JP2020182485A - Method and pharmaceutical composition for continuously growing motor neuron precursor cell - Google Patents

Abstract

To provide a method for continuously growing a motor neuron precursor cell.SOLUTION: There is provided a method comprising: a step for acquiring motor neuron precursor cells, in which the motor neuron precursor cells are acquired from a vertebrate animal; a step for producing a culture niche, in which the culture niche includes an olfactory nerve sheath cell; a step for culturing on the culture niche, the motor neuron precursor cells, in which the motor neuron precursor cells are continuously grown on the culture niche so that the motor neuron precursor cells have differentiation ability to a matured motor nerve cell and do not differentiate to the matured motor nerve cell, wherein a culture density of the motor neuron precursor cells is 100 cells/mL or greater and the motor neuron precursor cells contact the olfactory nerve.SELECTED DRAWING: None

Description

The present invention relates to a method for maintaining the growth of neural stem cells, particularly to a method for sustainably growing motor neuron progenitor cells, and a pharmaceutical composition.

Pluripotent embryonic stem cells can be used as a material to produce the desired cell community and have been a promising cell replacement therapy to date (Kozubenko Net al., 2010; Mandai M et al., 2010; Boddington). SE et al., 2010). In general, in the differentiation step of embryonic stem cells, nerve inducing factors and region forming factors are provided in order to induce cell differentiation and cell fate conversion, and to induce cell fate conversion in the developmental stage. Includes feeding embryonic singles, whereby embryonic stem cells can reproduce early development in the body of an individual in vitro (Mugruma K et al., 2012; Willerth SM, 2011). For example, by suppressing BMP signals, primordial neuroepithelial progenitor cells (EPCs) can be generated in both single embryos and embryonic stem cells. Spinal motor neurons can be generated by further supplying EPCs with sonic hedgehog (SHH) and retinoic acid (RA). As shown in previous studies, neurons derived from embryonic stem cells resemble normal neurons in the embryo and function in the normal physiology of the neurons, such as the release of neurotransmitters and the development of action potentials. Has. By transplanting neurons derived from embryonic stem cells into disease model animals, their motor ability and behavior can be restored, but the success rate depends on the high activity and high survival rate of the transplanted neurons (Lopez-Gonzelez R). et al., 2009; Harper JM et al., 2004; Chiba S et al., 2003).

Because of the physiological and pathological importance of motor neurons in the spinal cord and brainstem, they have been extensively studied as a special neuron population (Lopez-Gonzelez R et al., 2012; Chipman PH et al., 2012; Jessell TM). et al., 2011; Thonhof JR et al., 2009). During development, specific transcription factors (lineage-specific transcription factors) of multiple motor neurons were discovered (Chipman PH et al., 2012; Wu CY et al., 2012; Takazawa T et al. Compared to Wada T et al., 2009), periventricular and hippocampal neural stem cell populations, there have been few studies on the molecular basis for self-renewal and maintenance of motor neurons. As is clear from the results of studies on recombinant mice, sonic hedgehog and its downstream Gli pathway are important factors in motor neuron progenitor cell growth (Wu SM et al., 2012; Oh S et al., 2009; Ruiz. i Altaba A, 1998), however, it is currently unclear whether the growth of the motor neuron progenitor cell population is linked to other secretory factors. In addition, the low yield and purity of the motor neuron population limits research on motor neuron proliferation. A hybrid cell line of immortalizing motor neurons can be prepared and obtained by fusing spinal cord neurons with mouse N18 neuroblasts (Raimondi A et al., 2006; Cashman NR et al., 1992). Hybrid motor neuron cell lines are multinucleated, have abnormal genes, can proliferate sustainably, and cannot reflect the typical morphology and function of motor neurons, such as those with elongated axons and conduction action potentials.

Olfactory nerve sheath cells (OECs) are glial cells distributed in the olfactory nerve cell fibers, similar to Schwann cells of the peripheral nervous system (Mackay-Sim A et al., 2011; Su Z et al., 2010; Raisman G et al., 2007). Olfactory nerve sheath cells induce olfactory nerve axons to grow through stromal tissue and secrete multiple neurotrophic factors that protect olfactory neurons. To identify olfactory nerve sheath cells, they are recognized by the expression of glial fibrillary acidic proteins (GFAP), s100, p75 and intermediate filament proteins (nestin intermediate filaments). Co-culture of olfactory nerve sheath cells and neural stem cells can promote the differentiation of neural stem cells and the formation of neurites, but cannot promote the growth and replication of neural stem cells.

Stem cell transplantation is expected as an effective treatment method for degenerative neurological diseases and central nervous system lesions. Specifically, stem cell transplantation is the delivery of stem cells to or near the injured central nervous system to regenerate injured central nervous system neurons. As can be seen from previous studies, transplantation of olfactory nerve sheath cells or neural progenitor cells can result in brain damage such as amyotrophic lateral sclerosis (ALS) and spinal cord injury (Mackay-Sim A et al., It is useful for improving the function of experimental rodents suffering from 2011). However, in previous studies, the experimental time was only a few months, and only the effect of temporarily improving the symptoms related to nerve damage was achieved, and the short-term therapeutic effect was highly associated with the low survival rate of transplanted cells. .. Specifically, since the subject causes local inflammation and rejection of the transplanted cells, it is often impossible to provide suitable niches and growth factors for the survival of the transplanted cells, and the transplanted cells cannot replicate. Or when cell activity is reduced, the transplanted cell cannot integrate with the host cell. Therefore, the low survival rate of transplanted cells may lead to unsustainable therapeutic effects.

A main object of the present invention is to culture motor neuron progenitor cells in a niche constructed of olfactory nerve sheath cells so that the motor neuron progenitor cells maintain self-renewal for a long period of time and have the ability to induce differentiation into mature neurons. To provide a method for the sustainable growth of motor neuron progenitor cells, thereby effectively exerting a protective effect on motor neurons.

Another object of the present invention is to provide a pharmaceutical composition comprising an effective amount of motor neuron progenitor cells and at least one pharmaceutically acceptable carrier. By predicting the pharmaceutical composition to a patient suffering from a disease related to motor neuron injury such as stroke, spinal cord injury, neurodegenerative disease, etc., the effect of recovering the nerve function of the subject and promoting the growth of the motor neuron is achieved.

In order to achieve the above object, in one embodiment of the present invention, motor neuron progenitor cells are cultured in a culture niche constructed of olfactory nerve sheath cells and suitable for growth, and the motor neuron progenitor cells self-renew. Provided is a method for sustainably maintaining the activity of motor neuron progenitor cells so as to maintain and have the ability to differentiate into mature motor neurons.

Preferably, the culture niche is a medium containing olfactory nerve sheath cells.

Here, the motor neuron progenitor cells are cultured at a low density.

Here, at least one of the motor neuron progenitor cells is inoculated into the olfactory nerve sheath cells and cultured.

Preferably, the culture niche is a medium that has been pretreated with olfactory nerve sheath cells but does not contain olfactory nerve sheath cells, in which motor neuron progenitor cells are cultured at high density.

According to the method according to the present invention, the motor neuron progenitor cells can be continuously expanded and cultured for 10 generations or more in the culture niche, and one motor neuron progenitor cell can form a cell community.

Another embodiment of the invention discloses a pharmaceutical composition comprising an effective amount of motor neuron progenitor cells and at least one pharmaceutically acceptable carrier. The motor neuron progenitor cells are pretreated in a niche constructed of olfactory nerve sheath cells.

Preferably, the motor neuron progenitor cells are pretreated with olfactory nerve sheath cells.

Preferably, the motor neuron progenitor cells are pretreated with a medium obtained by previously treating the olfactory nerve sheath cells.

Preferably, the pharmaceutical composition further comprises olfactory nerve sheath cells.

Preferably, the motor neuron progenitor cells and the olfactory nerve sheath cells are pretreated in equal proportions.

The pharmaceutical composition according to the present invention has an application for treating a motor neuron disease.

Preferably, the disease is stroke, spinal cord injury, degenerative neurological disease, amyotrophic lateral sclerosis or any disease in which motor neurons die.

The beneficial effect of the present invention is that the method and pharmaceutical composition provided in the present invention for the continuous growth of motor neuron progenitors have the ability of motor neuron progenitors to maintain self-renewal for a long period of time and to induce differentiation into mature neurons. It is intended to effectively achieve a protective effect on motor neurons, repair or regenerate a target nerve injury site, and effectively treat a motor neuron disease.

It is the outline of the 4th day when the olfactory nerve sheath cells were cultured. It is the outline of the 7th day when the olfactory nerve sheath cells were cultured. It is the result of each immune cell staining of the olfactory nerve sheath cell of the olfactory bulb, the olfactory nerve sheath cell of the olfactory mucosa, and the fibroblast of the offspring hamster. The olfactory nerve sheath cells stained with the p75 antibody are analyzed by the fluorescence activated cell selection technique, where red indicates the olfactory nerve sheath cells stained with the p75 antibody and blue indicates the unstained cells. It is a growth curve diagram of an olfactory nerve sheath cell. It is a schematic diagram of the co-culture process of HB9 :: GFP embryonic stem cell and mouse osteoblast line PA6. Day 8 HB9 :: GFP ⁺ cells co-cultured with a mouse osteoblast line. It is a schematic diagram of the process of culturing HB9 :: GFP embryonic stem cells under different treatment conditions. It is the result of a cell community formed by inoculating one HB9 :: GFP ⁺ cell into an olfactory nerve sheath cell and culturing it for one week. This is the result after inoculating one HB9 :: GFP ⁺ cell into olfactory nerve sheath cells treated with mitomycin C and culturing for one week. This is the result after inoculating PA6 cells with HB9 :: GFP ⁺ cells proliferated in olfactory nerve sheath cells and culturing them. It is the analysis result of the CD11b expression of each group rat by the immunostaining method. It is the statistical analysis result of the expression of CD11b of each group rat, and here, * indicates P <0.05, ** indicates P <0.01. It is the analysis result of choline acetyltransferase expression of motor neurons of each group rat by immunostaining method. It is the result of statistical analysis of choline acetyltransferase expression in motor neurons of each group rat, where * indicates P <0.05 and ** indicates P <0.01.

Unless otherwise noted, the meanings of the technical and scientific terms used in the specification and claims of the invention are similar to those of ordinary skill in the art. In case of contradiction, the content of the present invention is used as a reference.

"HB9 :: GFP embryonic stem cell" is an undifferentiated and non-fluorescent embryonic stem cell, and has a foreign gene having an HB9 gene promoter and a green fluorescent protein (GFP) on its chromosome.

"HB9 :: GFP ⁺ cells" are motor neuron progenitor cells or mature motor neurons derived from embryonic stem cells and have green fluorescence. Expression of the green fluorescent protein is regulated by the motor neuron-specific promoter HB9 (Miles GB et al., 2004; Witchtale Het al., 2002). HB9 is a specific transcription factor for motor neurons (Arber S et al., 1999) and therefore expression of green fluorescent protein can only be detected in motor neurons (Miles GB et al., 2004; Soundararan P et al., 2006). In the examples of the present invention, for HB9 :: GFP ⁺ cells, the effect of olfactory nerve sheath cells on the proliferation efficiency of motor neurons is examined, and the proliferation ability of motor neurons co-cultured with olfactory nerve sheath cells is quantified.

"Effective amount" means the amount of a compound or active ingredient required to achieve the expected specific effect and is expressed as a weight ratio to the composition. Those skilled in the art of the present invention should understand that the effective amount depends on the method of investment for achieving a specific effect. Generally, the amount of the active ingredient or compound in the composition is from about 1% to about 100%, preferably about 30% to about 100% by weight of the composition.

"Pharmaceutically acceptable carrier" means any carrier that can be used in a pharmaceutical product, which carrier may be solid, semi-solid or liquid, depending on the form of the composition. By way of example, carriers include, but are not limited to, gelatin, emulsifiers, hydrocarbon mixtures, water, glycerin, saline, buffered saline, lanolin, paraffin, beeswax, cymethicone, ethanol.

A "pharmaceutical composition" comprises an effective amount of a compound or active ingredient necessary to achieve a particular effect, and at least one carrier. Those skilled in the art of the present invention should understand that the dosage form of the composition differs depending on the method of investment for achieving a specific effect, and examples thereof include tablets, powders, injections, and the like. , The carrier is solid, semi-solid or liquid depending on the dosage form of the composition.

"Injection" means a means of delivering a drug to a specific site, a specific cell, a specific target of a subject, or a method of contacting the subject to act, and in general, the injection method is oral administration, It includes, but is not limited to, application administration, spray administration, inhalation administration, injection administration, and the like.

Hereinafter, in order to explain the efficacy of the present invention in detail, some examples will be described in detail, but the examples are merely examples for explaining, and any term used is the specification of the present invention. And does not limit the scope or meaning of the claims.

In the following, all examples related to animal testing will pass the examination of the logic committee of Taichung Veterans General Hospital, Taiwan. Unless otherwise noted, all basal media and additive components for culturing and differentiating stem cells in the following examples are commercially available products (Invitrogen).

Example 1: Maintenance and differentiation of embryonic stem cells HB9 recombinant embryonic stem cells (hereinafter abbreviated as HB9 :: GFP embryonic stem cells) isolated from HB9 :: GFP recombinant mice from Columbia University in the United States. Obtained, the cells can be differentiated into motor neuron progenitor cells and mature motor neurons (hereinafter abbreviated as HB9 :: GFP ⁺ cells).

The HB9 :: GFP embryonic stem cells were kept in high glucose DMEM medium containing mouse embryonic fibroblasts treated with mitomycin C, 15% bovine fetal serum, 2 mM glutamine, 0.1 mM non-. Essential amino acids, 1 mM pyruvate, 0.1 mM 2-mercaptoethanol (2-mercaptoethanol, Sigma-Aldrich) and 1000 U / ml leukemia inhibitor (Chemicon) are added.

The detailed method of neural differentiation is known to those skilled in the art based on the contents of the prior art, and is serum-free embryoid-body-like (SFEB) (Watanabe K et al.). , 2005), neurobasal / N2B27 medium (Ying QL et al., 2003) and stromal cell-derived inducing activity methods (stroma cell-developed inducing activity methods, SDIA methods) (Kawataka.

The first day when embryonic stem cell differentiation begins is day 0, and on days 3-5, 0.1 μM retinoic acid (Sigma-Aldrich) is added to the differentiation medium, and 200 μM is added to each of 5-7 days of culturing. Substitute with exogenous sonic hedgehog (R & D Systems) or 2 μM 2,6,9-trisubstituted purine compounds (Purmorphamine, PU, Tocris).

Example 2: Culture and purification of olfactory nerve sheath cells Approximately 250 to 300 grams of SD rats (Sprague-Dawley Rat) are used to isolate olfactory nerve sheath cells from the olfactory mucosa (OM) or olfactory bulb of the rats. did. The olfactory nerve sheath cells were continuously cultured in a selective medium, and the cell outlines on days 4 and 7 were observed under a microscope, and the results are shown in FIG. As is clear from FIG. 1, the olfactory nerve sheath cells are typical spindle-shaped.

Next, olfactory nerve sheath cells and fibroblasts of offspring hamsters (baby hamster kidney fibroblast cells, BHK-21 cells) separated from the olfactory mucosa and the olfactory bulb were analyzed by immunocellular staining. Each of the above cells was fixed with 4% paraformal dye, penetrated with 0.3% Triton (Triton-X 100), and then immunoreactive with S100 and p75 primary antibodies. It was then washed with 0.1% Tween-20 phosphate buffer, further reacted with a suitable secondary antibody with a fluorescent label, subjected to nuclear reaction staining with DAPI, and finally with an upright microscope (Nikon ECLIPSE 80I). Alternatively, the immunostaining result is observed with a confocal microscope (LSM510 Meta, Zeiss), and the result is shown in FIG. Here, in the figure, the upper red color is the result of staining with the p75 antibody, the lower red color in the figure is the result of immunostaining with S100, and the blue color in the figure is the result of DAPI staining.

The olfactory nerve sheath cells are stained with p75 antibody and analyzed by a fluorescence activated cell selection technique (Flow cytosequence Activated Cell Sorter, FACS), and then dead cells are removed with trypan blue to remove the olfactory nerve. The number of sheath cells was analyzed, the growth curve was recorded, and the results are shown in FIGS. 3A and 3B.

As is clear from the results of FIG. 2, almost all cultured olfactory nerve sheath cells show olfactory nerve sheath cell markers such as p75 and s100. In contrast, offspring hamster fibroblasts do not show p75 and s100 antigens. It is shown that this culture method can generate high-purity olfactory nerve sheath cells. In addition, as is clear from the results of FIG. 3A, most of the olfactory nerve sheath cells can be stained with the p75 antibody (red part in the figure) as compared with the unstained olfactory nerve sheath cells (blue part in the figure). Furthermore, the olfactory nerve sheath cells were able to proliferate, and the time for doubling the number of cells (a double time) was about 28 to 32 hours.

Example 3: Co-culture of mouse osteoblastic cell line and motor neurons derived from embryonic stem cells As shown in FIG. 4, mouse-derived HB9 :: GFP embryonic stem cell and mouse osteoblastic cell line in Example 1 ( PA6 cells) are co-cultured by the following culture process disclosed in the prior literature (Pan HC et al., 2011). Contains 10% KSR (Knockout serum replacement) medium on days 0-3, KSR medium containing retinoic acid on days 3-5, retinoic acid and 2,6,9-3 substituted purine compounds on days 5-7. It was co-cultured using NB medium (neurobasal medium, Invitrogen). Next, the cells were cultured in NB medium, and the HB9 :: GFP ⁺ cells on the 8th day were observed and shown in FIG.

As is clear from the results of FIG. 5, the HB9 :: GFP ⁺ cells co-cultured with the mouse osteoblast line PA6 can differentiate into motor neurons and express green fluorescent protein, which is typical of motor neurons. It has an outer shape. In addition, as can be seen from previous studies by the inventors of the present application, motor neurons derived from mouse embryonic stem cells can express choline acetyltransferase and motor neuron specific protein MNR2 (Pan HC et al., 2011). It was revealed that HB9 :: GFP embryonic stem cells differentiate into functional mature motor neurons after co-culturing with mouse osteoblast line PA6.

Example 4: Maintenance of HB9 :: GFP ⁺ cell self-renewal ability by olfactory nerve sheath cells As shown in FIG. 6, first, HB9 :: GFP embryonic stem cells by the method disclosed in Example 1 or Example 3. Was cultured for 5 days. At this time, green fluorescence begins to be reflected in differentiated embryonic stem cells, and the cells exhibiting green fluorescence are oval and have no neurites. Therefore, the green fluorescent cells at this stage are motor neuron progenitor cells and are cultured for 8 days. Different from mature motor neurons.

On day 5 of culture, single HB9 :: GFP ⁺ cells exhibiting green fluorescence were screened using a flow cytometer (Influx, nozzle 100 m, 25 psi, Becton-Dickinson). Next, cell culture was performed under a low density condition of 100 cells / mL, and a single HB9 :: GFP ⁺ cell was inoculated into olfactory nerve sheath cells, PA6 cells and Matrigel, respectively, and cultured for 1 week.

After culturing for 1 week, a single HB9 :: GFP ⁺ cell can form a colony only with olfactory nerve sheath cells as shown in FIG. 7, and the cells formed by the HB9 :: GFP ⁺ cell. The cell community can express green fluorescent protein in olfactory nerve sheath cells for more than 10 generations, whereas the HB9 :: GFP ⁺ cells are uniformly inoculated into PA6 cells or Matrigel. It was found that although it could be distributed and differentiated into mature motor nerve cells, it could not self-replicate and grow.

In addition, a single selected HB9 :: GFP ⁺ cell was inoculated into olfactory nerve sheath cells treated with mitomycin C to impair their ability to replicate. After culturing for 1 week, the results are as shown in FIG. As can be seen from the results of FIG. 8, olfactory ensheathing cells can not cell replication reduces the growth efficiency of the HB9 :: GFP ⁺ cells, HB9 :: GFP ⁺ cells can only passaged five generations following.

In addition, a single selected HB9 :: GFP ⁺ cell was cultured under a high density condition of 10000 cells / mL and cultured in the olfactory nerve sheath cells for 1 day without contact with the olfactory nerve sheath cells. It was co-cultured with a medium (conditional media). The culture solution was changed every 2 days, and the cells were cultured for 2 weeks. From this culture result, it was found that a single HB9 :: GFP ⁺ cell can form a cell community, replicate, and continue to subculture without contact with olfactory nerve sheath cells. ..

As shown in the above results, specific niches capable of retaining self-renewal of motor neuron progenitor cells by co-culturing with healthy olfactory nerve sheath cells or by culturing the olfactory nerve sheath cells in a cultured conditioned medium. ) Is an important niche factor for retaining HB9 :: GFP ⁺ cells.

Example 5: Differentiation ability of motor neurons
Single fifth generation HB9 :: GFP ⁺ cells obtained by co-culturing with olfactory nerve sheath cells were selected using a flow cytometer (Influx, nozzle 100 μm, 25 psi, Becton-Dickinson). When the selected HB9 :: GFP ⁺ cells were inoculated into PA6 cells, cultured for 3 days, and then observed, most of the cells rapidly extended axons and matured motor neurons as shown in FIG. And show a typical morphology as a differentiated motor neuron. As shown in FIG. 9, HB9 :: GFP ⁺ cells proliferating in olfactory nerve sheath cells still maintain their differentiation ability and can differentiate into mature motor neurons.

Example 6: Creation of Spinal Cord Injury Animal Model In this example, a method for creating a spinal cord injury animal model is described in the prior art (Cheng FC, et al., 2012; Cheng FC et al., 2010; Yang DY et al., 2012) may be referred to.

Using 250-300 grams of SD rats, anesthesia was introduced with 4% isoflurane and then maintained anesthetized with 1-2% isoflurane. It contacted the right brachial plexus through a horizontal incision from the sternum parallel to the clavicle to the axilla. The pectoralis major muscle was removed to obtain a complete head vein. The blood vessels under the clavicle were fixed and the lower torso was dissected. After removing the C7 nerve root from the spinal cord for 5 minutes using forceps, the wound was sutured and used for preparation of a spinal cord injured rat.

Example 7: Animal test The spinal cord injured cells prepared in Example 6 are divided into 4 groups, and 2 weeks after the spinal cord injury, complete vertebral resection is performed on each of the rats with spinal cord T7-T8 to cause injury. Cell transplantation under different conditions was performed at the ventral horn and the contralateral healthy site, of which 2 μl phosphate buffer was used in the first group and 5x10 ⁵ cells in the second group. The olfactory nerve sheath cells were transplanted, and in the third group, 5x10 ⁵ HB9 :: GFP ⁺ cells were transplanted, and in the fourth group, 2.5x10 and 2.5x10 using ⁵ transplanted olfactory nerve sheath cells. ^Five HB9 :: GFP ⁺ cells are pretreated for one day to replicate HB9 :: GFP ⁺ cells and then transplant.

Microinjection is used as a means of cell transplantation. Microinjection on the right side of the spinal column injects cells into the white matter 0.75 mm from the midline of the spine T8 and T9 and 1.2 mm deep over 20 minutes and holds for 5 minutes after the injection is complete. Microinjection on the left side of the spinal column then injects the cells 0.5 mm from the midline of the spine T8 and T9 and 1.2 mm deep into the anterior horn over 20 minutes, after the injection is complete. It was held for 5 minutes. One week after transplantation into each group of rats, each group of rats was anesthetized, perfused with 25 ml of phosphate buffer and 100 ml of 4% paraformaldehyde, and the spinal cord was removed for immunohistochemical staining. Then, the expression of CD11b and choline acetyltransferase was observed, and the results are shown in FIGS. 10A, 10B and 11A, 11B.

Since microglial cells of spinal cord injured rats are overactivated and cause nerve cell inflammation, microglial cells express a large amount of CD11b and motor neurons are destroyed. As is clear from the results of FIGS. 10A, 10B and 11A, 11B, the rats in the first group had much higher CD11b expression levels than the other groups, and choline acetyltransferase was used to generate motor neurons in the anterior horn of the spinal cord. As a result of calibration, it was shown that the motor neurons of the first group of rats were seriously damaged. Compared to group 1 rats, group 4 rats had significantly reduced CD11b expression and were able to detect a large number of motor neurons expressing choline acetyltransferase, so that most of their endogenous motor neurons were not damaged. I showed that. In addition, as is clear by comparing the rats in the 4th group with the rats in the 2nd group or the 3rd group, the motor neurons and the olfactory nerve sheath are compared with the case where the motor neurons or the olfactory nerve sheath cells are transplanted alone. Transplantation of both cells has a synergistic repair effect on the host motor neurons.

As is clear from the results of the above examples, the method of sustainably maintaining the activity of the motor neuron progenitor cells according to the present invention is such that the motor neuron progenitor cells self-replicate in a niche in which healthy olfactory nerve sheath cells are present. It enables the maintenance of ability and provides motor neurons with a better protective effect than the prior art. Furthermore, the motor neuron progenitor cells obtained in the culture process can differentiate into mature motor neurons even under differentiation conditions, and contribute to the growth of motor neurons and the recovery of neural function. According to the above method, the pharmaceutical composition and treatment method provided in the present invention transplant a motor neuron progenitor cell having self-renewal ability and differentiation ability into a subject, first replicate the motor neuron progenitor cell, and then By repairing or regenerating the target nerve damage site, the effect of effectively treating motor neuron disease is realized.

Although the present invention has been described in detail with reference to the above embodiments, any simple modifications or changes made to the examples of the specification by those skilled in the art without deviating from the spirit of the present invention are claimed in the present invention. Should be included.

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Claims (9)

A method for the continuous growth of motor neuron progenitor cells in vitro.
A step of acquiring a motor neuron progenitor cell, which is a step a obtained by the motor neuron progenitor cell from a vertebrate,
A step of preparing a culture niche, wherein the culture niche includes step b containing olfactory nerve sheath cells, and
A step of culturing the motor neuron progenitor cells in the culture niche, in which the motor neuron progenitor cells are continuously proliferated in the culture niche and have the ability to differentiate into mature motor neurons, and are mature motor neurons. A method including step c in which the culture density of the motor neuron progenitor cells is 100 cells / mL or more and the cells come into contact with the olfactory nerve sheath cells. The method for continuously growing motor neuron progenitor cells in vitro according to claim 1, wherein in step c, at least one motor neuron progenitor cell is cultured in an olfactory nerve sheath cell. The motor neuron progenitor cell is a cell obtained by inducing differentiation of a pluripotent stem cell, and is characterized in that the cell morphology is oval and there is no neurite. A method of sustainably growing motor neuron progenitor cells in vitro as described. A method for the continuous growth of motor neuron progenitor cells in vitro.
A step of acquiring a motor neuron progenitor cell, which is a step a obtained by the motor neuron progenitor cell from a vertebrate,
A step of preparing a culture niche, wherein the culture niche is a culture niche used for culturing olfactory nerve sheath cells, and the culture niche does not contain olfactory nerve sheath cells.
A step of culturing the motor neuron progenitor cells in the culture niche, in which the motor neuron progenitor cells are continuously proliferated in the culture niche and have the ability to differentiate into mature motor neurons, and are mature motor neurons. A method including step c in which the culture density of the motor neuron progenitor cells is 100 cells / mL or more and the cells come into contact with the olfactory nerve sheath cells. The motor neuron progenitor cell is a cell obtained by inducing differentiation of a pluripotent stem cell, and is characterized in that the cell morphology is oval and there is no neurite. A method of sustainably growing motor neuron progenitor cells in vitro as described. A pharmaceutical composition comprising an effective amount of motor neuron progenitor cells and at least one pharmaceutically acceptable carrier.
A pharmaceutical composition, wherein the motor neuron progenitor cell is a motor neuron progenitor cell cultured by the method of claim 1 or 4. The pharmaceutical composition according to claim 6, further containing olfactory nerve sheath cells. Use in the manufacture of a drug for treating a motor neuron disease of the pharmaceutical composition according to claim 6. 8. Claim 8 is characterized in that the disease is selected from the group consisting of stroke, spinal cord injury, degenerative neurological disease, amyotrophic lateral sclerosis, and a disease symptomatic of death of motor neurons. Described use.