WO2023246644A1 - 一种用于治疗肌萎缩侧索硬化症的脊髓祖细胞及其诱导分化方法和用途 - Google Patents
一种用于治疗肌萎缩侧索硬化症的脊髓祖细胞及其诱导分化方法和用途 Download PDFInfo
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Definitions
- the present invention relates to the field of neurobiology, and in particular to a spinal cord progenitor cell used to treat amyotrophic lateral sclerosis and its induction differentiation method and use.
- ALS Amyotrophic Lateral Sclerosis
- ALS amyotrophic Lateral Sclerosis
- Lou Gehrig's disease is a rapidly progressive neurodegenerative disease characterized by motor Selective degeneration of upper motor neurons in the cortex and lower motor neurons in the brainstem and ventral horn of the spinal cord.
- the global incidence of amyotrophic lateral sclerosis (ALS) is estimated at 2/100,000.
- the disease usually appears between the ages of 45 and 75, with a slightly greater proportion of men than women.
- the disease develops rapidly, starting with focal muscle weakness and gradually progressing to affect most of the skeletal muscles.
- leading to complete paralysis After the onset of the disease, the rapid progression of the disease limits survival to 3-5 years, and respiratory muscle paralysis gradually affects the individual's survival, eventually leading to death. There is currently no satisfactory treatment.
- Stem cell therapy for amyotrophic lateral sclerosis was originally proposed as an ALS therapeutic agent to replenish progressively lost motor nerve (MN) populations.
- Stem cells have the ability to self-renew and maintain an undifferentiated state. When they divide, the mother cells remain stem while the daughter cells can differentiate, such as embryonic stem cells (ESC). This type of stem cell is extremely totipotent and can theoretically differentiate into any type of cell; while multiple Competent stem cells (PSC) can differentiate into a limited number of specific cell types depending on induction methods.
- Neural progenitor cells are multipotent stem cells that can differentiate into neurons or glial cells.
- ALS stem cell therapy is to use ESC or PSC to differentiate to produce motor neurons and then transplant them into ALS patients to replace the lost motor neurons and grow and extend in the body to treat the disease.
- these motor neurons need to be effectively connected to pre-existing neural circuits and need to achieve efficient projection of axons, but this projection often requires long distances.
- transplanted stem cells play a supportive role by providing a nourishing and neuroprotective microenvironment, improving the adverse conditions of the diseased motor neurons, thereby slowing down neurodegeneration and neuronal death.
- Transplanted stem cells with this ability secrete neurotrophic factors, differentiate into non-diseased non-neuronal cells such as astrocytes and microglia, or differentiate into synaptic contacts with diseased motor neurons. Regulatory neurons (Thomson JA et al., Embryonic stem cell lines derived from human blastocysts. Science. 1998; 282(5391):1145–7). In animal models, preclinical studies of stem cell therapy have shown good therapeutic effects, demonstrating the potential applicability of stem cells to treat ALS.
- MNP motor neuron progenitor cells
- hESC human embryonic stem cells
- hNSC human neural stem cells
- Tator CH Isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord. J Vis Exp. 2015;99:e52732).
- hNSCs are multipotent stem cells derived from the spinal cord or brain of aborted fetuses that can self-renew and differentiate into astrocytes, neurons and oligodendrocytes in vitro.
- iPSCs induced pluripotent stem cells
- MSCs adult mesenchymal stem cells
- GRNP glial-rich neural progenitor cells
- the spinal cord progenitor cells of the present invention exhibit Olig2 + and Nkx2.2 + .
- the spinal cord progenitor cells of the present invention exhibit SOX2 + and Nestin + .
- the spinal cord progenitor cells of the present invention exhibit SOX1 + , SOX2 + , and Nestin + .
- the spinal cord progenitor cells of the present invention highly express HOXC6, can express HOXC8 and HOXC9, and do not express HOXC10.
- the spinal cord progenitor cells of the present invention after 40-60 days of natural differentiation, at least exhibit the characteristics of TUJ1 + , Vglu1 + , and GAD67 + ; preferably, they also exhibit the characteristics of TH + and CHAT + feature.
- the second object of the present invention is to provide a method for inducing differentiation of the spinal cord progenitor cells, including:
- iNSC induced neural stem cells
- the final concentrations of CHIR99021, SB431542, RA, bFGF, DAPT, LDN, and Purmorphamine added to the second differentiation medium are 1-5 ⁇ M, 8-12 ⁇ M, 0.05-2 ⁇ M, and 70 ⁇ M, respectively. -130ng/mL, 70-130ng/mL, 0.02-0.5 ⁇ M, and 0.1-2 ⁇ M.
- the final concentrations of CHIR99021, SB431542, RA, bFGF, DAPT, LDN-193189, and Purmorphamine added to the second differentiation medium are 4 ⁇ M, 10 ⁇ M, 0.1 ⁇ M, 100 ng/mL, 100 ng/mL, and 0.1 respectively. ⁇ M, and 0.5 ⁇ M.
- half of the medium of the cells is exchanged every other day at each stage. Specifically, during the first stage of differentiation culture, the second stage of differentiation culture, or the maintenance culture stage, the cells were half-exchanged every other day.
- the first stage of differentiation culture is carried out for 7 days.
- the second stage of differentiation culture is carried out for 10 days.
- the maintenance culture can last for more than 40 days, preferably can last for 40-150 days, more preferably can last for 40-70 days, and further more preferably can last for 40-60 days.
- the maintenance culture can be expanded for at least 5 generations, preferably the maintenance culture can be expanded for 5-10 generations, and more preferably the maintenance culture can be expanded for at least 10 generations.
- the fourth object of the present invention is to provide the use of the spinal cord progenitor cells for preparing medical supplies for treating amyotrophic lateral sclerosis, and the medical supplies are medicines or medical devices.
- Figure 3 is an RNA sequencing diagram of spinal cord progenitor cells.
- A Hierarchical clustering of fetal brain and differentiated spinal cord progenitor cells, revealed into two major clusters.
- F Schematic diagram of HOX family gene expression in the spinal cord, cervical spine, thoracic spine, and lumbar spine.
- Figure 5 is a diagram showing the survival and migration identification of spinal cord progenitor cells in the spinal cord of immunodeficient mice at different time points.
- F Migration length of cells in the cervical spinal cord at different time points.
- G Migration length of cells in the thoracic spinal cord at different time points.
- Figure 7 shows the results of the effect of spinal cord progenitor cells on ALS SOD1 G93A mice.
- iNSC Induced neural stem cells
- Y. Yuan et al. Y.
- PBMCs peripheral blood mononuclear cells
- the reaction system can be amplified as required, and a 10 ⁇ l system can use up to 500ng of total RNA; (2) The reaction system is 5 ⁇ PrimeScript Buffer (2 ⁇ l), PrimeScript RT Enzyme MixI (0.5 ⁇ l), Oligo dT Primer (0.5 ⁇ l), Random 6mers (0.5 ⁇ l), total RNA, RNase Free dH2O (added to 10 ⁇ l); (3) Program settings are as follows: 37°C, 15 minutes; 85°C, 5 seconds; 4°C, ⁇ . When using Gene specific primer, it is recommended that the reverse transcription reaction conditions be set to 42°C for 15 minutes. When there is non-specific amplification in the PCR reaction, raising the temperature to 50°C will improve it. The above reaction is the reaction condition of TB Green qPCR analysis method.
- the experimental groups are as shown in Table 1; (2) Fix the rats on the operating table and shave their backs. Use a scalpel to cut open the back skin at T11-T12 in rats and T13-L1 in mice, peel off the muscle tissue at both ends of the spinal cord, and expose the spine; (3) Use a rongeur to gently Gently remove the lamina, taking care to avoid damaging the spinal cord tissue and exposing the spinal cord; (4) Adjust the position of the spinal cord of the large (small) mouse, fold it with gauze and raise the exposed spinal cord to keep it level; (5) Use a microsyringe to absorb For cell suspension, inject 3 ⁇ l per site in rats and 0.5 ⁇ l in each site in mice.
- the rotarod test can be used to evaluate the coordination, balance and limb strength of mice. It is performed as follows: Place the mouse on a rotary rod instrument with a diameter of 3.0cm, and adjust the speed to 15rpm to record the longest drop. incubation period. The recording time is 180 seconds, if it exceeds 180 seconds, the boundary value should be recorded as 180 seconds, if it is less than 180 seconds, it should be recorded as the actual time. Starting from the 70th day after birth, the mice practiced at a speed of 12 rpm for 5 days to adapt to exercise and obtain basic exercise values. Each mouse was tested three times each time and the results were recorded. Each experimental mouse was measured once a week. The longer the incubation period, the better the animal's motor coordination, limb strength and balance.
- iNSCs are induced to differentiate into spinal cord progenitor cells (scNPCs).
- iNSC cells differentiated into spinal cord progenitor cells of PAX6 + , SOX1 + , Nkx6.1 + , SOX2 + , DBX2 + , OLIG2 + , PAX3 + , and Nkx2.2 + ( Figure 1B), in which PAX7 was not expressed but PAX6 Keep expressing.
- Dbx2 + and Nkx6.1 + cells could be observed in clumps of cells (Fig. 1B ).
- the expression of Dbx2 + can prove that the cells belong to the p0 region of the ventral spinal cord. Evx1/2 + (marking V0 interneurons in the spinal cord region) can also be observed.
- Each mature spinal neuronal subclass expresses a subset of neurotransmitters (Fig. 2C).
- Cholinergic spinal motor neurons express choline acetyltransferase (CHAT); differentiated sensory neurons and V2a interneurons in areas dI1-3 of the dorsal spinal cord are glutamatergic neurons that express Vglut1 + ; dorsal spinal cord dI4
- Regionally differentiated somatosensory associated neurons and dI6 interneurons as well as V1 interneurons are GABAergic neurons that express GAD67 + .
- GAD67 + GABA neurons express PAX2 and CHX10, indicating that they are V1-derived interneurons or V2a interneurons (Fig. 2B,C).
- V1-derived interneurons or V2a interneurons Fig. 2B,C
- Purmorphamine 500nM neurons express Islet1 + . This demonstrated the generation of spinal motor neurons among the induced cells.
- culturing iNSC-induced spinal cord progenitor cells in the presence of RA, SB, CHIR, LDN, bFGF, Purmorphamine, and DAPT can generate several types of human spinal cord neurons in vitro, including somatosensory interneurons and spinal motor neurons. Yuan.
- iNSC-induced spinal cord progenitor cells was compared with RNA gene expression of human aborted fetal central nervous system tissue and aborted fetal spinal cord.
- the stemness markers of stem cells: SOX1, SOX2, PAX6, PAX7, and PAX3 were compared.
- PAX6, PAX3, and SOX2 are highly expressed in both the first generation of spinal cord progenitor cells and the fifth generation of spinal cord progenitor cells.
- FOXG1 forebrain marker
- OTX2 midbrain marker
- GSX2 is a ventral telencephalon marker that can guide the development of the ventral ganglion. It is also a marker for interneuron progenitor cells and regulates and regulates the ventral glial progenitor cell population in the spinal cord.
- scNPC p1, scNPC p5, and cells naturally differentiated for 7 days all express GSX2, indicating that they have the potential to develop into oligodendrocytes.
- HOX family genes were compared. Spinal cord neural progenitor cells induced to develop neural stem cells expressed cervical and thoracic spinal cord HOX genes but not lumbar HOX genes, indicating that the cells acquired cervical Identity.
- the gene expression of naturally differentiated spinal cord progenitor cells at 7 days more closely matched that of human fetal spinal cord tissue, but was separate from that of human fetal brain tissue.
- SD rats were selected as the research subjects.
- the inventor Before transplantation, the inventor previously used the AAVSI-Pur-CAG-EGFP plasmid to knock in the EGFP expression cassette driven by the CAG promoter, so that the iNSCs were labeled with green fluorescence. iNSCs labeled with green fluorescence were induced to differentiate into spinal cord progenitor cells.
- SD rats Three days before cell transplantation, SD rats were injected with the immunosuppressant cyclosporine A. The injection dose is 10mg/kg/day.
- the transplanted cell types were spinal cord progenitor cells with green fluorescence obtained through induced differentiation, and the T11-T13 position of the rat spinal cord was selected for transplantation.
- One week, two weeks and two months after transplantation the rats were perfused and the cells were collected to observe the survival of the cells in the rats.
- the transplanted cells were stained with HU (mouse anti-human nuclear antibody) and GFP fluorescence to facilitate the distinction between transplanted cells and mouse autologous cells.
- HU-positive cells show red fluorescence
- GFP-positive cells show green fluorescence. When cells have both red and green fluorescence, they are viable cells.
- mice were perfused and sampled two weeks after the cell transplantation it was found that a large area of cells had died, but a small number of cells were still alive. Two months after transplanting the cells, it was found that all the cells had died. After two months of transplantation, mice were stained for CD68 and TNF ⁇ inflammatory factors, and a large amount of TNF ⁇ infiltration was found. It was initially suspected that the death of spinal cord progenitor cells was related to immune rejection.
- C6-C7 and thoracolumbar spinal cord (T12-L1) segments were transplanted simultaneously. Observe the survival and migration of cells in the cervical spinal cord and thoracolumbar spinal cord.
- cells at 5 time points were also selected: undifferentiated spinal cord progenitor cells (scNPC P1), natural differentiation for 7 days (D7), natural differentiation for 10 days (D10), Cells that naturally differentiated for 12 days (D12) and 14 days (D14).
- scNPC P1 undifferentiated spinal cord progenitor cells
- D7 natural differentiation for 7 days
- D10 natural differentiation for 10 days
- D12 Cells that naturally differentiated for 12 days
- D14 14 days
- mice transplanted with spinal cord progenitor cells scNPC P1
- the transplanted cells were found only in the neck. This shows that the cells are indeed more suitable for survival in the cervical spinal cord.
- the length of cell migration to the left and right was measured. It was found that cells that had naturally differentiated for 12 days (D12) migrated longest in the neck, while cells that naturally differentiated for 10 days (D10) migrated longest in the thoracic spinal cord. long. And the cells can grow in large areas in both the substantia nigra and the white matter of the spinal cord. The results are shown in Figure 5.
- the cells were stained for KI67 and OCT4.
- the cells transplanted into the mouse spinal cord at all time points were able to colonize and survive, and the KI67 and OCT4 staining were negative, proving that there was no tumor tissue formation (see Figure 6A).
- synapses are formed between differentiated neurons and glial cells or between neurons, and whether neuronal networks are formed in mice.
- mice in this experiment were subjected to cell transplantation from the age of 50 days. Before the cell transplantation, the mice were injected with immunosuppressants three days in advance. The selected immunosuppressant was cyclosporine A. The injection dose is 10mg/kg/day. First, the weight changes of the mice were monitored. The weight can reflect the general health status and onset time of the mice, and is an objective evaluation of the development of the disease in the mice. The mice in this experiment received cell transplantation on the 50th day.
- mice were selected to recover for 20 days, and the mice were treated starting from the 70th day. Weight and behavioral testing. The body weight of the mice was measured once a week, see Table 2. The weight of the mice in the cell transplantation group reached the maximum at 105 days, and their weight gradually began to decrease after 105 days. The mice in the control group reached their maximum weight at 98 days and began to lose weight after 98 days. In the short period of time recorded, the weight of the mice in the control group dropped by approximately 0.9g, while the weight of the mice in the cell transplantation group dropped by approximately 0.76g. The onset time of SOD1 mice is about 90 to 100 days. According to two consecutive weight losses, the mice can be recorded as having developed the disease. According to the number of days for mice to lose weight in Table 2, it can be observed that the onset of disease in the mice in the cell transplantation group was delayed by 7 days compared with the mice in the control group. See Table 2 and Figure 7A.
- mice were first practiced at a speed of 12 rpm for 5 days to make the mice accustomed to exercise and obtain a basic exercise value of the mice.
- Each rotarod experiment was repeated 3 times for each mouse. Each time is 20 minutes apart, the best result is recorded, and the longest incubation period of the fall is recorded. Taking 180 seconds as the cut-off value, if it exceeds 180 seconds, it will be recorded as 180 seconds, if it is less than 180 seconds, it will be recorded as the actual time.
- Each experimental mouse will be measured once a week. The longer the incubation period, the better the mouse's motor coordination, limb strength and balance ability.
- mice in the cell transplantation group As can be seen from Table 3, starting from day 98 of the mice in the cell transplantation group, the latency period of the mice's rotarod movement gradually shortened. Starting from the 77th day of the control group, the incubation period of the mice's rotarod movement gradually shortened, and by the 105th day, there were already mice in the control group that could not complete the test. Although the incubation period of the mice in the cell transplantation group decreased at 84 days, the exercise time of the mice returned to 180 seconds at 91 days. This may be related to the early surgical recovery, and the post-traumatic recovery of the surgery still has an impact. Behavioral testing of mice. The mice in the control group began to be unable to complete the rotarod movement at about 105 days.
- the rotarod movement function of the cell transplantation group decreased significantly slower than that of the control mice. It was inferred based on the body weight test. The onset period of the mice was about 105 days in the cell transplantation group, while the onset in the control group was about 98 days. The trauma of surgery affected their behavioral tests, but from the point of view of onset, in the control group, the onset of disease occurred at 98 days, and the detection of rotarod movement plummeted by 105 days. See Table 3 and Figure 7B.
- mice's front and rear limbs were painted with black and red non-toxic dyes respectively, and the mice were placed on a narrow runway (50cm long, 7cm wide, and 10cm high).
- the runway covered a Use a piece of white paper to record the walking steps of the mouse's forelimbs and hindlimbs.
- the step length of mice in the cell transplantation group decreased starting from 112 days.
- the incubation period of the mice in the control group dropped from 105 days to 91 days, it returned to the average value at 98 days. This may be related to the early recovery from surgery and the post-traumatic stress of the surgery. The recovery status still affects the behavioral testing of mice.
- mice in the control group began to show a decrease in footprint length at about 105 days.
- the step length of the cell transplantation group did not change compared with that of the control mice.
- the cells The transplanted group of mice developed disease at approximately 105 days, while the mice in the control group developed disease at approximately 98 days.
- the trauma of the surgery affected their behavioral testing, but from the point of view of onset, the control group had onset at 98 days, and the footprint length test plummeted by 105 days.
- the cell transplantation group declined more slowly than the control group, and the decline time was 7 days later than the control group. See Table 4, Table 5 and Figure 7C, Figure 7D.
- the pairwise comparison results indicate that the forepaw gait at 91d is significantly lower than that at 112d and 119d; the forepaw gait at 98d The forepaw gait of 119d is significantly lower than that of 119d; the forepaw gait of 105d is significantly lower than that of 119d; the forepaw gait of 112d is significantly lower than that of 119d.
- the pairwise comparison results indicate that the hind paw gait at 91 days is significantly lower than that at 112 days and 119 days; the hind paw gait at 98 days The hind paw gait of 119d is significantly lower than that of 119d; the hind paw gait of 105d is significantly lower than that of 119d; the hind paw gait of 112d is significantly lower than that of 119d.
- induced neural stem cells can be induced to differentiate into PAX6 + , SOX1 + , NKX6.1 + , SOX2 + , DBX2 + , by adding RA, SB, CHIR, LDN, bFGF, Purmorphamine, and DAPT small molecules.
- the spinal cord progenitor cells obtained were identified through genetic testing as progenitor cells in the cervical segment of the spinal cord.
- spinal cord progenitor cells were transplanted into immunodeficient mice (NOD SCID) at different time points, it was found that the cells survived and differentiated into TUJ1 + neurons in vivo, with a differentiation efficiency of 70%. In the long-extending axonal region, cells can be found. NF-L + neurons. Staining for some markers of spinal cord progenitor cell differentiation subtypes found that a few appeared: OLIG2 + , PAX2 + , and LHX5 + .
- synaptic protein marker SYN The positive co-localization of synaptic protein marker SYN and the cell's own fluorescent GFP indicates that synapses may be formed between differentiated cells and Vglut1 may be secreted.
- SOD1 ALS model mice After the cells were transplanted into SOD1 ALS model mice, it was found that the cell transplantation group lost weight more slowly than the control group, the onset time was delayed, and the stride length shortened more slowly.
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Abstract
本发明提供了一种用于治疗肌萎缩侧索硬化症的脊髓祖细胞，所述脊髓祖细胞表现为PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、或Nkx2.2+中的至少任意两者，以及表现为PAX7-，并且所述脊髓祖细胞由人诱导神经干细胞分化得到，还提供了该脊髓祖细胞的诱导分化方法。所述脊髓祖细胞可用于制备用于治疗肌萎缩侧索硬化症的药品或医疗器械。本发明的脊髓祖细胞在移植后表现出良好的ALS治疗活性，为ALS的治疗提供了一种新的治疗用品和方案。
Description
本发明涉及神经生物学领域，具体涉及一种用于治疗肌萎缩侧索硬化症的脊髓祖细胞及其诱导分化方法和用途。
肌萎缩侧索硬化症(Amyotrophic Lateral Sclerosis，ALS)，俗称为“渐冻症”，也被称为卢伽雷氏病(Lou Gehrig syndrome)，是一种快速进展的神经退行性疾病，其特征是运动皮层中的上运动神经元以及脑干和脊髓腹角的下运动神经元的选择性退化。全球肌萎缩侧索硬化症(ALS)发病率估计为2/10万。这种疾病通常在45到75岁期间出现，在所有患者中男性患者的比例略大于女性，该病发病速度快，起初只是局灶性肌肉无力，逐渐进行性进展，影响大部分骨骼肌肉，最终导致完全瘫痪。当疾病发作后，疾病的迅速进展将生存期限制在3-5年内，由于呼吸肌麻痹受累逐渐影响到了个体的生存，最终导致死亡。至今尚无令人满意的治疗手段。
干细胞治疗肌萎缩侧索硬化症，最初被提议是作为一种ALS治疗剂来补充逐渐丢失的运动神经(MN)群体。干细胞具有自我更新和维持未分化状态的能力。当它们分裂时，母细胞保持干性，而子细胞则可以分化，例如：胚胎干细胞(ESC)这种类型的干细胞是具有极高的全能性，理论上可以分化成任何类型的细胞；而多能干细胞(PSC)根据诱导方法可以分化成有限的特定细胞类型。神经祖细胞(NPC)是可以分化成神经元或神经胶质细胞的多能干细胞。ALS干细胞治疗的最初概念是使用ESC或PSC分化产生运动神经元后移植到ALS患者体内，弥补丢失的运动神经元，并在体内生长延伸进而治疗疾病。要想实现这一目标，这些运动神经元需要与先前存在的神经回路有效连接，并且需要实现轴突的有效投射，但是这种投射通常需要很长的距离。既要与内源性神经元和肌肉发生突触连接，同时还要耐受患病处各种炎性细胞因子浸润的不利微环境，这对移植的细胞来说是一个挑战。基于当前研究进展，这一治疗策略暂时存在诸多困难，短期内难以实现(N.J.Maragakis,Stem cells and the ALS neurologist,Amyotroph Lateral Scler,11(5)(2010)417-23)。继而又有研究者提出通过移植的细胞释放神经保护性营养因子或利用移植细胞的免疫调节功能来改善ALS患者神经系统中的不利毒性微环境，以期有效防止或延缓现有运动神经元的退化。其中移植的干细胞通过提供滋养、神经保护微环境来发挥支持作用，改善患病处运动神经元的不利条件，从而减缓神经退行性病变和神经元的死亡。具有这种能力的移植干细胞会分泌神经营养因子，分化为未患病的非神经元细胞，例如星形胶质细胞和小胶质细胞，或分化为与患病运动神经元突触产生联系的调节性神经元(Thomson JA等，Embryonic stem cell lines derived from human blastocysts.Science.1998；282(5391):1145–7)。在动物模型中，干细胞治疗的临床前研究表现出较好的治疗效果，证明干细胞治疗ALS的潜在适用性。
已有研究将来自人类胚胎干细胞(hESC)经定向分化得到的运动神经元祖细胞(MNP)移植到SOD1G93A小鼠的腰椎椎管内，该细胞能够在SOD1G93A小鼠体内存活、分化和进行有益的神经营养因子释放，之后也在其他的运动神经元疾病如运动神经元变性(MND)模型小鼠体内得到证实(Rossi SL等，Histological and functional benefit following transplantation of motor neuron progenitors to the injured rat spinal cord.PLoS One.2010；5(7):e11852)。Wyatt等人将hESC衍生的MNP直接移植到接受过免疫抑制剂治疗的SOD1G93A小鼠、脊髓性肌萎缩(SMA)Δ7SMN幼鼠和脊髓损伤(SCI)大鼠的脊髓中，证明移植细胞在体内分化成了一个运动神经元细胞群混合体，其中包含了成熟和未成熟的运动神经元，而且移植的细胞能够通过主动释放神经营养因子例如神经营养因子3(NT-3)和神经生长因子(NGF)来减少移植部位附近的运动神经元损失(Wyatt TJ等，Human motor neuron progenitor transplantation leads to endogenous neuronal sparing in 3 models of motor neuron loss.Stem Cells Int.2011；2011:207230)，但是该研究分化细胞的轴突没有到达损伤外围，也未发现分化的细胞整合到了现有的神经回路中。尽管围绕ESC的治疗潜力引起了人们的兴趣，但使用人类胚胎干细胞仍存在一些问题阻碍无法回避，包括伦理问题、致瘤风险和自体移植的不可能性，因而hESC在实验向临床转化过程中仍存在较大阻碍亟待解决。
为了降低致瘤风险，研究者开始考虑使用人类神经干细胞(hNSC)，该细胞已经具有一定分化程度，其移植后在人体内形成畸胎瘤的倾向是比较小的(Mothe A等，Tator CH.Isolation of neural stem/progenitor cells from the periventricular region of the adult rat and human spinal cord.J Vis Exp.2015；99:e52732)。hNSC是源自流产胎儿脊髓或大脑的多能干细胞，能够在体外自我更新并分化成星形胶质细胞、神经元和少突胶质细胞。多项研究已经证实在ALS小鼠动物模型中将hNSC移植到脊柱、鞘内或颅内的安全性和治疗潜力(Xu L等，Human neural stem cell grafts in the spinal cord of SOD1 transgenic rats:differentiation and structural integration into the segmental motor circuitry.J Comp Neurol.2009；514(4):297–309；Yan J等，Extensive neuronal differentiation of human neural stem cell grafts in adult rat spinal cord.PLoS Med.2007；4(2):e39)。已经有I期临床试验用人类的神经干细胞移植治疗ALS，在这项试验中的主要目的是评估将人类神经干细胞(hNSC)移植到肌萎缩侧索硬化症(ALS)患者的脊髓中的可行性和安全性，18名确诊为ALS的患者接受了通过显微注射的方式将hNSC移植到腰椎或颈脊髓灰质束中的手术。通过临床、心理、神经放射学和神经生理学评估对移植前后的患者进行监测(L.Mazzini等，Results from Phase I Clinical Trial with Intraspinal Injection of Neural Stem Cells in Amyotrophic Lateral Sclerosis:A Long-Term Outcome,Stem Cells Transl Med 8(9)(2019)887-897)。尽管有上述积极的实验结果，但由于可用于移植的细胞数量有限，从流产胎儿体内提取神经干细胞也存在着伦理挑战，临床转化依然存在诸多限制。
为解决神经干细胞的来源问题并降低或消除免疫原性，考虑使用了诱导多能干细胞(iPSC)和成体间充质干细胞(MSC)。有研究者从人iPSC中分化出富含胶质的神经祖细胞(GRNP)后并移植到ALS小鼠体内，细胞存活并产生了星形胶质细胞，延长了mSOD1小鼠的生存期，改善了ALS模型小鼠的运动功能，上调神经营养因子和减少了胶质细胞形成(Kondo T等，Focal transplantation of human iPSC-derived glial-rich neural progenitors improves lifespan of ALS mice.Stem Cell Reports.2014 Aug 12；3(2):242–249)。然而，使用iPSC存在诸如重编程效率、表观遗传记忆和安全性等问题仍待解决或优化。另外，已有研究者将MSC使用在ALS小鼠动物模型中并发现可以延缓疾病的进展并增加了动物存活率(H.Magota等，Intravenous infusion of mesenchymal stem cells delays disease progression in the SOD1G93A transgenic amyotrophic lateral sclerosis rat model,Brain research 1757(2021)147296)，但是，由于MSC为成体干细胞，分化潜力有限，在彻底治愈ALS方面存在困难。
鉴于现有研究中所使用细胞存在诸如上述内容展现的不足，需要进一步寻找更适合的多能干细胞来源，用于移植治疗ALS。
发明内容
为解决上述现有技术中存在的问题，本发明的目的在于提供一种全新的细胞，用于治疗肌萎缩侧索硬化症。
根据本发明的第一方面，本发明的第一个目的在于提供一种脊髓祖细胞，所述脊髓祖细胞的特征表现为PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、或Nkx2.2+中的至少任意两者，以及表现为PAX7-，并且所述脊髓祖细胞由人诱导神经干细胞分化得到，所述人诱导神经干细胞由人外周血单个核细胞诱导产生。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞表现为Dbx2+和Nkx6.1+。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞表现为Olig2+和Nkx2.2+。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞表现为SOX2+和Nestin+。优选地，本发明所述的脊髓祖细胞表现为SOX1+、SOX2+、和Nestin+。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞表现为PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、和Nkx2.2+，以及表现为PAX7-，并且所述脊髓祖细胞由人诱导神经干细胞分化得到，所述人诱导神经干细胞由人外周血单个核细胞诱导产生。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞高表达HOXC6，能够表达HOXC8和HOXC9，不表达HOXC10。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞自然分化7天后，表现出LHX1+、LHX5+、EVX1/2+、EN1+、Islet1+、CHX10+、PAX2+的特征。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞自然分化40-60天后，至少表现出TUJ1+、Vglu1+、GAD67+的特征；优选地，还另外表现出TH+、CHAT+的特征。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞表达SOX1、SOX2、PAX6、PAX3，但不表达FOXG1和OTX2。优选地，本发明所述的脊髓祖细胞还另外表达GSX2。进一步优选地，本发明所述的脊髓祖细胞高比例表达PAX6、PAX3、SOX2。
根据本发明的一个实施方式，本发明所述的脊髓祖细胞用于治疗肌萎缩侧索硬化症。
在本发明中，术语“高比例表达”的含义是指分化后的脊髓祖细胞群中表达PAX6、PAX3、SOX2蛋白的细胞数量占脊髓祖细胞总细胞的数量的比例不低于90％。
根据本发明的第二方面，本发明的第二个目的在于提供制备所述脊髓祖细胞的诱导分化方法，包括：
(1)准备诱导神经干细胞(iNSC)，并悬浮培养，其中所述iNSC由人外周血单个核细胞诱导产生；
(2)准备N2B27基础培养基，所述N2B27基础培养基包含DMEM/F12、NeuralBasal-A、N2添加剂、B27添加剂、GlutaMAX、青链霉素、胰岛素；
(3)解离所述iNSC，离心，并在第一分化培养基中进行第一阶段的分化培养，所述第一分化培养基为在所述N2B27基础培养基中添加CHIR-99021、SB431542、和RA得到的培养基；
(4)在第一阶段的分化培养进行6-8天后，将所述第一分化培养基更换为第二分化培养基进行第二阶段的分化培养，所述第二分化培养基为在所述N2B27基础培养基中添加CHIR99021、SB431542、RA、bFGF、DAPT、LDN、和Purmorphamine得到的培养基；
(5)在第二阶段分化培养进行9-12天后获得所述脊髓祖细胞。
根据本发明的一个实施方式，所述N2B27基础培养基的组成为：DMEM/F12:NeuralBasal-A＝1:1、N2添加剂0.5-2X、B27添加剂0.5-2X、GlutaMAX0.5-2X、青链霉素0.5-2X、胰岛素2.5-10μg/mL。
根据本发明的一个实施方式，所述N2B27基础培养基的组成为：DMEM/F12:NeuralBasal-A＝1:1、N2添加剂1X、B27添加剂1X、GlutaMAX 1X、青链霉素1X、胰岛素5μg/mL。
根据本发明的一个实施方式，所述第一分化培养基中添加的CHIR-99021、SB431542、和RA的终浓度分别为1-5μM、8-12μM、和0.02-0.5μM。优选地，所述第一分化培养基中添加的CHIR-99021、SB431542、和RA的终浓度分别为3μM、10μM、和0.1μM。
根据本发明的一个实施方式，所述第二分化培养基中添加的CHIR99021、SB431542、RA、bFGF、DAPT、LDN、和Purmorphamine的终浓度分别为1-5μM、8-12μM、0.05-2μM、70-130ng/mL、70-130ng/mL、0.02-0.5μM、和0.1-2μM。优选地，所述第二分化培养基中添加的CHIR99021、SB431542、RA、bFGF、DAPT、LDN-193189、和Purmorphamine的终浓度分别为4μM、10μM、0.1μM、100ng/mL、100ng/mL、0.1μM、和0.5μM。
根据本发明的一个实施方式，本发明在获得所述脊髓祖细胞后进行维持培养以进行传代，用于所述维持培养的scNPC维持培养基为在N2B27基础培养基中添加CHIR99021、SB431542、和Purmorphamine得到的培养基。优选地，所述scNPC维持培养基中添加的CHIR99021、SB431542、和Purmorphamine的终浓度分别为1-5μM、1-3μM、和0.1-0.5μM。进一步优选地，所述scNPC维持培养基中添加的CHIR99021、SB431542、和Purmorphamine的终浓度分别为3μM、2μM、和0.2μM。
根据本发明的一个实施方式，本发明在所述制备脊髓祖细胞的诱导分化方法中在各阶段均隔天对细胞进行半换液。具体地，在进行第一阶段的分化培养、第二阶段的分化培养、或者在维持培养阶段均隔天对细胞进行半换液。
根据本发明的一个实施方式，所述第一阶段的分化培养进行7天。根据本发明的另一实施方式，所述第二阶段的分化培养进行10天。
根据本发明的一个实施方式，所述维持培养能够持续40天以上，优选地能够持续40-150天，更优选地能够持续40-70天，进一步更优选地能够持续40-60天。根据本发明的另一实施方式，所述维持培养能够至少扩增5代，优选地所述维持培养能够扩增5-10代，更优选地所述维持培养能够至少扩增10代。
根据本发明的第三方面，本发明的第三个目的在于提供一种医疗用品，包含根据本发明所述的脊髓祖细胞，所述医疗用品为药品或医疗器械。优选地，所述医疗用品用于治疗治疗肌萎缩侧索硬化症。
根据本发明的第四方面，本发明的第四个目的在于提供所述脊髓祖细胞用于制备治疗肌萎缩侧索硬化症的医疗用品的用途，所述医疗用品为药品或医疗器械。
本发明具有良好的技术效果：本发明建立了一种新的培养系统，能从iNSC中产生人类脊髓神经细胞，诱导来的脊髓祖细胞包括所有的脊髓腹侧NPC类型(p0-pMN)，并且可以产生广泛的已鉴定的脊髓神经元表型与主要神经胶质细胞类型。创建这种脊髓神经元类型的临床作用十分重要，可以用于建立疾病建模和药物筛查，疾病治疗等各种方向。本发明提供的脊髓祖细胞及其诱导分化方法，具有新颖的生物标记物组合特征和RNA表达特点，并且在移植后能够表现出治疗ALS的活性，为ALS的治疗提供了一种新的治疗用品和方案。另外，本发明的脊髓祖细胞的来源为人外周血单个核细胞(PBMC)，该细胞来源容易，成本低，没有致瘤性，为采用自体PBMC制备诱导神经干细胞并进一步按本发明的方法获得自体脊髓祖细胞从而降低异体细胞的免疫排斥反应提供了可能性。
附图用来提供对本申请技术方案的进一步理解，并且构成说明书的一部分，与本申请的实施例一起用于解释本申请的技术方案，但并不构成对本申请技术方案的限制。
图1为脊髓祖细胞分化鉴定图。A：脊髓祖细胞分化流程图，将神经干细胞消化之后，-1天加入神经干细胞培养基培养。从第0天开始换成N2B27培养基并加入RA、SB、CHIR，7天之后更换分化小分子为RA、SB、CHIR、LDN、bFGF、Purmorphamine、DAPT继续分化，10天之后更换分化小分子为SB、CHIR、Purmorphamine。B：神经诱导后20天，细胞分化为PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、Nkx2.2+、PAX7-脊髓腹侧祖细胞，标尺＝100微米。C：发育中的脊髓祖细胞区域标记物表达模式示意图。D：qPCR显示的诱导神经干细胞神经诱导后18天内各种HOX基因的表达(每个基因n＝3；三次独立重复实验)。E：P1代细胞SOX1、SOX2、PAX6、Ki-67免疫染色，标尺＝100微米。F：P5代细胞SOX1、SOX2、PAX6、Ki-67免疫染色，标尺＝100微米。G：P1和P5Ki-67阳性百分比。H：SOX1、SOX2、PAX6、Nkx2.2在P1和P5脊髓祖细胞的基因表达分析图(每个基因n＝3；三次独立重复实验)。
图2为脊髓祖细胞自然分化亚型图。A：在N2B27培养基中对脊髓祖细胞进行自然分化，7天后对自然分化的细胞进行免疫组化染色，观察到了LHX1+、LHX5+、EVX1/2+、EN1+、Islet1+、CHX10+、PAX2+的阳性细胞，标尺＝100微米。B：V0-V3中间神经元的标记表达示意图。C：脊髓神经递质表达模式的示意图。D：自然分化40天、50天、60天后对细胞进行TUJ1、Vglu1、GAD67、TH、CHAT免疫荧光染色，发现TUJ1+、Vglu1+、GAD67+、TH+、CHAT+细胞，标尺＝100微米。E：自然分化14天TUJ1+和GFAP+百分比。
图3为脊髓祖细胞的RNA测序图。A：胎儿脑和分化的脊髓祖细胞的分层聚类，揭示为两个主要集群。B-E：由RNA-seq确定的脊髓祖细胞SOX1、SOX2、PAX6、PAX7、PAX3、OTX2、GSX2、FOXG1、HOXA、HOXB、HOXC簇的表达。对于每个基因，将表达水平标准化为具有最高表达水平(100)的细胞样品，n＝3。F：脊髓颈椎、胸椎、腰椎在HOX家族基因表达示意图。
图4为脊髓祖细胞在SD大鼠体内存活鉴定图。A：使用AAVSI-Pur-CAG-EGFP质粒，敲入由CAG启动子驱动的EGFP表达盒，使iNSC带有绿色荧光标记。B：移植位点示意图。C：移植后1周处死大鼠取材染色结果发现细胞大量存活(HU：红色、GFP：绿色)。D：移植后2周处死大鼠取材染色结果发现细胞少量细胞存活(HU：红色、GFP：绿色)。E：两个月时取材鉴定染的情况发现细胞死亡大量炎性细胞浸润，从左向右依次为(HU：红色、GFP：绿色；TNFα：红色、GFP：绿色；CD68：红色、GFP：绿色)，标尺＝100微米。
图5为不同时间点的脊髓祖细胞在免疫缺陷小鼠脊髓中的存活迁移鉴定图。A：自然分化7天细胞移植到小鼠脊髓颈段和胸段存活情况(GFP：绿色，Stem121：红色)，标尺＝100微米。B：自然分化10天细胞移植到小鼠脊髓颈段和胸段存活情况细胞移植到(GFP：绿色，Stem121：红色)，标尺＝100微米。C：自然分化12天细胞移植到小鼠脊髓颈段和胸段存活情况细胞移植到(GFP：绿色，Stem121：红色)，标尺＝100微米。D：自然分化14天细胞移植到小鼠脊髓颈段和胸段存活情况细胞移植到(GFP：green，Stem121：red)，标尺＝100微米。E：脊髓祖细胞(scNPC P1)细胞移植到小鼠脊髓颈段和胸段存活情况细胞移植到(GFP：green，Stem121：red)。标尺＝100微米。F：不同时间点细胞在颈段脊髓迁移长度。G：不同时间点细胞在胸段脊髓迁移长度。
图6为脊髓祖细胞在免疫缺陷小鼠脊髓中的安全性分化鉴定图。A：scNPC移植体内2月成瘤性实验免疫组化染色结果。未见到OCT4阳性细胞；未见到KI67阳性细胞。B：从左向右：scNPC在体内自然分化为OLIG2+、NG2+、PAX2+、LHX5+、TUJ-1阳性早期神经元；GFAP未见表达。C：NF-L+、Vglut1+、突触蛋白Synapsin+表达。D：移植细胞在免疫缺陷小鼠体内所分化的TUJ1神经元和GFAP细胞的比例。
图7为脊髓祖细胞对ALS SOD1 G93A小鼠的影响的结果图。A：体重折线分析图，其中p＝0.023<0.05，成对比较结果提示，70d时的体重显著地低于84d时的体重，细胞移植组(n＝8)，对照组(n＝7)。B：旋转棒运动分析折现图，时间的主效应显著，F＝3.383，p＝0.062>0.05，细胞移植组(n＝8)，对照组(n＝7)；C：前爪步态分析折线图，p＝0.000<0.001，成对比较结果提示显著，细胞移植组步态变化下降缓慢，细胞移植组(n＝8)，对照组(n＝7)；D：后爪步态分析折线图，p＝0.000<0.001，成对比较结果提示成对比较结果提示显著，细胞移植组步态变化下降缓慢。细胞移植组(n＝8)，对照组(n＝7)。
下面将参照附图来详细描述本发明的各种示例性实施例。对示例性实施例的描述仅仅是说明性的，并不作为对本发明及其应用或使用的任何限制。本发明可以以许多不同的形式实现，不限于这里所述的实施例。提供这些实施例是为了使本发明透彻且完整，并且向本领域技术人员充分表达本发明的范围。应注意到：除非另外具体说明，否则在这些实施例中描述的技术手段应被解释为仅是示例性的，而非限制性的。
实施例1脊髓祖细胞(scNPC)的制备
1.1人诱导神经干细胞的制备
采用诱导神经干细胞(iNSC)作为分化制备脊髓祖细胞的起始细胞，其中iNSC可采用任何已知方法获得的人诱导神经干细胞，例如根据发明人先前的中国专利201810372724.7中公开的诱导方法及其获得的细胞。在本实施例中，示例性地使用了Y.Yuan等报导的方法(Y.Yuan等，Dopaminergic precursors differentiated from human blood-derived induced neural stem cells improve symptoms of a mouse Parkinson's disease model，Theranostics,8(17)(2018)4679-4694)，通过仙台病毒感染将人外周血单个核细胞(PBMC)重编程为人iNSC：将300万个人外周血单个核细胞悬浮在SCF培养基中，然后使用编码OCT3/4、SOX2、KLF4和c-MYC(OSKM)的仙台病毒(Life Technologies)感染(MOI＝10)。两天后，将这些细胞以2×105/孔的密度置于在PDL/Laminin包被的12孔板上的NSC培养基中，该NSC培养基按DMEM/F12:Neurobasal(1:1)、1×N2、1×B27、2mM GlutaMAX、1％NEAA(Life Technologies)、10ng/mL重组人白血病抑制因子(Millpore)、3μM CHIR99021(Gene Operation)和2μM SB431542(Gene Operation)配制。每隔一天更换一次培养基。十天后，上皮样克隆出现在培养物中。在第32至35天，克隆大到足以被拾取并转移到PDL/Laminin包被的96孔板上进行扩增。为了灭活残留的仙台病毒，当iNSC处于第三代时，将培养箱温度从37℃提高到39℃一周。在较高温度下培养一周导致大约一半的iNSC死亡，并且存活的细胞在PDL/Laminin包被的24孔板中传代，然后在细胞群足够大时转移到PDL/Laminin包被的6孔板上，获得人iNSC。
1.2脊髓祖细胞(scNPC)的分化
取实施例1.1中悬浮培养的iNSC细胞，按照如下方法将其分化为scNPC：(1)准备N2B27基础培养基，其配制方法为(按300mL计算)：DMEM/F12(Invitrogen，11320-033)144mL、NeuralBasal-A(Life Technologies，10888-022)144mL、N2添加剂(Invitrogen，17502048)3mL、B27添加剂(Invitrogen，17504044)6mL、GlutaMAX(Gibco，35050)3mL、青链霉素(Invitrogen，15140-122)3mL、胰岛素(Roche，12585014)5μg/mL，即按照浓度计算，DMEM/F12:NeuralBasal-A＝1:1、N2添加剂1X、B27添加剂1X、GlutaMAX 1X、青链霉素1X、胰岛素5μg/mL，一次性滤器过滤后4℃冰箱保存，可放置1个月。(2)将低黏附6孔板中悬浮培养的iNSC细胞收集到15mL离心管里自然沉降，吸走培养基上清，每孔加入1mL DPBS(Invitrogen，C14190500BT)清洗残留培养基，吸走DPBS，每孔加入1mL Accutase(Invitrogen,A11105-01)，CO2培养箱37℃消化3分钟，加入3mL iNSC培养基终止消化。(3)将混合液体放入低速离心机，离心管配平后以1200RMP离心3分钟后去除上清，轻弹离心管底部，在离心管内加入12mL的N2B27分化培养基(D0-D7)，混匀细胞和培养基后，均分在低粘附6孔板中，隔天对细胞进行半换液，所述N2B27分化培养基(D0-D7)为通过在N2B27基础培养基中加入CHIR99021(Operation，04-0004，终浓度3μM)、SB431542(OperationGene，R0101000，终浓度10μM)、全反式维甲酸RA(Sigma-Aldrich，R316091，终浓度0.1μM)得到。(4)培养7天后，将培养基更换为N2B27分化培养基(D7-D17)，隔天对细胞进行半换液，所述N2B27分化培养基(D7-D17)为在N2B27基础培养基中加入CHIR99021(Operation，04-0004，终浓度4μM)、SB431542(OperationGene，R0101000，终浓度10μM)、全反式维甲酸(RA)(Sigma-Aldrich，R316091，终浓度0.1μM)、bFGF(Peprotech，100-18B，终浓度100ng/mL)、DAPT(Sigma-Aldrich，D5942，终浓度100ng/mL)、LDN(Selleck，1062368-24-4，终浓度0.1μM)、Purmorphamine(Selleck，483367-10-8，终浓度0.5μM)得到。(5)诱导10天后，即在细胞分化的第17天，将培养基更换为scNPC维持培养基，隔天对细胞进行半换液，所述scNPC维持培养基为在N2B27基础培养基中加入CHIR99021(Operation，04-0004，终浓度3μM)、SB431542(OperationGene，R0101000，终浓度2μM)、Purmorphamine(Selleck，483367-10-8，终浓度0.2μM)得到，由此获得的分化细胞为即为脊髓祖细胞。上述分化方法的主要步骤如图1A所示。
实施例2脊髓祖细胞(scNPC)的鉴定和表征
2.1 scNPC的鉴定及免疫组织化学染色
采用如下方法对scNPC进行鉴定及免疫组织化学染色：(1)将12mm的玻璃片用75％酒精浸泡1分钟，超净台内在酒精灯前燃烧去除残留酒精后置于24孔板的孔中，先用DPBS(Invitrogen，C14190500BT)清洗一遍，然后用0.5mL PDL工作液(50μg/mL)包被，然后加入0.5mL Laminin工作液，37℃下处理2小时后弃去液体，将scNPC按2×104个细胞/孔接种在包被好的培养板孔内，培养2-3天，当细胞融合度达到70-80％左右，从培养箱中拿出，准备固定，染色；(2)细胞用DPBS室温洗3次，每次5分钟；(3)使用4％多聚甲醛(PFA)室温固定10分钟；(4)吸去4％PFA，用PBST(0.01mM PBS中加入0.3％Triton X-100室温放置)作为Wash Buffer洗3遍，每次5分钟；(5)细胞用封闭液(Wash Buffer中加入3％驴血清)室温孵育2小时；(6)细胞换一抗混合液：抗体稀释液中(Wash Buffer中加入1％驴血清)分别加入1:500小鼠抗Nestin抗体(BD bioscience)、1:500山羊抗SOX1抗体(Santa cruz)、1:1000山羊抗SOX2抗体(Santa cruz)、1:500兔抗Olig2抗体(Millipore)、1:500兔抗PAX6抗体(ebioscience)、1:200小鼠抗PAX7抗体(DSHB)、1:300兔抗DBX2抗体(ProSci Incorporated)、1:100小鼠抗Nkx2.2抗体(DSHB)、1:100小鼠抗Nkx6.1抗体(DSHB)、1:300小鼠抗IRX3抗体(Kerafast)、1:300小鼠抗PAX3(DSHB)，每个孔分别设立阴性对照，不加一抗，只加入抗体稀释液，加完后放在4℃冰箱孵育过夜；(7)弃一抗混合液，用Wash Buffer室温洗3遍，每次5分钟；(8)细胞换二抗混合液：Antibody Buffer中分别加入对应的二抗，室温避光孵育2小时；(9)弃二抗混合液，用Wash Buffer室温避光洗3遍，每次5分钟；(10)弃液体，用DAPI核染液(0.01mM PBS中加入1/500体积1mg/mL DAPI)室温避光孵育10分钟，弃液体，用0.01mM PBS室温避光洗3遍，每次5分钟；(11)用眼科直镊小心夹起载有细胞的圆玻璃片，在载玻片上涂上防止猝灭剂PVA，将载有细胞侧的圆玻璃片贴在PVA上封片，防止气泡生成(PVA化冻后离心去除里面的小泡)，室温避光晾干；(12)共聚焦显微镜拍照。
2.2 scNPC增殖能力的鉴定
采用如下方法鉴定scNPC的增殖能力：分别取scNPC的P1、P5、P10三个代次(P1指第1代次，P5指第5代次，P10指第10代次)的细胞，均以2×104的起始细胞种植在PDL/Laminin包被的24孔板内，每个代次的细胞做3个重复。培养2-3天，当细胞融合度达到70-80％左右，从培养箱中拿出，准备固定，对细胞进行KI67(1:500兔抗KI67，Millipore)染色，染色方法同实施例2.1部分所述。
2.3 scNPC的自然分化
采用如下方法自然分化scNPC：(1)将scNPC P1以2×104接种在铺有PDL/Laminin的玻璃片上，使用脊髓祖细胞培养基培养1天；(2)1天后更换培养基为N2B27基础培养基进行分化，隔日半换液，大概在分化第8天左右开始，会有部分死亡细胞沉积在玻璃片上，此时，每次换液时，均需要用DPBS(Invitrogen，C14190500BT)洗一遍玻璃片，然后更换培养基；(3)分别分化至7天、14天、21天、30天、40天、60天时将细胞固定。
2.4 scNPC自然分化鉴定细胞表达marker及统计学分析
按如下方法鉴定自然分化的scNPC表达生物标志物的情况：(1)在脊髓祖细胞自然分化的第5天、第10天、第13天、第15天、第18天及第24天，各取8个孔的细胞用DPBS(Invitrogen，C14190500BT)洗三遍，然后4％PFA室温固定10分钟，然后加入DPBS可放入4℃冰箱保存；(2)选取需要的细胞孔用PBST(Wash Buffer中加入3％驴血清)清洗三遍，然后室温下加入封闭液(Wash Buffer中加入3％驴血清)封闭2小时；(3)加入一抗：1:100兔抗PAX2(R&D)、1:300兔抗EN1(abgent)、1:300小鼠抗EVX1/2(DSHB)、1:300小鼠抗ISL1(DSHB)、1:300小鼠抗CHX10(Santa cruz)、1:100小鼠抗LHX5(DSHB)、1:100小鼠抗LHX1(DSHB)、1:500鸡抗GAD67(abcam)、1:500兔抗VGLUT1(Proteintech)、1:200山羊抗CHAT(Millipore)染色，染色方法同实施例2.1部分所述；(4)共聚焦显微镜采用相同的扫描参数(激光器强度、电压值、去背景值、Pinhole值、放大倍数、分辨率)扫描，每个样品随机取3或9个视野，共做2至3组独立实验；(5)用ImageJ软件计算每个视野下GFAP，LHX5，TUJ-1，LHX1，ISL1、EN1、EVX1/2、CHX10阳性细胞数量和DAPI阳性细胞数量，计算每个视野中阳性细胞与细胞核的百分率。
2.5 scNPC总RNA提取
采用如下方法提取scNPC的总RNA：(1)除去培养基上清，细胞按照1×106加入1mL Trizol(Invitrogen)后，室温放置5分钟，使其充分裂解(注：此时可放入-70℃长期保存)；(2)将裂解好的细胞液体放入高速低温离心机中，配平，12,000RPM，4℃条件，离心5分钟；(3)弃沉淀收集上清至新的1.5EP管中，按200μl氯仿/mL Trizol加入氯仿，振荡混匀后室温静置15分钟，将震荡均匀的液体，放置低温高速离心机中。12,000RPM，4℃，离心15分钟；(4)取出离心机的样品，吸取上层水相，按500μl异丙醇/mL Trizol加入异丙醇混匀，室温静置5-10min，将混合了异丙醇的液体放入高速低温离心之中，12,000RPM，4℃，离心10分钟；(5)弃上清，RNA沉淀可见于管底。按1mL75％乙醇/mL Trizol加入乙醇，温和振荡，悬浮沉淀，8,000g，4℃，离心5分钟；(6)尽量弃上清，室温晾干5-10分钟，用50μl水溶解RNA；(7)RNA溶解液放入55-60℃下5-10分钟，变性，放入-70℃长期保存。RNA测定浓度：A260/A280＝1.8-2.0RNA。
2.6二代测序准备
悬浮细胞收集用DPBS缓冲液(Invitrogen，C14190500BT)快速洗一次，每5×106个细胞加入1mL Trizol试剂，用枪头反复抽打细胞，直至看不见成团的细胞块，整个溶液呈清亮而不粘稠的状态；液氮速冻之后放入-80℃冰箱中保存，干冰运输。
2.7反转录
采用如下方法进行反转录：(1)计算所需cDNA的用量，根据用量来反转录相应RNA。反应体系可按需求相应放大，10μl体系可最大使用500ng的总RNA；(2)反应体系为5×PrimeScript Buffer(2μl)、PrimeScript RT Enzyme MixⅠ(0.5μl)、Oligo dT Primer(0.5μl)、Random 6mers(0.5μl)、总RNA、RNase Free dH2O(添加至10μl)；(3)程序设定如下：37℃，15分钟；85℃，5秒；4℃，∞。应用Gene specific primer时，建议反转录反应条件设置为42℃，15分钟。PCR反应有非特异性扩增时，将温度升到50℃会有改善。以上反应为TB Green qPCR分析法反应条件。
2.8 RT PCR反应
根据反应所需用量配置总反应液。试剂盒为Master，2×conc.(避光保存，避免反复冻融)，Water，PCR grade。反应体系为：Water，PCR grade(3μl)、PCR Primer(10μM，2μl)、Master，2×conc.(10μl)、cDNA(5μl)，总体积为20μl。使用引物为：HOXC4：正向：gggtgaatttcaggggaaatgagg，反向：ctcaaactgaacagctctgagagg；HOXC5：正向：atcaagatctggttccagaaccgc，反向：aggaaaagcgcttttgtctgtggg；HOXC6：正向：ttagcaccgtcagtgttcctatcc，反向：tatacaggagggtaacacgaaggg；HOXC8：正向：aggaacctgatggaaacctgaagg，反向：atcaaacagcgaaggagaggaagg；HOXC9：正向：tagagttagttctacccagcgagg，反向：acctggaccaaatacgatacaggg；HOXC10：正向：ctcacacacagcattctgttctcc，反向：acacgaacactagccgaactttcc；PAX6：正向：TCTTTGCTTGGGAAATCCG，反向：CTGCCCGTTCAACATCCTTAG；SOX1：正向：GCACAACTCGGAGATCAG，反向：CTTCTTGAGCAGCGTCTT；SOX2：正向：GCCGAGTGGAAACTTTTGTCG，反向：GGCAGCGTGTACTTATCCTTCT；Nkx2.2：正向：AAACCATGTCACGCGCTCA，反向：GGCGTTGTACTGCATGTGCT；GAPDH：正向：GTGGACCTGACCTGCCGTCT，反向：GGAGGAGTGGGTGTCGCTGT。所使用的程序为：预孵化，95℃，5分钟，4.4℃/s；扩增，95℃，10秒，4.4℃/s，引物依赖，5-20秒，2.2℃/s(Tm≥50℃)，1.5℃/s(Tm＜50℃)，72℃，5-30秒，4.4℃/s；熔解曲线，95℃，5秒，4.4℃/s，65℃，1分钟，2.2℃/s，97℃，持续，每℃采集5-10；冷却，40℃，10秒，1.5℃/s。
实施例3脊髓祖细胞(scNPC)的体内移植
3.1移植细胞的准备
按照如下方法准备移植细胞：(1)将细胞从37℃的CO2培养箱取出，去培养基，用DPBS(Invitrogen，C14190500BT)清洗细胞除去残留培养基，在长有脊髓祖细胞或自然分化7天、10天、12天、14天的脊髓祖细胞的6孔板中加入Accutase(Invitrogen，A11105-01)1mL/孔并加入200μl 500ng/mL的DNase溶液，消化3分钟后，将细胞吹打为单细胞悬液，加入3mL DMEM/F12培养基终止反应，而后，转移至15mL离心管后，用细胞网筛将液体过滤，去除死细胞和未消化开的细胞团；(2)将细胞计数后，配平放入低速离心机，以1200RMP的速度离心3分钟；(3)弃上清，加入1mL细胞重悬液重悬，转移至1.5mL EP管中，以1200RMP的速度离心3分钟；(4)弃上清，加入相应细胞重悬液重悬细胞，使细胞浓度为1×105/μl，放置于冰上待用。
3.2大鼠及小鼠的胸椎脊髓内移植
按照如下方法向Sprague Dawley(SD)大鼠(北京维通利华动物实验技术有限公司)、免疫缺陷小鼠(北京维通利华动物实验技术有限公司)、SOD1G93A小鼠(ALS模型小鼠，由首都医科大学宣武医院郭艳苏教授赠与)的胸椎脊髓内移植scNPC：(1)麻醉准备：配制1％戊巴比妥钠溶液，将大鼠或者小鼠从笼中取出，对其体重进行称量，按照30mg/kg的剂量对大鼠进行腹腔注射麻醉，小鼠按照50mg/kg的剂量进行腹腔注射麻醉，实验分组如表1所示；(2)将鼠固定于手术台，剃除背部毛，清理出待手术部位视野，在大鼠T11-T12处，小鼠T13-L1处，用手术刀剖开背部皮肤，剥离脊髓两端肌肉组织，暴露脊柱；(3)用咬骨钳轻轻将椎板去除，注意避免损伤到脊髓组织，暴露脊髓；(4)调节大(小)鼠脊髓的位置，用纱布折叠后将脊髓暴露出垫高，保持水平；(5)用微量注射器吸取细胞悬液，大鼠每位点注射3μl，小鼠每位点注射0.5μl，将微量注射器安装于立体定位仪上，调整微量注射器至合适位置，使针尖对准脊髓中线(脊髓表面中间有一条静脉血管)；(6)大鼠注射位点以及深度：向脊髓左及右侧移动0.5mm，然后下调注射器，以针尖接触到脊髓组织为0点，下行1.5mm，脊髓左右两侧各三针，一共注射6针，小鼠注射位点以及深度：向脊髓左及右侧移动0.3mm，后下调注射器，以针尖接触到脊髓组织为0点，下行0.8mm，脊髓左右两侧各三针，一共注射6针，深度0.8mm；(7)注射细胞悬液，注射速度约为1μl/min。完成注射后留针时间为5分钟，之后缓慢将注射器移出脊髓组织；(8)缝合大(小)鼠的皮肤切口，碘伏消毒处理缝合伤口，将大(小)鼠放在电热毯上保持体温，待大(小)鼠苏醒后放回笼中。
表1大鼠及小鼠的胸椎脊髓内移植的实验分组表
3.3小鼠的颈椎脊髓内移植
按照如下方法向免疫缺陷小鼠的颈椎脊髓内移植scNPC：(1)将小鼠称重，按照50mg/kg的剂量腹腔注射1％戊巴比妥钠溶液进行麻醉；(2)将小鼠固定于鼠板上，剃除背部毛，对皮肤进行消毒，用手触摸颈部节段找到需要手术的部位，在C6-C7处用手术剪剪开背部皮肤，在用手术刀剥离脊柱两边的肌肉组织，暴露脊柱，咬骨钳除去椎板，暴露脊髓；(3)固定显微注射器，注射位点以及深度：向脊髓左及右侧移动0.5mm，后下调注射器，以针尖接触到脊髓组织为0点，下行1mm。脊髓左右两侧各三针，一共注射6针；(4)注射细胞悬液0.5μl，缓慢推动针管平均速度为1μl/min。完成注射后不要立即拔针，停留5分钟后，缓慢将显微注射器移出脊髓；(5)缝合小鼠的皮肤切口，碘伏消毒处理缝合伤口。将小鼠放在电热毯上保持体温，待小鼠苏醒后放回笼中。
实施例4移植效果的评估方法
4.1大鼠、小鼠的脊髓组织灌注及切片
按如下方法进行大鼠和小鼠的脊髓组织灌注及切片制备：(1)在细胞移植1周、2周、2月后，将鼠麻醉，大头针固定，剪开皮肤，暴露心脏，剪开右心耳；(2)灌注泵的注射针刺入跳动的左心室，用止血钳固定。灌注生理盐水至右心耳流出的液体变淡；(3)灌注4％PFA，灌注速度为7×RMP，先出现鼠尾巴翘起，然后观察肝脾等内脏呈白色，四肢肌肉组织僵硬灌注即可停止；(4)用剪刀将大(小)鼠的脊髓取下，剥离皮肤肌肉组织，完整取出脊髓，观察脊髓组织有无明显肿胀，局部有无隆起，有无肿瘤形成，后放入含有4％PFA的50mL离心管中，置于4℃冰箱浸泡固定2天；(5)将鼠脊髓转移至含有组织脱水剂的50mL离心管中，置于4℃冰箱放置直至组织沉底；(6)取出脊髓，将脊髓放置在盛有OCT包埋剂的塑料盒中，将盒置于干冰上速冻，沿矢状切面位对脊髓进行连续切片，大鼠20μm厚度，小鼠12μm厚度，切完后贴于粘附性载玻片上，按顺序放入切片盒，-20℃冰箱保存。
4.2大鼠、小鼠的脊髓免疫组化染色鉴定
按照如下方法进行大鼠和小鼠的脊髓免疫组化染色鉴定：(1)每次染色取出一张脊髓切片，用PBS室温洗1次，除去组织周围的包埋剂。(2)将清洗过的脊髓组织，用蜡笔涂圈，加入Blocking Buffer室温孵育2小时。(3)脊髓组织切片换一抗混合液：Antibody Buffer中加入1:500鼠抗STEM121(Cellartis)、1:500兔抗GFP(Genscript)、1:500兔抗GFAP(中山金桥)、1:400兔抗NEUN抗体(Millipore)、1:500鼠抗TUJ-1(Millipore)、1:500鼠抗OCT4(Santa cruz)、1:500兔抗KI67(Millipore)、1:500抗人核抗体(Human nuclei antibody，HNA，Millipore)、1:500兔抗NG2(Millipore)、1:100鼠抗LHX5(DSHB)、1:200兔抗PAX2(R&D)，在4℃冰箱孵育过夜；(4)弃一抗混合液，用Wash Buffer室温洗3次，每次5分钟，共3遍；(5)更换二抗混合液：Antibody Buffer中加入相对应种属的二抗，室温避光孵育2小时；(6)弃二抗混合液，用Wash Buffer室温避光洗5分钟，共3遍；(7)弃液体，用DAPI核染液室温避光孵育10分钟；(8)弃液体，用PBS室温避光洗5分钟，共3遍；(9)弃液体，待脑片中水分挥发后滴加防止猝灭剂PVA封片，室温避光晾干；(10)共聚焦显微镜拍照；(11)用GraphPad5软件，计算不同时间点不同marker阳性细胞数均数及标准误，并进行统计作图。
4.3旋转棒实验
旋转棒实验可用于评估小鼠的协调性、平衡性和肢体力量，按如下方法进行：将小鼠放置在直径为3.0cm的旋转杆仪器上，将速度调整至15rpm，以记录最长的下落潜伏期。记录时间为180秒，如果超过180秒，则应将边界值记录为180秒，如果小于180秒，则应记录为实际时间。从出生后第70天开始，小鼠以每分钟12rpm的速度练习5天，以适应运动并获得基本的运动值。每只鼠每次测试3次并记录结果，每只实验小鼠每周测量一次。潜伏期越长，动物的运动协调能力、肢体力量和平衡能力越好。
4.4体重检测
体重是反映小鼠健康状况的重要指标之一。可间接反映小鼠的各种生理功能，从而判断其发病时间点。从出生后第10周开始记录每只小鼠的体重，每周2次，直置小鼠死亡。
4.5步态分析
步态分析可以检测小鼠肢体的运动功能。小鼠前肢和后肢分别涂有黑色和红色无毒染料。小鼠被放置在一条狭窄的跑道上(长50厘米，宽7厘米，高10厘米)，跑道上覆盖着一条同等长度的白纸，促使小鼠在跑道上行走，采集小鼠的足印，记录小鼠前肢后肢行走的步长。实验是在没有外部影响的情况下进行的，小鼠首次被放置在一个新环境中60分钟。
实施例5移植结果
5.1 iNSC诱导分化为脊髓祖细胞(scNPC)
诱导20天后，iNSC细胞分化为PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、Nkx2.2+的脊髓祖细胞(图1B)，其中PAX7不表达而PAX6持续表达。在培养第17天后，成团的细胞中，可以观察到Dbx2+和Nkx6.1+细胞(图1B)。Dbx2+的表达可以证明细胞为脊髓腹侧p0区域。还可以观察到Evx1/2+(标记V0中间神经元脊髓区域)。也可以观察到Olig2+和Nkx2.2+阳性细胞，这表明细胞分别产生了pMN结构域和p3结构域。这些结果表明，通过该方案诱导的iNSC获得了的脊髓位置细胞的特性。WNT信号和Sonic Hedgehog(SHH)信号是发育中脊髓中的有效有丝分裂原，而SMAD2/3对中胚层谱系的启动和维持至关重要。因此，WNT/SHH的持续激活和SMAD2/3的抑制可以指定细胞分化为脊髓NSC并在较长时间内维持细胞干性。在这种定义的条件下，表达SOX1+、SOX2+、Nestin+脊髓祖细胞可以在体外扩增至少10代，但因为5代以后的表达量不稳定，取5代以内的细胞进行保存使用(图1G和H)。WNT信号的持续激活导致中胚层方向的诱导分化倾向，但是通过添加SB-431542，连续阻断TGF-β和SMAD2/3信号传导，最终成功阻断了中胚层诱导。事实上，几乎所有诱导出的脊髓祖细胞在连续传代中表达神经干细胞标记SOX2、神经中间丝巢蛋白(Nestin)(图1E和F)。上述结果表明腹侧脊髓组织结构域的诱导成功。对细胞进行了HOX基因表达含量的鉴定。qPCR分析清楚地检测到HOXC6的高表达和HOXC8、HOXC9的表达，但没有检测到HOXC10，这表明诱导的腹侧脊髓组织对应于颈侧脊髓水平(图1D)。因为FGF在诱导和调节HOX激活中起关键作用，所以随着bFGF在第7天的添加，HOXC6的表达出现波动，HOXC8、HOXC9的表达出现上调、HOXC10的表达仍然不明显。在Purmorphamine 500nM的条件下诱导了头侧颈椎节段的脊髓腹侧祖细胞。
5.2脊髓祖细胞亚型的鉴定
为了检测iNSC诱导分化的腹侧脊髓祖细胞是否可以产生脊髓中间神经元或脊髓运动神经元的亚型。将分化好的脊髓祖细胞，去掉分化小分子，在N2B27培养基中进行自然分化，七天后对自然分化的细胞进行免疫组化染色，观察到了LHX1+、LHX5+、EVX1/2+、EN1+、Islet1+、CHX10+的阳性细胞(图2A)。免疫荧光染色显示大多数细胞表达Lhx1+、LHX5+、PAX2+、EVX1/2+、EN1+，表明存在V0或V1中间神经元(图2B)。V2b中间神经元的出现需要Notch信号，而Notch抑制后V2a中间神经元的比例会增加。对分化的脊髓祖细胞进行了p2结构域的鉴定，观察Chx10(V2a中间神经元标记)细胞和GATA3(V2b中间神经元标记)细胞的表达量(图2A)。因为在诱导的过程中添加了Notch信号抑制剂，所以免疫荧光结果显示了有大量Chx10+细胞，而GATA3+细胞几乎未检测到(图2G-J)。同时对pMN区域进行了鉴定，发现ISL1+。所以由iNSC诱导分化的腹侧脊髓祖细胞是可以产生脊髓中间神经元或脊髓运动神经元的亚型的。每个成熟的脊髓神经元亚类会表达一组的神经递质(图2C)。胆碱能脊髓运动神经元表达胆碱乙酰转移酶(CHAT)；背侧脊髓dI1-3区域分化的感觉神经元和V2a中间神经元是表达Vglut1+的谷氨酸能神经元；背侧脊髓dI4区域分化的体感关联神经元和dI6中间神经元以及V1中间神经元是表达GAD67+的GABA能神经元。对脊髓祖细胞进行了自然分化，取分化了40天、50天、60天的细胞进行了免疫荧光染色，证实细胞产生了Tuj1+神经元群，但是对于GFAP+神经胶质细胞的产生几乎没有(图2D、E)。发现在Tuj1+神经元中，主要群体是Vglut1+神经元和GABA+神经元(图2D)，部分还会分泌TH和CHAT。Vglut1+神经元又表达LHX1、LHX5、EN1、EVX1/2，表明它们是源自V2-V3的中间神经元(图2B、C)。GAD67+的GABA神经元表达PAX2、CHX10，表明它们是V1衍生的中间神经元或V2a中间神经元(图2B、C)。在Purmorphamine 500nM的条件下，神经元是表达Islet1+。这证明了诱导的细胞中有脊髓运动神经元的产生。总之，在添加RA、SB、CHIR、LDN、bFGF、Purmorphamine、DAPT的条件下培养由iNSC诱导的脊髓祖细胞能够在体外产生若干种类型的人类脊髓神经元，包括体感中间神经元和脊髓运动神经元。
5.3脊髓祖细胞的RNA测序
为了进一步检测分化的细胞的性能以及确定它沿头尾轴的区域特性。将iNSC诱导的脊髓祖细胞的基因表达与人类流产胎儿中枢神经系统组织的RNA、流产胎儿脊髓的RNA基因表达进行了比较。选取三个不同代次细胞：scNPC p1、scNPC p5、自然分化7天的细胞，每个细胞取3个孔(6孔板)提取RAN。对于干细胞的干性标记物：SOX1、SOX2、PAX6、PAX7、PAX3进行了对比。PAX6、PAX3、SOX2无论在脊髓祖细胞第1代还是脊髓祖细胞第5代都高比例表达。而在流产胎儿脑组织中高比例表达的FOXG1(前脑标志物)和OTX2(中脑标志物)在脊髓祖细胞中都不表达。GSX2腹侧端脑标记物，可指导发育为腹侧神经节隆，也是中间神经元祖细胞的标记物，规范和调控脊髓腹侧胶质祖细胞群。scNPC p1、scNPC p5、自然分化7天的细胞都表达GSX2，说明有发育为少突胶质细胞的潜力。为评测头尾轴的区域特性，将HOX家族基因进行了比较，诱导神经干细胞发育的脊髓神经祖细胞表达颈部和胸部脊髓HOX基因，但不表达腰部HOX基因，这表明细胞获得了颈部的同一性。此外，自然分化7天的脊髓祖细胞与人类胎儿脊髓组织的基因表达更紧密接近，但与人类胎儿脑组织的基因表达分开。这些结果证实该脊髓神经干细胞表现出脊髓而非大脑的身份。结果如图3所示。
5.4脊髓祖细胞在SD大鼠体内存活检测
为了检测细胞在普通大鼠体内脊髓的存活的情况，选择了SD大鼠作为研究的对象。在移植前发明人先前使用AAVSI-Pur-CAG-EGFP质粒，敲入由CAG启动子驱动的EGFP表达盒，使iNSC带有绿色荧光标记。将带有绿色荧光标iNSC诱导分化为脊髓祖细胞。在细胞移植的前三天，对SD大鼠进行了环孢素A免疫抑制剂的注射。注射剂量为10mg/kg/天。移植的细胞类型为诱导分化获得的带有了绿色荧光的脊髓祖细胞，并选取大鼠脊髓T11-T13的位置进行移植。在移植后一周、两周以及两个月，分别对大鼠进行灌注取材，观察细胞在大鼠体内的存活情况。取材后，对移植的细胞进行了HU(鼠抗人核抗体)和GFP荧光染色，方便区别移植细胞和小鼠自体的细胞。HU阳性的细胞显示红色荧光，GFP阳性细胞显示绿色荧光，当细胞同时存在红色和绿色荧光时，即为存活的细胞。发现，细胞在移植一周后还有大量的存活，部分轴突开始延伸。当在细胞移植二周后，对老鼠进行灌注取材时，发现细胞已经出现了大面积死亡，仍然有少量细胞存活。在移植细胞两个月之后，取材发现细胞已经全部死亡。经过对移植两个月的老鼠进行了CD68和TNFα炎性因子的染色，发现了大量TNFα的浸润，初步怀疑脊髓祖细胞的死亡与免疫排斥有关。对于细胞的死亡有两种猜测，因为在移植之前，免疫抑制剂的使用只选择了单一的环孢素A，它只是选择性抑制辅助性T淋巴细胞的增殖及功能，而对B细胞和巨噬细胞来说，并没有有效的抑制，而移植的细胞为干性细胞在生长的过程中受到了炎性细胞的影响而死亡。分析另外一种可能性的根据是来自前期做RT-PCR中HOX基因的结果，表示分化的脊髓细胞以颈段细胞为主，胸段位置的微环境可能不利于细胞的存活。结果如图4所示。
5.5不同时间点的脊髓祖细胞在免疫缺陷小鼠脊髓中的存活迁移
为了确定细胞最佳移植时间点以及获得体内的安全性，再一次选择合适的动物模型进行进一步的细胞移植治疗。在早先的实验中，用了普通的SD大鼠作为移植的对象，结果发现大多数细胞在两月的时候已经死亡。因此选取免疫缺陷小鼠(Noer SCID Beige)进行移植，此种小鼠为T淋巴细胞和B淋巴细胞系统双重缺陷的小鼠，能够满足移植细胞的需要。在本次移植中，总结了前一次移植实验细胞死亡的原因：首先在测序结果表明由iNSC诱导而来的脊髓祖细胞是更偏向颈段脊髓细胞，所以这一次选择了在小鼠颈段脊髓(C6-C7)处和胸腰段脊髓(T12-L1)段同时移植。观察细胞在颈段脊髓和胸腰段脊髓存活迁移情况。本次移植为了选择最佳时间点的细胞，还选取了5个时间点的细胞：未自然分化的脊髓祖细胞(scNPC P1)、自然分化7天(D7)、自然分化10天(D10)、自然分化12天(D12)、自然分化14天(D14)的细胞。在细胞移植两个月后，处死小鼠并灌注取材，发现5个时间点的细胞在所有移植动物中存活，并在移植后2月后生长迁移。scNPC自然分化第7天和第14天的细胞移植入小鼠脊髓体内后，免疫组织化学染色存活的细胞较scNPC自然分化第10天和第12天细胞明显减少。移植脊髓祖细胞(scNPC P1)的小鼠，只在颈部发现了移植的细胞。这说明该细胞确实更适合在颈段脊髓生存。以移植位点为中心，测量了细胞向左右迁移的长度，发现在颈部自然分化12天(D12)的细胞迁移最长，而在胸段脊髓处自然分化10天(D10)的细胞迁移最长。并且细胞无论在脊髓黑质还是白质中都能大面积生长。结果如图5所示。
5.6脊髓祖细胞在免疫缺陷小鼠脊髓中的无致瘤性并且能发育成神经元
对细胞进行了KI67和OCT4染色，所有时间点移植入小鼠脊髓体内的细胞能够定植存活，且KI67，OCT4染色均为阴性，证明无肿瘤组织形成(见图6A)。
与体外数据一致，对移植2个月后小鼠灌注取脊髓组织进行免疫组织化学染色鉴定结果表明，脊髓祖细胞可以在体内分化成为大量TUJ1+的神经元，分化效率达70％；NEUN阳性的成熟神经元在免疫染色中并未观察到，在延伸较长的轴突区域，经免疫荧光染色可以发现NF-L+神经元(见图6C)。对脊髓祖细胞分化亚型部分标记物染色发现有少数出现：OLIG2+、PAX2+、LHX5+(见图6B)。说明本实验中移植的细胞大多数还停留在祖细胞阶段没有进一步分化。在胶质细胞标记物染色中发现了NG2+细胞而对于星形胶质细胞(GFAP)染色，几乎没有。
为了进一步明确分化的神经元与胶质细胞之间或者神经元之间有无形成突触，是否在小鼠体内形成神经元网络。选择轴突延伸较多的NF-L+区域，进行了神经递质Vglut1和Synapsin免疫组织荧光染色，检测有无递质的产生和突触蛋白Synapsin表达，结果发现在移植细胞的周围有Vglut1阳性信号的包裹并且有突触蛋白Synapsin的表达(见图6C)。突触蛋白标记SYN以及GFP阳性共定位，说明分化的细胞之间可能有突触形成。
5.7脊髓祖细胞对ALS SOD1 G93A小鼠的影响
在检测脊髓祖细胞在大鼠体内存活实验的同时，鉴定了脊髓祖细胞对ALS SOD1G93A小鼠发病的影响。本实验的小鼠是从日龄50天进行细胞移植工作，在细胞移植之前，提前三天，对小鼠进行了免疫抑制剂的注射，选择的免疫抑制剂为环孢素A。注射剂量为10mg/kg/天。首先监测了小鼠的体重变化，体重可以反映小鼠的一般健康状况和发病时间点，而且是对小鼠病情发展进程的一个客观评价。本实验的小鼠是在50天接受了细胞移植，因为是有创性手术，会被对小鼠的生命状态发生影响，所以选择小鼠康复了20天，从第70天开始对小鼠进行体重、行为学的检测。每周对小鼠进行1次体重测量，见表2。细胞移植组的小鼠在105天时体重达到最大值，从105天之后体重逐渐开始下降。对照组的小鼠在98天时体重达到了最大值，在98天之后体重开始下降。在记录的较短时间内，对照组小鼠体重约下降了0.9g，而细胞移植组的小鼠体重约下降了0.76g。SOD1小鼠的发病时间约在90天到100天，根据体重连续下降两次可以记为老鼠已经发病。根据表2的小鼠体重下将的天数，可以观察出细胞移植组的小鼠比对照组的小鼠疾病发生延迟了7天。参见表2和图7A。
表2体重的两因素重复测量方差分析结果
表2中，经两因素重复测量方差分析检验，组别和时间的交互作用不显著，F＝2.420，p＝0.119>0.05；组别的主效应不显著，F＝0.155，p＝0.700>0.05；时间的主效应显著，F＝4.655，p＝0.023<0.05，成对比较结果提示，70d时的体重显著地低于84d时的体重。细胞移植组(n＝8)，对照组(n＝7)。
从小鼠出生后的第70天开始，将小鼠先以12rpm的速度练习5天，使小鼠习惯运动并获得一个小鼠的基础运动值，每次转棒实验每只鼠重复3次，每次间隔20分钟，取其最好一次成绩记录，记录坠落的最长潜伏期。以180秒为分界值，超过180秒时按照180秒记录，不足180秒时按实际时间记录，每只实验鼠每周测定1次。潜伏期越长，表明小鼠的运动协调能力、肢体力量以及平衡能力越好。从表3可见，细胞移植组的小鼠从98天开始，小鼠转棒运动的潜伏期逐渐缩短。对照组的小鼠从77天开始，小鼠转棒运动的潜伏期逐渐缩短，到达105天时对照组的小鼠已经有不能完成检测的鼠出现。细胞移植组的小鼠虽然在84天时出现了潜伏期下降的情况，但是在91天时又老鼠运动时间又恢复到了180秒，这可能和前期的手术康复有关，手术的创伤后恢复情况，还是有影响到小鼠的行为学检测。对照组小鼠大约在105天开始出现不能完成转棒运动的小鼠，相比之下从91天到105天，细胞移植组比对照组小鼠转棒运动功能明显下降缓慢，根据体重检测推断的小鼠发病期，细胞移植组小组大约105天发病，而对照组小鼠在98天左右发病。手术的创伤影响了他们行为学的检测，但是从发病点来看，在对照组在98天时发病，到105天时转棒运动的检测是直线下降的。参见表3和图7B。
表3旋转棒的两因素重复测量方差分析结果
表3中，经两因素重复测量方差分析检验，组别和时间的交互作用不显著，F＝1.175，p＝0.399>0.05；组别的主效应不显著，F＝0.622，p＝0.446>0.05；时间的主效应不显著，F＝3.383，p＝0.062>0.05。细胞移植组(n＝8)，对照组(n＝7)。
从小鼠出生后的第91天开始，小鼠的前后肢分别涂上黑色和红色的无毒染料，将小鼠置于一条狭窄的跑道上(长50cm，宽7cm，高10cm)，跑道覆盖一张白纸，记录小鼠前肢后肢行走的步长。从表4、5可见，细胞移植组的小鼠从112天开始，步长减少。对照组的小鼠从105天开始，细胞移植组的小鼠虽然在91天时出现了潜伏期下降的情况，但是在98天时又恢复到了平均值，这可能和前期的手术康复有关，手术的创伤后恢复情况，还是有影响到小鼠的行为学检测。对照组小鼠大约在105天开始出现足印长度减少，相比之下从91天到105天，细胞移植组比对照组小鼠步长没有变化，根据体重检测推断的小鼠发病期，细胞移植组小组大约105天发病，而对照组小鼠在98天左右发病。手术的创伤影响了他们行为学的检测，但是从发病点来看，在对照组在98天时发病，到105天足印长度的检测是直线下降的。测足印长度这项指标中，对照组相比，细胞移植组下降的缓慢且下降时间比对照组晚7天。参见表4、表5和图7C、图7D。
表4前爪步态的两因素重复测量方差分析结果
表4中，经两因素重复测量方差分析检验得，组别和时间的交互作用不显著，F＝2.813，p＝0.078>0.05；组别的主效应不显著，F＝1.207，p＝0.290>0.05；时间的主效应显著，F＝15.463，p＝0.000<0.001，成对比较结果提示，91d的前爪步态显著地低于112d、119d时的前爪步态；98d的前爪步态显著地低于119d时的前爪步态；105d的前爪步态显著地低于119d时的前爪步态；112d的前爪步态显著地低于119d时的前爪步态。细胞移植组(n＝8)，对照组(n＝7)。
表5后爪步态的两因素重复测量方差分析结果
表5中，经两因素重复测量方差分析检验得，组别和时间的交互作用不显著，F＝0.207，p＝0.929>0.05；组别的主效应不显著，F＝1.244，p＝0.284>0.05；时间的主效应显著，F＝22.943，p＝0.000<0.001，成对比较结果提示，91d的后爪步态显著地低于112d、119d时的后爪步态；98d的后爪步态显著地低于119d时的后爪步态；105d的后爪步态显著地低于119d时的后爪步态；112d的后爪步态显著地低于119d时的后爪步态。细胞移植组(n＝8)，对照组(n＝7)。
5.8针对移植结果的分析
根据上述试验结果可以看出，通过添加RA、SB、CHIR、LDN、bFGF、Purmorphamine、DAPT小分子可以将诱导神经干细胞诱导分化为PAX6+、SOX1+、NKX6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、NKX2.2+的脊髓腹侧祖细胞。脊髓祖细胞自然分化后可以得到LHX1+、LHX5+、EVX1/2+、EN1+、Islet1+、CHX10+的V0、V1、V2中间神经元细胞和运动神经元细胞。经基因检测鉴定发现得到的脊髓祖细胞为脊髓颈段祖细胞。将不同时间点的脊髓祖细胞移植到免疫缺陷小鼠(NOD SCID)体内发现细胞存活，在体内分化成为TUJ1+的神经元，分化效率达70％，在延伸较长的轴突区域，可以发现NF-L+神经元。对脊髓祖细胞分化亚型部分标记物染色发现有少数出现：OLIG2+、PAX2+、LHX5+。突触蛋白标记SYN以及细胞自身荧光GFP阳性共定位，说明分化的细胞之间可能有突触形成并且有Vglut1分泌。将细胞移植到SOD1的ALS模型小鼠体内后，发现细胞移植组比对照组体重减轻缓慢，发病时间点推迟，步距变短发生缓慢。这些结果反映了本发明诱导分化的脊髓祖细胞对于肌萎缩侧索硬化症的良好治疗作用。
Claims (12)
- 一种脊髓祖细胞，所述脊髓祖细胞表现为PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、或Nkx2.2+中的至少任意两者，以及表现为PAX7-，并且所述脊髓祖细胞由人诱导神经干细胞分化得到，所述人诱导神经干细胞由人外周血单个核细胞诱导产生。
- 根据权利要求1所述的脊髓祖细胞，其中所述脊髓祖细胞表现为PAX7-和以下中的任一者：(1)Dbx2+和Nkx6.1+；或者(2)Olig2+和Nkx2.2+；或者(3)SOX2+和Nestin+；或者(4)SOX1+、SOX2+、和Nestin+；或者(5)PAX6+、SOX1+、Nkx6.1+、SOX2+、DBX2+、OLIG2+、PAX3+、和Nkx2.2+。
- 根据权利要求1或2中任一项所述的脊髓祖细胞，其中所述脊髓祖细胞自然分化7天后，表现出LHX1+、LHX5+、EVX1/2+、EN1+、Islet1+、CHX10+、以及PAX2+的特征。
- 根据权利要求1或2中任一项所述的脊髓祖细胞，其中所述脊髓祖细胞自然分化40-60天后，表现出TUJ1+、Vglu1+、GAD67+、TH+、以及CHAT+的特征。
- 根据权利要求1或2中任一项所述的脊髓祖细胞，其中所述脊髓祖细胞表达SOX1、SOX2、PAX6、PAX3，但不表达FOXG1和OTX2。
- 根据权利要求5所述的脊髓祖细胞，其中所述脊髓祖细胞高比例表达PAX6、PAX3、SOX2。
- 一种根据权利要求1-6中任一项所述的脊髓祖细胞的诱导分化方法，包括：(1)准备诱导神经干细胞(iNSC)，并悬浮培养，其中所述iNSC由人外周血单个核细胞诱导产生；(2)准备N2B27基础培养基，所述N2B27基础培养基包含DMEM/F12、NeuralBasal-A、N2添加剂、B27添加剂、GlutaMAX、青链霉素、胰岛素；(3)解离所述iNSC，离心，并在第一分化培养基中进行第一阶段的分化培养，所述第一分化培养基为在所述N2B27基础培养基中添加CHIR-99021、SB431542、和RA得到的培养基；(4)在第一阶段的分化培养进行6-8天后，将所述第一分化培养基更换为第二分化培养基进行第二阶段的分化培养，所述第二分化培养基为在所述N2B27基础培养基中添加CHIR99021、SB431542、RA、bFGF、DAPT、LDN、和Purmorphamine得到的培养基；(5)在第二阶段分化培养进行9-12天后获得所述脊髓祖细胞；其中所述N2B27基础培养基的组成为：DMEM/F12:NeuralBasal-A＝1:1、N2添加剂0.5-2X、B27添加剂0.5-2X、GlutaMAX 0.5-2X、青链霉素0.5-2X、胰岛素2.5-10μg/mL。
- 根据权利要求7所述的诱导分化方法，其中所述第一分化培养基中添加的CHIR-99021、SB431542、和RA的终浓度分别为1-5μM、8-12μM、和0.02-0.5μM。
- 根据权利要求7所述的诱导分化方法，其中所述第二分化培养基中添加的CHIR99021、SB431542、RA、bFGF、DAPT、LDN-193189、和Purmorphamine的终浓度分别为1-5μM、8-12μM、0.05-2μM、70-130ng/mL、70-130ng/mL、0.02-0.5μM、和0.1-2μM。
- 根据权利要求7所述的诱导分化方法，其中在获得所述脊髓祖细胞后进行维持培养以进行传代，用于所述维持培养的scNPC维持培养基为在N2B27基础培养基中添加CHIR99021、SB431542、和Purmorphamine得到的培养基。优选地，所述scNPC维持培养基中添加的CHIR99021、SB431542、和Purmorphamine的终浓度分别为1-5μM、1-3μM、和0.1-0.5μM。
- 一种医疗用品，包含根据权利要求1-6中任一项所述的脊髓祖细胞，所述医疗用品为药品或医疗器械。
- 根据权利要求1-6中任一项所述的脊髓祖细胞用于制备治疗肌萎缩侧索硬化症的医疗用品的用途，所述医疗用品为药品或医疗器械。
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