skin was prepared with a razor, the surgical area was disinfected, and a dorsal laminectomy was performed at the T9-T10 level using a fine-tip bite to fully expose the spinal cord. A 2 mm spinal cord segment was completely removed at the T10 level. Hemostasis was achieved with gelatin sponge, and the incision was sutured. The animals were divided into 4 groups (10 rats per group): the Sham group underwent laminectomy as a control; the SCI group underwent spinal cord injury without transplantation; the BSS group received only BSS scaffolds, and the BSS+iNs group received BSS neural network structures (2 mm in length and 3 mm in diameter) that were differentiated into neurons induced by CYSP. Penicillin was used to prevent infection after surgery, and manual bladder emptying was performed every day.

[0054] (2) Behavioral assessment. Using a double-blind method, the examiner performed a 21-point Basso, Beattie, Bresnahan (BBB) ​​motor score on the experimental animals at designated time points for up to 8 weeks to assess hindlimb motor function, including joint movement, stepping ability, coordination, and overall mobility. At least six rats were tested per treatment group, and each treatment course was tested three times.

[0055] (3) Tissue processing, H&E and Masson staining, and immunofluorescence labeling. After anesthesia, rats were perfused with 0.9% NaCl. The dissected spinal cord segments were fixed in 4% paraformaldehyde (PFA) at 4°C for 48 hours and then dehydrated in 30% (w / v) sucrose solution. The spinal cord segments containing the injured area were then excised, approximately 2 cm long, the dura mater was carefully removed, and embedded in OCT. 25 μm thick sections were cut using a cryostat (Leica CM1900) and mounted on gelatin-coated slides. Five sections from each area were stained with neurofilament-H (NF-H), GAP-43, NeuN, or GFAP and imaged using a 50x Leica microscope. Sagittal panoramic images of the spinal cord were created and exported using the Autopano Giga v3.0.0 software program (Kolor, Lyon, France).

[0056] (4) Hematoxylin and eosin (H&E) staining and Masson staining. Immunofluorescence labeling: Sections were first incubated in PBS containing 5% BSA and 0.3% Triton X-100 at room temperature for 1 hour, and then incubated with mouse anti-NF-H (1:200 dilution; Cell Signaling Technologies). Hoechst 33258 (1:100 dilution; Sigma-Aldrich) was used. Fluorescence micrographs were captured using a confocal microscope, and the stained areas in different tissue regions were measured using NIH ImageJ (v.1.51) software.

[0057] (5) Electrophysiological testing of rat hindlimbs. The electrophysiological activity of the rat hindlimbs was assessed 8 weeks after SCI. After anesthetizing the rats, bipolar stimulating electrodes were buried behind the head and neck, and recording electrodes were placed in the gastrocnemius muscles of the hindlimbs. An electromyogram / evoked potential meter (Nicolet, USA) was connected to detect the electromyogram of the rat hindlimbs. The peak amplitude and latency of the motor evoked potential (MEP) of the rats in each group were recorded and compared.

[0058] (6) Statistical analysis. All results are expressed as mean ± SEM. Statistical comparisons of the mean values ​​of each group were performed by t-test or analysis of variance followed by the Bonferroni-Dunn multiple comparison test (GraphPad Prism 7). *, #, P < 0.05; **, ##, P < 0.01 were considered significant.

[0059] To establish the SCI model, 8-week-old SD rats underwent complete spinal cord transection at the T9-T10 segment, and a 2mm spinal cord tissue segment was removed. The BSS neural network structure (BSS+iNs) carrying CYSP-induced neurons was transplanted into the SCI injury site. The BBB score was used to evaluate the hind limb motor function at different time points (0d, 1d, 1w, 2w, 4w, 6w, 8w). One day after modeling, the hind limb paws of the model group rats were everted and dragged forward, with a score of 0, while the rats in the sham group showed normal ( Figure 6 A). The SCI group showed partial self-recovery 1 week after modeling, but the score was still low, with only occasional slight movement of the hind limb joints. By the end of 8 weeks, the score was still within 5 points, with only hind limb joint movement. In contrast, the BSS+iNs group showed a significantly higher BBB score than the SCI and BSS groups 1 week after transplantation. Over time, the recovery of hind limb motor function was significantly enhanced, reaching 12.0±0.6 points, as shown by more frequent weight-bearing movements of the paws and occasional coordinated movements of the forelimbs and hindlimbs ( Figure 6B). The BSS transplantation group alone also showed a significantly higher BBB score than the SCI group after 4 weeks, but the score (8.3±0.8) was still significantly lower than that of the BSS+iNs group. Electrophysiological activity of the hind limbs showed that the peak amplitude of the gastrocnemius motor evoked potential in the BSS+iNs group was significantly higher than that in the SCI and BSS groups, indicating the effectiveness of nerve conduction after electrical stimulation ( Figure 6 C). H&E staining showed that 8 weeks after injury (SCI group), a large area of ​​cavitation formed at the injury site and extended into normal tissue. However, in the BSS+iNs group, the transplantation of the biomimetic material BSS and the neural network structure significantly reduced the area of ​​cavitation in the injured spinal cord cavity, not only filling the gap but also inhibiting further expansion of the injury. The implanted neurons in the BSS further inhibited the expansion of the injury and promoted regeneration ( Figure 6 DE). Masson staining showed extensive collagen deposition at the injury site. After neural network structure transplantation (BSS+iNs group), collagen deposition was significantly improved ( Figure 6 The results showed that the neural network structure can effectively fill the damaged cavity and inhibit collagen deposition, which is beneficial to the connection of the neural network.

[0060] Rebuilding neural network connections is the key to SCI repair. NF-H tissue immunofluorescence staining showed that 8 weeks after SCI, there was less regeneration of nerve fibers in the injured area, and they were distributed in a dotted pattern in the glial scar area. In contrast, the transplantation of biomimetic silk fibroin scaffolds and silk fibroin neural network structures carrying CYFP-induced iNs significantly promoted the regeneration of nerve fibers. A large number of fibrous NF-H regenerations were observed near the injured area ( Figure 7 A). Transplanted iNs labeled with GFP (located within the neural network or migrating to the surrounding tissue) also showed NF-H positivity, indicating the survival of the transplanted cells and the formation of NF-H-indicated neurofilament bridges with uninjured tissue areas ( Figure 7 A, E). At the same time, the positive increase in GAP43 (an indicator of newly formed nerve fibers) also indicates that the transplantation of BSS and BSS+iN plays an important role in the functional repair and regeneration of the nervous system ( Figure 7 B, F). To further investigate neuronal survival in the spinal cord, immunofluorescence staining for NeuN, a specific marker for mature neurons, was performed. The results showed that BSS and BSS+iN transplantation significantly promoted host neuronal survival, with iN transplantation further improving neuronal survival. Furthermore, transplanted iNs were positive for NeuN, Islet1, and CHAT, indicating that they survived transplantation, integrated well into the host spinal cord tissue, and had the potential to differentiate into motor neuron-like cells in vivo. Figure 7C, G, J, and K). Furthermore, immunofluorescence staining for GFAP, a specific marker for glial scarring, revealed that transplantation of BSS and BSS+iNs significantly inhibited its expression and suppressed glial scar formation. These results suggest that transplantation of the spinal cord biomimetic scaffold BSS promotes SCI repair by inhibiting glial scarring, rescuing host neurons, and promoting nerve fiber regeneration. The transplanted iNs not only survived and differentiated but also established a neural network, further promoting nerve fiber regeneration and neuronal survival at the injury site, elucidating the mechanism by which they promote recovery of hindlimb motor function in SCI rats.

[0061] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for inducing mesenchymal stem cell reprogramming, characterized in that: The method comprises: inoculating mesenchymal stem cells into a culture carrier containing a neuron induction medium, and then adding a composition for induction culture; wherein the composition comprises a GSK-3β inhibitor CHIR99021, a ROCK inhibitor Y27632, a TGF-β inhibitor SB431542, and a smooth muscle receptor agonist purmorphamine, and the composition induces the reprogramming of mesenchymal stem cells into cholinergic motor neurons and glutamatergic interneurons.

2. The method according to claim 1, characterized in that The culture carrier comprises a silk fibroin scaffold or a culture dish coated with poly-D-lysine, wherein the silk fibroin scaffold is prepared by physically shearing and freeze-drying tussah silk.

3. The method according to claim 1, characterized in that The concentration ratio of CHIR99021:Y27632:SB431542:purmorphamine is (1-5):(1-5):(1-5):(1-5).

4. The method according to claim 1, wherein The neuron induction medium is prepared by adding auxiliary materials to F12 medium, and the auxiliary materials are selected from one or more of insulin, transferrin, sodium selenite, progesterone, putrescine, basic fibroblast growth factor, valproic acid, vitamin C and forskolin.

5. A composition for inducing mesenchymal stem cell reprogramming, characterized in that: The composition comprises a GSK-3β inhibitor CHIR99021, a ROCK inhibitor Y27632, a TGF-β inhibitor SB431542 and a smoothened receptor agonist purmorphamine. The composition induces mesenchymal stem cells to reprogram into cholinergic motor neurons and glutamatergic interneurons.

6. The composition according to claim 5, characterized in that The mesenchymal stem cells are derived from bone marrow.

7. Use of the composition according to claim 5 or 6 in preparing a product for treating spinal cord injury, characterized in that: The spinal cord injury treatment product also includes a silk fibroin scaffold.

8. The use according to claim 7, characterized in that The compression modulus of the silk fibroin scaffold is 40 kPa-50 kPa.

9. The use according to claim 7, characterized in that The spinal cord injury treatment product can inhibit glial scarring and induce mesenchymal stem cells to reprogram into cholinergic motor neurons and glutamatergic interneurons.

10. The use according to claim 7, characterized in that The spinal cord injury treatment product inhibits the precipitation of collagen in the damaged cavity caused by spinal cord injury.

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