Use of motor neuron progenitor cell population in treating spinal cord injury and mechanism thereof

Abstract

New uses of populations of motor neural progenitor cells are provided, including reducing reactive astrocytes, reducing glial scars, inhibiting reactive astrocyte-mediated axonal regeneration impairment, neuronal and / or oligodendrocyte death, priming for altering protein factor expression profiles in cerebrospinal fluid, promoting axonal regeneration and / or recovery, reducing demyelination, and promoting remyelination and / or increasing myelin thickness, among others.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine and provides new uses for motor neural progenitor cell populations, including reducing reactive astrocytes, reducing glial scarring, inhibiting reactive astrocyte-mediated axonal regeneration impairment, neuronal and / or oligodendrocyte death, altering the protein factor expression profile in cerebrospinal fluid, promoting axonal regeneration and / or recovery, reducing demyelination, and promoting myelin regeneration and / or increasing myelin thickness. Background Technology

[0002] According to WHO statistics, there were approximately 15.4 million people with spinal cord injury (SCI) worldwide in 2021, with about 900,000 new cases. External forces cause primary mechanical injury to the spinal cord through vertebral fractures or displacement compressing the spinal cord, excessive flexion / extension / rotation leading to spinal cord traction, or direct impact causing contusions. This results in axonal rupture, vascular tears, gray matter hemorrhage, and white matter tract damage. Initial mechanical injury causes abnormalities in spinal cord vascular tissue and blood supply. Secondary injuries may include local hemorrhage, ischemia, immune cell infiltration, and the release of inflammatory cytokines, ultimately leading to secondary neuronal death. Therefore, SCI usually irreversibly reduces the patient's quality of life and places a heavy burden on families and society.

[0003] Cell transplantation therapy is highly anticipated as a potential treatment for spinal cord injury (SCI) due to its potential to regenerate and replenish damaged neural tissue. Currently, the limited number of clinical trials focused on mesenchymal stem cell (MSC) transplantation and neuronal transplantation. In addition, studies have been reported on transplantation of oligodendrocyte precursors, neural stem cells (NSCs) or neural progenitor cells (NPCs), Schwann cells, motor neurons, or motor neuron precursors (Li J et al., Cell transplantation for spinal cord injury: a systematic review. Biomed ResInt. 2013;2013:786475. doi: 10.1155 / 2013 / 786475.). Transplanted undifferentiated donor cells, which depend on the internal environment of the injured spinal cord to regulate their survival and differentiation, may be more promising than transplanted mature neurons. Transplanted NPCs primarily differentiate into oligodendrocytes in vivo, thereby regenerating myelin and improving nerve conduction (Salewski RP et al., Transplantation of Induced Pluripotent Stem Cell-Derived Neural Stem Cells Mediate Functional Recovery Following Thoracic Spinal Cord Injury Through Remyelination of Axons. Stem Cells Transl Med. 2015 Jul;4(7):743-54); they can also migrate long distances, integrate into the spinal cord, and differentiate into mature neurons and glial cells, thereby reconstructing synapses and restoring some motor function (Kobayashi Y et al., Pre-evaluated safe human iPSC-derived neural stem cells promote functional recovery after spinal cord injury in common marmoset without tumorigenicity. PLoS One. 2012;7(12):e52787). In addition, NPCs can secrete neurotrophic factors to regulate immunopathological events following spinal cord injury (SCI).

[0004] However, a major obstacle to cell transplantation therapy for SCI is the formation of glial scars at the injury site. During mechanical injury or degenerative changes, neural tissue forms scar tissue containing dense glial cells but lacking functional neurons. In the early stages of injury, this scar tissue can close open wounds and act as a protective barrier for remaining neural tissue. However, in the later stages of the disease, it becomes a major factor hindering neural tissue regeneration, recanalization, and functional recovery (Yang T et al., Dissecting the Dual Role of the Glial Scar and Scar-Forming Astrocytes in Spinal Cord Injury. Front Cell Neurosci. 2020 Apr 3;14:78. doi: 10.3389 / fncel.2020.00078). Studies have been reported on the elimination of glial scars through surgical excision or cellular and molecular biological methods (Rasouli A et al., Resection of glial scar following spinal cord injury. JOrthop Res. 2009 Jul;27(7):931-6. doi: 10.1002 / jor.20793), but it is difficult to avoid causing secondary harm to the subjects and the treatment effect is not good.

[0005] The formation of glial scars is closely related to reactive astrocytes. As the main constituent cells of glial scars, reactive astrocytes can act as a physical barrier by extending large, star-shaped processes around the injury site. They can also secrete various neurodepressant factors (such as chondroitin sulfate proteoglycan (CSPG) and lipid transporter-2 (LCN2)) and inflammatory cytokines (such as TNFα and IL-1β), creating a local toxic microenvironment unfavorable to neuronal survival and regeneration (Zhang L et al., Development of Neuroregenerative GeneTherapy to Reverse Glial Scar Tissue Back to Neuron-Enriched Tissue. FrontCell Neurosci. 2020 Nov 5;14:594170). Therefore, a new therapeutic strategy is needed to reduce reactive astrocytes during the tissue recovery period, achieving early protection of the integrity of damaged neural tissue and subsequent repair and regeneration. Summary of the Invention

[0006] Through in-depth research, the inventors of this application have discovered that, following the acute phase of spinal cord mechanical injury (e.g., the subacute or chronic phase), transplantation of motor progenitor cell populations derived from pluripotent stem cells induced in vitro can effectively reduce reactive astrocytes at the site of injury. In test animals, a significant reduction in the area of ​​glial scars, a significant increase in the number of regenerated axons in the damaged descending tracts, and a substantial recovery of limb motor function were observed.

[0007] Furthermore, the inventors of this application discovered that after transplantation of motor progenitor cell populations during the subacute or chronic phase, the protein factor expression profile in the cerebrospinal fluid of the test animals underwent significant changes. The concentrations of various neurotrophic factors and growth factors increased significantly, while the concentrations of neuroinhibitory factors and inflammatory cytokines decreased. The resulting local environment not only directly benefits neuronal regeneration, growth, and tissue repair but also reduces the stress and toxic factor secretion of reactive astrocytes, further promoting the repair of damaged sites.

[0008] Therefore, the object of the present invention is to provide the use of motor progenitor cell populations in the preparation of a drug for (1) reducing reactive astrocytes, (2) reducing glial scarring, (3) inhibiting reactive astrocyte-mediated axonal regeneration impairment, (4) inhibiting reactive astrocyte-mediated neuronal and / or oligodendrocyte death, (5) altering the protein factor expression profile in cerebrospinal fluid, (6) promoting axonal regeneration and / or recovery, and (7) reducing demyelination, promoting myelin regeneration, and / or increasing myelin thickness.

[0009] In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs that reduce subacute-phase reactive astrocytes. In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs that reduce chronic-phase reactive astrocytes. In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs that reduce both subacute-phase and chronic-phase reactive astrocytes.

[0010] In some embodiments, the motor progenitor cell populations disclosed herein or the drugs disclosed herein reduce the number of reactive astrocytes in the subacute and / or chronic phases by about 1% to about 100%. In some embodiments, the motor progenitor cell populations disclosed herein or the drugs disclosed herein reduce the number of reactive astrocytes in the subacute and / or chronic phases by approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, and approximately 46%. %, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%. In some embodiments, reducing the number of reactive astrocytes means reducing the number of reactive astrocytes present in damaged central nervous system tissue (e.g., damaged spinal cord tissue or damaged brain tissue). In some embodiments, reducing the number of reactive astrocytes means reducing the number of reactive astrocytes present in any one or any combination of damaged cervical spinal cord, damaged thoracic spinal cord, damaged lumbar spinal cord, or any combination thereof.

[0011] In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs for reducing glial scars resulting from brain injuries. In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs for reducing glial scars resulting from spinal cord injuries. In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs for reducing glial scars resulting from both brain and spinal cord injuries.

[0012] In some embodiments, reducing glial scars means reducing any or any combination of the area, length, volume, thickness, or mass of the glial scar. In some embodiments, the motor progenitor cell populations disclosed herein or the drugs disclosed herein reduce any or any combination of the area, length, volume, thickness, or mass of the glial scar by at least about 50% to at least about 100%. In preferred embodiments, any or any combination of the area, length, volume, thickness, or mass of the glial scar is reduced by at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 70 ... 2%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%. In a more preferred embodiment, the area of ​​the glial scar is reduced by at least about 60%. In the most preferred embodiment, the area of ​​the glial scar is reduced by at least about 90%.

[0013] In some embodiments, the area of ​​the glial scar is automatically identified and measured using default parameters in ImageJ software. In other embodiments, the area of ​​the glial scar is measured by manual delineation by someone skilled in the art.

[0014] In some embodiments, the motor progenitor cell populations disclosed herein are used to prepare drugs that alter the expression profile of protein factors in cerebrospinal fluid. In some embodiments, the protein factors are selected from brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMP-4, BMP-5), β-nerve growth factor (beta-NGF), epidermal growth factor (EGF) and its receptor (EGFR), and endothelial growth factor (EG-VEGF). (PK1), fibroblast growth factor (FGF-4, FGF-7 (KGF)), growth differentiation factor 15 (GDF-15), glial cell-derived neurotrophic factor (GDNF), auxin, hepatocyte growth factor (HB-EGF, HGF), insulin-like growth factor binding protein (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), insulin-like growth factor (IGF-1), insulin, macrophage colony-stimulating factor receptor (MCSFR), nerve growth factor receptor (NGFR (TNFRSF16)), neurotrophic factors (NT-3, NT-4), osteoprotegerin (TNFRSF11B)), platelet-derived growth factor (PDGF-AA), placental growth factor (PLGF), stem cell factor (SCF) and its receptor (SCF R (CD117 / c-kit)), transforming growth factor (TGF alpha, TGF beta1, TGF beta2) 3) Any one or any combination of vascular endothelial growth factors (VEGF-A, VEGFR2, VEGFR3, VEGF-D). In a preferred embodiment, the protein factor is selected from brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenetic protein (BMP-4, BMP-5), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), stem cell factor (SCF) and its receptor (SCFR(CD117 / c-kit)), transforming growth factor (TGF alpha, TGF beta 1, TGF beta 3), and vascular endothelial growth factors (VEGF-A, VEGFR2, VEGFR3, VEGF-D). In a more preferred embodiment, the protein factor is selected from any one or any combination of BDNF, bFGF, BMP-4, BMP-5, EGF, IGFBP-3, IGFBP-4, stem cell factor receptor, TGFbeta 3, and VEGF.

[0015] In some embodiments, altering the protein factor expression profile in cerebrospinal fluid (CSF) involves increasing and / or decreasing the concentration of protein factors in the CSF. In some embodiments, altering the protein factor expression profile in CSF involves increasing and / or decreasing the concentration of cytokines (such as IL-10 and IL-4) in the CSF. In some embodiments, altering the protein factor expression profile in CSF involves increasing and / or decreasing the concentration of extracellular matrix proteins (such as laminin, fibronectin, and hyaluronic acid) in the CSF.

[0016] In some embodiments, reactive astrocytes are astrocytes expressing any one of the markers selected from AQP4, GLT-1, VIMENTIN, GLAST, ALDH1L1, Lcn2, Steap4, Cxc110, guanylate-binding protein 2 (GBP2), GFAP, S100beta, and A2B5, or any combination thereof. In some embodiments, reactive astrocytes are AQP4-positive astrocytes. In some embodiments, reactive astrocytes are GLT-1-positive astrocytes. In some embodiments, reactive astrocytes are VIMENTIN-positive astrocytes. In some embodiments, reactive astrocytes are GLAST-positive astrocytes. In some embodiments, reactive astrocytes are ALDH1L1-positive astrocytes. In some embodiments, reactive astrocytes are Lcn2-positive astrocytes. In some embodiments, the reactive astrocytes are Steap4-positive astrocytes. In some embodiments, the reactive astrocytes are Cxc110-positive astrocytes. In some embodiments, the reactive astrocytes are guanylate-binding protein 2 (GBP2)-positive astrocytes. In some embodiments, the reactive astrocytes are GFAP-positive astrocytes. In some embodiments, the reactive astrocytes are S100beta-positive astrocytes. In some embodiments, the reactive astrocytes are A2B5-positive astrocytes.

[0017] In some embodiments, the motor progenitor cell population disclosed herein comprises at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, and at least about 99% of motor progenitor cells. In some embodiments, the motor progenitor cells are cells that express the markers OLIG2 and HOXB4, and hardly express the marker OCT4.

[0018] In some embodiments, the motor progenitor cell population disclosed herein comprises a cell population with a non-motor progenitor cell content of less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, and less than about 1%. In some embodiments, the non-motor progenitor cells are selected from vascular leptomeningeal cells (VLMCs).

[0019] In some embodiments, motor neural progenitor cells are obtained through in vitro induced pluripotent stem cell differentiation. In some embodiments, the pluripotent stem cells are selected from at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells. In a preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are derived from mammals. In a preferred embodiment, the pluripotent stem cells are derived from primates. In a more preferred embodiment, the pluripotent stem cells are human pluripotent stem cells.

[0020] In another aspect, the present invention also provides the use of motor neuron progenitor cell populations or motor neuron cell populations in the preparation of medicaments for (1) reducing reactive astrocytes, (2) reducing glial scarring, (3) inhibiting reactive astrocyte-mediated axonal regeneration impairment, (4) inhibiting reactive astrocyte-mediated neuronal and / or oligodendrocyte death, (5) altering the protein factor expression profile in cerebrospinal fluid, (6) promoting axonal regeneration and / or recovery, and (7) reducing demyelination, promoting myelin regeneration, and / or increasing myelin thickness.

[0021] In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs that reduce reactive astrocytes in the subacute phase. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs that reduce reactive astrocytes in the acute phase. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs that reduce reactive astrocytes in the chronic phase. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs that reduce reactive astrocytes in both the subacute and acute phases. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs that reduce reactive astrocytes in both the subacute and chronic phases.

[0022] In some embodiments, the motor neuron progenitor cell population or motor neuron cell population disclosed herein, or the drug disclosed herein, reduces the number of reactive astrocytes in the subacute, chronic, and / or acute phases by about 1% to about 100%. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein, or the drugs disclosed herein, reduce the number of reactive astrocytes in the subacute, chronic, and / or acute phases by approximately 1%, approximately 2%, approximately 3%, approximately 4%, approximately 5%, approximately 6%, approximately 7%, approximately 8%, approximately 9%, approximately 10%, approximately 11%, approximately 12%, approximately 13%, approximately 14%, approximately 15%, approximately 16%, approximately 17%, approximately 18%, approximately 19%, approximately 20%, approximately 21%, approximately 22%, approximately 23%, approximately 24%, approximately 25%, approximately 26%, approximately 27%, approximately 28%, approximately 29%, approximately 30%, approximately 31%, approximately 32%, approximately 33%, approximately 34%, approximately 35%, approximately 36%, approximately 37%, approximately 38%, approximately 39%, approximately 40%, approximately 41%, approximately 42%, approximately 43%, approximately 44%, approximately 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%. In some embodiments, reducing the number of reactive astrocytes means reducing the number of reactive astrocytes present in damaged central nervous system tissue (e.g., damaged spinal cord tissue or damaged brain tissue). In some embodiments, reducing the number of reactive astrocytes means reducing the number of reactive astrocytes present in any one or any combination of damaged cervical spinal cord, damaged thoracic spinal cord, damaged lumbar spinal cord, or any combination thereof.

[0023] In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs for reducing glial scars resulting from brain injuries. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs for reducing glial scars resulting from spinal cord injuries. In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs for reducing glial scars resulting from both brain and spinal cord injuries.

[0024] In some embodiments, reducing glial scars means reducing any or any combination of the area, length, volume, thickness, or mass of the glial scar. In some embodiments, the motor nerve progenitor cell populations or motor neuron cell populations disclosed herein, or the drugs disclosed herein, reduce any or any combination of the area, length, volume, thickness, or mass of the glial scar by about 50% or at least about 100%. In preferred embodiments, any or any combination of the area, length, volume, thickness, or mass of the glial scar is reduced by at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 70 ... 2%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%. In a more preferred embodiment, the area of ​​the glial scar is reduced by at least about 60%. In the most preferred embodiment, the area of ​​the glial scar is reduced by at least about 90%.

[0025] In some embodiments, the area of ​​the glial scar is automatically identified and measured using default parameters in ImageJ software. In other embodiments, the area of ​​the glial scar is measured by manual delineation by someone skilled in the art.

[0026] In some embodiments, the motor neuron progenitor cell populations or motor neuron cell populations disclosed herein are used to prepare drugs that alter the expression profile of protein factors in cerebrospinal fluid. In some embodiments, the protein factors are selected from brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMP-4, BMP-5), β-nerve growth factor (beta-NGF), epidermal growth factor (EGF) and its receptor (EGFR), endothelial growth factor (EG-VEGF(PK1)), fibroblast growth factor (FGF-4, FGF-7(KGF)), growth differentiation factor 15 (GDF-15), glial cell-derived neurotrophic factor (GDNF), auxin, and hepatocyte growth factor (HB-EGF, HGF). Insulin-like growth factor binding protein (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), insulin-like growth factor (IGF-1), insulin, macrophage colony-stimulating factor receptor (MCSFR), nerve growth factor receptor (NGFR (TNFRSF16)), neurotrophic factors (NT-3, NT-4), osteoprotegerin (TNFRSF11B)), platelet-derived growth factor (PDGF-AA), placental growth factor (PLGF), stem cell factor (SCF) and its receptor (SCF R (CD117 / c-kit)), transforming growth factor (TGF alpha, TGF beta 1, TGF beta 3), vascular endothelial growth factor (VEGF-A, VEGFR2, VEGFR3, VEGF-D) or any combination thereof. In a preferred embodiment, the protein factor is selected from any one or any combination of brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMP-4, BMP-5), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), stem cell factor (SCF) and its receptor (SCF R (CD117 / c-kit)), transforming growth factor (TGF alpha, TGF beta 1, TGF beta 3), and vascular endothelial growth factor (VEGF-A, VEGFR2, VEGFR3, VEGF-D). In a more preferred embodiment, the protein factor is selected from any one or any combination of BDNF, bFGF, BMP-4, BMP-5, EGF, IGFBP-3, IGFBP-4, stem cell factor receptor, TGF beta 3, and VEGF.

[0027] In some embodiments, altering the protein factor expression profile in cerebrospinal fluid (CSF) involves increasing and / or decreasing the concentration of protein factors in the CSF. In some embodiments, altering the protein factor expression profile in CSF involves increasing and / or decreasing the concentration of cytokines (such as IL-10 and IL-4) in the CSF. In some embodiments, altering the protein factor expression profile in CSF involves increasing and / or decreasing the concentration of extracellular matrix proteins (such as laminin, fibronectin, and hyaluronic acid) in the CSF.

[0028] In some embodiments, reactive astrocytes are astrocytes expressing any one of the markers selected from AQP4, GLT-1, VIMENTIN, GLAST, ALDH1L1, Lcn2, Steap4, Cxc110, guanylate-binding protein 2 (GBP2), GFAP, S100beta, and A2B5, or any combination thereof. In some embodiments, reactive astrocytes are AQP4-positive astrocytes. In some embodiments, reactive astrocytes are GLT-1-positive astrocytes. In some embodiments, reactive astrocytes are VIMENTIN-positive astrocytes. In some embodiments, reactive astrocytes are GLAST-positive astrocytes. In some embodiments, reactive astrocytes are ALDH1L1-positive astrocytes. In some embodiments, reactive astrocytes are Lcn2-positive astrocytes. In some embodiments, the reactive astrocytes are Steap4-positive astrocytes. In some embodiments, the reactive astrocytes are Cxc110-positive astrocytes. In some embodiments, the reactive astrocytes are guanylate-binding protein 2 (GBP2)-positive astrocytes. In some embodiments, the reactive astrocytes are GFAP-positive astrocytes. In some embodiments, the reactive astrocytes are S100beta-positive astrocytes. In some embodiments, the reactive astrocytes are A2B5-positive astrocytes.

[0029] In some embodiments, the motor progenitor cell population or motor neuron cell population disclosed herein is a cell population comprising at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of motor progenitor cells. In some embodiments, the motor progenitor cell population is cells expressing the marker MNX1 (HB9). In some embodiments, the motor neuron cell population is cells expressing the markers CHAT, MNX1, and ISLET1.

[0030] In some embodiments, the motor neural progenitor cell population disclosed herein comprises a cell population with a non-motor neural progenitor cell content of less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, and less than about 1%. In some embodiments, the non-motor neural progenitor cells are selected from vascular leptomeningeal cells (VLMCs).

[0031] In some embodiments, the motor neuron cell populations disclosed herein are cell populations with a nonmotor neuron cell content of less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, and less than about 1%. In some embodiments, the nonmotor neuron cells are selected from vascular leptomeningeal cells (VLMCs).

[0032] In some embodiments, motor neuron progenitor cells and motor neurons are obtained through in vitro induced pluripotent stem cell differentiation. In some embodiments, the pluripotent stem cells are selected from at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells. In a preferred embodiment, the pluripotent stem cells are induced pluripotent stem cells. In some embodiments, the pluripotent stem cells are derived from mammals. In a preferred embodiment, the pluripotent stem cells are derived from primates. In a more preferred embodiment, the pluripotent stem cells are human pluripotent stem cells.

[0033] In some embodiments, the drugs disclosed herein are administered during the subacute and / or chronic phases of spinal cord injury or brain injury. In preferred embodiments, the drugs disclosed herein are administered during the subacute phase of spinal cord injury or brain injury. In some embodiments, the drugs disclosed herein are used to (1) reduce reactive astrocytes formed during the subacute phase, (2) reduce glial scarring formed during the subacute phase, (3) inhibit reactive astrocyte-mediated axonal regeneration impairment during the subacute phase, (4) inhibit reactive astrocyte-mediated neuronal death and / or oligodendrocyte death during the subacute phase, (5) alter the protein factor expression profile in the cerebrospinal fluid during the subacute phase, (6) promote axonal regeneration and / or recovery during the subacute phase, and (7) reduce demyelination, promote myelin regeneration, and / or increase myelin thickness during the subacute phase. In some embodiments, the drugs disclosed herein are administered during the chronic phase of spinal cord injury or brain injury. In some embodiments, the drugs disclosed herein are used to (1) reduce reactive astrocytes formed in the chronic phase, (2) reduce glial scars formed in the chronic phase, (3) inhibit reactive astrocyte-mediated axonal regeneration impairment in the chronic phase, (4) inhibit reactive astrocyte-mediated neuronal death and / or oligodendrocyte death in the chronic phase, (5) alter the protein factor expression profile in the cerebrospinal fluid in the chronic phase, (6) promote axonal regeneration and / or recovery in the chronic phase, and (7) reduce demyelination in the chronic phase, promote myelin regeneration, and / or increase myelin thickness. In some embodiments, the drugs disclosed herein are used to (1) reduce reactive astrocytes formed in the acute phase, (2) reduce glial scars formed in the acute phase, (3) inhibit reactive astrocyte-mediated acute axonal regeneration impairment, (4) inhibit reactive astrocyte-mediated acute neuronal death and / or oligodendrocyte death, (5) alter the protein factor expression profile in the cerebrospinal fluid in the acute phase, (6) promote axonal regeneration and / or recovery in the acute phase, and (7) reduce acute demyelination, promote myelin regeneration, and / or increase myelin thickness.

[0034] In some embodiments, the drug disclosed herein is administered by any of the methods selected from intrathecal injection, intramedullary injection, intraventricular injection, or intravenous injection. In some embodiments, the drug disclosed herein may be selected from intrathecal injection formulations, intramedullary injection formulations, intraventricular injection formulations, or intravenous injection formulations.

[0035] In some respects, this document also provides pharmaceutical compositions comprising the motor progenitor cell populations disclosed herein and pharmaceutically acceptable excipients.

[0036] In some respects, this document also provides methods selected from (1) to (6) for (1) reducing reactive astrocytes, (2) reducing glial scar formation, (3) inhibiting reactive astrocyte-mediated axonal regeneration impairment, (4) inhibiting reactive astrocyte-mediated neuronal death and / or oligodendrocyte death, (5) altering the protein factor expression profile in cerebrospinal fluid, (6) promoting axonal regeneration and / or recovery, and (7) reducing demyelination, promoting myelin regeneration, and / or increasing myelin thickness, the methods comprising administering to a subject in need the motor progenitor cell population or the pharmaceutical composition provided herein. Attached Figure Description

[0037] Figure 1 The flow cytometry results of the exemplary motor neural progenitor cell population prepared in Example 1 are shown.

[0038] Figure 2 The results of immunofluorescence staining of the exemplary motor nerve progenitor cell population prepared in Example 2 with DAPI, anti-HB9 monoclonal antibody, and anti-OLIG2 monoclonal antibody are shown.

[0039] Figure 3 Immunofluorescence staining was performed on the exemplary motor neural progenitor cell population prepared in Example 2 to reveal HB9. + OLIG2 + HB9 + OLIG2 - HB9 - OLIG2 + Statistical results of cells;

[0040] Figure 4 The results of immunofluorescence staining of the exemplary motor neuron cell population prepared in Example 3 with DAPI, anti-CHAT monoclonal antibody, and anti-HB9 monoclonal antibody are shown.

[0041] Figure 5 The results of immunofluorescence staining of lesion areas in an SCI mouse model after intrathecal injection of the exemplary motor progenitor cell population prepared in Example 1 are shown. Figure 5 A to 5C represent the control group, low-dose treatment group, and high-dose treatment group, respectively. Red represents the fluorescence emitted by BDA particles, green represents GFAP-positive neuronal axons, blue represents DAPI-positive live cells, and the dashed area represents the damaged area surrounded by GAFP-positive cells (which are GAFP-negative). Figure 5 D shows the statistical results for the area of ​​GAFP negative regions in 5A to 5C respectively;

[0042] Figure 6 The results of tracing the corticospinal tract in the lesion region with BDA after intrathecal injection of the exemplary motor progenitor cell population prepared in Example 1 are shown. Figure 6 A to 6C represent the control group, low-dose treatment group, and high-dose treatment group, respectively. In the figure, a is the center of the spinal cord lesion, b is the descending region of the lesion center, red is the fluorescence emitted by BDA particles, and green represents NeuN positive neurons.

[0043] Figure 7 The BMS scores of the SCI mouse control group, high-dose treatment group, and low-dose treatment group are shown.

[0044] Figure 8 shows the results from the 3-dose group ( Figure 8A , 8B ), 2-dose group ( Figure 8C ) and the single-dose group ( Figure 8D Representative transmission electron microscopy (TEM) images of an individual; arrows indicate regenerated axons;

[0045] Figure 9 The BBB scores of the SCI rat control group, the single-dose group, the double-dose group, and the triple-dose group are shown.

[0046] Figure 10 This shows that in SD rats, BDNF ( Figure 10 A), bFGF ( Figure 10 B), BMP4 Figure 10 C), BMP5 Figure 10 D), BMP7 Figure 10 E), IGFBP3 ( Figure 10 F), IGFBP4 ( Figure 10 G), EGF ( Figure 10 H), SCF-RF ( Figure 10 I), TGFb3 ( Figure 10 J) and VEGF Figure 10 The change in cerebrospinal fluid concentration over time for K). Detailed Implementation

[0047] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available. Unless otherwise specified, the quantitative experiments in the following examples are the average values ​​of three replicate experiments.

[0049] definition

[0050] As used in the specification of this invention, the following words and phrases are generally considered to have the meanings set forth below, unless otherwise specified in the context in which they are used.

[0051] As used herein, the terms “comprising” or “including” mean that a composition and method comprises the described components, but does not exclude other components. “Mainly composed of”, when used to define a composition and method, should exclude any other components that are obviously essential to the composition. Therefore, compositions defined herein as mainly composed of these components will not exclude trace contamination from separation and purification methods and pharmaceutically acceptable carriers such as phosphate-buffered saline, preservatives, etc. “Composed of” should exclude trace components of other ingredients used in the application of the compositions of the invention and of substantial process. Examples defined by these provisional terms are within the scope of this invention.

[0052] Unless otherwise stated herein, the listing of numerical ranges herein is intended only as a shorthand method for individually referring to each individual value falling within that range, and each individual value is incorporated into the specification as if it were listed separately herein. For example, in this specification, if the concentration range is expressed as 1 to 10 μM, it is intended to explicitly list values ​​such as 2 to 9 μM, 5 to 6 μM, or 1 to 5 μM. These are merely examples of specific intentions, and all possible combinations of values ​​between and including the listed minimum and maximum values ​​will be considered explicitly stated in this disclosure. The use of the term “about” to describe a particular listed quantity or range of quantities means indicating that a value very close to the listed quantity is included in that quantity, such as values ​​that can or naturally be taken into account due to manufacturing tolerances, instrumental and human errors in the formation of the measurement. For example, a numerical value described herein as “about” means covering ±10% of the indicated value. In some cases, “about” may mean ±20%, or ±5%, or ±1%. A value or parameter described herein as “about” includes the value or parameter itself.

[0053] As used herein, the terms “first” and “second” are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Unless otherwise expressly stated, a feature defined as “first” or “second” may explicitly or implicitly include at least one of that feature.

[0054] As used herein, the terms “treatment” and “improvement” are used interchangeably to refer to a method of obtaining a beneficial or desired outcome, including but not limited to therapeutic benefits and / or preventive benefits. A therapeutic benefit means the eradication or reduction of an underlying disease being treated. A therapeutic benefit can be achieved by eradicating or reducing one or more physiological symptoms associated with an underlying disease, thereby resulting in an improvement observed in the patient. For the purposes of this invention, therapeutic benefits include, but are not limited to: symptom reduction, disease severity reduction, delay or slowing of disease progression, improvement or mitigation of a disease state, and remission (whether partial or complete), whether detectable or undetectable. For preventive benefits, the pharmaceutical compositions disclosed herein may also be administered to patients at risk of developing a specific disease or to patients reporting one or more physiological symptoms of a disease, even if the disease has not yet been diagnosed. As used herein, the term “prevention” means a reduction in the frequency of the disease or condition in a treated sample relative to an untreated control sample, or a delay in the onset and / or reduction in the severity of one or more symptoms of a disease or condition relative to an untreated control sample.

[0055] In some respects, the term "treatment" includes both preventative and therapeutic treatment. Generally, treatment is preventative (i.e., protecting the subject from developing the unwanted condition) if administered before the clinical manifestation of an unwanted condition (e.g., a hallmark behavior such as tremor, or the observation of typical pathological molecular markers); and therapeutic (i.e., aiming to reduce, improve, or stabilize the existing unwanted condition or its side effects) if administered after the manifestation of the unwanted condition.

[0056] As used herein, the term "pharmaceutical-acceptable carrier" refers to various compounds used in the preparation of pharmaceutical compositions that are generally safe and non-toxic and do not possess biologically or otherwise undesirable properties.

[0057] As used in this article, the term "astrocytocyte" refers to a type of glial cell widely distributed in the central nervous system (CNS) of mammals. It mediates the formation of neurovascular units, forms the blood-brain barrier together with pericytes and endothelial cells, provides structural and metabolic support to neurons, and plays a crucial role in regulating neuronal survival, morphology, axonal growth, synapse formation, and ion channel distribution (Chiareli RA et al., The Role of Astrocytes in the Neurorepair Process. Front Cell Dev Biol. 2021 May 25;9:665795. doi:10.3389 / fcell.2021.665795). Astrocytes lack Nissl bodies in their cytoplasm but contain abundant fibrils composed of glial fibrillary acid proteins (GFAP).

[0058] As used herein, the term "reactive astrocyte" refers to astrocytes in a reactive state activated by pathological conditions such as injury or local inflammation. Reactive astrocytes are derived from protoplasmic astrocytes through morphological and molecular transformation, highly express reactive astrocyte markers, and can also secrete inflammatory cytokines (such as TNF-α, IL-1β, IFN-γ, MCP-1, MIP-1α, RANTES, and MMPs).

[0059] As used in this article, the term "glial scar" refers to a dense, boundary-shaped structure that forms near the injured tissue after spinal cord injury. Glial scars are primarily formed by reactive astrocytes, oligodendrocyte precursors, and microglia that are rapidly activated in response to injury signals near the injury site, effectively sealing off the entire injured area. Glial scars can be easily identified by manually delineating their area or by using software (such as ImageJ) to automatically recognize them.

[0060] The terms “acute phase,” “subacute phase,” and “chronic phase” have their commonly understood meanings in this article (Chandra, J., Sheerin, F., Lopez de Heredia, L. et al. MRI in acute and subacute post-traumatic spinal cord injury: pictorial review. Spinal Cord 50, 2–7 (2012)). Specifically, for humans, the term “acute phase” typically refers to the first 0–72 hours (within 3 days) after injury, characterized by direct mechanical injury and the ensuing local ischemia, hemorrhage, swelling, and inflammation. Spinal cord nerve cells suffer initial physical damage, accompanied by loss of nerve conduction function. Pathological changes in the acute phase include local hemorrhage and edema, acute nerve cell damage and death, and the initial initiation of an inflammatory response (such as leukocyte infiltration). Clinical manifestations include acute pain, loss of movement and sensation, and loss of reflexes. The term “subacute phase” typically refers to 3 days to 6 weeks after spinal cord injury. During the subacute phase, neuronal necrosis expands, glial response intensifies, local inflammation persists in the spinal cord, and glial cells begin to proliferate, forming glial scars. Simultaneously, neural circuits and axons in the injured area begin to exhibit preliminary self-repair and regeneration during the subacute phase, although the repair process is limited and slow. Histologically, "cavities" or cystic changes appear around the injured area during the subacute phase. The inflammatory response reaches its peak, with increased secretion of cytokines and other inflammatory mediators. Clinical manifestations of the subacute phase include significant motor and sensory loss, although some reflex recovery may occur. The term "chronic phase" typically refers to spinal cord injury lasting more than 6 weeks. In some implementations, the chronic phase refers to 6 weeks to 6 months after spinal cord injury. During this stage, the loss of neurological function in the injured area has stabilized, glial scars gradually form, and nerve conduction function almost completely ceases to recover. Damaged spinal cord tissue has undergone long-term adaptive changes in structure and function. Nerve tissue and cells at the injury site generally experience atrophy and degeneration, with significant glial scarring and glial cell proliferation forming a physical barrier that significantly hinders nerve regeneration and makes reconstructing neural pathways difficult. Simultaneously, long-term inflammation may also lead to further degeneration of the nervous system. Clinical manifestations in the chronic phase include stable neurological function loss; patients may develop complete or incomplete paraplegia, sensory loss, bladder and bowel dysfunction, etc. Non-human animal models can also be classified into stages based on these characteristics.

[0061] As used herein, the term "BMP inhibitor" refers to a protein, peptide, or small molecule compound capable of inhibiting the activity of the BMP signaling pathway. Non-limiting examples include those disclosed in WO2011 / 149762, Chambers et al., Nat Biotechnol. 2009 Mar, 27(3):275-80, Kriks et al., Nature. 2011 Nov 6, 480(7378): 547-51, and Chambers et al., Nat Biotechnol. 2012 Jul 1, 30 (7): 715-20, all of which are incorporated herein by reference in their entirety. In some embodiments, the BMP inhibitor may be selected from DMH-1 or its active derivatives, or pharmaceutically acceptable salts thereof. Derivatives of these small molecule inhibitors can be readily obtained by those skilled in the art through substituent modification, substitution, or addition to the core backbone of the inhibitor.

[0062] "DMH-1" refers to the molecule with CAS number 1206711-16-1-41-9 and name 4-[6-[4-(1-methoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline. In in vitro kinase assays, DMH-1 selectively inhibits the bone morphogenetic protein (BMP) type I receptor activin receptor-like kinase 2 (ALK2) receptor. Its selectivity for ALK-2 is 6 times that of ALK-1 and 19 times that of ALK-3, with no significant inhibitory effect on AMPK, ALK5, KDR (VEGFR-2), or PDGFRβ receptors. DMH-1 has been reported to block BMP4-induced phosphorylation of Smads 1, 5, and 8 in HEK293 cells (Neely et al., ACSChem.Neurosci., 2012;3:482).

[0063] As used herein, the term "Nodal inhibitor" may also be referred to as "TGFβ / Activin-Nodal inhibitor," which is a class of SMAD inhibitors. In some embodiments, TGF-β1 inhibitors can inactivate receptors including TGFβ, Nodal, and / or Activin, and / or block signal transduction pathways by blocking these receptors and their downstream effectors. Non-limiting examples of TGF-β1 inhibitors include those disclosed in WO / 2010 / 096496, WO / 2011 / 149762, WO / 2013 / 067362, WO / 2014 / 176606, WO / 2015 / 077648, Chambers et al., NatBiotechnol. 2009 Mar, 27(3):275-80, Kriks et al., Nature. 2011 Nov 6, 480 (7378): 547-51, and Chambers et al., NatBiotechnol. 2012 Jul 1, 30 (7): 715-20, all of which are incorporated herein by reference in their entirety. In some embodiments, the TGF-β1 inhibitor may be selected from SB431542 or its active derivatives (such as A83-01), or pharmaceutically acceptable salts thereof. “SB431542” refers to the small molecule compound with the CAS number 301836-41-9, the name 4-[4-(1,3-benzodioxolane-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide, and the molecular formula C22H18N4O3.

[0064] It has been reported that reducing the activity of glycogen synthase kinase 3β (GSK3β) can activate Wnt signaling. Therefore, the term "GSK3 inhibitor" in this article can be used interchangeably with "Wnt activator". Non-limiting examples of GSK3β inhibitors include CHIR99021, WNT1, WNT5A, WNT3A, CHIR98014, AMBMP hydrochloride, LP 922056, lithium, deoxycholic acid, BIO, or SB-216763, as well as those described in WO2011 / 149762, WO13 / 067362, Chambers et al., NatBiotechnol. 2012 Jul 1, 30 (7): 715-20, Kriks et al., Nature. 2011 Nov 6, 480(7378): 547-51, and Calder et al., J Neurosci. 2015 Aug 19, 35(33):11462-81, all of which are incorporated herein by reference in their entirety.

[0065] In some embodiments, the GSK3 inhibitor may be selected from CHIR99021 or its active derivatives, or pharmaceutically acceptable salts thereof. “CHIR99021” refers to a small molecule inhibitor of the IUPAC name 6-(2-(4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)pyrimidin-2-ylamino)ethylamino)nicotinonitrile, also known as “aminopyrimidine” or “3-[3-(2-carboxyethyl)-4-methylpyrrole-2-methylene]-2-indolone”.

[0066] As used herein, the term “SHH” is used interchangeably with “Sonic hedgehog” and refers to a protein of one of at least three proteins in the mammalian signaling pathway family known as hedgehog factors. SHH regulates the expression of certain downstream genes by interacting with the transmembrane molecules Patched (PTC) and Smoothened (SMO) (see Gilbert, 2000 Developmental Biology (Sunderland, Mass., Sinauer Associates, Inc., Publishers)). Non-limiting examples of SHH activators include those described in WO10 / 096496, WO13 / 067362, Chamberset et al., Nat Biotechnol. 2009 Mar, 27(3):275-80, and Kriks et al., Nature. 2011 Nov6, 480 (7378): Those in 547-51. In some embodiments, the SHH activator may be selected from SAG (N-methyl-N'-(3-pyridylphenyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane) or its active derivatives, or pharmaceutically acceptable salts thereof.

[0067] As used herein, the term "ROCK inhibitor" is an abbreviation for "Rho-associated kinase inhibitor," referring to any substance that inhibits or reduces the function of Rho-associated kinases or their signaling pathways in cells. In some embodiments, the ROCK inhibitor may be selected from Y27632 or its active derivatives, or pharmaceutically acceptable salts thereof (e.g., Y27632 dihydrochloride 2HCl).

[0068] The term "cell population" generally refers to a group of cells. A cell population can consist of cells with a common phenotype (e.g., pluripotent stem cells), or may contain at least a subset of cells with a common phenotype. Cells are considered to have a common phenotype when they are substantially similar or identical in one or more provable characteristics, including but not limited to morphological appearance, the presence, absence, or level of expression of specific cellular components or products (e.g., RNA, proteins, or other markers), activity of certain biochemical pathways, proliferative capacity and / or kinetics, differentiation potential and / or response to differentiation signals, or behavior during in vitro culture (e.g., adhesion, non-adhesion, monolayer growth, proliferation kinetics, etc.). Thus, such provable characteristics can define a cell population or its fraction.

[0069] The preferred embodiments for carrying out the present invention will now be described. It should be noted that the embodiments described below are examples illustrating representative embodiments of the present invention, but the present invention is not limited to these embodiments.

[0070] Two or more of the methods described below can be combined, and such combinations are also included in this invention.

[0071] Example

[0072] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0073] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following examples are commercially available.

[0074] Experimental materials, reagents, instruments and experimental methods

[0075] Cell lines:

[0076] According to the manufacturer's instructions, induced pluripotent stem cells (iPSCs) were prepared by reprogramming human PBMC cells using the CTS™ CytoTune™-iPS 2.1 Sendai Virus Reprogramming Kit (CTS™ CytoTune™-iPS 2.1 Sendai Reprogramming Kit, catalog number: A34546).

[0077] When iPSC cells reach 70-80% confluence using StemFit medium, TrypLE digestion solution is added, and the cells are incubated at 37°C for 15 minutes (12-16 minutes is also acceptable) to digest them into complete single cells. The cells are then resuspended in StemFit medium supplemented with 10 μM Y27632 (purchased from Sigma), counted, and an appropriate amount of StemFit medium is added. The cells are then seeded into T75 cell culture flasks pre-coated with Laminin at a density of 5000 cells / cm². The wells are then incubated at 37°C with 5% CO₂ for 24 hours.

[0078] Culture medium:

[0079] First culture medium: neural basal medium containing 2 μM SB431542 (purchased from Selleck, catalog number S106707), 2 μM DMH-1 (purchased from Selleck, catalog number S714602) and 3 μM CHIR99021 (purchased from Tocris, catalog number 442310).

[0080] Second culture medium: neural basal medium containing 2 μM SB431542, 2 μM DMH-1, 1 μM CHIR99021, 0.1 μM retinoic acid (purchased from Selleck, catalog number S106707), 10 μM Y-27632 and 0.5 μM SAG (purchased from Tocris, catalog number 4366).

[0081] The third culture medium was a neural basal medium containing 2 μM SB431542, 2 μM DMH-1, 3 μM CHIR99021, 0.1 μM retinoic acid, 10 μM Y-27632 and 0.5 μM SAG.

[0082] Fourth culture medium: neural basal medium containing 2 μM SB431542, 2 μM DMH-1, 3 μM CHIR99021, 0.1 μM retinoic acid, 0.5 μM SAG and 0.1 μM compound E (purchased from Tocris, catalog number 6476).

[0083] Fifth culture medium: neural basal medium containing 0.1 μM compound E, 0.2 μM SAG, 0.5 μM retinoic acid, and 10 μM Y-27632.

[0084] The sixth culture medium was a basal medium for neural differentiation containing 10 ng / mL BDNF (purchased from Peprotech, catalog number 450-02-1MG), 10 ng / mL GDNF (purchased from Peprotech, catalog number 450-10-1MG), 0.2 μM SAG, 0.5 μM retinoic acid, and 0.2 mM AA (L-ascorbic acid, purchased from Sigma).

[0085] Neural basal culture medium: DMEM / F12 (Gibco, catalog number 10565042) containing 0.5% (v / v) N2 supplement (Gibco), 1% (v / v) non-essential amino acid (Gibco) and 1% (v / v) B-27.

[0086] Neurobasal (Gibco) containing 1% (v / v) N2 supplement (Gibco) and 2% (v / v) B-27 (Thermo, catalog number A3653401).

[0087] Antibody:

[0088] Antibody Name Supplier Code

[0089] Antibody name Item number supplier NeuN antibody Ab177487 Abcam GFAP antibody ab68428 Abcam Rabbit II Anti- ab150079 Abcam Mouse secondary antibody ab150113 Abcam

[0090] Chemical reagents:

[0091] Reagent Name Supplier Code

[0092] Reagent Name Item number supplier BDA N7167 Thermofisher Streptavidin - Alexa Flour647 S32357 Thermofisher

[0093] Flow cytometry:

[0094] Transfer cells to 1.5 mL centrifuge tubes, centrifuge and discard the supernatant. Resuspend cells in staining solution (PBS buffer containing 0.5% BSA) and aliquot into four new centrifuge tubes. Add isotype control, OLIG2, OCT4, and HOXB4 antibodies to the four centrifuge tubes respectively, mix well, and incubate for 25 min (20-30 min is also acceptable). Wash once with staining solution, centrifuge, discard the supernatant, and analyze using flow cytometry.

[0095] Immunofluorescence:

[0096] Equal amounts of cells from the experimental group and the control group were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. After washing with paraformaldehyde, the cells were fixed at room temperature for 15 minutes. After washing three times with PBS, the cells were permeabilized with PBST (0.2% Triton + PBS) for 10 minutes. After washing once with PBS, blocking buffer (PBST + 3% donkey serum) was added, and the cells were incubated at room temperature for 30 minutes. The cells were then incubated with the corresponding primary antibody overnight at 4°C. After washing three times with PBS, the cells were incubated with the corresponding secondary antibody at 37°C in the dark for 1 hour. After washing three times with PBS, the cells were stained with DAPI for 5 minutes. After washing three times with PBS, the cells were observed under a fluorescence microscope and photographed in the dark.

[0097] ImageJ Analysis

[0098] Twelve weeks after cell injection, mice were anesthetized with 1% tribromoethanol. The heart was perfused with 0.9% saline, and the spinal cord was quickly removed after lamina resection and placed in pre-cooled 2.5% glutaraldehyde fixative. Sections of 2-3 mm were taken from the lesion center and the boundaries of the lesions at the beak and tail. The removed tissue was trimmed into approximately 1 mm blocks, dispersed, dehydrated, infiltrated, and embedded. Semi-thin and ultrathin sections were prepared by Beijing Hengdao Medical Laboratory Co., Ltd. for imaging with a transmission electron microscope (JEOL-1400flash, Japan) at 8000x magnification. The commissioning party was unaware of the sample information. From the photographs, approximately 30 images were randomly selected from each group, and approximately 10 images from each animal were randomly selected. ImageJ analysis was used to calculate the total number of axons, myelinated axons, unmyelinated and myelinated axons, and fiber diameters in all photographs, and the G-ratio (axon diameter / fiber diameter) was obtained. At least 1000 axons were measured in each image set. All data are expressed as mean ± SD and statistically analyzed using GraphPad Prism. G-ratio is widely used as a functional and structural index of the relative amount of myelin sheath (i.e., axonal myelin formation) present on the axon. A higher G-ratio value indicates a thinner myelin sheath relative to the axon diameter; conversely, a lower G-ratio value indicates a thicker myelin sheath relative to the axon diameter.

[0099] Example 1

[0100] This embodiment describes an exemplary method for inducing human pluripotent stem cells to differentiate into motor neural progenitor cells in vitro.

[0101] Differentiation of pluripotent stem cells into neural epithelial cells (D0~D6)

[0102] After 24 hours of cell adhesion and growth, the culture medium was replaced with the first culture medium (representing day 0 of differentiation, D0). Cells were cultured at 37°C in a 5% CO2 incubator for 6 days, with the medium changed every two days.

[0103] Differentiation of neuroepithelial cells into motor neuron progenitor cells (D6-D12)

[0104] On day 6 of differentiation, the supernatant of the cell culture flask was aspirated, and the cells were washed once with 1×DPBS buffer. TrypLE digestion solution was added, and the flask was incubated in a 37°C incubator for 15 minutes (12-16 minutes is also acceptable). The cells were gently shaken to completely detach from the bottom of the culture dish. Negative neural basal medium was added to neutralize and digest the cells, resulting in a cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, and the supernatant was aspirated. Secondary culture medium was added, and the cells were gently pipetted 1-2 times to disperse them into single cells as much as possible. The cells were seeded at a density of 50,000 cells / cm² into Laminin-coated T75 cell culture flasks and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 2 days, and the cells were cultured for another 6 days to obtain motor neural progenitor cells (iPSC-MNP).

[0105] The prepared iPSC-MNPs were analyzed by flow cytometry, and the results are as follows: Figure 1 As shown, it can be seen that in the obtained MNP cell population, more than 90% of the cells express the motor neural progenitor cell markers OLIG2 and HOXB4, and almost none express the pluripotent stem cell marker OCT4.

[0106] Example 2

[0107] This embodiment describes an exemplary method for inducing motor neural progenitor cells to differentiate into motor neural precursor cells in vitro.

[0108] Differentiation of motor progenitor cells into motor nerve precursor cells

[0109] (D12-D18)

[0110] After discarding the cell culture supernatant of the MNPs prepared in Example 1, the cells were washed once with 1×DPBS buffer, then TrypLE digestion solution was added, and the cells were incubated in a 37°C incubator for 18 minutes (14-20 minutes is also acceptable). The cells were gently agitated to completely detach from the bottom of the culture dish, and then added to neural basal medium for neutralization and digestion to obtain a cell suspension. The cell suspension was transferred to a 50 mL centrifuge tube, centrifuged at 400 g for 3 min, the supernatant was discarded, and the third culture medium was added. The cells were then centrifuged at a rate of 100,000 cells / cm³. 2 The cells were seeded at the appropriate density into Laminin-coated T75 cell culture flasks and cultured at 37°C in a 5% CO2 incubator. The medium was changed every 2 days for 4 days. The medium was then replaced with a fourth medium and cultured at 37°C in a 5% CO2 incubator for 2 days to obtain motor nerve progenitor cells.

[0111] For the iPSC-MNPs prepared in Example 1 and the motor neural progenitor cells prepared in Example 2, immunofluorescence staining was performed using anti-HB9 monoclonal antibody (1:500) and anti-OLIG2 monoclonal antibody (1:500) as primary antibodies, and corresponding antibodies diluted 1:500 (Jackson Lab) as secondary antibodies. The cells were observed under a fluorescence microscope and photographed in the dark. The results are shown in [the figures are missing from the original text]. Figure 2 and 3 .

[0112] As shown in the figure, compared with iPSC-MNP, the majority (approximately 84%) of the motor progenitor cells obtained after further differentiation expressed the marker HB9, while no longer expressing the MNP marker OLIG2. HB9+OLIG2- cells accounted for approximately 76% of the population. In contrast, the iPSC-MNP cell population was predominantly composed of HB9-OLIG2+ cells. This indicates that the iPSC-MNP cell population of this application can effectively differentiate into a high-purity motor progenitor cell population.

[0113] Example 3

[0114] This embodiment describes an exemplary method for inducing the differentiation of motor nerve progenitor cells into motor neurons in vitro.

[0115] Differentiation of motor nerve progenitor cells into motor neurons

[0116] (D18-D27)

[0117] After discarding the culture supernatant of the motor neural progenitor cells prepared in Example 2, the cells were washed once with 1×DPBS buffer, then 0.75×TrypLE digestion solution was added. The cells were incubated at 37°C for 8 minutes (6-10 minutes is also acceptable), gently agitating to ensure complete detachment from the bottom of the culture dish. Neural basal medium was added for neutralization and digestion to obtain a cell suspension. The cell suspension was transferred to a 15 mL centrifuge tube, centrifuged at 400 g for 3 min, the supernatant was discarded, and the fifth culture medium was added. The cells were then centrifuged at 100,000 cells / cm³. 2 The cells were seeded at the specified density into Matrigel-coated 24-well cell culture plates and cultured at 37°C with 5% CO2 for 3 days. On day 21, the culture medium was replaced with medium VI and cultured at 37°C with 5% CO2. The medium was partially changed every three days, and the culture was continued for 6 days to obtain a mature motor neuron cell population.

[0118] The obtained motor neuron cell populations were subjected to immunofluorescence staining. Anti-HB9 monoclonal antibody (1:500) and anti-CHAT monoclonal antibody (1:500) were used as primary antibodies, and the corresponding antibodies diluted 1:500 (Jackson Lab) were used as secondary antibodies. The cells were observed under a fluorescence microscope and photographed in the dark. The results are shown in [the image / image / etc.]. Figure 4 .

[0119] As shown in the figure, most cells in the obtained motor neuron cell population expressed the characteristic marker combination of motor neurons, HB9 and CHAT, with double-positive cells accounting for over 90%. This indicates that high-purity mature motor neurons can be differentiated from the motor neuron progenitor cell population of this application.

[0120] Example 4

[0121] This embodiment describes an exemplary method for preparing an animal model of spinal cord injury.

[0122] SCI rat model

[0123] Rats were anesthetized with Supra-50 (40-50 mg / kg, 50 mg / mL, intramuscular injection). After the corneal reflex disappeared, the animals were fixed in a prone position, and the skin was prepared at T11 of the spine. Centered on the T11 spinous process, the skin was longitudinally incised along the midline of the back. The subcutaneous tissue, fascia, and muscles on both sides of the spinous process were dissected layer by layer to expose the T11 spinous process. The accessory tissues were dissected to fully expose the dorsal structures of the spine. After removing the spinous process with bone forceps, the lamina posterior to T11 was carefully removed along the interlaminar space to fully expose the spinal cord and dura mater.

[0124] The spinal cord was struck using a spinal cord impactor (manufacturer: Shenzhen Ruiwode Life Technology Co., Ltd., model: 68099) (impact parameters: using a cylindrical flat-headed hammer with an outer diameter of 2 mm, impact speed of 2 m / s, impact depth of 1.0 mm, and dwell time of 3 s).

[0125] After causing spinal cord injury in rats, the muscles, fascia, and skin were sutured layer by layer from the inside out. The sutured incision was disinfected with iodine and the rats were placed in a clean rearing cage.

[0126] SCI mouse model

[0127] Mice were weighed and intraperitoneally injected with 1.25% tribromoethanol at a rate of 150 μL / 10 g. Mice were secured to a surgical mat, and ophthalmic scissors were used to cut the skin along the spine to fully expose the target area, T9-T13 of the spine. The T10 lamina was located, and the lamina and muscle were dissected layer by layer using Venus scissors to expose the lamina. Finally, the lamina was removed to expose the spinal cord.

[0128] After aiming the strike at the exposed spinal cord, the strike force of the spinal cord striker and the stereotactic instrument were adjusted (strike force 150 kdyn, strike depth 0.75 mm). After the strike, tail tremors, spinal cord congestion and swelling at the strike site were observed in the mouse.

[0129] After suturing the inner lining and outer skin completely, wait for the mouse to wake up.

[0130] Example 5

[0131] This example describes the administration of drugs to an SCI animal model.

[0132] iPSC-MNP cells prepared using the same method as in Example 1 were processed at 1.0 × 10⁻⁶. 8 A concentration of 1 cell / mL was prepared by suspending the human serum albumin in sodium lactate Ringer's solution for administration.

[0133] SCI rat model

[0134] The day of model establishment was designated as day 0 (modeling day 0). On day 7, rats with a BBB score of 0 to 1 were randomly assigned to groups and administered the drug via intrathecal injection. Administration was performed using a high-performance microsyringe with a 26G bevel needle. The treatment group received a dose of 1.05 × 10⁻⁶. 6 One cell per animal, and the negative control group was given an equal volume of cell-free preservation solution.

[0135] In the treatment groups, the single-dose group was administered the drug on day 8, the double-dose group on day 8 and day 22, the negative control group on day 8, day 22, and day 36, respectively.

[0136] Animals were weighed weekly before and after administration. During the experiment, all groups showed a trend of increasing body weight, and no adverse effects of modeling or administration on animal body weight were observed. Clinical observation, in vivo imaging, and BBB scores were performed on the test animals before and after administration.

[0137] SCI mouse model

[0138] Mice were deeply anesthetized after modeling, and the skin of their lumbar region was surgically cut open to expose the lumbar vertebrae. Using a 10 μL Hamilton microsyringe, the needle was inserted perpendicularly into the interspinous space (L5 and L6) on the dorsal side to administer the drug intrathecally. After the injection, the skin opening was sealed with biomedical adhesive.

[0139] The injection volume was 10 μL. The dosage in the low-dose group was 1.0 × 10⁻⁶. 6 1 cell / animal, injection concentration of 2.0 × 10⁻⁶ 7 Cells / mL; 5.0 × 10⁻⁶ in the high-dose group.6 1.0 × 10⁻⁶ cells / animal, injection concentration 1.0 × 10⁻⁶ 8 Cells / mL.

[0140] The day of model establishment is designated as day 0. Drug administration is performed on day 9, and the day of drug administration is designated as day 0. Continuous observation continues until day 50-day 56 (i.e., continuous observation until week 8 post-treatment).

[0141] Animals were weighed weekly before and after administration. During the experiment, the body weight of all groups showed an increasing trend, and no adverse effects of modeling and administration on animal body weight were observed.

[0142] Example 6

[0143] This embodiment describes the repair of glial scars in a mouse SCI model after administration of the motor progenitor cells of this application.

[0144] SCI mice were prepared using the same method as in Example 5, including a control group, a high-dose treatment group, and a low-dose treatment group (n=5 per group). At the end of week 8 (d65) following the injection of motor progenitor cells, two intramedullary injections of 10% biotin-conjugated BDA were performed at approximately 0.5 cm (0.5–1.0 cm) above the site of spinal cord injury to assess the integrity of the corticospinal tract (CST). Two weeks after injection (d79), spinal cord tissue from the cervical to lumbar region was collected and subjected to immunofluorescence staining.

[0145] Collected cervical-thoracic-lumbar spinal cord sections were fixed with 4% paraformaldehyde. Then, the vertebrae were dissected, the spinal cord was removed, embedded in OCT, and sagittal sections with a thickness of 20 μm were prepared. One section was selected from every 5–7 consecutively collected sections, and immunofluorescence staining was performed using rabbit anti-astrocytocyte marker GFAP monoclonal antibody as the primary antibody and goat anti-rabbit Alexa Flour 488 (green fluorescence) and streptavidin Alexa Flour 647 (red fluorescence) as secondary antibodies. The results are shown in [the image / image / etc.]. Figure 5 .

[0146] Figure 5 Anti-GFAP staining results showed that, compared with the control group, the area of ​​glial scars in the spinal cord tissue of mice in both the high-dose and low-dose groups was significantly reduced (P < 0.01). This indicates that the application of motor progenitor cells in the subacute phase of spinal cord injury can reduce reactive astrocytes and the glial scar area they form, reduce or even potentially eliminate obstacles to the later repair process of damaged nerve tissue, and promote axonal regeneration and functional recovery.

[0147] Example 7

[0148] This example describes the application of motor progenitor cells to promote corticospinal tract / axon regeneration in SCI model mice.

[0149] SCI mouse control, high-dose treatment, and low-dose treatment groups (n=5 per group) were prepared using the same method as in Example 5. Sagittal sections of spinal cord tissue were prepared and stained with fluorescence using the same method as in Example 6. The only difference was the use of rabbit anti-mouse NeuN (neuronal marker) monoclonal antibody as the primary antibody. Additionally, the cells were incubated with DAPI (blue fluorescence) to visualize live cells. Images taken under a fluorescence microscope are shown below. Figure 6 In the figure, the GFAP-negative area (GFAP-) represents the cavity area of ​​the glial scar region; red fluorescence shows BDA molecules transported in nerve cells, green fluorescence shows motor neurons, and blue fluorescence shows living cells.

[0150] Depend on Figure 6 As can be seen, dense BDA-positive nerve fibers were observed ascending from the site of spinal cord injury (lesion center) in the control group, high-dose group, and low-dose group. However, at the lesion center (a) and descending from the lesion center (b), almost no BDA-positive axons were observed in the control group, while the number of BDA-positive axons was significantly increased in both the high-dose and low-dose groups. This indicates that the administration of motor progenitor cells during the subacute phase can effectively promote axonal regeneration and elongation of motor neurons.

[0151] Example 8

[0152] This example describes the BMS score results in SCI model mice before and after administration of motor neural progenitor cells.

[0153] SCI mice (n=15 per group) were prepared using the same method as in Example 5. The BMS main scoring system (BMS score) was used to assess the motor function (walking and limb coordination) of both hind limbs in mice of the control, high-dose, and low-dose treatment groups. The scores were determined by Basso, DM et al. (2006). Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mousestrains. J. Neurotrauma 23, 635–659. The scores were assessed on days 1 (d8), 3 (d6), 7 (d2), 9 (d0), 7, 14, 21, 28, 35, and 50 (the final experimental endpoint). Two researchers participated in each assessment, recording their scores and averaging them. When the scores of the left and right hind limbs of the same mouse differed, the average score was taken as the mouse's final BMS score. Results are shown in […]. Figure 7 .

[0154] The results showed that the initial BMS scores of the control group, high-dose group, and low-dose group were 0.77±0.7528, 0.833±0.7237, and 0.967±0.7432, respectively, with no statistically significant differences. At the experimental endpoint (D50), the hind limb motor function of the control group showed some self-recovery, but the degree of recovery varied greatly among individuals (BMS score 2.467±1.2315). The degree of motor function recovery in the high-dose group and the low-dose group was statistically significantly higher than that in the control group, and the differences among individuals within the group were relatively small (BMS scores 3.333±1.1286 and 3.4667±1.3425, respectively). In addition, the high-dose group showed statistically significantly higher recovery than the control group at each observation after cell injection (D7, D14, D21, D28, D35, D50), and the low-dose group showed statistically significantly higher recovery at each observation after cell injection (D7, D14, D21, D35) (P<0.05), but there was no significant difference between the two dose groups.

[0155] In actual observation, all individuals in the three groups of mice exhibited slight ankle joint movement or inability to move before receiving cell injection, with no foot contact with the ground or walking, and the inability to lift the paws after contact with the ground. At the experimental endpoint (D50), most mice in the control group were still unable to walk (9 / 15), with a few recovering to the point of being able to walk on their dorsiflexion (3 / 15) or complete plantar walking (3 / 15). In contrast, the number of mice unable to walk was significantly reduced in both treatment groups (4 / 15 in both groups), while the number of recovered individuals (dorsiflexion or plantar walking) was significantly increased (11 / 15 in both groups). Furthermore, in the high-dose group, the recovered individuals were predominantly plantar walking, which showed a higher degree of recovery (7 / 11 vs 5 / 11), indicating that the therapeutic effect of motor neural progenitor cells showed a certain dose-dependent effect.

[0156] Example 9

[0157] This example describes the evaluation of spinal cord tissue morphology after administration of motor progenitor cells using transmission electron microscopy (TEM).

[0158] SCI rats were prepared in the same manner as in Example 5, including a control group, a high-dose treatment group, and a low-dose treatment group (n=3 per group). The BBB scores of each group were measured one day before electron microscopy sampling, as shown in Table 1 below.

[0159] Table 1: BBB scores in SCI rat model after intrathecal MNP administration

[0160] Group Animal number BBB rating Model control group Y24-2023 1.0 1-dose group Y24-2027 5.5 2-dose group Y24-2030 13.5 3-dose group Y24-2035 11.0 Y24-2036 7.8

[0161] Twelve weeks after cell injection, mice were anesthetized with 1% tribromoethanol. The heart was perfused with 0.9% saline, and the spinal cord was quickly removed after lamina resection and placed in pre-cooled 2.5% glutaraldehyde fixative. Sections of 2-3 mm were taken from the lesion center (T10) and the boundaries of the lesion at the beak and tail. The removed tissue was trimmed into blocks approximately 1 mm in length, dispersed, dehydrated, infiltrated, and embedded. Semi-thin and ultrathin sections were prepared by Beijing Hengdao Medical Laboratory Co., Ltd. for imaging with a transmission electron microscope (JEOL-1400flash, Japan) at 8000x magnification. The commissioning party was unaware of the sample information. Exemplary electron micrographs of the sections are shown in [image description missing]. Figures 8A to 8D As shown in the figure, different degrees of axonal regeneration were observed in the damaged area (where axonal demyelination was present) in each treatment group (locations indicated by arrows).

[0162] Transmission electron microscopy (TEM) images of the SCI mouse model were captured and analyzed using the same method. Results showed that vacuolated damaged myelin sheaths were observed in both the control and treatment groups following spinal cord contusion. However, after 5 weeks of treatment (D35), compared to the control group, the treatment group showed a significant increase in the number of myelin-encased axons in the anterior horn and corticospinal tract region of the spinal cord, indicating that administration of motor progenitor cells significantly improved demyelination of anterior horn neurons (motor neurons). It was also observed that the integrity and number of myelinated axons in the descending corticospinal tract region located in the lateral columns of the spinal cord were significantly higher in the treatment group than in the control group. The myelinated axons in the treatment group had thicker myelin sheaths compared to the control group, with a statistically significant difference. Simultaneously, the treatment group exhibited significantly more myelin regeneration than the control group.

[0163] Myelin sheaths encapsulate axons, providing electrical insulation, improving the speed and accuracy of nerve conduction, and simultaneously providing nutritional support and protection. Therefore, myelin regeneration plays a crucial role in reconstructing the neuronal signal transduction system. The above observations consistently indicate that the administration of motor progenitor cells can significantly alleviate demyelination caused by spinal cord contusion and, to some extent, promote myelin regeneration. This suggests that the motor progenitor cells described in this application may enhance myelin regeneration at the damaged site by differentiating oligodendrocytes and / or paracrine secretion of oligodendrocyte-promoting factors.

[0164] Example 10

[0165] This example describes HE staining of a cross section of the spinal cord.

[0166] SCI mouse control, high-dose treatment, and low-dose treatment groups (n=4-5 per group) were prepared using the same method as in Example 5. Normal healthy mice were also prepared as a healthy control group. On day 50 (D50) after injection of motor progenitor cells, deeply anesthetized mice underwent PFA cardiac perfusion. Spinal cord sections from the injury site were collected, fixed in 4% paraformaldehyde, and then embedded in OCT. Frozen sections of the spinal cord cross-sections were prepared with a thickness of 20 μm. Sections were collected continuously, and one section was selected every 5-7 sections for HE staining. The integrity (preserved area) of the spinal cord cross-section at the lesion center (0 mm), 0.2 mm and 0.3 mm above and below the lesion center, and ±3 mm, ±6 mm, and ±9 mm away from the lesion center were assessed to evaluate the repair of damaged tissue.

[0167] The results showed that the spinal cord of normal mice had normal morphology, dense and orderly tissue, clear gray and white matter boundaries, regular and plump cell shapes, and clear cell nuclei; after spinal cord injury, the damaged spinal cord tissue would atrophy. HE staining to determine the preserved area after injury (i.e., the cross-sectional area of ​​the spinal cord) showed that the cross-sectional area at the lesion center (0 mm) and at 0.2 mm and 0.3 mm above and below the lesion center in the treatment group mice was significantly larger than that in the control group (P < 0.05), while the cross-sectional area of ​​the spinal cord tissue far from the lesion center (±3 mm, ±6 mm, and ±9 mm) was not different from that in the control group mice.

[0168] Example 11

[0169] This example describes the BBB scores of SCI model rats before and after administration of motor neural progenitor cells.

[0170] SCI rats were prepared into a control group and a treatment group (n=4 per group) using the same method as in Example 5. Based on the BBB scoring system described in Basso DM et al., A sensitive and reliable locomotor rating scale for open field testing in rats. J Neurotrauma. 1995 Feb;12(1):1-21, behavioral scores were assessed on the joint activity and motor function (coordination of walking and limb movement) of both hind limbs of rats in the control and treatment groups on days 3 (d3), 7 (d7), the day of administration of motor progenitor cells (d8), 7 (d15), 14 (d22), 21 (d29), 28 (d36), 35 (d43), 42 (d50), 49 (d57), and 56 (d64) after modeling. Each hind limb was scored individually, and approximately 5 minutes of video recording showing bilateral hind limb activity was recorded for each animal. Two people participated in each scoring session, and the scores from both individuals were recorded and averaged. When the scores of the left and right hind limbs of the same rat differed, the average score was taken as its final BBB score. Results were shown in... Figure 9 .

[0171] As shown in the figure, spinal cord injury significantly reduced the BBB score in the model animals, indicating successful model establishment and severe impairment of hind limb motor function. Before cell injection, the baseline BBB scores of rats in the control and treatment groups were essentially the same: control group: 0.25±0.29; single-dose group: 0.375±0.25; double-dose group: 0.32±0.25; triple-dose group: 0.25±0.29. The score of the control group rats increased slightly over time, possibly because the damaged spinal cord tissue still retained a weak spontaneous recovery capacity. In the three treatment groups, the BBB scores of the single-dose group were similar to those of the control group throughout the observation window, with no statistically significant difference observed (P>0.05); the BBB scores of the double-dose group were significantly higher than those of the control group and the single-dose group starting from 3 weeks after cell injection (D21 to D56) during the same period (P<0.05~P<0.01); the BBB scores of the triple-dose group also showed a trend of being higher than those of the control group and the single-dose group during the same period from 3 to 7 weeks after cell injection (D21 to D49), but there was still no statistically significant difference (P>0.05), while at 8 weeks after injection (D56), the BBB scores were also statistically significantly higher than those of the control group and the single-dose group during the same period (P<0.01). At the experimental endpoint (D56), the control group scored 4.1±2.6 points, which was considered to be the spontaneous recovery process after spinal cord injury in SD rats; the scores of the treatment groups were 4.1±3.5 points (1 dose), 10.9±4.4 points (2 doses), and 10.5±2.7 points (3 doses).

[0172] Example 12

[0173] This example describes the results of measuring secretory protein factors in rat cerebrospinal fluid before and after administration of motor neuron progenitor cells.

[0174] SCI rats were treated three times (n=3 per group) using the same method as in Example 5, except that the third administration was performed on day 21. Following the manufacturer's instructions, 40 key protein factors in rat cerebrospinal fluid were quantitatively measured using a multichannel sandwich ELISA protein quantification chip (protein chip kit purchased from RayBiotech, Inc., catalog number QAH-GF-1) at different time points on the day of administration and after administration (D3, D7, D17, D21, D31, D35), using an InnoScan300 Microarray Scanner fluorescence scanner.

[0175] The protein factors measured included: brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMP-4, BMP-5, BMP-7), β-nerve growth factor (beta-NGF), epidermal growth factor (EGF) and its receptor (EGFR), and endothelial growth factor (EG-VEGF). (PK1), fibroblast growth factor (FGF-4, FGF-7 (KGF)), growth differentiation factor 15 (GDF-15), glial cell-derived neurotrophic factor (GDNF), auxin, hepatocyte growth factor (HB-EGF, HGF), insulin-like growth factor binding protein (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), insulin-like growth factor (IGF-1), insulin, macrophage colony-stimulating factor receptor (MCSFR), nerve growth factor receptor (NGFR (TNFRSF16)), neurotrophic factors (NT-3, NT-4), osteoprotegerin (TNFRSF11B)), platelet-derived growth factor (PDGF-AA), placental growth factor (PLGF), stem cell factor (SCF) and its receptor (SCF R (CD117 / c-kit)), transforming growth factor (TGF alpha, TGF beta 1, TGF beta) 3) Vascular endothelial growth factors (VEGF-A, VEGFR2, VEGFR3, VEGF-D), etc. Results showed... Figure 10 A to 10F.

[0176] Figure 10 The results showed that after cell transplantation, BDNF ( Figure 10 A), BMP5 ( Figure 10 D), IGFBP4 ( Figure 10 G), EGF ( Figure 10 H), SCF-RF ( Figure 10 I), TGFb3 ( Figure 10 J) and VEGF Figure 10 The concentration of bFGF (β-FGF) peaked on day 7 after administration, with abundances of approximately 1.0 to 5.0 pg / ml, 200 to 2500 pg / ml, 500 to 4000 pg / ml, 0.2 to 1.8 pg / ml, 5.0 to 30.0 pg / ml, 25.0 to 250.0 pg / ml, and 25.0 to 250.0 pg / ml, respectively; Figure 10 B), BMP4 Figure 10 C), IGFBP3 ( Figure 10The expression levels of F were generally better than before cell application, with abundances reaching approximately 10.0–70.0 pg / ml, 200–1400 pg / ml, and 200–1000 pg / ml, respectively. Protein factors whose expression levels did not differ significantly included BMP7 (…). Figure 10 E).

[0177] Therefore, the application of motor progenitor cells can significantly upregulate the expression levels of most protein factors, especially growth factors that nourish neurons and / or glial cells, thus having a positive effect on related biological processes such as repair, regeneration, growth, and axonal extension of neurons and / or glial cells.

[0178] Sample name or code Sample grouping E24-909 Comparison E24-910 Comparison E24-911 Comparison E24-912 D3 E24-913 D3 E24-914 D3 E24-915 D7 E24-916 D7 E24-917 D7 E24-918 D17 E24-919 D17 E24-920 D17 E24-921 D21 E24-922 D21 E24-923 D21 E24-924 D31 E24-925 D31 E24-926 D31 E24-927 D35 E24-928 D35 E24-929 D35

[0179] Example 13

[0180] This embodiment describes single-cell sequencing of host spinal cord nerve tissue after transplantation of motor nerve progenitor cells.

[0181] A 10-week-old SCI mouse model was prepared using the same method as described in Example 4. Following the intrathecal injection method described in Example 5, motor progenitor cells prepared using the method described in Example 1 were injected into the L5 segment one week after injury (d7), serving as the treatment group (n=3); a cell-free carrier was injected at the same location, serving as the control group (n=3). Intact spinal cord segments from T10 to L6 were harvested at weeks 1 (d14), 3 (d28), and 5 (d42) post-transplantation and flash-frozen in liquid nitrogen. Three spinal cord samples from each group were pooled and subjected to single-cell transcriptome sequencing. Data sets of human transplanted cells and mouse spinal cord tissue cells were identified by comparison with human and mouse reference genomes, respectively, followed by downstream dimensionality reduction clustering analysis.

[0182] The results showed that, in both the control and treatment groups, the proportion of spinal cord neurons in the total cells of mice gradually decreased over time. However, the proportion of neurons in the treatment group was higher than that in the control group at each measurement. This suggests that motor progenitor cells can reduce SCI-induced neuronal loss and death.

[0183] Furthermore, significant differences in glial cells were observed between the two groups of mice. The proportion of microglia increased over time in both the treatment and control groups, with the proportion consistently lower in the treatment group than in the control group at each measurement, indicating lower inflammation and fewer activated and recruited microglia in the treatment group. Conversely, the proportion of astrocytes was lower in the control group at the initial stage (week 1) and gradually increased over time, while in the treatment group it was higher at week 1 and then gradually decreased. This suggests that the treatment group mice will form smaller glial scars later on with almost no loss of the initial protective effect of astrocytes, which is more conducive to the self-repair of the damaged host nervous system and avoids the enlargement of glial scars hindering the reconnection of ascending and descending axons in the host spinal cord.

[0184] In addition, axonal regeneration is usually accompanied by angiogenesis. It was observed that the proportion of vascular leptomeningeal cells and vascular epithelial cells in the treatment group mice was greater than that in the control group in each measurement. Sufficient angiogenesis can improve local blood supply, which may be beneficial for axonal regeneration.

[0185] Single-cell nuclear transcriptome sequencing was then performed. Cell suspensions were loaded onto a Chromium single-cell controller using a single-cell 3' library and a 10× Genomics gel bead kit and a Chromium single-cell microarray kit, generating single-cell nuclear gel beads in an emulsion according to the manufacturer's protocol. Approximately 20,000 cells were added to each channel, with an estimated 10,000 target cells to be recovered. Captured nuclei were lysed, and the released RNA was barcoded via reverse transcription in a single GEM. Reverse transcription was performed at 53°C for 45 min on a Bio-Rad thermal cycler, followed by 5 min at 85°C and incubation at 4°C. Complementary DNA (cDNA) was generated, amplified, and its quality was assessed using an Agilent 4200. snRNA-seq libraries were constructed using a single-cell 3' library and a gel bead kit according to the manufacturer's instructions. Finally, the library was sequenced using an Illumina Novaseq 6000 sequencer, with a sequencing depth of at least 100,000 reads per cell and a paired-end read strategy of 150 bp (performed by Beijing Novogene Biotechnology Co., Ltd.). For 10× Genomics data, the Cell Ranger single-cell toolkit (version 3.0.0) provided by 10× Genomics was used to align reads and generate a gene-cell unique molecular identifier (UMI) matrix for each sample. The read data were plotted using the corresponding mouse (Mus musculus) reference genome. Different samples were merged using the cellranger aggr function. The obtained raw_feature_bc_matrix was loaded, and further downstream analysis was performed using the Seurat R package (v 4.4.0).

[0186] snRNA-seq is a technique that can directly measure transcriptome information at single-cell resolution. By comparing differences in the nuclear transcriptomes of various cell types at the single-cell level, different cell subpopulations can be identified. Viruses carrying luciferase genes driven by constitutive promoters (such as the EF1α promoter or ubiquitin promoter) were added to a secondary culture medium to transfect D10 MNP cells. After one day of differentiation and culture, the medium was replaced with a virus-free secondary medium, and cultured for another day to obtain motor progenitor cells XS228-Luc+ carrying the reporter gene Luc. snRNA-seq results showed that one week after intrathecal injection of XS228-Luc+ motor progenitor cells carrying the reporter gene Luc, 22.9% of the detected human cells were still identified as MNPs, maintaining the potential for further differentiation and expansion; another 11.3% differentiated into immature neurons, 25.7% differentiated into astrocytes, and 37.7% differentiated into mature neurons.

[0187] Example 14

[0188] Motor neural progenitor cells prepared using the same method as in Example 2 were suspended in lactated Ringer's solution containing human serum albumin to prepare a formulation for administration. This formulation was administered to SCI rat and SCI mouse models using the same method as in Example 5. Animals were weighed weekly before and after administration. During the experiment, the body weight of all groups showed an increasing trend, and no adverse effects of modeling or administration on animal body weight were observed. Clinical observation, in vivo imaging, and behavioral scoring (BBB score for rats, BMS score for mice) were performed on the test animals before and after administration.

[0189] Example 15

[0190] Motor neurons prepared using the same method as in Example 3 were suspended in lactated Ringer's solution containing human serum albumin to prepare a formulation for administration. This formulation was administered to SCI rat and SCI mouse models using the same method as in Example 5. Animals were weighed weekly before and after administration. During the experiment, the body weight of all groups showed an increasing trend, and no adverse effects of modeling or administration on animal body weight were observed. Clinical observation, in vivo imaging, and behavioral scoring (BBB score for rats, BMS score for mice) were performed on the test animals before and after administration.

[0191] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of motor nerve progenitor cell populations selected from any one of (a) to (g), (a) Preparation of a drug for reducing reactive astrocytes, (b) Preparation of a drug for reducing glial scars, (c) To prepare a drug for inhibiting reactive astrocyte-mediated axonal regeneration disorder. (d) To prepare drugs for inhibiting reactive astrocyte-mediated neuronal and / or oligodendrocyte death, (e) Preparation of drugs for altering the expression profile of protein factors in cerebrospinal fluid. (f) Prepare drugs for promoting axonal regeneration and / or recovery, or (g) Prepare drugs for reducing demyelination, promoting myelin regeneration and / or increasing myelin thickness.

2. The use according to claim 1, wherein, The reduction of reactive astrocytes refers to the reduction of reactive astrocytes in the subacute and / or chronic phases. Optionally, the reactive astrocytes are astrocytes expressing any one of the following markers or any combination thereof: AQP4, GLT-1, VIMENTIN, GLAST, ALDH1L1, Lcn2, Steap4, Cxc110, guanylate-binding protein 2 (GBP2), GFAP, S100beta, A2B5, or other markers selected from AQP4, GLT-1, VIMENTIN, GLAST, ALDH1L1, Lcn2, Steap4, Cxc110, guanylate-binding protein 2 (GBP2), GFAP, S100beta, A2B5.

3. The use according to claim 1, wherein, The gelatinous scars mentioned are gelatinous scars caused by brain injury or spinal cord injury. Optionally, the reduction of gelatinous scars means reducing any one or any combination of the area, length, volume, thickness, or mass of the gelatinous scars, preferably by at least about 50%, more preferably by at least about 60%, and most preferably by at least about 90%.

4. The use according to claim 1, wherein, The protein factors mentioned are selected from brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMP-4, BMP-5), β-nerve growth factor (beta-NGF), epidermal growth factor (EGF) and its receptor (EGFR), and endothelial growth factor (EG-VEGF). (PK1), fibroblast growth factor (FGF-4, FGF-7 (KGF)), growth differentiation factor 15 (GDF-15), glial cell-derived neurotrophic factor (GDNF), auxin, hepatocyte growth factor (HB-EGF, HGF), insulin-like growth factor binding protein (IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6), insulin-like growth factor (IGF-1), insulin, macrophage colony-stimulating factor receptor (MCSFR), nerve growth factor receptor (NGFR (TNFRSF16)), neurotrophic factors (NT-3, NT-4), osteoprotegerin (TNFRSF11B)), platelet-derived growth factor (PDGF-AA), placental growth factor (PLGF), stem cell factor (SCF) and its receptor (SCF R (CD117 / c-kit)), transforming growth factor (TGF alpha, TGF beta 1, TGF beta) 3) Any one or any combination of vascular endothelial growth factors (VEGF-A, VEGFR2, VEGFR3, VEGF-D).

5. The use according to any one of claims 1 to 4, wherein, The motor neural progenitor cell population comprises at least about 70%, preferably at least about 75%, more preferably at least about 80%, further preferably at least about 85%, and most preferably at least about 90% of motor neural progenitor cells.

6. The use according to any one of claims 1 to 4, wherein, The motor progenitor cells are cells that express the markers OLIG2 and HOXB4, but hardly express the marker OCT4.

7. The use according to any one of claims 1 to 4, wherein, The motor neural progenitor cells were obtained through in vitro induced differentiation of pluripotent stem cells; Optionally, the pluripotent stem cells are selected from at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells, preferably induced pluripotent stem cells; Optionally, the pluripotent stem cells are derived from mammals, preferably from primates, and more preferably from humans.

8. The use according to any one of claims 1 to 4, wherein, The drug is administered during the subacute and / or chronic phases of spinal cord injury or brain injury, preferably during the subacute phase. Optionally, the subacute phase is 3 days to 6 weeks after spinal cord injury or brain injury; Optionally, the chronic period is more than 6 weeks after spinal cord injury or brain injury, preferably 6 weeks to 6 months. Optionally, the drug is a drug administered by any one of the following methods: intrathecal injection, intramedullary injection, intraventricular injection, or intravenous injection.

9. A method for treating spinal cord injury or brain injury, characterized in that, The method includes administering a population of motor neural progenitor cells or a pharmaceutical composition containing a population of motor neural progenitor cells to a subject with a spinal cord injury or brain injury who requires such treatment. Optionally, the motor progenitor cells are cells that express markers OLIG2 and HOXB4, and hardly express marker OCT4. Optionally, the motor neural progenitor cells are obtained by in vitro induced differentiation of pluripotent stem cells; Optionally, the pluripotent stem cells are selected from at least one of embryonic stem cells, parthenogenetic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose stem cells, and umbilical cord blood stem cells, preferably induced pluripotent stem cells; Optionally, the pluripotent stem cells are derived from mammals, preferably from primates, and more preferably from humans; Optionally, the application occurs during the subacute and / or chronic phase of spinal cord injury or brain injury, preferably during the subacute phase; Optionally, the administration is selected from any one of intrathecal injection, intramedullary injection, intraventricular injection, or intravenous injection.

10. The method according to claim 9, wherein, After the subject was administered the motor neural progenitor cell population or the pharmaceutical composition, (1) Reactive astrocytes decreased. (2) Reduced formation of collagen scars, (3) Reactive astrocyte-mediated axonal regeneration impairment is inhibited. (4) Reactive astrocyte-mediated neuronal death and / or oligodendrocyte death is suppressed. (5) Changes in the expression profile of protein factors in cerebrospinal fluid, (6) Increased axonal regeneration and / or recovery, or (7) Decreased demyelination, increased myelin regeneration and / or increased myelin thickness.

Images