Method for repairing spinal cord injury by inducing spinal cord GABAergic neural precursor cells through human pluripotent stem cells and application

Abstract

The invention provides a method for producing functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells, which successfully realizes directional differentiation of the human pluripotent stem cells by adopting a feeder-layer-free culture method based on a three-stage in-vitro induced differentiation strategy. The invention further provides application of the cells obtained through the method in treatment of spinal cord injury, a novel strategy based on the functional spinal cord GABAergic neural precursor cells is provided, a new thought and means are provided for treatment of spinal cord injury, and stem cell treatment is expected to proceed towards clinical precision and industrial application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and in particular relates to a method for repairing spinal cord injury by human pluripotent stem cell induced spinal cord GABAergic neural precursor cells and application thereof. BACKGROUND

[0002] Most of the existing stem cell differentiation culture techniques use embryoid body (EB) method and monolayer method. Embryoid body EB method is a classical embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC) differentiation method, which simulates embryonic development process by aggregating cells to form embryoid bodies, thereby inducing cells to differentiate into multiple cell types. Monolayer method is to culture cells in monolayer form on culture substrate, and guide cells to differentiate in a specific direction by adding specific growth factors or induction factors. In monolayer method, feeder layer culture method is a traditional embryonic stem cell culture method, which uses treated feeder layer cells (such as mouse embryonic fibroblasts) to provide support and nutrition for stem cells and maintain their undifferentiated state.

[0003] With the development of technology, feeder-free culture method has been developed, which avoids the use of feeder layer cells by using specific culture medium and substrate, simplifies the culture process and reduces the cost. More importantly, animal-free feeder-free culture method completely avoids the use of animal-derived components, which not only reduces the risk of pathogen contamination, but also is more suitable for clinical application.

[0004] Spinal cord injury (SCI) is a common traumatic disease, commonly seen in young male population. It is one of the main causes of disability, and the number of patients in chronic phase is relatively large in clinic. Spinal cord injury often causes chronic motor dysfunction or even paralysis, accompanied by complications such as muscle spasm and pain, which often further aggravate motor dysfunction. At present, there is no effective method to repair damaged spinal cord, and existing therapies include stabilizing the disease, surgical decompression, blood pressure management, drug treatment and rehabilitation training, etc.

[0005] In recent years, cell transplantation has been proposed as a potential therapeutic strategy for repairing damaged spinal cord. However, the cavity region formed after spinal cord injury is usually accompanied by complex pathological processes such as inflammation, apoptosis and oxidative stress. This microenvironment is not conducive to the survival and differentiation of transplanted cells, and may even be toxic to transplanted cells, making it difficult for transplanted cells to effectively colonize and function in the cavity region.

[0006] It has been shown that the subacute phase (2 weeks to 3 months after injury) after spinal cord injury is the best time window for cell transplantation. However, the formation of cavities is usually more obvious in the chronic phase, and the microenvironment for transplanted cells is more complex at this time, making treatment more difficult. Therefore, it is urgent to develop a new treatment strategy to effectively address the various dysfunctions caused by traumatic spinal cord injury in the subacute and chronic phases, including pain, spasticity, motor and autonomic dysfunction, etc. SUMMARY

[0007] In view of the problems existing in the prior art, the purpose of the present application is to provide a technology for directing inducing functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells, and specifically discloses a method for repairing spinal cord injury using functional spinal cord GABAergic neural precursor cells and applications thereof.

[0008] Specifically, the present application relates to the following aspects:

[0009] 1. A method for producing functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells, comprising:

[0010] differentiating and culturing the human pluripotent stem cells to obtain spinal cord neuroepithelial cells;

[0011] differentiating and culturing the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells;

[0012] differentiating and culturing the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells;

[0013] wherein, in the step of differentiating and culturing human pluripotent stem cells to obtain spinal cord neuroepithelial cells, the marker for obtaining the spinal cord neuroepithelial cells is determined by satisfying the following conditions:

[0014] flow cytometry detection of differentiated spinal cord neuroepithelial cells shows that the SOX1 positive rate is at least 90%, preferably at least 95%;

[0015] wherein, the method is a feeder-free culture method.

[0016] 2. The method of item 1, wherein, in the step of differentiating and culturing human pluripotent stem cells to obtain spinal cord neuroepithelial cells, comprising:

[0017] differentiating and culturing the human pluripotent stem cells in a one-stage differentiation culture medium containing a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, and a TGF-β signaling pathway inhibitor to obtain the spinal cord neuroepithelial cells;

[0018] Preferably, the Wnt signaling pathway agonist is CHIR99021, the BMP signaling pathway inhibitor is DMH1, and the TGF-β signaling pathway inhibitor is SB431542.

[0019] Further preferably, 1 μM-10 μM CHIR99021, 1 μM-10 μM DMH1, and 1 μM-10 μM SB431542 are contained in the one-stage differentiation medium.

[0020] Further preferably, the one-stage differentiation medium further comprises a NIM basal medium, which is a medium comprising 45%-50% DMEM / F12, 45%-50% Neurobasal, 0.5%-4% N-2 supplement, and 0.5%-4% NEAA.

[0021] 3. The method according to item 2, wherein the seeding density of the human pluripotent stem cells is 20,000 cells / cm 2 - 40,000 cells / cm 2 ;

[0022] Preferably, the human pluripotent stem cells are subjected to differentiation culture for 5-8 days in the one-stage differentiation medium to obtain the spinal cord neuroepithelial cells.

[0023] 4. The method according to any one of items 1-3, wherein in the step of subjecting the spinal cord neuroepithelial cells to differentiation culture to obtain early spinal cord GABAergic neural precursor cells, the marker for obtaining the early spinal cord GABAergic neural precursor cells is determined by satisfying the following condition:

[0024] The differentiated early spinal cord GABAergic neural precursor cells are subjected to flow cytometry detection, and the double positive rate of ASCL1 and HoxB4 is at least 90%, preferably at least 95%.

[0025] 5. The method according to item 4, wherein in the step of subjecting the spinal cord neuroepithelial cells to differentiation culture to obtain early spinal cord GABAergic neural precursor cells, the method comprises:

[0026] subjecting the spinal cord neuroepithelial cells to differentiation culture in a two-stage differentiation medium comprising a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, a TGF-β signaling pathway inhibitor, a retinoic acid signaling agonist, an SHH signaling inhibitor, and a B27 supplement to obtain the early spinal cord GABAergic neural precursor cells;

[0027] Preferably, the Wnt signaling pathway agonist is CHIR99021, the BMP signaling pathway inhibitor is DMH1, the TGF-β signaling pathway inhibitor is SB431542, the retinoic acid signaling agonist is retinoic acid, and the SHH signaling inhibitor is cyclopamine.

[0028] Further preferably, 1 μM-10 μM CHIR99021, 1 μM-10 μM DMH1, 1 μM-10 μM SB431542, 0.01 μM-2 μM retinoic acid, 0.1 μM-2 μM cyclopamine, and 1x B27 supplement are contained in the two-stage differentiation medium.

[0029] Further preferably, the two-stage differentiation medium further comprises a NIM basal medium, which is a medium comprising 45%-50% DMEM / F12, 45%-50% Neurobasal, 0.5%-4% N-2 supplement, and 0.5%-4% NEAA.

[0030] 6. The method of item 5, wherein the seeding density of the spinal cord neuroepithelial cells is 20,000 cells / cm 2 - 40,000 cells / cm 2 ;

[0031] Preferably, the spinal cord neuroepithelial cells are subjected to a 5-8 day differentiation culture in the two-stage differentiation medium to obtain the early spinal cord GABAergic neural precursor cells.

[0032] 7. The method of any one of items 1-6, wherein in the step of culturing the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells, the marker for obtaining the functional spinal cord GABAergic neural precursor cells is determined by satisfying the following condition:

[0033] The differentiated functional spinal cord GABAergic neural precursor cells are subjected to flow cytometry detection, and the PTF1A positive rate is at least 90%.

[0034] 8. The method of item 7, wherein in the step of culturing the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells, the method comprises:

[0035] The early spinal cord GABAergic neural precursor cells are subjected to a differentiation culture in a three-stage differentiation medium comprising a retinoic acid signaling agonist, a SHH signaling inhibitor, and a B27 supplement to obtain the functional spinal cord GABAergic neural precursor cells.

[0036] Preferably, the retinoic acid signaling agonist is retinoic acid, and the SHH signaling inhibitor is cyclopamine.

[0037] Further preferably, the three-stage differentiation medium comprises 0.01 μM-2 μM retinoic acid, 0.1 μM-2 μM cyclopamine, and 1x B27 supplement.

[0038] Further preferably, the three-stage differentiation medium further comprises NIM basal medium, which is a medium comprising 45%-50% DMEM / F12, 45%-50% Neurobasal, 0.5%-4% N-2 supplement, and 0.5%-4% NEAA.

[0039] 9. The method of item 8, wherein the early spinal cord GABAergic neural precursor cells are seeded at a density of 200,000 cells / mL-400,000 cells / mL.

[0040] Preferably, the early spinal cord GABAergic neural precursor cells are differentiated in the three-stage differentiation medium for 5-8 days to obtain the functional spinal cord GABAergic neural precursor cells.

[0041] 10. The method of any one of items 1-9, wherein the method further comprises:

[0042] The human pluripotent stem cells are subjected to expansion treatment before being subjected to differentiation culture.

[0043] 11. The method of any one of items 1-10, wherein the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

[0044] 12. A composition comprising the functional spinal cord GABAergic neural precursor cells produced by the method of any one of items 1-11.

[0045] 13. The composition of item 12, wherein the composition further comprises any one or two or more of fibrinogen, BDNF, VEGF, and MDL28170.

[0046] Preferably, the composition further comprises thrombin.

[0047] 14. The composition of item 12 or 13, wherein the functional spinal cord GABAergic neural precursor cells are present at a concentration of 2 x 10 4 -2 x 10 6 .

[0048] 15. A medical product comprising the composition of any one of items 12-14.

[0049] 16. The medical product according to item 15, wherein the medical product is a drug or a medical device.

[0050] Preferably, the medical device is a biomaterial scaffold, a gel, or a bio-ink for 3D printing.

[0051] 17. Use of the functional spinal cord GABAergic neural precursor cells produced according to the method of any one of items 1-11, or the composition according to any one of items 12-14, in the manufacture of a medicament for treating a spinal cord injury.

[0052] 18. The use according to item 17, wherein the spinal cord injury is a traumatic spinal cord injury.

[0053] Preferably, the traumatic spinal cord injury is a complete injury or a total transection injury.

[0054] Further preferably, the treating a spinal cord injury comprises promoting synapse formation after a spinal cord injury, promoting angiogenesis, relieving pain, relieving muscle spasm, or improving bladder function.

[0055] Beneficial effects:

[0056] (1) Based on the industrial GMP-level production mode, the present application successfully differentiates human iPSCs into spinal cord dorsal dI4 / dIL A region-specific GABAergic neural precursor cells using a three-stage in vitro induction and differentiation strategy, and high-purity and high-yield cells are obtained at each stage of differentiation. In the entire differentiation process, the present application uses a feeder-free monolayer culture method, which is different from the traditional embryoid body EB method, and uses all animal-free small molecules and reagents to ensure the safety and applicability in clinical applications. In addition, due to the similarity in biological characteristics between human iPSCs and human ESCs, the differentiation scheme of the present application is also applicable to human ESCs.

[0057] (2) For traumatic spinal cord injury (including subacute and chronic periods), the present application develops a composition for treating spinal cord injury, which can effectively improve sensory (such as pain and spasm), motor dysfunction, and autonomic dysfunction (such as bladder dysfunction) caused by spinal cord injury through cell transplantation technology, while effectively repairing damaged neural circuits and blood-spinal cord barriers, promoting axonal regeneration, synapse formation, and angiogenesis. The present application provides a new treatment strategy based on functional spinal cord GABAergic neural precursor cells for the treatment of spinal cord injury, which is expected to promote the clinical precision and industrial application of stem cell therapy. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figures 1A-1BResults of flow cytometry detection of human iPSC phenotype. Among them, Figure 1A Results of detection of the proportion of Nanog and OCT4 positive cells; Figure 1B Results of detection of the proportion of SSEA4 and TRA-1-60 positive cells. E1 / E2 / Q5 is a circle gate strategy, E1 gate represents the main cell colony, E2 gate represents the cell colony of E1 gate after being removed of adhesion; T1 and T2 represent two different detection index experiments of the same sample; C1 and C2 represent the corresponding negative control groups.

[0059] Figure 2 Results of flow cytometry detection of the phenotype of spinal cord neuroepithelial cells at the first stage of differentiation. E1 / E2 / R3 is a circle gate strategy, E1 gate represents the main cell colony, E2 gate represents the cell colony of E1 gate after being removed of adhesion, and R3 represents the proportion of SOX1 positive cells of the adhesion-removed cells; test represents the cell group dyed by adding anti-SOX1 antibody; UN represents the corresponding negative control group.

[0060] Figure 3 Results of flow cytometry detection of the phenotype of cells at the second stage of differentiation. E1 / E2 / Q3 is a circle gate strategy, E1 gate represents the main cell colony, and E2 gate represents the cell colony of E1 gate after being removed of adhesion; test represents the cell group dyed by adding anti-ASCL1 and anti-HoxB4 antibodies; UN represents the corresponding negative control group.

[0061] Figure 4 Results of flow cytometry detection of the phenotype of cells at the third stage of differentiation. E1 / E2 / R3 is a circle gate strategy, E1 gate represents the main cell colony, E2 gate represents the cell colony of E1 gate after being removed of adhesion, and R3 represents the proportion of PTF1A positive cells of the adhesion-removed cells; test represents the cell group dyed by adding anti-PTF1A antibody; UN represents the corresponding negative control group.

[0062] Figures 5A-5B Results of detection of synapse formation at 24 weeks after transplantation of functional spinal cord GABAergic neural precursor cells. Among them, Figure 5A Results of detection of synapse formation between human neurons; Figure 5B Results of detection of synapse formation between human neurons and rat host neurons; the arrow indicates synapse formation.

[0063] Figures 6A-6C Results of detection of angiogenesis at 24 weeks after transplantation of functional spinal cord GABAergic neural precursor cells. Among them, Figure 6A Results of immunofluorescence staining of RECA-1 and laminin expression; Figure 6B Results of immunofluorescence staining of RECA-1 and collagen type IV expression; Figure 6CFigure 6 shows the results of immunofluorescent staining for RECA-1 and PDGFRβ expression.

[0064] Figures 7A-7D Figure 7 shows the effects of transplantation of functional spinal cord GABAergic neural precursor cells on pain after transplantation. In this figure, Figure 7A Figure 8 shows the results of detection of the mechanical withdrawal threshold of the hind paw of rats within 24 weeks after transplantation. Figure 7B Figure 9 shows the results of statistics of the mechanical withdrawal threshold of the hind paw of rats at 24 weeks after transplantation. Figure 7C Figure 10 shows the results of detection of the thermal withdrawal latency of the hind paw of rats within 24 weeks after transplantation. Figure 7D Figure 11 shows the results of statistics of the thermal withdrawal latency of the hind paw of rats at 24 weeks after transplantation. The medium group represents the control group injected with solvent without cells; the dl4 group represents the group of transplantation of functional spinal cord GABAergic neural precursor cells. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0065] Figures 8A-8B Figure 12 shows the effects of transplantation of functional spinal cord GABAergic neural precursor cells on improvement of bladder function after transplantation. In this figure, Figure 8A Figure 13 shows the results of evaluation of the urination ability of rats after transplantation. Figure 8B Figure 14 shows the results of detection of the residual urine volume in the bladder of rats after transplantation. The medium group represents the control group injected with solvent without cells; the dl4 group represents the group of transplantation of functional spinal cord GABAergic neural precursor cells. *p<0.05, **p<0.01. DETAILED DESCRIPTION

[0066] The present application is further illustrated by the following examples, which are not to be construed as limiting the application.

[0067] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are described below. However, if there is a conflict between the definitions in this specification and those in the materials, methods, and examples, the definition in this specification controls. Unless otherwise indicated, materials, methods, and examples are illustrative only and not intended to be limiting.

[0068] DEFINITIONS

[0069] As used herein, the term "multipotent stem cell" or "pluripotent stem cell" refers to a class of stem cells with unlimited self-renewal capacity and multilineage differentiation potential, which can differentiate into cell types of all three germ layers (endoderm, mesoderm, ectoderm) of the human body. It includes embryonic stem cells (ESC) and induced pluripotent stem cells (iPSC).

[0070] As used herein, the term "about" or "approximately," when applied to one or more values of interest, means values that are similar to a specified reference value or within an acceptable error range of a specified value, as determined by one of ordinary skill in the art, which will depend at least in part on how the value is measured or determined, e.g., the limitations of the measurement system. In certain aspects, the term "about" means a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the recited reference value, unless otherwise stated or otherwise evident from context (unless the number exceeds 100%), unless the number of digits in the reference value is otherwise specified. Alternatively, "about" can mean within less than 3 or more than 3 standard deviations, per practices in the art. Alternatively, the term "about" can mean within an order of magnitude, preferably within 5-fold and more preferably within 2-fold, of a value.

[0071] As used herein, the terms "comprising", "including", "containing", are interchangeable and are meant to encompass both the open and the semi-closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0072] As used herein, the term "DMEM / F12" is a mixture of two culture media Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Medium, which is commercially available.

[0073] As used herein, the term "N-2 supplement" and its non-commercially available formulation, referred to as "hormone mix", means a hormone mix comprising transferrin, insulin, putrescine, selenium and progesterone. For example, the N-2 supplement can comprise 10 mg / ml transferrin, 2.5 mg / ml insulin, 1 mg / ml putrescine, 1 ul / ml 15 selenium, 1 ul / ml progesterone. The N-2 supplement can be commercially available or can be prepared.

[0074] As used herein, the term "SB431542" also includes SB431542 and salts thereof, particularly pharmaceutically acceptable salts. SB431542 is a TGF-beta receptor kinase inhibitor, SB-431542 has inhibitory effects on ALK4, ALK5 and ALK7 activities with IC50 values of 1 µM, 0.75 µM and 2 µM, respectively, and its chemical structural formula is (CAS No.: 301836-41-9):

[0075]

[0076] As used herein, the term "CHIR99021" is an aminopyrimidine derivative, which is a potent and selective inhibitor of glycogen synthase kinase 3 (GSK-3). CHIR99021 has IC50 values of 6.7 nM and 10 nM for the inhibition of GSK-3β and GSK-3α, respectively. GSK-3 is a serine / threonine kinase and a key inhibitor of the Wnt signaling pathway, so CHIR99021 activates the Wnt / β-catenin signaling pathway by inhibiting GSK-3β and GSK-3α. In addition, CHIR99021 also shows selectivity for other kinases, which is more than 500 times selective for GSK-3 than for CDC2, ERK2 and other protein kinases. CHIR99021 can induce autophagy and enhance the self-renewal capacity of mouse and human embryonic stem cells. CHIR99021 plays an important role in various biological processes such as stem cell culture, maintenance and self-renewal, reprogramming and differentiation, and its molecular structure is:

[0077]

[0078] As used herein, the term "DMH1" is a selective BMP-1 receptor inhibitor, and its molecular structure is:

[0079]

[0080] As used herein, the term "retinoic acid (RA)" is also known as tretinoin, which is a metabolite of vitamin A in the body. Current research suggests that the biological activity of vitamin A is closely related to retinoic acid. Two active forms of retinoic acid are of great interest, namely all-trans retinoic acid (ATRA, RA) and 9-cis retinoic acid.

[0081] As used herein, the term "Cyclopamine" is an antagonist of the Hedgehog pathway with an IC50 of 46 nM in cell experiments, and is also a selective Smoothened ("Smo") inhibitor, and its molecular structure is as follows:

[0082]

[0083] As used herein, the term "SHH" refers to Sonic Hedgehog, Hedgehog (HH) is an evolutionarily highly conserved gene family, which in mammals includes three homologous genes: Sonic Hedgehog (SHH), Desert Hedgehog (DHH) and Indian Hedgehog (IHH). Among them, the SHH signaling pathway plays a very important role in the process of tissue and organ formation.

[0084] As used herein, the term "B27" is a nutrient factor containing many added ingredients, which is often used for primary culture of neural stem cells.

[0085] As used herein, the term "Neurobasal" is a basal medium developed to meet the special needs of neural cell culture.

[0086] As used herein, the term "spinal cord injury" refers to a disease caused by damage to the spinal cord due to trauma, inflammation, tumors, etc., resulting in dysfunction of motor, sensory, sphincter, autonomic nerves, etc. below the injury plane.

[0087] As used herein, the term "complete injury" also known as complete spinal cord injury, refers to a type of spinal cord injury in which motor, sensory, sphincter function is completely lost below the injury plane, suggesting that complete transverse damage occurs at the spinal cord injury plane.

[0088] As used herein, the term "incomplete injury" also known as incomplete spinal cord injury, refers to a type of spinal cord injury in which partial sensory or motor function is retained below the injury plane, suggesting that complete transverse damage does not occur at the spinal cord injury plane.

[0089] As used herein, the term "pharmaceutically acceptable carrier" is art-recognized. It refers to a pharmaceutically-acceptable material, composition or carrier, with which any subject composition, or component thereof, is combined for

[0090] Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laureate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances used in pharmaceutical formulations; and (22) natural high molecular weight biomaterial carriers.

[0091] As used herein, the term "treat" a disorder or disease means to cure as well as to ameliorate at least one symptom of the disorder or disease. Those of skill in the art will appreciate that the term "treat" means to reduce, ameliorate or maintain the symptoms of an existing medical condition in a subject after administration as compared to a subject not administered.

[0092] As used herein, the term "differentiate" refers to the process by which a pluripotent stem cell transforms into a target cell type in a particular context.

[0093] As used herein, the term "vitronectin", also known as S-protein or serum spreading factor, is a multifunctional glycoprotein found in plasma and extracellular matrix. It is composed of two single-chain glycoproteins (65 kD and 75 kD). Vitronectin binds to specific cell surface receptors such as integrins αVβ3 and αVβ5 through Arg-Gly-Asp (RGD) sequence mediation. It can promote endothelial cell adhesion, regulate cell adhesion, and play a key role in tissue remodeling; promote cell stretching and proliferation, and regulate the differentiation of various normal cells and cancer cells. In addition, vitronectin can bind to various ligands such as glycosaminoglycans, collagen, plasminogen and urokinase receptor, and stabilize the inhibitory conformation of plasminogen activator inhibitor-1. By locating in the extracellular matrix and binding to plasminogen activator inhibitor-1, vitronectin can regulate the degradation of proteins in the matrix. It also binds to complement, heparin and thrombin-antithrombin III complex, indicating that it is involved in the regulation of immune response and clot formation.

[0094] Methods of generating functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells

[0095] In a first aspect, the present application provides a method for producing functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells, comprising:

[0096] differentiating the human pluripotent stem cells to obtain spinal cord neuroepithelial cells;

[0097] differentiating the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells;

[0098] differentiating the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells.

[0099] In some embodiments, the human pluripotent stem cells are human induced pluripotent stem cells, which can be obtained commercially, for example, from Wuhan Hongchen Innovative Biotechnology Co., Ltd.

[0100] In the present application, the term "human embryonic stem cell" refers to an embryonic stem cell isolated or obtained from a human embryo that has not developed in vivo, i.e., a human embryo that is less than 14 days old after fertilization, or an established human embryonic stem cell line that is commercially available. Human embryonic stem cells are capable of dividing for long periods of time in culture without differentiating and developing into cells and tissues of the three primary germ layers. For example, the human embryonic stem cell line can be SA01 (SA001), SA02 (SA002), ES01 (HES-1), ES02 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6), BG01 (BGN-01), BG02 (BGN-02), BG03 (BGN-03), TE03 (I3), TE04 (I4), TE06 (I6), UC01 (HSF1), UC06 (HSF6), WA01 (H1), WA07 (H7), WA09 (H9), WA13 (H13), WA14 (H14), Q-2.

[0101] In the present application, the term "human induced pluripotent stem cell" can be abbreviated as "iPS" cell or "iPSC" and generally refers to a type of pluripotent stem cell that is artificially prepared from a non-pluripotent cell. The way to obtain the induced pluripotent stem cell can be to introduce specific transcription factors to reprogram a terminally differentiated somatic cell. For example, the terminally differentiated somatic cell can be a fibroblast, a hematopoietic stem cell, a muscle cell, a neuron, an epidermal cell, etc.

[0102] In some embodiments, the functional spinal cord GABAergic neural precursor cells are generated from the human pluripotent stem cells using a feeder-free culture method.

[0103] In the present application, the term "feeder-free culture method" refers to a culture method in which feeder cells are not used during the cell culture process. Traditionally, in certain types of cell culture, especially stem cell culture, feeder cells are used to provide essential growth factors, extracellular matrix, and other support conditions to maintain the undifferentiated state of stem cells or to promote the differentiation of a specific type of cell.

[0104] In some embodiments, the feeder-free culture is achieved using VTN-N (vitronectin).

[0105] Stage one differentiation, human pluripotent stem cells are differentiated into spinal cord neuroepithelial cells

[0106] In the one-stage differentiation, i.e., in the step of culturing the human pluripotent stem cells to obtain spinal cord neuroepithelial cells, the marker for determining that the spinal cord neuroepithelial cells are obtained is that the following conditions are met:

[0107] The differentiated spinal cord neuroepithelial cells are subjected to flow cytometry to detect SOX1 positivity at a rate of at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0108] The method of flow cytometry to detect SOX1 positivity is routine in the art. In some embodiments, the SOX1 positivity is at least 95%. In some embodiments, the SOX1 positivity is at least 96%. In some embodiments, the SOX1 positivity is at least 97%.

[0109] In the present application, the spinal cord neuroepithelial cells refer to a product of differentiation of a pluripotent stem cell, which can be differentiated into various types of neural lineage cells, and is a state between stem cells and adult cells, without the totipotency of stem cells.

[0110] In some embodiments, in the step of culturing the human pluripotent stem cells to obtain spinal cord neuroepithelial cells, the human pluripotent stem cells are cultured in a one-stage differentiation medium comprising a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, and a TGF-β signaling pathway inhibitor to obtain the spinal cord neuroepithelial cells.

[0111] The Wnt signaling pathway agonist can comprise a GSK-3 inhibitor. Non-limiting examples of GSK-3 inhibitors include CHIR99021, Wnt / β-catenin signaling agonist 6-bromo-iridium-3'-oxime, or a combination thereof. In some embodiments, the Wnt signaling pathway agonist is CHIR99021. In the one-stage differentiation medium, about 1 μΜ to about 10 μΜ of the Wnt signaling pathway agonist can be included, such as about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, or about 10 μΜ.

[0112] Non-limiting examples of the BMP signaling pathway inhibitors include DMH1, dorsomorphin, LDN193189, dominant negative BMP, truncated BMP receptor, soluble BMP receptor, BMP receptor-Fc chimera, noggin, follistatin, gremlin, cerberus / DAN family proteins, ventropin, high dose activin, amnionless, or a combination thereof. DMH1 blocks BMP signaling by inhibiting activin receptor-like kinase 2 (ALK2). Dorsomorphin and LDN193189 each affect Smad-dependent and Smad-independent BMP signaling triggered by BMP2, BMP6, or GDF5. In some embodiments, the BMP signaling pathway inhibitor is DMH1. In the one-stage differentiation medium, about 1 μΜ to about 10 μΜ of the BMP signaling pathway inhibitor can be included, such as about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, or about 10 μΜ.

[0113] Non-limiting examples of the TGF-β signaling pathway inhibitors include SB431542, SB505124, A83-01, or a combination thereof. SB431542 inhibits TGF-β / Activin / Nodal signaling by inhibiting activin receptor-like kinases 4, 5, and 7 (ALK4, 5, 7). SB505124 and A83-01 are additional small molecule inhibitors of activin receptor-like kinase 5 (ALK5), also known as transforming growth factor-alpha type I receptor kinase, which can also be used to inhibit TGF-β / Activin / Nodal signaling. In some embodiments, the TGF-β signaling pathway inhibitor is SB431542. In the one-stage differentiation medium, about 1 μΜ to about 10 μΜ of the TGF-β signaling pathway inhibitor can be included, such as about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, or about 10 μΜ.

[0114] In some embodiments, in the one-stage differentiation medium, about 1 μΜ to about 10 μΜ of CHIR99021, about 1 μΜ to about 10 μΜ of DMH1, and about 1 μΜ to about 10 μΜ of SB431542 are included. In some embodiments, in the one-stage differentiation medium, 3 μΜ of CHIR99021, 2 μΜ of DMH1, and 2 μΜ of SB431542 are included.

[0115] In some embodiments, the one-stage differentiation medium further comprises a NIM basal medium, which is a medium comprising 45-50% DMEM / F12, 45-50% Neurobasal, 0.5-4% N-2 supplement, 0.5-4% NEAA. For example, 45%, 46%, 47%, 48%, 49% or 50% DMEM / F12; 45%, 46%, 47%, 48%, 49% or 50% Neurobasal; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4% N-2 supplement; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4% NEAA. In some embodiments, the NIM basal medium is a medium comprising 49% DMEM / F12, 49% Neurobasal, 0.1% N-2 supplement, 0.1% NEAA.

[0116] In some embodiments, the human pluripotent stem cells are differentiation cultured in a NIM medium (comprising 49% DMEM / F12, 49% Neurobasal, 0.1% N-2 supplement, 0.1% NEAA) spiked with 3 mM CHIR99021, 2 mM DMH1 and 2 mM SB431542 to obtain the spinal cord neuroepithelial cells.

[0117] In some embodiments, the human pluripotent stem cells are seeded at a density of 20,000 cells / cm2 2 - 40,000 cells / cm2 2 , preferably at a density of 20,000 cells / cm2 2 .

[0118] The present application does not limit the specific type of culture consumables used. In the present application, the seeding density of the human pluripotent stem cells is calculated based on the area of the culture bottom, which is applicable to various types of culture consumables. If large-scale, standardized production is required according to GMP standards, consumables that meet GMP requirements, such as T25, T75 flasks, etc., can be selected to meet the production needs of clinical-grade cell products.

[0119] In some embodiments, the human pluripotent stem cells are differentiation cultured for 5-8 days in the one-stage differentiation medium to obtain the spinal cord neuroepithelial cells. For example, the human pluripotent stem cells are differentiation cultured for 5 days, 6 days, 7 days or 8 days; preferably, the human pluripotent stem cells are differentiation cultured for 6 days, 7 days or 8 days; further preferably, the human pluripotent stem cells are differentiation cultured for 7 days.

[0120] During the one-stage differentiation culture, the medium can be replaced / supplemented according to the culture condition of the human pluripotent stem cells and the content change of nutrients and cell secretions in the medium. Generally, the content of nutrients and / or cell secretions in the medium can be determined according to the color change of the medium.

[0121] Stage two differentiation, spinal cord neuroepithelial cells are differentiated into early spinal cord GABAergic neural precursor cells

[0122] In the two-stage differentiation, i.e. in the step of differentiating culture of the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells, the marker for obtaining the early spinal cord GABAergic neural precursor cells is determined by satisfying the following conditions:

[0123] The differentiated early spinal cord GABAergic neural precursor cells are subjected to flow cytometry detection, and the double positive rate of ASCL1 and HoxB4 is at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0124] The method for flow cytometry detection of the double positive rate of ASCL1 and HoxB4 is routine in the art. In some embodiments, the double positive rate of ASCL1 and HoxB4 is at least 95%. In some embodiments, the double positive rate of ASCL1 and HoxB4 is at least 96%. In some embodiments, the double positive rate of ASCL1 and HoxB4 is at least 97%. In some embodiments, the double positive rate of ASCL1 and HoxB4 is at least 98%.

[0125] In some embodiments, in the step of differentiating culture of the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells, the spinal cord neuroepithelial cells are subjected to differentiating culture in a two-stage differentiation medium comprising a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, a TGF-β signaling pathway inhibitor, a retinoic acid signaling agonist, an SHH signaling inhibitor, and a B27 supplement, to obtain the early spinal cord GABAergic neural precursor cells.

[0126] The Wnt signaling pathway agonist can comprise a GSK-3 inhibitor. Non-limiting examples of GSK-3 inhibitors include CHIR99021, Wnt / β-catenin signaling agonist 6-bromo-iridium-3'-oxime, or a combination thereof. In some embodiments, the Wnt signaling pathway agonist is CHIR99021. In the two-stage differentiation medium, about 1 μM to about 10 μM of the Wnt signaling pathway agonist can be included, such as about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, or about 10 μM.

[0127] Non-limiting examples of the BMP signaling pathway inhibitors include DMH1, dorsomorphin, LDN193189, dominant negative BMP, truncated BMP receptor, soluble BMP receptor, BMP receptor-Fc chimera, noggin, follistatin, gremlin, cerberus / DAN family proteins, ventropin, high dose activin, amnionless, or a combination thereof. In some embodiments, the BMP signaling pathway inhibitor is DMH1. In the two-stage differentiation medium, about 1 μΜ to about 10 μΜ of the BMP signaling pathway inhibitor can be included, such as about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, or about 10 μΜ.

[0128] Non-limiting examples of the TGF-β signaling pathway inhibitors include SB431542, SB505124, A83-01, or a combination thereof. In some embodiments, the TGF-β signaling pathway inhibitor is SB431542. In the two-stage differentiation medium, about 1 μΜ to about 10 μΜ of the TGF-β signaling pathway inhibitor can be included, such as about 1 μΜ, about 2 μΜ, about 3 μΜ, about 4 μΜ, about 5 μΜ, about 6 μΜ, about 7 μΜ, about 8 μΜ, about 9 μΜ, or about 10 μΜ.

[0129] Retinoic acid signaling agonists can include, for example, retinoic acid, EC23, or a combination thereof. In some embodiments, the retinoic acid is all-trans retinoic acid. Retinoic acid activates the retinoic acid signaling by binding to the nuclear hormone receptor retinoic acid receptor (RAR). In the two-stage differentiation medium, about 0.01 μΜ to about 2 μΜ of the retinoic acid signaling agonist can be included, such as about 0.01 μΜ, about 0.02 μΜ, about 0.03 μΜ, about 0.04 μΜ, about 0.05 μΜ, about 0.08 μΜ, about 0.1 μΜ, about 0.2 μΜ, about 0.3 μΜ, about 0.4 μΜ, about 0.5 μΜ, about 0.8 μΜ, about 1 μΜ, about 1.5 μΜ, or about 2 μΜ.

[0130] SHH signaling inhibitors can include, for example, cyclopamine. Cyclopamine inhibits SHH signaling by directly targeting a component of the SHH signaling pathway, Smo. Other small molecule inhibitors of Smo can also be used to inhibit SHH signaling, such as SANT-1. In the two-stage differentiation medium, about 0.1 μΜ to about 2 μΜ of the SHH signaling inhibitor can be included, such as about 0.1 μΜ, about 0.2 μΜ, about 0.3 μΜ, about 0.4 μΜ, about 0.5 μΜ, about 0.8 μΜ, about 1 μΜ, about 1.5 μΜ, or about 2 μΜ.

[0131] In some embodiments, in the two-stage differentiation medium, comprises about 1 μΜ - about 10 μΜ CHIR99021, about 1 μΜ - about 10 μΜ DMH1, about 1 μΜ - about 10 μΜ SB431542, about 0.01 μΜ - about 2 μΜ retinoic acid, about 0.1 μΜ - about 2 μΜ cyclopamine, and 1x B27 supplement. In some embodiments, in the two-stage differentiation medium, comprises 1 μΜ CHIR99021, 2 μΜ DMH1, 2 μΜ SB431542, 0.1 μΜ retinoic acid, 0.5 μΜ cyclopamine, and 1x B27 supplement.

[0132] In some embodiments, in the two-stage differentiation medium, further comprises a NIM basal medium, which is a medium comprising 45% - 50% DMEM / F12, 45% - 50% Neurobasal, 0.5% - 4% N-2 supplement, 0.5% - 4% NEAA. For example, 45%, 46%, 47%, 48%, 49%, or 50% DMEM / F12; 45%, 46%, 47%, 48%, 49%, or 50% Neurobasal; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% N-2 supplement; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% NEAA. In some embodiments, the NIM basal medium is a medium comprising 49% DMEM / F12, 49% Neurobasal, 0.1% N-2 supplement, 0.1% NEAA.

[0133] In some embodiments, the spinal cord neuroepithelial cells are differentiated in a NIM medium (comprising 49% DMEM / F12, 49% Neurobasal, 0.1% N-2 supplement, 0.1% NEAA) spiked with 1 μΜ CHIR99021, 2 μΜ DMH1, 2 μΜ SB431542, 0.1 μΜ retinoic acid, 0.5 μΜ cyclopamine, and 1x B27 supplement to obtain the early spinal cord GABAergic neural precursor cells.

[0134] In some embodiments, the spinal cord neuroepithelial cells are seeded at a density of 20000 cells / cm 2 - 40000 cells / cm 2 , preferably at a density of 30000 cells / cm 2 .

[0135] The present application is not limited to the specific type of culture consumables used. In the present application, the seeding density of the spinal cord neuroepithelial cells is calculated based on the area of the culture substrate, which is applicable to various types of culture consumables. If large-scale, standardized production is required according to GMP standards, consumables that meet GMP requirements, such as T25, T75 culture flasks, etc., can be used to meet the production needs of clinical-grade cell products.

[0136] In some embodiments, the spinal cord neuroepithelial cells are subjected to 5-8 days of differentiation culture in the two-stage differentiation medium to obtain the early spinal cord GABAergic neural precursor cells. For example, the spinal cord neuroepithelial cells are subjected to 5 days, 6 days, 7 days, or 8 days of differentiation culture; preferably, the spinal cord neuroepithelial cells are subjected to 6 days, 7 days, or 8 days of differentiation culture; further preferably, the spinal cord neuroepithelial cells are subjected to 7 days of differentiation culture.

[0137] During the two-stage differentiation culture, the medium can be replaced / supplemented according to the culture condition of the spinal cord neuroepithelial cells and the change in the content of nutrients and cell secretions in the medium. Generally, the content of nutrients and / or cell secretions in the medium can be determined according to the color change of the medium.

[0138] Stage three differentiation, early spinal cord GABAergic neural precursor cells are differentiated into functional spinal cord GABAergic neural precursor cells Expansion treatment prior to differentiation

[0139] In the three-stage differentiation, i.e., the step of subjecting the early spinal cord GABAergic neural precursor cells to differentiation culture to obtain the functional spinal cord GABAergic neural precursor cells, the criteria for determining that the functional spinal cord GABAergic neural precursor cells have been obtained are as follows:

[0140] The differentiated functional spinal cord GABAergic neural precursor cells are subjected to flow cytometry detection, and the PTF1A positive rate is at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0141] The method for flow cytometry detection of the PTF1A positive rate is routine in the art.

[0142] In some embodiments, in the step of subjecting the early spinal cord GABAergic neural precursor cells to differentiation culture to obtain the functional spinal cord GABAergic neural precursor cells, the early spinal cord GABAergic neural precursor cells are subjected to differentiation culture in a three-stage differentiation medium comprising a retinoic acid signal agonist, an SHH signal inhibitor, and a B27 supplement to obtain the functional spinal cord GABAergic neural precursor cells.

[0143] Retinoic acid signaling agonists can include, for example, retinoic acid, EC23, or a combination thereof. In some embodiments, the retinoic acid is all-trans retinoic acid. In the three-stage differentiation medium, about 0.01 μΜ to about 2 μΜ of a retinoic acid signaling agonist can be included, for example, about 0.01 μΜ, about 0.02 μΜ, about 0.03 μΜ, about 0.04 μΜ, about 0.05 μΜ, about 0.08 μΜ, about 0.1 μΜ, about 0.2 μΜ, about 0.3 μΜ, about 0.4 μΜ, about 0.5 μΜ, about 0.8 μΜ, about 1 μΜ, about 1.5 μΜ, or about 2 μΜ.

[0144] SHH signaling inhibitors can include, for example, cyclopamine. In the three-stage differentiation medium, about 0.1 μΜ to about 2 μΜ of a SHH signaling inhibitor can be included, for example, about 0.1 μΜ, about 0.2 μΜ, about 0.3 μΜ, about 0.4 μΜ, about 0.5 μΜ, about 0.8 μΜ, about 1 μΜ, about 1.5 μΜ, or about 2 μΜ.

[0145] In some embodiments, in the three-stage differentiation medium, about 0.01 μΜ to about 2 μΜ of retinoic acid, about 0.1 μΜ to about 2 μΜ of cyclopamine, and 1x B27 supplement are included. In some embodiments, in the three-stage differentiation medium, 0.1 μΜ of retinoic acid, 0.5 μΜ of cyclopamine, and 1x B27 supplement are included.

[0146] In some embodiments, in the three-stage differentiation medium, a NIM basal medium is also included, the NIM basal medium being a medium comprising 45% to 50% DMEM / F12, 45% to 50% Neurobasal, 0.5% to 4% N-2 supplement, 0.5% to 4% NEAA. For example, 45%, 46%, 47%, 48%, 49%, or 50% DMEM / F12; 45%, 46%, 47%, 48%, 49%, or 50% Neurobasal; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% N-2 supplement; 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% NEAA. In some embodiments, the NIM basal medium is a medium comprising 49% DMEM / F12, 49% Neurobasal, 0.1% N-2 supplement, 0.1% NEAA.

[0147] In some embodiments, the early spinal cord GABAergic neural precursor cells are differentiated in a NIM medium (containing 49% DMEM / F12, 49% Neurobasal, 0.1% N-2 supplement, 0.1% NEAA) supplemented with 0.1 μΜ retinoic acid, 0.5 μΜ cyclopamine and 1x B27 supplement to obtain the functional spinal cord GABAergic neural precursor cells.

[0148] In some embodiments, the early spinal cord GABAergic neural precursor cells are seeded at a density of 200,000 cells / mL to 400,000 cells / mL, preferably at a density of 300,000 cells / mL.

[0149] The present application does not limit the specific type of culture consumables used. In the present application, the seeding density of the early spinal cord GABAergic neural precursor cells is calculated in "cells / mL", which is applicable to various types of culture consumables. Specifically, the total amount of cells seeded can be adjusted according to the culture volume, so as to adapt to different culture systems. If large-scale, standardized production according to GMP standards is required, consumables that meet GMP requirements, such as T25, T75 flasks, etc., can be used to meet the production needs of clinical-grade cell products.

[0150] In some embodiments, the early spinal cord GABAergic neural precursor cells are differentiated in the three-stage differentiation medium for 5 to 8 days to obtain the functional spinal cord GABAergic neural precursor cells. For example, the early spinal cord GABAergic neural precursor cells are differentiated for 5 days, 6 days, 7 days or 8 days; preferably, the early spinal cord GABAergic neural precursor cells are differentiated for 6 days, 7 days or 8 days; further preferably, the early spinal cord GABAergic neural precursor cells are differentiated for 7 days.

[0151] During the three-stage differentiation culture, the medium can be replaced / supplemented according to the culture condition of the early spinal cord GABAergic neural precursor cells and the change in the content of nutrients and cell secretions in the medium. Generally, the content of nutrients and / or cell secretions in the medium can be determined according to the color change of the medium.

[0152] Compositions, methods of treating spinal cord injury, and uses

[0153] In the above method for producing functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells, the human pluripotent stem cells can further be subjected to expansion treatment before being differentiated; preferably, the expansion treatment is performed at least once.

[0154] In some embodiments, the expansion process is a passaging expansion, and the culturing continues for 7 days after the passaging to complete the expansion process. In some embodiments, the seeding density of the human pluripotent stem cells is 10000 cells / cm 2 .

[0155] The present application does not limit the specific type of culture consumables used. In the present application, the seeding density of the human pluripotent stem cells is calculated based on the area of the culture bottom, which is applicable to various types of culture consumables. If the production is to be scaled up and standardized according to the GMP standard, consumables that meet the GMP requirements, such as T25, T75 culture flasks, can be selected to meet the production needs of clinical-grade cell products.

[0156] During the passaging expansion process, the culture medium can be replaced / supplemented according to the culture condition of the human pluripotent stem cells and the change in the content of nutrients and cell secretions in the culture medium. Generally, the content of nutrients and / or cell secretions in the culture medium can be determined according to the color change of the culture medium.

[0157] In some embodiments, the human pluripotent stem cells are subjected to one passaging expansion before being subjected to differentiation culture. That is, the human pluripotent stem cells are cultured for 7 days after resuscitation to perform one passaging expansion, and the culturing continues for 7 days after the passaging to complete the expansion process.

[0158] Formulation components

[0159] In a second aspect, the present application provides a composition comprising the functional spinal cord GABAergic neural precursor cells produced by the method of the first aspect of the present application.

[0160] In some embodiments, the functional spinal cord GABAergic neural precursor cells are present in an amount of 2 x 10 4 -2 x 10 6 .

[0161] In some embodiments, the composition further comprises any one or two or more of fibrinogen, BDNF, VEGF, MDL28170, such as any one, two, three, or all four. In some embodiments, the composition further comprises thrombin.

[0162] In the present application, the inventors found that when using fibrin to load functional spinal cord GABAergic neural precursor cells and growth factors, the cell colonization, survival, differentiation and maturation can be effectively promoted, thereby significantly promoting the reconstruction of damaged nerve circuits and angiogenesis after spinal cord injury. In the physiological process, fibrinogen can be converted to fibrin under the action of thrombin and the like, forming a stable network structure and participating in hemostasis and wound healing. In tissue engineering, fibrinogen not only provides growth space and suitable microenvironment for cells by forming a three-dimensional network structure, but also promotes the proliferation and differentiation of endothelial cells and vascular smooth muscle cells, thereby promoting the formation of new blood vessels. Moreover, it is derived from the human body, has higher safety and clinical application potential.

[0163] In some embodiments, the composition comprises a first composition and a second composition. In some embodiments, the first composition comprises the functional spinal cord GABAergic neural precursor cells, fibrinogen, BDNF, VEGF and MDL28170; and the second composition comprises thrombin.

[0164] In some embodiments, the first composition and the second composition are solutions.

[0165] In the first composition, the concentration of fibrinogen can be 10-200 mg / mL, the concentration of BDNF can be 10-100 μg / mL, the concentration of VEGF can be 5-50 μg / mL, and the concentration of MDL28170 can be 10-100 μM. For example, the concentration of fibrinogen can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 150 mg / mL or 200 mg / mL; the concentration of BDNF can be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL or 100 μg / mL; the concentration of VEGF can be 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL or 50 μg / mL; and the concentration of MDL28170 can be 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM or 100 μM.

[0166] In the second composition, the concentration of the thrombin can be 10-200 U / mL. For example, the concentration of the thrombin can be 10 U / mL, 20 U / mL, 30 U / mL, 40 U / mL, 50 U / mL, 60 U / mL, 70 U / mL, 80 U / mL, 90 U / mL, 100 U / mL, 150 U / mL or 200 U / mL.

[0167] In some embodiments, the first composition is prepared by adding fibrinogen, BDNF, VEGF and MDL28170 into a cell suspension containing the functional spinal cord GABAergic neural precursor cells, thereby obtaining the first composition.

[0168] In some embodiments, the second composition is prepared by dissolving the thrombin in a second solution, thereby obtaining the second composition.

[0169] In some embodiments, the second composition is prepared by dissolving the thrombin in a second solution, thereby obtaining the second composition.

[0170] In some embodiments, the second solution can be any solution that can dissolve the thrombin and has good biocompatibility, such as a protein solution, a physiological saline solution, or a sterile double distilled water solution.

[0171] In some embodiments, the composition is in the form of an injection. In some embodiments, the first composition and the second composition are injectable solutions.

[0172] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0173] In some embodiments, the dosage form of the pharmaceutical composition is an injection, a capsule, a tablet or a granule. Preferably, the dosage form of the pharmaceutical composition is an injection.

[0174] In some embodiments, the dosage form of the pharmaceutical composition is an injection, a capsule, a tablet or a granule. Preferably, the dosage form of the pharmaceutical composition is an injection.

[0175] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0176] In some embodiments, the medical product is a drug or a medical device.

[0177] The medical device can be, for example, a biomaterial scaffold, a gel, or a biological ink for 3D printing.

[0178] In a fifth aspect, the present application provides use of the functional spinal cord GABAergic neural precursor cells produced according to the method of the first aspect of the present application, the composition of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application in the manufacture of a medicament for treating spinal cord injury.

[0179] In some embodiments, the spinal cord injury is a traumatic spinal cord injury.

[0180] In some embodiments, the traumatic spinal cord injury is a complete injury or a total transection injury.

[0181] The treatment of the spinal cord injury may, for example, comprise promoting synapse formation after spinal cord injury, promoting angiogenesis, relieving pain, relieving muscle spasm, or improving bladder function.

[0182] In a sixth aspect, the present application provides use of the functional spinal cord GABAergic neural precursor cells produced according to the method of the first aspect of the present application, the composition of the second aspect of the present application, or the pharmaceutical composition of the third aspect of the present application in the treatment of spinal cord injury.

[0183] In some embodiments, the spinal cord injury is a traumatic spinal cord injury.

[0184] In some embodiments, the traumatic spinal cord injury is a complete injury or a total transection injury.

[0185] The treatment of the spinal cord injury may, for example, comprise promoting synapse formation after spinal cord injury, promoting angiogenesis, relieving pain, relieving muscle spasm, or improving bladder function.

[0186] In a seventh aspect, the present application also provides a method for treating spinal cord injury, comprising: using the functional spinal cord GABAergic neural precursor cells produced by the method of the first aspect of the present application, using the composition of the second aspect of the present application, or using the pharmaceutical composition of the third aspect of the present application for treatment.

[0187] In some embodiments, the spinal cord injury is a traumatic spinal cord injury. In some embodiments, the traumatic spinal cord injury is a complete injury or a total transection injury.

[0188] The treatment of the spinal cord injury may, for example, comprise promoting synapse formation after spinal cord injury, promoting angiogenesis, relieving pain, relieving muscle spasm, or improving bladder function.

[0189] Embodiments

[0190] The present application will be described below in conjunction with specific examples, but the scope of the present application is not limited thereto. If not otherwise specified, the reagents and instruments used in the following examples are all conventional reagents and instruments in the art, which can be obtained by commercial means. The methods used are all conventional experimental methods, and the skilled person in the art can undoubtedly implement the described schemes and obtain the corresponding results according to the content of the examples.

[0191] Example 1 Preparation of functional spinal cord GABAergic neural precursor cells

[0192] 1.1 Human iPSC resuscitation

[0193] (1) Preparation of TeSR TM AOF complete medium (stemcell, 100-0401): TeSR TM AOF basal medium and TeSR TM AOF 20X supplement is mixed at a ratio of 19:1;

[0194] (2) Dilute VTN-N (Life Technologies, CTS279S3) and DPBS (Life Technologies, 14190144) at a ratio of 1:99, and add 100 μL / cm 2 to a T25 culture flask, cross-shaking to ensure that the Matrigel covers the bottom, and transfer to 37°C for incubation for 1 h or more;

[0195] (3) Take out the frozen human iPSC (purchased from Wuhan Hongchen Innovative Biotechnology Co., Ltd.), and after thawing, use a 1 mL gun tip to transfer the cell suspension to a 15 mL centrifuge tube containing 5 mL DMEM / F12 (Life Technologies, 11330032), and centrifuge at 150g at room temperature for 3 min;

[0196] (4) After centrifugation, discard the supernatant, and add 1 mL of TeSR TM AOF complete medium to the centrifuge tube, mix well by blowing, and ensure that the size of the cell clumps is 50 μm-200 μm;

[0197] (5) Count the cells, take 10 μL of cell suspension into a 200 μL centrifuge tube, add 10 μL of AO / PI staining solution (Countstar, RE010212) to the centrifuge tube, mix well, take 20 μL of cell-staining solution mixture into a cell counting plate, and place the cell counting plate in a cell counter, select the "AO / PI fluorescence counting-iPSC" program for counting;

[0198] (6) According to the density of living cells and the cell seeding density (10000 cells / cm 2) Calculate the volume of cell suspension required and transfer to the prepared T25 flask above, add TeSR TM AOF complete medium: TeSR

[0199] 1.2 Human iPSC subculture expansion

[0200] (1) After human iPSC resuscitation or subculture, when the cells are observed under a microscope to be undifferentiated, well-shaped and the cell confluence is more than 85% at day 7, the cells are subcultured and expanded;

[0201] (2) Prepare TeSR TM AOF complete medium: TeSR TM AOF basal medium: TeSR TM AOF 20X supplement is mixed at a ratio of 19:1;

[0202] (3) Dilute VTN-N and DPBS at a ratio of 1:99, and add 100 μL / cm 2 to the flask (T25 flask / T175 flask), cross-shaking to ensure that the Matrigel covers the bottom, and transfer to 37°C for incubation for 1 h or more;

[0203] (4) Use 40 μL / cm 2 of ReLeSR TM to digest the cells, and when the cells are observed under a microscope to retract and detach from the flask wall, transfer to a biological safety cabinet and add 2 volumes of DMEM / F12 to stop digestion. Centrifuge the cells and aspirate the supernatant, add 1 mL (T25 flask) / 3 mL (T175 flask) of DMEM / F12, mix by blowing and ensure that the cell clumps are 50 μm-200 μm in size;

[0204] (5) Count the cells, place the cell counting plate in the cell counter, and select the “AO / PI fluorescence counting-iPSC” program to count;

[0205] (6) Calculate the volume of cell suspension required according to the live cell density and the cell seeding density (10000 cells / cm 2 ), and transfer to the prepared VTN-N coated flask above, add TeSR TMAOF complete medium to 5 mL (T25 flask) / 30 mL (T175 flask), add 5 μL / 30 μL of 10 mM Y27632 to a final concentration of 10 μM, and place in an incubator at 37°C, 5% CO2 for culture. Change the medium on the 2nd day, 4th day, 5th day, and 6th day of culture.

[0206] 1.3 Human iPSC differentiation plating

[0207] (1) Prepare TeSR TM AOF complete medium: TeSR TM AOF basal medium with TeSR TM Mix AOF 20X supplement at a ratio of 19:1;

[0208] (2) Dilute VTN-N and DPBS at a ratio of 1:99, and add 100 μL / cm 2 to the culture flask (T25 flask / T175 flask), cross-shake to ensure that the Matrigel covers the bottom, and transfer to 37°C for incubation for 1 h or more;

[0209] (3) Take the human iPSCs for differentiation from the incubator (use once-passaged expanded human iPSCs for improved yield, i.e., human iPSCs that have been passaged once after 7 days of culture after resuscitation and continue to be cultured for 7 days after the passage), gently shake the culture flask to completely suspend the unattached cells, discard the culture medium, and then add 40 μL / cm 2 of DPBS to rinse the cell surface, and discard the rinse;

[0210] (4) Digest the cells using 40 μL / cm 2 of ReLeSR TM digestive solution, count the digested cells, and according to the live cell density calculation, transfer the cell suspension (1.5×10 6 cells) to a 15 mL centrifuge tube, and detect the iPSC phenotype by flow cytometry;

[0211] (5) Calculate the required cell suspension volume according to the live cell density and cell seeding density (20000 cells / cm 2 ), and transfer to the VTN-N-coated culture flask prepared above, and supplement with TeSR TM AOF complete medium to 5 mL (T25 flask) / 30 mL (T175 flask), add 10 mM Y27632 (5 μL (T25 flask) / 30 μL (T175 flask)) to a final concentration of 10 μM, and place in an incubator at 37°C, 5% CO2 for culture, which is the 0th day of differentiation culture (i.e., the first stage of differentiation D0).

[0212] 1.4 Spinal cord neuroepithelial cell differentiation

[0213] (1) Prepare the neural induction medium (NIM) and spinal cord neuroepithelial cell differentiation medium (referred to as stage 1 medium) according to Table 1 and Table 2.

[0214] Table 1 NIM medium preparation information table

[0215] DMEM / F12 Neurobasal N-2 supplement NEAA Ratio (v / v) Formulation components 49％ 49％ 1％ 1％

[0216] Note: The NIM medium is stored at 2-8°C for no more than two weeks after preparation; Neurobasal (Life Technologies, A1371201), N-2 supplement (Life Technologies, A1370701), NEAA (Life Technologies, 11140050).

[0217] Table 2 Spinal cord neuroepithelial cell differentiation stage medium preparation information table

[0218] NIM SB431542 DMH1 CHIR99021 Final concentration N / A 2 μΜ 2 μΜ 3 μΜ Figure 2

[0219] Note: SB431542 (MCE, HY-10431G), DMH1 (MCE, HY-12273), CHIR99021 (MCE, HY-10182G).

[0220] (2) When the cells in Example 1, 1.3, are cultured to the second day (i.e., stage 1 differentiation D1), observe the cell adhesion under a microscope, gently shake the culture bottle, and discard the old medium. Add 40 μL / cm 2 DMEM / F12 rinse the cells once, then add 5 mL (T25 culture bottle) / 30 mL (T175 culture bottle) stage 1 medium, and continue to culture in the incubator;

[0221] (3) Change the medium every other day (i.e., stage 1 differentiation D3 and D5), and observe the cell growth state under a microscope and take pictures;

[0222] (4) When the differentiation culture reaches D7, the stage 1 differentiation is completed, and the cell growth state is observed under a microscope and photographed.

[0223] 1.5 Early spinal cord GABAergic neural precursor cell induction and differentiation

[0224] (1) Dilute VTN-N and DPBS at a ratio of 1:99, and dilute according to 100 μL / cm 2Add to the culture flask (T25 flask / T175 flask), cross-shaking, make sure the substrate gel covers the bottom, and transfer to 37℃ incubation for 1h or more;

[0225] (2) Prepare the neural induction medium (NIM) and spinal cord GABAergic neural precursor cell induction differentiation medium (referred to as two-stage medium) according to Table 1 and Table 3.

[0226] Table 3 Preparation information table of spinal cord GABAergic neural precursor cell induction differentiation medium

[0227]

[0228] Note: B27 supplement (Life Technologies, A5047601), retinoic acid (MCE, HY-14649G), cyclopamine (MCE, HY-17024).

[0229] (3) Transfer the cells to be treated (cells cultured to D7 in Example 1 1.4) to the biosafety cabinet, discard the old culture medium, and add 40μL / cm 2 of DPBS to rinse the cells once;

[0230] (4) Digest the cells with 40μL / cm 2 of ReLeSR TM digestive solution, and transfer to the 37℃ incubator for 2min. When the edge of the cell clone curls, transfer to the biosafety cabinet and add 2 times the volume of DMEM / F12 to stop digestion. Gently blow off the cell clone, mix well, and transfer to a 15mL centrifuge tube. Centrifuge at 150g for 3min, and discard the supernatant;

[0231] (5) Add 2mL of one-stage medium to resuspend the cell pellet (the number of blows should not exceed 5 times), mix gently, and detect the proportion of SOX1 positive cells (using anti-SOX1 antibody for detection (BD, 562224)) by flow cytometry (as shown in Formulation components );

[0232] (6) Discard the VTN-N working solution in the coated culture flask in step (1), and add one-stage medium. Mix well, calculate the volume of the required cell suspension at a cell seeding density of 30000 cells / cm 2 , and transfer to the culture flask. Add one-stage medium to 5mL (T25 flask) / 30mL (T175 flask), and place in a 37℃, 5% CO2 incubator for culture. This is the 0th day of differentiation culture (i.e. two-stage differentiation D0);

[0233] (7) The next day after differentiation culture (i.e., the second stage of differentiation D1), observe the cell adhesion under a microscope, gently shake the culture bottle, and discard the old culture medium. Add 40 μL / cm 2 of DMEM / F12 to rinse the cells once, then add 5 mL (T25 culture bottle) / 30 mL (T175 culture bottle) of the second stage of culture medium, and continue to culture in the incubator;

[0234] (8) Change the medium every other day (i.e., the second stage of differentiation D3 and D5), observe the cell growth state under a microscope, and take pictures;

[0235] (4) When the differentiation culture reaches D7, the second stage of differentiation is completed, observe the cell growth state under a microscope, and take pictures.

[0236] 1.6 Induced differentiation and proliferation of spinal cord GABAergic neural precursor cells

[0237] (1) Prepare the neural induction medium (NIM) and the induced differentiation and proliferation medium of spinal cord GABAergic neural precursor cells (referred to as the third stage of culture medium) according to Table 1 and Table 4.

[0238] Table 4 Preparation information table of the induced differentiation and proliferation medium of spinal cord GABAergic neural precursor cells

[0239] NIM B27 supplement (50x) Retinoic acid Cyclopamine Final concentration N / A 0.1 μΜ 1× 0.5 μΜ Figure 3

[0240] (2) Transfer the cells to be treated (cells cultured to D7 in Example 1 1.5) to a biosafety cabinet, aspirate the old culture medium, and add 40 μL / cm 2 of DPBS to rinse the cells once;

[0241] (3) Digest the cells with 40 μL / cm 2 of ReLeSR TM digestive solution, transfer to a 37°C incubator for 2 min, when the edge of the cell clone appears to be curled, transfer to a biosafety cabinet, and add 2 times the volume of DMEM / F12 to stop digestion, gently blow off the cell clone, mix well, transfer to a 15 mL centrifuge tube, and centrifuge at 150 g for 3 min, and aspirate the supernatant;

[0242] (4) Add the third stage of culture medium to resuspend the cell pellet (the number of blows should not exceed 5 times), gently mix, and detect the proportion of ASCL-1 (use anti-ASCL-1 antibody for detection (Santa Cruz, sc-374104 AF647)) and Hoxb4 (use anti-Hoxb4 antibody for detection (Santa Cruz, sc-365927 AF488)) positive cells by flow cytometry (as shown in Figure 4 );

[0243] (5) Calculate the volume of cell suspension required with a cell seeding density of 3 x 10 5 cells / mL, and transfer to a new culture flask, add three-stage medium to 10 mL (T25 culture flask) / 60 mL (T175 culture flask), and place in a 37°C, 5% CO2 incubator for culture, which is the 0th day of differentiation culture (i.e., three-stage differentiation D0);

[0244] (6) Observe the aggregation of neural spheres every day, and repeatedly blow and beat 4-6 times to blow off the cells adhering to the surface of the neural spheres;

[0245] (7) Perform semi-replacement every other day (i.e., three-stage differentiation D2, D4, D6), mix the cells, transfer the cell suspension to a 15 mL centrifuge tube, repeatedly blow and beat the cell suspension 4-6 times, stand for 2-3 min, and after the neural spheres completely settle, discard half of the culture medium, and add the same volume of fresh three-stage medium;

[0246] (8) When the differentiation culture reaches D7, the three-stage differentiation is completed;

[0247] (9) Transfer the cell suspension to a 50 mL centrifuge tube, repeatedly blow and beat the cell suspension 4-6 times, centrifuge at 150 g for 3 min, and discard the supernatant;

[0248] (10) Add 1 mL (T25 culture flask) / 5 mL (T175 culture flask) DMEM / F12 to rinse the cells, centrifuge at 150 g for 3 min, and discard the supernatant;

[0249] (11) Add 1 mL (T25 culture flask) / 5 mL (T175 culture flask) preheated Accutase digestion solution (stemcell, 07920), incubate in a 37°C incubator, and blow and beat the cell suspension once in the middle;

[0250] (12) Digest the neural spheres into neural spheres of 50-200 μM in size, add 2 times the volume of digestion solution of DMEM / F12 to mix to terminate the digestion, centrifuge at 300 g for 5 min, discard the supernatant, and detect the proportion of PTF1A positive cells (use anti-PTF1A antibody for detection (Santa Cruz, sc-393011PE)) by flow cytometry (as shown in Figures 1A-1B ).

[0251] (13) Calculate the volume of the required freezing solution (Sartorius, 05-714-1A) with a freezing density of 3 x 10 6 cells / 0.5 mL, and after freezing overnight at -80°C, transfer to liquid nitrogen for storage.

[0252] 1.7 Detection of markers in each stage of differentiation culture

[0253] The human iPSC phenotype was detected by flow cytometry when the human iPSCs were differentiated and plated, and the results are shown in Table 1. The positive rates of the four specific indicators, SSEA4 (99.76%), TRA-1-60 (97.66%), Nanog (96.48%), and OCT4 (95.13%), were all above 90%. Among them, the anti-SSEA4 antibody (BioLegend, 330418), the anti-TRA-1-60 antibody (BioLegend, 330610), the anti-Nanog antibody (BioLegend, 674010), and the anti-OCT4 antibody (BioLegend, 653704) were used for detection. Figure 2

[0254] After the completion of the first stage of differentiation, the spinal cord neuroepithelial cell phenotype was detected by flow cytometry, and the results are shown in Table 2. The positive rate of the SOX1 specific indicator was 97.47%. Figure 3

[0255] After the completion of the second stage of differentiation, the cell phenotype (early spinal cord GABAergic neural precursor cells, i.e., early spinal cord GABAergic neural precursor cells) was detected by flow cytometry, and the results are shown in Table 3. The double-positive rate of the ASCL1 and HoxB4 specific indicators was 98.42%. Figure 4

[0256] After the completion of the third stage of differentiation, the cell phenotype (functional spinal cord GABAergic neural precursor cells, i.e., late spinal cord GABAergic neural precursor cells) was detected by flow cytometry, and the results are shown in Table 4. The positive rate of the PTF1A specific indicator was 90.91%. Figure 5A

[0257] As shown in the following Table 5 statistical results, after one passage amplification and three stages of differentiation, the initial 1×10 6 iPSCs in a T175 culture flask can proliferate and differentiate into about 1.1×10 9 product spinal cord GABAergic neural precursor cells, with an amplification factor of about 1100 times.

[0258] Table 5 Detection of cell amplification factor at each stage of differentiation culture

[0259]

[0260] Example 2 Transplantation of functional spinal cord GABAergic neural precursor cells

[0261] 2.1 Transplantation scheme of functional spinal cord GABAergic neural precursor cells

[0262] ​​​​Nude rats (athymic, T-lymphocyte depleted, immunocompromised Nude rats, SPF grade adult females) were anesthetized by intramuscular injection of tiletamine-zolazepam (Zoletil 50, Virbac S.A.) (20 mg / kg) and tiletamine (2.5 mg / kg); atropine (0.05 mg / kg) was injected intramuscularly to reduce rat airway glandular secretions; ceftriaxone (50 mg / kg) was injected intraperitoneally to prevent infection; erythromycin ointment was applied to both eyes of the rat to prevent injury during anesthesia.

[0263] The rat was placed on a heating pad to maintain body temperature at 37°C, the back skin was disinfected with iodophor twice, then disinfected with 75% alcohol, and then the skin, fascia, and muscle were cut layer by layer, the muscles on both sides were further stretched with a muscle stretcher, the first spinal cord injury operation area was exposed, a mosquito hemostat was used to tear the dural patch covering the surface of the spinal cord, a surgical microscope was used to focus the surgical field of view, an insulin needle was used to pick the dura mater, and the cell injection site of the spinal cord was exposed. The rat was clamped with a spinous process clamp on both sides of the T7 and T10 laminae and transferred to the rat spinal column fixator of the stereotactic injection device, and the spinal cord was kept in the middle horizontal position.

[0264] Functional spinal GABAergic neural precursor cells (differentiated according to the method in Example 1) were collected on the day of surgery. The amount of cells transplanted into each nude rat was 1.2 million. The functional spinal GABAergic precursor cells were prepared into a suspension with a cell density of 2 million / μL, and before transplanting the cells, 10 μL of the cells were mixed by blowing with a 10 μL pipette. Then, 2 μL of the cell suspension (containing about 40 million cells) was taken up with a 10 μL Hamilton microsyringe, and the solvent of the suspension contained 100 mg / mL fibrinogen (human fibrinogen, Sigma, F3879), 50 μg / mL BDNF (Peprotech), 10 μg / mL VEGF (Peprotech), and 50 μM MDL28170 (Sigma). The fibrinogen, BDNF, VEGF, and MDL28170 were prepared using sterile double distilled water. The 2 μL of the cell suspension was transferred into a glass electrode with a 40 μm inner diameter and a 100 μm outer diameter connected to a pulled tip using a compression fitting (RN Compression Fitting, 1 mm), and then the glass electrode containing the cell suspension was connected to a 100 μL Hamilton microsyringe containing a certain volume of double distilled water, mounted on a stereotaxic injection device and connected to a microsyringe pump. Under a surgical microscope, three injection sites were located at the center of the lesion, 2 mm rostral, and 2 mm caudal, and the glass electrode tip was first moved to the dorsal surface of the spinal cord at the center of the lesion injection site, and then slowly inserted into the spinal cord tissue with a depth of 1 mm. Using the microsyringe system, 2 μL of the cell suspension was accurately delivered to the target area of the spinal cord at a constant rate of 500 nl / min, and after the injection was completed, the needle was maintained in place for 1 minute to ensure that the cells were fully deposited, and then the glass electrode was slowly withdrawn. Using the same procedure, the other two sites were injected. After the cell injection was completed, 2 μL of thrombin (human thrombin, Sigma, T7009, 100 U / mL, prepared using sterile double distilled water) was injected at each injection site. The control group was injected with a solvent without cells.

[0265] After hemostasis in the surgical area, the muscle was sutured with 4-0 needle thread in an interrupted manner and the fascia was sutured in a continuous manner. Before skin suturing, the skin was disinfected with iodophor, and then the skin was sutured with 3-0 needle thread in an interrupted manner. After the surgery was completed, the rats were subcutaneously injected with 5 mL of compound normal saline and the bladder was pressed to empty the urine. The rats were routinely cared for after the surgery, and ceftriaxone sodium (50 mg / kg) was injected intraperitoneally every day for 5 days after the surgery to prevent infection. Meloxicam (0.2 mg / kg) was subcutaneously injected every day for 3 days after the surgery to prevent inflammation and relieve pain.

[0266] 2.2 Promotion of synapse formation after transplantation of functional spinal GABAergic neural precursor cells

[0267] At 24 weeks post-transplantation, tissue samples were taken from the transplantation site for further analysis of the integration of transplanted cells and their functional properties using immunoelectron microscopy.

[0268] Using immunoelectron microscopy, typical synaptic structures were observed between transplanted human neurons (as shown in Figure 5B ) and between human neurons and rat host neurons (as shown in Figures 6A-6C ). This detection method, which uses human-specific antibodies (mouse anti-stem121 antibody, Takara, Y40410) to recognize and locate transplanted human cells, and mouse anti-human synaptophysin antibodies (Invitrogen, 14-6525-80) to specifically label the synaptic protein components of human neurons, combined with the high-resolution imaging technology of electron microscopy, can clearly demonstrate the morphological characteristics of synapses.

[0269] This result not only confirms that transplanted human neurons successfully survive and integrate in the host body, but also shows that they can form effective neural connections with host neurons, providing an important structural basis for subsequent functional recovery.

[0270] 2.3 Functional spinal cord GABAergic neural precursor cell transplantation promotes angiogenesis

[0271] At 24 weeks post-transplantation, samples were taken from the transplantation site for immunofluorescence staining. The antibodies used included rabbit anti-laminin polyclonal antibody (Sigma-Aldrich, L9393), rabbit anti-type IV collagen polyclonal antibody (Abeam, ab254263), mouse anti-RECA1 monoclonal antibody (Bio-Rad, MCA970R), and rabbit anti-PDGFRβ polyclonal antibody (Abeam, ab32570).

[0272] The results, as shown in Figures 7A-7B , found that rat endothelial cell marker (RECA-1) positive host capillaries were visible inside the graft; the structural components of the blood-spinal cord barrier (BSCB), including the basement membrane (laminin, type IV collagen) and pericytes (PDGFRβ positive cells), were comparable to the complete gray matter, indicating that a normal vascular network with complete BSCB function was formed inside the graft, effectively supporting the metabolic needs of transplanted cells.

[0273] 2.4 Functional spinal cord GABAergic neural precursor cell transplantation relieves pain

[0274] Mechanical pain threshold of the hind paw was measured using an electronic Von Frey apparatus until 24 weeks after transplantation. Three days before the experiment, rats were acclimated in a transparent plexiglass chamber (20 cm x 10 cm x 12.5 cm) for 45 min each day; the chamber was empty at the bottom and had a movable floor at the top, which was placed on a metal grid rack with an inclined mirror underneath to observe the hind paw; the apparatus was calibrated before the experiment, and a 1-mm diameter probe was installed; during acclimation, the experimenter held the apparatus and lightly touched the center of the hind paw palm with the probe; the test was performed in a quiet room, and rats were acclimated in the chamber for 15 min on the test day; during measurement, the probe was lightly touched to the center of the hind paw palm and gradually increased in force until the rat withdrew the hind paw or exhibited a jump or other reaction, at which point the stimulation was stopped, and the maximum value recorded by the apparatus was the mechanical pain threshold.

[0275] Results, as shown in Figures 7C-7D Figure 6, showed that the mechanical withdrawal force of the hind paw decreased two weeks after spinal cord injury and before transplantation, which proved the occurrence of mechanical hyperalgesia; in the control group (medium group) of rats injected with solvent without cells, the mechanical withdrawal force remained unchanged; in rats receiving functional spinal GABAergic neural precursor cell transplantation (dI4 group), the mechanical withdrawal force gradually increased; at 8 weeks after transplantation, a significant difference was detected; at 24 weeks after transplantation, the mechanical withdrawal force of rats in the dI4 group was significantly higher than that of rats in the medium group. These results showed that the transplanted functional spinal GABAergic neural precursor cells could alleviate mechanical hyperalgesia.

[0276] Further, the thermal pain latency of the hind paw was measured using a plantar test apparatus until 24 weeks after transplantation. Three days before the experiment, rats were acclimated in a transparent plexiglass chamber for 45 min each day; the chamber was placed on a transparent glass plate, and the thermal pain excitation light source was located below the glass plate, which was focused by a lens to increase the temperature; during the formal test, rats were also acclimated for 30 min, and the illumination intensity of the light source located below the glass plate was set to 10% of the maximum intensity, the heating intensity was set to 30% of the maximum intensity, and the cutoff time was set to 20 s to protect the hind paw; during acclimation, the center of the hind paw palm was stimulated with thermal pain; the test was performed in a quiet room, and the focused light spot was placed on the center of the hind paw palm, the excitation light was turned on until the rat withdrew the hind paw or exhibited licking or other reactions, the excitation light was stopped, and the latency (s) was recorded; each hind paw was measured 5 times with an interval of at least 1 min.

[0277] Results, as shown in Figures 8A-8BAs shown, the rats receiving functional spinal GABAergic neural precursor cell transplantation also showed a higher proportion of spontaneous micturition (p<0.05) and a significantly reduced amount of residual urine in the bladder (p<0.05) compared to the medium group.

[0278] 2.5 Functional spinal GABAergic neural precursor cell transplantation improves bladder function

[0279] After spinal cord injury, the impairment of bladder function can be manifested as a decreased ability to empty the bladder, and this functional deficit can be quantified by measuring the amount of residual urine. To assess the severity of urinary retention after spinal cord injury, the weight of the urine discharged was measured and recorded once a week in the morning after the bladder was manually pressed routinely. In addition, the spontaneous micturition function of the rats was monitored at the time when the bladder was most full in the morning every day. If the bladder was not full and no urine was discharged when the bladder was pressed, and if this phenomenon occurred for 3 consecutive days, it was considered that the rat had recovered the spontaneous micturition function.

[0280] Results as shown in Figure 6, rats receiving functional spinal GABAergic neural precursor cell transplantation showed a higher proportion of spontaneous micturition (p<0.05) and a significantly reduced amount of residual urine in the bladder (p<0.05) compared to the medium group. Figure 8A Figure 8B ​

[0281] Example 3 Optimization of key process parameters (CPP) at each stage of differentiation culture

[0282] 3.1 Optimization of process parameters in spinal neural epithelial cell differentiation

[0283] During the first stage of differentiation, the seeding density and culture time of human iPSCs were mainly optimized, and by using the optimized method, the number and quality of cells harvested in the first stage of differentiation were ensured. The optimization test was carried out in a T25 culture flask, and the culture method was the same as 1.3-1.4 in Example 1.

[0284] (1) Optimization of cell seeding density in the first stage of differentiation

[0285] The seeding densities of 20,000 cells / cm 2 , 30,000 cells / cm 2 , and 40,000 cells / cm 2 were selected, and finally it was found that the seeding density of 20,000 cells / cm 2 ​​​The cell viability of the cells harvested in the first stage of differentiation was best and the expansion fold was the largest (between 16-21 folds) at the seeding density of 30000 cells / cm

[0286] Table 6 Optimization of the seeding density in the first stage of differentiation

[0287]

[0288] (2) Optimization of the culture time in the first stage of differentiation

[0289] The culture time was designed to harvest the cells at D6 / D7 / D8 / D9, and it was finally found that the positive cell rate of the cells harvested at different days had no obvious influence; the number of the cells harvested at D6 / D7 / D8 was increased in turn, and the cell viability was decreased at D9 due to the limited culture space, so the first stage of differentiation was selected to harvest the cells at D6 / D7 / D8 (as shown in Table 7).

[0290] Table 7 Optimization of the culture time in the first stage of differentiation

[0291]

[0292] 3.2 Optimization of the process parameters in the induction and differentiation of the early spinal cord GABAergic neural precursor cells

[0293] In the second stage of differentiation, the seeding density of the cells, the culture time and the key small molecules cyclopamine and retinoic acid were mainly optimized, and through the use of the optimized method, the purity and yield of the cells harvested in the second stage of differentiation were ensured. The optimization test was carried out in a T25 culture flask, and the culture method was the same as that in Example 1.5.

[0294] (1) Optimization of the seeding density of the cells in the second stage of differentiation

[0295] The seeding density was selected to be 20000 cells / cm 2 , 30000 cells / cm 2 , 40000 cells / cm 2 , and finally it was found that the cell viability of the cells harvested in the second stage of differentiation was best and the expansion fold was the largest (between 2.5-6 folds) at the seeding density of 30000 cells / cm 2

[0296] Table 8 Optimization of the seeding density of the cells in the second stage of differentiation

[0297]

[0298] (2) Optimization of the culture time in the second stage of differentiation

[0299] ​The culture time was designed to harvest cells at D6 / D7 / D8 / D9, and it was finally found that the positive cell rate of cells harvested at different days had no significant effect; the number of cells harvested at D6 / D7 / D8 had no significant change, while at D9, due to limited culture space, the cell viability decreased, so the two-stage differentiation selected cells harvested at D6 / D7 / D8 (as shown in Table 9).

[0300] Table 9 Optimization of culture time in two-stage differentiation

[0301]

[0302] (3) Optimization of key small molecules in two-stage differentiation

[0303] The concentration of key small molecules cyclopamine / retinoic acid was designed as follows: low concentration group 0.1 μM / 0.02 μM; medium concentration group 0.5 μM / 0.1 μM; high concentration group 2.5 μM / 0.5 μM. It was finally found that in the low concentration group, the cell ratio decreased; while in the high concentration group, the cell viability decreased, so the medium concentration group 0.5 μM / 0.1 μM was selected as the final concentration combination (as shown in Table 10).

[0304] Table 10 Optimization of key small molecules in two-stage differentiation

[0305]

[0306] 3.3 Optimization of process parameters in early spinal cord GABAergic neural precursor cell induction and differentiation proliferation

[0307] In three-stage differentiation, the cell seeding density, culture time and key small molecules cyclopamine / retinoic acid were mainly optimized, and by using the optimized method, the purity of cells harvested in three-stage differentiation was ensured. The optimization test was carried out in T25 culture flask, and the culture method was the same as 1.6 in Example 1.

[0308] (1) Optimization of cell seeding density in three-stage differentiation

[0309] The seeding density was selected as 200,000 cells / mL, 300,000 cells / mL, and 400,000 cells / mL, and it was finally found that at a seeding density of 300,000-400,000 cells / mL, the cell viability of cells harvested in three-stage differentiation was the best, and the expansion ratio was the largest (between 1.2-1.6 times) (as shown in Table 11).

[0310] Table 11 Optimization of cell seeding density in three-stage differentiation

[0311]

[0312] (2) Optimization of culture time in three-stage differentiation

[0313] The culture time was designed to harvest cells at D6 / D7 / D8 / D9, and it was finally found that the positive cell rate of cells harvested at different days had no significant effect; the number of cells harvested at D6 / D7 / D8 increased in turn, while at D9, due to limited culture space, the number of cells decreased, so the three-stage differentiation selected cells harvested at D6 / D7 / D8 (as shown in Table 12 below).

[0314] Table 12 Optimization of culture time in three-stage differentiation

[0315]

[0316]

[0317] (3) Optimization of key small molecules in three-stage differentiation

[0318] The concentration of key small molecules cyclopamine / retinoic acid was designed as follows: low concentration group 0.1 μM / 0.02 μM; medium concentration group 0.5 μM / 0.1 μM; high concentration group 2.5 μM / 0.5 μM. It was finally found that in the low concentration group, the cell ratio decreased; while in the high concentration group, the cell viability decreased, so the medium concentration group 0.5 μM / 0.1 μM was selected as the final concentration combination (as shown in Table 13 below).

[0319] Table 13 Optimization of key small molecules in three-stage differentiation

[0320]

[0321] The above is only the preferred embodiment of the present application, and is not intended to limit the other forms of the present application, and any skilled person in the art can use the above disclosed technical content to make changes or modifications to equivalent embodiments. However, any simple modification, equivalent change and modification made to the above embodiments without departing from the technical solution content of the present application, according to the technical essence of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. A method for producing functional spinal cord GABAergic neural precursor cells from human pluripotent stem cells, comprising: differentiating the human pluripotent stem cells to obtain spinal cord neuroepithelial cells; differentiating the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells; differentiating the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells; wherein, in the step of differentiating human pluripotent stem cells to obtain spinal cord neuroepithelial cells, the marker for obtaining the spinal cord neuroepithelial cells is determined by satisfying the following condition: flow cytometry detection of differentiated spinal cord neuroepithelial cells shows that the positive rate of SOX1 is at least 90%, preferably at least 95%; wherein, the method is a feeder-free culture method.

2. The method of claim 1, wherein, In the step of differentiating human pluripotent stem cells to obtain spinal cord neuroepithelial cells, comprising: differentiating the human pluripotent stem cells in a one-stage differentiation medium comprising a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor and a TGF-β signaling pathway inhibitor to obtain the spinal cord neuroepithelial cells; preferably, the Wnt signaling pathway agonist is CHIR99021, the BMP signaling pathway inhibitor is DMH1, and the TGF-β signaling pathway inhibitor is SB431542; further preferably, the one-stage differentiation medium comprises 1 μM-10 μM CHIR99021, 1 μM-10 μM DMH1 and 1 μM-10 μM SB431542; further preferably, the one-stage differentiation medium further comprises NIM basal medium, and the NIM basal medium is a medium comprising 45%-50% DMEM / F12, 45%-50% Neurobasal, 0.5%-4% N-2 supplement, and 0.5%-4% NEAA.

3. The method of claim 2, wherein, The seeding density of the human pluripotent stem cells is 20000 cells / cm 2 - 40000 cells / cm 2 ; preferably, the human pluripotent stem cells are differentiated in the one-stage differentiation medium for 5-8 days to obtain the spinal cord neuroepithelial cells.

4. The method of any one of claims 1-3, wherein, In the step of differentiating the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells, the marker for obtaining the early spinal cord GABAergic neural precursor cells is determined by satisfying the following condition: flow cytometry detection of differentiated early spinal cord GABAergic neural precursor cells shows that the double positive rate of ASCL1 and HoxB4 is at least 90%, preferably at least 95%.

5. The method of claim 4, wherein, In the step of differentiating the spinal cord neuroepithelial cells to obtain early spinal cord GABAergic neural precursor cells, comprising: differentiating the spinal cord neuroepithelial cells in a two-stage differentiation medium comprising a Wnt signaling pathway agonist, a BMP signaling pathway inhibitor, a TGF-β signaling pathway inhibitor, a retinoic acid signaling agonist, a SHH signaling inhibitor, and a B27 supplement to obtain the early spinal cord GABAergic neural precursor cells. Preferably, the Wnt signaling pathway agonist is CHIR99021, the BMP signaling pathway inhibitor is DMH1, the TGF-β signaling pathway inhibitor is SB431542, the retinoic acid signaling agonist is retinoic acid, and the SHH signaling inhibitor is cyclopamine. Further preferably, the two-stage differentiation medium comprises 1 μM-10 μM CHIR99021, 1 μM-10 μM DMH1, 1 μM-10 μM SB431542, 0.01 μM-2 μM retinoic acid, 0.1 μM-2 μM cyclopamine, and 1x B27 supplement. Further preferably, the two-stage differentiation medium further comprises a NIM basal medium, which is a medium comprising 45%-50% DMEM / F12, 45%-50% Neurobasal, 0.5%-4% N-2 supplement, and 0.5%-4% NEAA.

6. The method of claim 5, wherein, The seeding density of the spinal cord neuroepithelial cells is 20000 cells / cm 2 - 40000 cells / cm 2 ; Preferably, the spinal cord neuroepithelial cells are subjected to differentiation culture for 5-8 days in the two-stage differentiation medium to obtain the early spinal cord GABAergic neural precursor cells.

7. The method of any one of claims 1-6, wherein, In the step of culturing the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells, the obtained functional spinal cord GABAergic neural precursor cells are identified by the following criteria: The differentiated functional spinal cord GABAergic neural precursor cells are subjected to flow cytometry detection, and the PTF1A positive rate is at least 90%.

8. The method of claim 7, wherein, In the step of culturing the early spinal cord GABAergic neural precursor cells to obtain the functional spinal cord GABAergic neural precursor cells, the following steps are included: The early spinal cord GABAergic neural precursor cells are subjected to differentiation culture in a three-stage differentiation medium comprising a retinoic acid signaling agonist, a SHH signaling inhibitor, and a B27 supplement to obtain the functional spinal cord GABAergic neural precursor cells. Preferably, the retinoic acid signaling agonist is retinoic acid, and the SHH signaling inhibitor is cyclopamine. Further preferably, the three-stage differentiation medium comprises 0.01 μM-2 μM retinoic acid, 0.1 μM-2 μM cyclopamine, and 1x B27 supplement. Further preferably, the three-stage differentiation medium further comprises a NIM basal medium, which is a medium comprising 45%-50% DMEM / F12, 45%-50% Neurobasal, 0.5%-4% N-2 supplement, and 0.5%-4% NEAA.

9. The method of claim 8, wherein, The early spinal cord GABAergic neural precursor cells are seeded at a density of 200,000 cells / mL-400,000 cells / mL. Preferably, the early spinal cord GABAergic neural precursor cells are subjected to differentiation culture for 5-8 days in the three-stage differentiation medium to obtain the functional spinal cord GABAergic neural precursor cells.

10. The method of any one of claims 1-9, wherein, The human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.

11. A composition comprising the functional spinal cord GABAergic neural precursor cells produced by the method of any one of claims 1-10; Preferably, the composition further comprises any one or more of fibrinogen, BDNF, VEGF, MDL28170; Further preferably, the composition further comprises thrombin; Still more preferably, the functional spinal GABAergic neural precursor cells are present in an amount of 2 x 10 4 -2 x 10 6 .

12. A medical product comprising the composition of claim 11; Preferably, the medical product is a pharmaceutical product or a medical device; Further preferably, the medical device is a biomaterial scaffold, a gel, or a bioink for 3D printing.

13. Use of the functional spinal cord GABAergic neural precursor cells produced by the method of any one of claims 1-10, or the composition of claim 11, in the manufacture of a medicament for treating spinal cord injury; Optionally, the spinal cord injury is a traumatic spinal cord injury; Preferably, the traumatic spinal cord injury is a complete injury or a total transection injury; Further preferably, the treating spinal cord injury comprises promoting synapse formation after spinal cord injury, promoting angiogenesis, relieving pain, relieving muscle spasm, or improving bladder function.

Images