JP2008119003A - Method for inducing differentiation from mesodermal stem cell, embryonic stem cell or immortalized mesodermal stem cell to nervous system cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inducing differentiation from mesodermal stem cells prepared from mononuclear cell fraction isolated from bone marrow fluid or umbilical blood, or embryonic stem (ES) cells to nerve cells or glial cells. <P>SOLUTION: This method for inducing the differentiation from the mesodermal stem cells or ES cells to the nerve cells or glial cells is provided by adding ischemic brain extract liquid to a basal culturing medium on culturing. The neural cells obtained by the above method has a neural regeneration potency in a brain infarction model, dementia model, spinal cord injury model and demyelination model. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for inducing differentiation from mesoderm stem cells or ES cells to neural cells, a method for inducing differentiation from immortalized mesoderm stem cells to neural cells, and use thereof.

  Oligodendrocytes (Archer, DR. Et al., Exp Neurol, 1994, 125, 268-77.), Blakemore, WF. And Crang, AJ., Dev Neurosci , 1988, 10, 1-11. (Non-patent document 2), Gumpel, M. et al., Ann New York Acad Sci, 1987, 495, 71-85. (Non-patent document 3)), or Schwann cells ( Blakemore, WF., Nature, 1977, 266, 68-9. (Non-Patent Document 4), Blakemore, WF. And Crang, AJ., Dev Neurosci, 1988, 10, 1-11. Honmou, O. et al., J Neurosci, 1996, 16, 3199-208. (Non-patent document 5)) or olfactory ensheating cells (Franklin, RJ. Et al., Glia, 1996, 17, 217-24. (Non-patent document 6), Imaizumi, T. et al., J Neurosci, 1998, 18 (16), 6176-6185. (Non-patent document 7), Kato, T. et al., When transplanted with myelinating cells such as Glia, 2000, 30, 209-218. (Non-patent Document 8)), remyelination occurs in animal models. And can restore electrophysiological function (Utzschneider, DA. Et al., Proc Natl Acad Sci USA, 1994, 91, 53-7. (Non-patent Document 9), Honmou, O. et al., J Neurosci, 1996, 16, 3199-208 (Non-patent Document 5)). Although it is not impossible to prepare such cells from patients or others and use them in cell therapy, there are many problems because tissue material must be collected from the brain or nerves.

  Neural progenitor cells or stem cells obtained from the brain are self-replicating and are known to differentiate into neurons and glial cells of various cell lineages (Gage, FH. Et al., Proc Natl Acad Sci USA, 1995, 92, 11879-83. (Non-Patent Document 10), Lois, C. and Alvarez-Buylla, A., Proc Natl Acad Sci USA, 1993, 90, 2074-7. Morshead, CM. Et al., Neuron, 1994, 13, 1071-82. (Non-patent Document 12), Reynolds, BA. And Weiss, S., Science, 1992, 255, 1707-10. )). When human neural stem cells collected from fetal tissues are transplanted into the brains of newborn mice, they differentiate into neurons and astrocytes (Chalmers-Redman, RM. Et al., Neurosci, 1997, 76, 1121-8. Non-Patent Document 14), Moyer, MP. Et al., Transplant Proc, 1997, 29, 2040-1. (Non-Patent Document 15), Svendsen, CN. Et al., Exp Neurol, 1997, 148, 135-46 (Non-patent document 16)), and can be remyelinated (Flax, JD. Et al., Nat Biotechnol, 1998, 16, 1033-9. (Non-patent document 17)). Transplantation of adult brain-derived neural progenitor cells into a demyelinated rodent spinal cord has been reported to remyelinate and restore impulse conduction (Akiyama, Y. et al. , Exp Neurol, 2001. (Non-patent document 18)).

  These studies are of great interest as they suggest that the cells may be used for neurological disease repair. (Akiyama, Y. et al., Exp Neurol, 2001. (Non-patent document 18), Chalmers-Redman, RM. Et al., Neurosci, 1997, 76, 1121-8. (Non-patent document 14), Moyer, MP. Et al., Transplant Proc, 1997, 29, 2040-1. (Non-patent document 15), Svendsen, CN. Et al., Exp Neurol, 1997, 148, 135-46. (Non-patent document 16), Yandava, BD. Et al., Proc Natl Acad Sci USA, 1999, 96, 7029-34.

  Already, the present inventors have extracted and cultured human adult-derived nerve cells from the brain, converted them into cell lines, and examined their functions, and found that these nerve cells have a self-replicating function and a multi-differentiation function. (Akiyama, Y. et al., Exp Neurol, 2001. (Non-patent Document 18)). That is, when a progenitor cell obtained from the brain is subjected to single cell expansion and subjected to clonal analysis in an in vitro system, self-replication ability (ie, , Proliferation ability) and pluripotency [i.e., differentiation into neurons (neuron), astrocytes (astrocytes), oligodendrocytes (oligodendrocytes)), These cells were confirmed to have neural stem cell properties.

  Actually, when transplantation experiments were performed on these cells using a rat ischemia model and a trauma model, very good engraftment rate, migration and differentiation were shown. In addition, when the cells were transplanted into a spinal cord demyelination model rat, it was found that a functional myelin sheath was formed. That is, in the spinal cord demyelination model rat, the demyelinated nerve axon is remyelinated and the nerve function is restored, and the effect of the transplantation treatment method of the cell is histologically, electrophysiologically, Confirmed by behavioral science.

  Judging from the above facts, collecting a small amount of neural tissue from the own cerebrum, extracting and culturing neural stem cells, and transplanting the obtained neural stem cells to the injury site of the spinal cord is an autologous transplantation. As a therapy, it is considered to be a highly applicable therapy.

  However, it is not easy to collect tissue containing neural stem cells from the cerebrum, even though neurological symptoms do not occur during collection. Therefore, it is extremely important to establish a safer and simpler autotransplantation therapy from the viewpoint of establishing a treatment for today's complex diseases. In contrast, the present inventors have developed a technique for collecting a mononuclear cell fraction from bone marrow cells, umbilical cord blood cells, or fetal liver cells, which is easier than a technique for collecting neural stem cells, in order to obtain donor cells. Developed (Japanese Patent Application 2001-160579). That is, the present inventors have found that the mononuclear cell fraction prepared from bone marrow cells has the ability to differentiate into nervous system cells. Furthermore, the present inventors have developed a cell fraction containing mesodermal stem cells isolated from the mononuclear cell fraction, a cell fraction containing stromal cells, and a cell fraction containing AC133 positive cells into neural cells. It was found to have differentiation potential.

Archer, DR. Et al., Exp Neurol, 1994, 125, 268-77. Blakemore, WF. And Crang, AJ., Dev Neurosci, 1988, 10, 1-11. Gumpel, M. et al., Ann New York Acad Sci, 1987, 495, 71-85. Blakemore, WF., Nature, 1977, 266, 68-9. Honmou, O. et al., J Neurosci, 1996, 16, 3199-208. Franklin, RJ. Et al., Glia, 1996, 17, 217-24. Imaizumi, T. et al., J Neurosci, 1998, 18 (16), 6176-6185. Kato, T. et al., Glia, 2000, 30, 209-218. Utzschneider, DA. Et al., Proc Natl Acad Sci USA, 1994, 91, 53-7. Gage, FH. Et al., Proc Natl Acad Sci USA, 1995, 92, 11879-83. Lois, C. and Alvarez-Buylla, A., Proc Natl Acad Sci USA, 1993, 90, 2074-7. Morshead, CM. Et al., Neuron, 1994, 13, 1071-82. Reynolds, BA. And Weiss, S., Science, 1992, 255, 1707-10. Chalmers-Redman, RM. Et al., Neurosci, 1997, 76, 1121-8. Moyer, MP. Et al., Transplant Proc, 1997, 29, 2040-1. Svendsen, CN. Et al., Exp Neurol, 1997, 148, 135-46. Flax, JD. Et al., Nat Biotechnol, 1998, 16, 1033-9. Akiyama, Y. et al., Exp Neurol, 2001. Yandava, BD. Et al., Proc Natl Acad Sci USA, 1999, 96, 7029-34.

  The present invention has been made in view of such a situation, and the object thereof is a method for inducing differentiation from a mesoderm stem cell or ES cell to a nervous system cell, and a sufficient amount of cells by proliferating the mesoderm stem cell. A method for acquiring and efficiently inducing differentiation into neural stem cells and neural cells, a neural cell obtained by the method, a composition for treatment of a nervous system disease containing the neural cell, and the composition It is to provide a method for treating a nervous system disease.

  The present inventors have newly found that mesoderm stem cells or ES cells contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood induce differentiation into neural cells in vitro. .

Specifically, mesoderm stem cells or ES cells prepared from a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood, culture medium 1 (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC 1%, bFGF (Basib fibroblast growth factor) 10 ng / ml added daily, EGF (Epidermal growth factor) 10 ng / ml added daily, or culture solution 2 (NPBM (Neural Progenitor Basal Medium), 2% Neural survival factors (Clonetics), 0.2% hEGF (human epidermal growth factor), 0.2% Gentamicine-amphotericinB, 0.2% hFGF (human fibroblast growth factor), bFGF 10 ng / ml added daily, EGF 10 ng / ml added daily) In Fig. 5, it was found that the mesoderm stem cells or the ES cells were induced to differentiate into neural stem cells when cultured at 37 ° C in 5% CO ₂ in a floating state.

  In addition, mesoderm stem cells or ES cells contained in a mononuclear cell fraction separated from bone marrow fluid or umbilical cord blood are cultured in culture solution 1 (DMEM 50%, F-12 50%, FSC 1%) or culture solution 2. (NPBM (Neural progenitor cell basal medium: Clonetics), 2% Neural survival factors (Clonetics), 0.2% hEGF (human Epidermal growth factor), 0.2% Gentamicine-amphotericinB, 0.2% hFGF (human fibroblast growth factor)) Thus, it was found that the mesoderm stem cells or the ES cells induce differentiation into neurons or glial cells.

  Furthermore, it has been found that the induction of differentiation from mesoderm stem cells or ES cells to neural cells is promoted by adding an ischemic brain extract to the culture medium.

  The nerve regeneration ability of the nervous system cells obtained by the above differentiation induction method was examined in cerebral infarction model, dementia model, spinal cord injury model, demyelination model, and neural stem cells extracted and cultured from the brain. It turns out that it has the same reproducibility.

  From the above results, it became possible to use neural cells obtained by the above differentiation induction method for the treatment of cerebral infarction, dementia, spinal cord injury, demyelinating disease and the like. In addition, the present invention is more general and is considered to be applicable to nerve transplantation / regeneration therapy for wide-area cranial nerve injury. That is, it can be applied to autotransplantation therapy for ischemic cranial nerve injury, traumatic cranial nerve injury, cranial nerve degenerative disease, and metabolic neurological disease of the central nervous system and peripheral nervous system.

  Furthermore, the differentiation induction method provides a clue to unravel the mechanism of differentiation from mesodermal stem cells or ES cells to nervous system cells. If genes that regulate such differentiation are identified and analyzed, mesoderm stem cells and ES cells can be efficiently and sufficiently transformed into neural cells using these genes. Therefore, it is highly expected that “gene therapy” for promoting regeneration of nerve tissue will be possible.

  Furthermore, the present inventors have succeeded in developing a method for inducing differentiation of the nervous system capable of stably proliferating cells in a large amount. Usually, mesodermal stem cells are useful in nerve regeneration medicine, but their growth under culture conditions is limited to some extent. However, as a result of research by the present inventors, when a viral vector incorporating an immortalizing gene such as telomerase is introduced into stromal cells in vitro, cell proliferation continues even after repeated cell division, resulting in a dramatic increase in cell life. The cell shape was found to be the same as that of the normal cell, despite the fact that the cell was elongated. Then, the present inventors have focused on the fact that the cells can be stably and proliferated by introducing an immortalizing gene into mesoderm stem cells and immortalizing the cells. As a result of further research, it was found that mesoderm stem cells immortalized by introducing an immortalizing gene can be efficiently induced to differentiate into neural stem cells and nervous system cells by culturing under appropriate conditions. .

  Specifically, the mesoderm stem cells immortalized by introducing the immortalization gene hTERT gene into mesoderm stem cells were successfully induced to differentiate into adipocytes, chondroblasts, osteoblasts, and the like. Furthermore, mesoderm stem cells immortalized by hTERT gene transfer were induced to differentiate into neural cells containing neural stem cells at a high rate under culture conditions, and these cells (neural stem cells induced to differentiate from mesoderm stem cells themselves or mesoderm stem cells) It was clarified that the demyelination site of the spinal cord was repaired by transplanting the neural cells differentiated from mesoderm stem cells and the neural cells induced to differentiate from mesoderm stem cells.

In addition, neural stem cells and nervous system cells obtained by differentiating mesoderm stem cells introduced with an immortalizing gene by the above-described method of the present invention are expected to be very useful for nerve regeneration.
When an oncogene or the like is introduced to immortalize the cell, transformation of the cell itself occurs. On the other hand, when the cell is immortalized by introducing an immortalizing gene in the present invention, In addition, the introduction of an immortalized gene allows the gene to be removed after sufficient growth has been obtained.
As described above, the present inventors have developed an efficient new method for inducing differentiation from mesoderm stem cells to neural cells and completed the present invention.

That is, the present invention provides a method for inducing differentiation from mesoderm stem cells or ES cells to neural cells, a method for immortalizing mesoderm stem cells, and further efficiently inducing differentiation into neural cells including neural stem cells, The obtained nervous system cell, a composition for the treatment of a nervous system disease containing the nervous system cell, and a method for treating a nervous system disease using the composition, more specifically,
[1] Mesodermal stem cells or ES cells contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from vertebrates are cultured in a basic culture solution at a temperature of 33 ° C to 38 ° C. A method of inducing into neural cells,
[2] A method for inducing mesoderm stem cells into neural cells, comprising the following steps (a) and (b):
(A) providing an immortalized mesoderm stem cell by highly expressing or activating the immortalizing gene;
(B) culturing the immortalized mesoderm stem cells of step (a) in a basic culture medium, thereby inducing the mesoderm stem cells into nervous system cells,
[3] The method according to [2], wherein the immortalizing gene is highly expressed or activated by introducing the immortalizing gene into a mesoderm stem cell.
[4] The method according to [3], wherein the mesoderm stem cell is immortalized by introducing the immortalization gene into the mesoderm stem cell using a retroviral vector,
[5] The method according to [4], wherein the retroviral vector is pBabe,
[6] The method according to any one of [2] to [5], wherein an immortalized gene can be removed from a nervous system cell by gene removal treatment, or
[7] The method according to [6], wherein the gene removal treatment of the immortalized gene is performed by sandwiching the immortalized gene into a loxP sequence or a loxP-like sequence and then treating with recombinase,
[8] The method according to any one of [2] to [7], wherein the immortalizing gene is any of a telomerase gene, a gene derived from telomerase, or a gene that regulates the expression or activity of telomerase,
[9] Mesodermal stem cells are derived from any of bone marrow fluid, umbilical cord blood, peripheral blood, skin, hair follicle cells, muscle tissue, ES cells or cells derived from ES cells, [2] to [8] A method according to any of the above,
[10] The method according to any one of [2] to [8], wherein the mesoderm stem cell is a mesoderm stem cell contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from a vertebrate. ,
[11] A mononuclear cell fraction is obtained by subjecting bone marrow fluid or umbilical cord blood collected from a vertebrate to density gradient centrifugation in a solution for a time sufficient for separation according to specific gravity at 2000 rpm, and after centrifugation, a specific gravity of 1.07 The method according to [1] or [10], which is a cell fraction that can be prepared by collecting a cell fraction contained in the range of g / ml to 1.1 g / ml,
[12] Mesodermal stem cells have the characteristics of SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD14 (-), CD34 (-), CD45 (-) The method according to any one of [1] to [11], which is a cell,
[13] The method according to any one of [2] to [12], wherein the culture is performed under conditions of 33 ° C to 38 ° C.
[14] The method according to any one of [1] to [13], wherein bFGF, EGF, or ischemic brain extract is added to the basic culture solution.
[15] The method according to [1] to [14], wherein the neural cells are selected from the group consisting of neural stem cells, neural progenitor cells, neural cells, and glial cells,
[16] A cell obtained by the method according to any one of [1] to [15],
[17] A composition for the treatment of nervous system diseases comprising the cell according to [16],
[18] Nervous system diseases include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke, brain tumors, higher dysfunction, mental illness, epilepsy, traumatic nervous system diseases, inflammatory The composition according to [17], which is selected from the group consisting of a disease, an infectious disease, and spinal cord infarction,
[19] A method for treating a nervous system disease, comprising transplanting the cell according to [16] or the composition according to [17] to a recipient,
[20] Nervous system diseases include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke, brain tumor, higher dysfunction, mental illness, epilepsy, traumatic nervous system disease, inflammatory The method for treatment according to [19], which is selected from the group consisting of diseases, infectious diseases, and spinal cord infarction,
[21] The therapeutic method according to [19] or [20], wherein the cells to be transplanted are derived from a recipient.

  In the present invention, first, mesodermal stem cells or ES cells contained in a mononuclear cell fraction separated from bone marrow fluid or umbilical cord blood collected from a vertebrate are used in a basic culture solution at 33 ° C. to 38 ° C. Provided is a method for induction into nervous system cells by culturing under conditions.

  In addition, the present invention provides a novel method capable of securing a large amount of mesoderm stem cells by introducing an immortalizing gene into mesoderm stem cells and efficiently inducing differentiation into neural stem cells and nervous system cells. In a preferred embodiment of the present invention, the mesoderm stem cell is immortalized by high expression or activation of the immortalizing gene in the mesoderm stem cell, and the cell is cultured to induce differentiation into a nervous system cell.

In the method of the present invention, first, immortalized mesodermal stem cells are provided by highly expressing or activating an immortalized gene (step (a)).
In the present invention, a mesoderm stem cell refers to a cell constituting a tissue that is developmentally classified as a mesoderm, and includes blood cells. In addition, mesoderm stem cells are cells that can copy (divide and proliferate) cells that have the same ability as self and have the ability to differentiate into all the cells that make up the mesoderm tissue. Point to. Mesodermal stem cells have, for example, the characteristics of SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD14 (-), CD34 (-), CD45 (-) Although it is a cell, it is not particularly limited to these markers. In addition, so-called mesenchymal stem cells are also included in the mesodermal stem cells of the present invention.

The cell related to the mesenchymal system means a mesenchymal stem cell, a mesenchymal cell, a progenitor cell of a mesenchymal cell, or a cell derived from a mesenchymal cell.
Mesenchymal stem cells are, for example, bone marrow, peripheral blood, skin, hair roots, muscle tissue, endometrium, blood, umbilical cord blood, and stem cells that can be obtained from initial cultures of various tissues. .
A progenitor cell of a mesenchymal cell means a cell that is differentiated from a mesenchymal stem cell and is in the process of differentiation into a mesenchymal cell.

  A mesenchymal cell is a cell that is generated by the differentiation of a mesenchymal stem cell and does not have the ability to differentiate in many directions like a stem cell, but has a differentiation ability and a proliferation ability in a certain direction. It is a cell that has stopped in the G0 phase in a normal state but can enter the G1 phase (start of division) by stimulation. Mesenchymal cells include, for example, stromal cells and cells having the properties of stromal cells. Mesenchymal cells are present in all organs such as subcutaneous tissues, lungs, and liver, and are present in mesenchymal tissues such as bone, cartilage, fat, tendon, skeletal muscle, and interstitial bone.

  Cells derived from mesenchymal cells are (1) cardiovascular cells such as endothelial cells or cardiomyocytes or progenitor cells of cardiovascular cells, and cells having properties as these cells, (2) bone A cell of any one of cartilage, tendon, or skeletal muscle, and a progenitor cell of any cell of bone, cartilage, tendon, skeletal muscle, or adipose tissue, and further, a cell having these cell properties, (3) Nervous cells or progenitor cells of neural cells, further cells having properties as these cells, (4) endocrine cells or progenitor cells of endocrine cells, further cells having properties as these cells, (5) hematopoiesis It includes cells or progenitor cells of hematopoietic cells, further cells having properties as these cells, and (6) hepatocytes or hepatocyte progenitor cells, and further cells having properties as these cells.

  The mesodermal stem cells of the present invention are prepared using, for example, vertebrate bone marrow fluid, umbilical cord blood, peripheral blood, skin, cutaneous root cells, muscle tissue, ES cells, or cells derived from ES cells (origin). be able to. For example, for cell collection from skin, Young et al. (Young et al. Anat Rec, 2001, 264 (1), 51-62, Campagnli et al. Blood, 2001, 98 (8), 2396-2402) Cell collection from muscle was performed by Asakura et al. (Asakura A et al. Differentiation, 2001, 68 (4-5), 245-253), and cell collection from adipose tissue was performed by Zuk et al. (Zuk PA et al. Tissue Eng, 7 (2), 211-228), and cell collection from synovium is performed according to the method of De Bari et al. (De Bari C et al. Arthritis Rheum, 2001, 44 (8), 1928-1942) be able to.

  The vertebrate in the present invention is preferably a mammal (for example, mouse, rat, rabbit, pig, dog, monkey, human etc.), but is not particularly limited.

  The bone marrow fluid used in the present invention can be collected, for example, by anesthetizing vertebrates (including humans) (local or general anesthesia), inserting a needle into the bone, and sucking with a syringe. Examples of the bone include, but are not limited to, the femur, sternum, and iliac bone forming the pelvis. In addition, it has become an established technique to puncture the umbilical cord directly at the time of birth, suck it with a syringe, and collect and store the umbilical cord blood.

  In addition, as a method for preparing ES cells in the present invention, methods well known to those skilled in the art (Doetschman TC, et al. J Embryol Exp Morphol, 1985, 87, 27-45, Williams RL et al., Nature, 1988, 336, 684-687). In the present invention, the ES cells prepared in this way can be induced to differentiate into neural cells under the conditions described in the examples.

  In the present invention, mesoderm stem cells are obtained by subjecting bone marrow fluid or umbilical cord blood collected from vertebrates to density gradient centrifugation in a solution for a time sufficient for separation according to the specific gravity at 900 g, and after centrifugation, the specific gravity is 1.07 g / It is also possible to prepare by collecting a cell fraction having a specific gravity contained in the range of ml to 1.1 g / ml. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for cells to occupy a position corresponding to the specific gravity in a solution for density gradient centrifugation, usually 10 to 30 minutes. Degree. The specific gravity of the cells to be collected may vary depending on the type of animal from which the cells are derived (eg, human, rat, mouse, etc.). As the solution for density gradient centrifugation, Ficol solution or Percol solution can be used, but is not limited thereto.

Specifically, bone marrow fluid (25 ml) or umbilical cord blood collected from vertebrates is first mixed with the same amount of PBS solution, centrifuged (900 g for 10 minutes), and the precipitated cells are mixed with PBS and collected ( The cell density is about 4 × 10 ⁷ cells / ml) to remove blood components. Thereafter, 5 ml of the mixture is mixed with Percol solution (1.073 g / ml) and centrifuged (900 g for 30 minutes) to extract the mononuclear cell fraction. For cell washing, extract the extracted mononuclear cell fraction, for example, culture medium 1 (DMEM (Dulbecco's Modified Eagles Medium-Low Glucose), 10% FBS (fetal bovine serum), 1% anti-biotic-antimycotic solution ), Medium 2 (MSCBM (Mesenchymal Stem Cell Basal Medium), 10% MCGS (Mesenchymal Cell Growth Supplement), 4 mM L-Glutamine, 1% Penicillin-Streptomycin) or Medium 3 (DMEM (sigma), 10% FBS ( gibco), 1% Penicillin-Streptomycin, 2 mM L-Glutamine (gibco)) and centrifuge (900 g for 15 minutes or 2000 rpm for 15 minutes). Next, the supernatant after centrifugation is removed, and the precipitated cells are collected and cultured (37 ° C., 5% CO ₂ in air).

  Further, the mesodermal stem cells are, for example, from the above mononuclear cell fraction, the above SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD14 (-), Cells having cell surface markers such as CD34 (−) and CD45 (−) can be obtained by selecting using an antibody. The selection method is not particularly limited, and examples thereof include a method using magnetic beads or a method using a normal cell sorter (FACS or the like).

  In the present invention, mesoderm stem cells can also be prepared from a mononuclear cell fraction (including the state of a culture solution). Mononuclear cell fraction is obtained by subjecting bone marrow fluid or umbilical cord blood collected from vertebrates to density gradient centrifugation in the solution for a time sufficient for separation according to the specific gravity at 900 g, and after centrifugation, the specific gravity from 1.07 g / ml It can be prepared by collecting the cell fraction contained in the range of 1.1 g / ml. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for cells to occupy a position according to the specific gravity in a solution for density gradient centrifugation. Usually, it is about 10 to 30 minutes. The specific gravity of the cell fraction to be collected is preferably in the range of 1.07 g / ml to 1.08 g / ml (for example, 1.077 g / ml). As the solution for density gradient centrifugation, Ficol solution or Percol solution can be used, but is not limited thereto.

  Specifically, first, bone marrow fluid (5-10 μl) collected from a vertebrate was mixed with a solution (2 ml of L-15, 3 ml of Ficol), centrifuged (900 g for 15 minutes), Extract the drawing (about 1 ml). This mononuclear cell fraction is mixed with a culture solution (NPBM 2 ml) for cell washing and centrifuged again (900 g for 15 minutes). Next, after removing the supernatant, the precipitated cells are collected.

The mononuclear cell fraction in the present invention can be used for the preparation of the following mesodermal stem cells even in the state of a culture solution. As the culture solution containing the mononuclear cell fraction, the mononuclear cell fraction is obtained, for example, in the above-mentioned culture solution 1, culture solution 2 or culture solution 3 at 37 ° C., 5% CO _2. Although it can prepare by culture | cultivating on the conditions of in air, it is not limited to this condition in particular.

  Moreover, there is no restriction | limiting in particular as a nervous system cell in this invention, A neural stem cell, a neural progenitor cell, a neural cell, or a glial cell etc. can be illustrated.

  In the present invention, the immortalization of cells usually means that the cells stop growing after repeating a certain number of divisions, but the cells still proliferate after repeating such a number of divisions. Means a cell. That is, the immortalizing gene in the present invention means a gene that controls the function of continuing cell division even after repeating such divisions. Examples of such a gene include a telomerase gene, a gene derived from telomerase, or a gene that regulates the expression or activity of telomerase (for example, the myc gene is said to increase telomerase activity). Yes, but not limited to these. In the present invention, a human telomerase gene (hTERT) can be particularly preferably used.

  In the present invention, examples of a method for highly expressing or activating an immortalized gene include a method of introducing an immortalized gene into mesoderm stem cells, but is not particularly limited to this method. As a method for introducing the immortalized gene into the mesoderm stem cells, various known methods can be used. For example, an immortalizing gene is incorporated into a plasmid vector, the vector is introduced into a mesoderm stem cell in the presence of calcium monophosphate, and transformed, or the immortalizing gene is combined with a vesicle such as a liposome, and a mesoderm stem cell. A method of introducing into a mesoderm stem cell by electroporation in the presence of an immortalizing gene, a method of introducing an immortalizing gene into various viral vectors and infecting a mesoderm stem cell, etc. Can be used. As an introduction method using a viral vector, there are a method using a retrovirus, an adenovirus, and an adeno-associated virus, and as a retrovirus vector, there is a method using a MoMLV virus. In the present invention, a pBabe vector can be preferably used.

In the present invention, the step of immortalizing mesoderm stem cells by the above-described method may not necessarily be included, and the mesoderm stem cells immortalized by the above-described method or the like are used for the following step (b). be able to.
In the method of the present invention, following the step (a), the mesoderm stem cells that have been immortalized are cultured in a basic culture medium to induce the mesoderm stem cells into nervous system cells (step (b)). .

In a preferred embodiment in this step, the mesoderm stem cells are induced into neural stem cells by culturing the immortalized mesoderm stem cells in a basic culture medium.
In a preferred embodiment of the present invention, the mesoderm stem cell or the ES cell is cultured in a basic culture solution at a temperature of 33 ° C to 38 ° C.

The basic culture solution in the present invention is not particularly limited as long as it is a normal culture solution used for cell culture, but preferably DMEM (Dulbecco's modified essential medium) or NPBM (Neural progenitor cell basal medium: Clonetics). It is. The other components of the basic culture solution are not particularly limited, and preferably include F-12, FCS, Neural survival factors (Clonetics) and the like. Concentrations in these culture solutions are, for example, 50% for F-12 and 1% for FCS. Further, the CO ₂ concentration in the culture solution is preferably 5%, but is not particularly limited.

  In a preferred embodiment of the present invention, bFGF (Basic fibroblast growth factor) or EGF (Epidermal growth factor) is added to the basic culture solution. In this case, these may be added alone or both may be added. The concentration of bFGF or EGF includes 1 ng / ml to 100 ng / ml, preferably 10 ng / ml. Although there is no restriction | limiting in particular as an addition time and the addition method, Preferably, the method of adding every day, cultivating the said mesodermal stem cell in this basic culture solution is mentioned. In addition, when mesoderm stem cells are induced to differentiate into neural stem cells, it is preferable to add bFGF and EGF to the basic culture solution.

  In the present invention, the ischemic brain extract is added to the basic culture solution containing the basic culture solution and other components to induce induction from the mesoderm stem cells to the nervous system cells including the neural stem cells. It is possible to promote. Normally, in the state where an extract from ischemic brain tissue is not added, differentiation into neural stem cells is required for one week or more. However, when added, induction is possible in only a few days, and the induction rate is very high. Become. The present invention also provides a method for promoting induction into neural cells including such neural stem cells.

  The ischemic brain extract in the present invention can be prepared, for example, by centrifuging a vertebrate ischemic brain pulverized solution. Specifically, the whole brain is extracted from a whole brain ischemia model animal (rat, etc.), and the prepared small section is added to NPBM (culture solution for neural stem cells) and mechanically pulverized with a homogenizer. Subsequently, the ischemic brain extract can be prepared by centrifuging at 300 g for 5 minutes or 800 rpm for 5 minutes, collecting the supernatant, and removing cellular components with a membrane filter, but is not limited to this method. The whole brain ischemia model animal can be prepared by anesthetizing an animal with Nembutal and then perfusing with physiological saline. The ischemic brain extract thus obtained is added to the basic culture solution containing the basic culture solution and other components. There is no restriction | limiting in particular as an addition time.

  In the present invention, the mesoderm stem cells or the ES cells are cultured under the above conditions to be induced into nervous system cells. Specific examples of the nervous system cells include neural stem cells, neural progenitor cells, neural cells, glial cells, and the like.

In addition, the culture temperature condition in the present invention is 33 ° C. to 38 ° C., preferably 37 ° C.
Other culture conditions are not particularly limited. The cells may be in a floating state (Neurosphere state) or attached to the culture vessel. Examples of the culture container include a non-coating dish.

  In addition, it is considered that the safety of the neural cells induced to differentiate by the method of the present invention is improved by removing the immortalizing gene. In the present invention, an immortalized gene introduced into a cell can be removed by a technique already established. For example, by configuring the immortalized gene so as to be sandwiched between loxP sequences or loxP-like sequences, it is possible to perform recombinase treatment such as Cre recombinase and specifically remove the immortalized gene.

  In the present invention, the culture solution of the mesoderm stem cell is a new culture solution (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC 1%) or culture solution 2 (NPBM (Neural progenitor cell basal medium). : Clonetics), 2% Neural survival factors (Clonetics), 0.2% Gentamycine-amphotericinB, 10 ng / ml hFGF) It is possible to induce differentiation into nerve cells and glial cells (without conversion to). Thus, a method of inducing differentiation from mesoderm stem cells directly into nerve cells or glial cells is also included in the present invention.

  The present invention provides a cell obtained by the above method of the present invention. The cells are neural cells, and examples thereof include, but are not limited to, neural stem cells, neural progenitor cells, neural cells, glial cells, and the like.

  The present invention also provides a composition for the treatment of nervous system diseases comprising cells obtained by the above method. The cells of the present invention can be used for transplantation as they are, but in order to improve the therapeutic efficiency by transplantation, it is also possible to transplant them as a composition to which various drugs are added or genes are introduced.

  In the preparation of the composition of the present invention, for example, (1) addition of a substance that improves the proliferation rate of the cell of the present invention or promotes further differentiation into a nervous system cell, or a gene having such an effect Introduction, (2) Addition of a substance that improves the survival rate of cells of the present invention in damaged nerve tissue, or introduction of a gene having such an effect, (3) Cells of the present invention receive from damaged nerve tissue Addition of substances that prevent adverse effects, or introduction of genes with such effects, (4) Addition of substances that extend the life of donor cells, or introduction of genes with such effects, (5) Cell cycle Addition of regulatory substances or introduction of genes with such effects, (6) Addition of substances aimed at suppressing immune reactions, or introduction of genes with such effects, (7) Active energy metabolism To Addition of a substance or introduction of a gene having such an effect, (8) Introduction of a substance that improves the migration ability of donor cells in the host tissue, or introduction of a gene having such an effect, (9) Blood flow Substances to be improved or introduction of genes having such effects (including angiogenesis induction), (10) Addition of substances having some therapeutic effect to host cranial nerves (target diseases include, for example, brain tumors, central and peripheral Demyelinating disease, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumor, higher dysfunction including dementia, mental illness, epilepsy, traumatic nervous system Disease (including head injury, brain contusion, spinal cord injury), inflammatory disease, infectious disease, etc.) or introduction of a gene having such an effect, but is limited to these Not to.

  The cells and compositions of the present invention can be used for the treatment of nervous system diseases by being transplanted into a recipient. Nervous system diseases to be treated include, for example, central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumor, dementia Higher dysfunction, including mental illness, epilepsy, traumatic nervous system disease (including head injury, brain contusion, spinal cord injury), inflammatory disease, infectious disease (eg Jacob disease), and spinal cord infarction But are not limited to these.

  According to the present invention, cells derived from a recipient, such as bone marrow fluid, umbilical cord blood, peripheral blood, skin, muscle tissue, adipose tissue, synovium, and hair root, can be transplanted as donor cells. (Autotransplantation therapy). This is preferable because there is little risk of rejection due to transplantation and there is no difficulty in using an immunosuppressant in combination. When autotransplantation therapy is difficult, cells derived from another person or other medical animals can be used.

In addition, the present inventors examined when the cell transplantation was attempted and how much therapeutic effect was obtained, and the transplantation treatment in the hyperacute phase (within several hours) was the best, and then the acute phase (several days later) ), And in the order of the chronic phase (after several weeks). Therefore, since transplantation treatment in the hyperacute phase is considered desirable, it is very useful to collect cells in advance, culture, gene transfer, proliferation, differentiation induction, and preservation. That is, it is also preferable to collect cells from a recipient in advance, introduce an immortalized gene by the method of the present invention, and then preserve the neural cells that have been induced to differentiate by culturing in preparation for transplantation. To be implemented. The following states can be considered as the types of cells to be stored. Those skilled in the art can appropriately select cells in an appropriate state according to the disease to be treated.
1: collected crude state (crude fraction), 2: purified to some extent, 3: purified cells cultured and expanded, 4: purified cells immortalized, increased: 5: nerve State induced to differentiate into stem cells, 6: State differentiated into nervous system cells). In addition, cells stored in a bone marrow bank, an umbilical cord blood bank, or the like can be used for transplantation. The cells may be stored frozen.

  Furthermore, in the present invention, mesoderm stem cells (including immortalized cells) are directly transplanted into a recipient so that the mesoderm stem cells transferred to the neural tissue are differentiated into neural cells and functional. It was clarified that reconstruction was seen. Therefore, a method of directly administering (transplanting) mesoderm stem cells to a recipient and inducing differentiation of the cells into neural cells in the recipient body is also included in the present invention as one embodiment of the present invention. The administration method in this case is not particularly limited, and examples thereof include local administration, intravenous administration, intraarterial administration, and cerebrospinal fluid administration (for example, lumbar puncture or intraventricular administration).

  For example, cells can be transplanted into a patient by storing the cells to be transplanted in a syringe in a suspended state using artificial cerebrospinal fluid or physiological saline, exposing the nerve tissue damaged by the surgery, and This can be done by direct injection with a syringe needle. Since the cells of the present invention have a high migration ability, they can move in the nervous system tissue. Therefore, it may be transplanted near the damaged site. An effect can also be expected by injection into cerebrospinal fluid. In this case, since cells can be injected with a normal lumbar puncture, there is no need for the patient's surgery, and only local anesthesia is required, which is preferable in that the patient can be treated in a hospital room. Furthermore, an effect can be expected by injection into an artery or vein. Therefore, it is preferable in that transplantation can be performed in the normal manner of blood transfusion and transplantation operation in a ward is possible.

  The cells of the present invention can also be used as a gene carrier (vector) because of their high migration ability. For example, brain tumors, central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumors, higher dysfunction including dementia, mental illness As a gene therapy vector for various neurological diseases such as epilepsy, traumatic nervous system diseases (including head injury, brain contusion, spinal cord injury), inflammatory diseases, infectious diseases (eg Jacob disease) Be expected.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] Preparation of mononuclear cell fraction and mesoderm stem cell (1) Mononuclear cell fraction and culture solution of the mononuclear cell fraction A cell sample collected from a mouse (and human) contains 3 ml of Ficol. Diluted in L-15 medium (2 ml) and centrifuged (2,000 rpm, 15 minutes). Cells are collected from the mononuclear cell fraction, suspended in 2 ml of serum-free medium (Neural Progenitor cell Maintenance Medium: NPMM), further centrifuged (2,000 rpm, 15 minutes), and the supernatant is removed. Removed and sedimented cells were collected. The cells were again suspended in NPMM to prepare a mononuclear cell fraction.

In addition, the mononuclear cell fraction was cultured in culture medium 1 (DMEM (Dulbecco's Modified Eagles Medium-Low Glucose) 10% FBS (fetal bovine serum), 1% anti-biotic-antimycotic solution), culture medium 2, and culture medium 3. Then, the cells were cultured under the conditions of 37 ° C. and 5% CO ₂ to prepare a culture solution of the mononuclear cell fraction.

(2) Mesodermal stem cells From the mononuclear cell fraction prepared by the method described in (1) above or the culture solution of the mononuclear cell fraction, SH2 (+), SH3 (+), SH4 (+), Cells having characteristics such as CD29 (+), CD44 (+), CD14 (-), CD34 (-), CD45 (-) were extracted using antibodies. As a sorting method, a method using magnetic beads or a method using a normal cell sorter (FACS or the like) was used.

[Example 2] Differentiation induction from mesoderm stem cells to neural cells (1) Induction from mesoderm stem cells to neural stem cells First, washing was performed, and then cells were treated with enzyme treatment (reagent (0.05% trypsin , 0.02% EDTA), room temperature, 5 minutes), an equal volume of the culture solution was added, and pipetting was performed several times to separate single cells. Then, it was centrifuged at 600 rpm for 5 minutes. The precipitated cells were sucked up with a pipette. Next, new culture medium 1 (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC 1%, Basib fibroblast growth factor (bFGF) 10 ng / ml added daily, Epidermal growth factor (EGF) 10 ng / ml added daily) or culture medium 2 (NPBM (Neural progenitor cell basal medium: Clonetics), 2% Neural survival factors (Clonetics), 0.2% hEGF (human Epidermal growth factor), 0.2% Gentamicine-amphotericinB, 0.2% hFGF (human fibroblast growth factor), Basib fibroblast growth factor (bFGF) 10 ng / ml added daily, Epidermal growth factor (EGF) 10 ng / ml added daily), suspended in culture container (on-treated polystyrene dish) In this state, the culture was continued at 5% CO ₂ and 37 ° C.

(2) Induction from mesoderm stem cells to nerve cells and glial cells The culture medium of cultured mesoderm stem cells is a new culture medium (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC 1% ) Or medium 2 (NPBM (Neural progenitor cell basal medium: Clonetics), 2% Neural survival factors (Clonetics), 0.2% hEGF (human Epidermal growth factor), 0.2% Gentamicine-amphotericinB, 0.2% hFGF) We succeeded in inducing differentiation from mesoderm stem cells directly into neurons and glial cells (without conversion to neural stem cells) by culturing for about 4 weeks.

Confirmation of differentiation induction from mesoderm stem cells to nervous system cells by the methods (1) and (2) above, disappears the markers of mesoderm cells, SH2 and SH3 (neutin, which is a marker of neural stem cells, is originally negative) ), Instead, it was carried out by nestin being a marker of neural stem cells being positive (FIG. 1).
The cultured cells also changed morphologically by induction of differentiation from mesoderm stem cells to neural cells. Specifically, cells that were single cells formed neurospheres (FIG. 1). It was also confirmed that nerve cells (NSE positive) and glial cells (GFAP positive) differentiate from these neural stem cells (FIG. 2).

[Example 3] Promotion of differentiation induction from mesoderm stem cells to nervous system cells using ischemic brain extract The ischemic brain is loaded with ischemic stress, and the ischemic brain extract is added to cultured mesoderm stem cells By doing so, it was found that mesoderm stem cells differentiate into neural stem cells at a high rate. According to this method, it was found that cultured mesoderm stem cells can be induced to neural stem cells at a high rate in just a few days.

(1) Preparation of ischemic brain extract First, the rat is deeply anesthetized with Nembutal, then the front chest is incised from the anterior abdominal wall to expose the heart and the ascending aorta. An incision is made in the apex and a tube is inserted from the left ventricle into the ascending aorta. Next, an incision was made in the right atrial appendage, and physiological saline was perfused from the tube for 3 minutes to perform sufficient systemic blood removal. After perfusion, it was left as it was for 4 to 5 hours to obtain a whole brain ischemia model. Next, the cerebrum and midbrain were removed from the above-mentioned whole brain ischemia model rat and cut into small pieces of about 1 to 2 mm with a pointed knife. The prepared small section was added to NPMM and mechanically pulverized with a homogenizer to prepare a turbid solution. Next, the turbid solution was transferred to a centrifuge tube, centrifuged at 800 rpm for 5 minutes, and the supernatant was collected. Cell components were removed with a 0.22 μm membrane filter to obtain an ischemic brain extract.

(2) Induction of differentiation of cultured mesoderm stem cells (MSC) into neural stem cells
The culture of MSC cells was continued under conditions of 100 mm non-coating dish (IWAKI), conditioning medium (3 types: separately), and 37 ° C., 5% CO ₂ . At 90% confluent, the culture was aspirated with a Pasteur pipette and rinsed 3 times with Dulbecco's PBS. 2 ml of 0.05% Trypsin and 0.02% EDTA in PBS were added and incubated at 37 ° C. for 2-5 minutes until the cells were detached. 2ml of conditioning medium was added to stop Trypsin reaction. The supernatant and detached cells were collected in a centrifuge tube with a Pasteur pipette, pipetted several times, and then centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and NPMM was added to resuspend. The cells were seeded in 50% NPMM and 50% ischemic brain extract medium warmed to 37 ° C., and suspended in a 100 mm non-coating dish (IWAKI) under conditions of 37 ° C. and 5% CO 2. bFGF 10 ng / ml and EGF 10 ng / ml were added every day.

[Example 4] Evaluation of nerve regeneration ability Regarding the nerve regeneration ability of neural cells obtained by the above differentiation induction method, cerebral infarction model (Fig. 3), dementia model (Fig. 3), spinal cord injury model (Fig. 4). As a result of the examination in the demyelination model (FIG. 5), it was found that the regenerative ability was comparable to that of neural stem cells extracted and cultured from the brain.

[Example 5] Differentiation induction from ES cells to neural cells Mouse ES cells were treated with 100 mm gelatin-coating dish (IWAKI), conditioning medium 20 ml (DMEM, 10% FCS, 100 μM 2-mercaptoethanol, 1000 UNITs / ml ESGRO (CHEMICON)), and the culture was continued under conditions of 37 ° C. and 5% CO ₂ . The culture solution was sucked with a Pasteur pipette at 90% confluent and rinsed 3 times with PBS. 2 ml of 0.25% Trypsin, 0.03% EDTA in PBS was added and incubated at 37 ° C. for 2-5 minutes until the cells detached. 400 μl of FCS was added to stop Trypsin reaction. The supernatant and detached cells were collected in a centrifuge tube with a Pasteur pipette, pipetted several times, and then centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and NPMM was added to resuspend. The cells were seeded in 50% NPMM and 50% ischemic brain extract medium that had been warmed to 37 ° C., and suspended in a 100 mm non-coating dish (IWAKI) under conditions of 37 ° C. and 5% CO ₂ . bFGF 10 ng / ml and EGF 10 ng / ml were added every day.

[Example 6] Introduction of hTERT gene into cells Hereinafter, a method for immortalizing cells by introducing the hTERT gene into stromal cells will be described.

1. Collection of primary stromal cells Mononuclear cells were separated from 10 ml of bone marrow fluid obtained from the iliac bones of healthy adult men, and the cells attached to the flask after overnight culture were used as stromal cells.
2. A gene encoding a catalytic activity subunit (hTERT) of human telomerase was used as a gene to be introduced into stromal cells. The sequence of hTERT is described, for example, in Science 277, p.955-959. Furthermore, a known ras gene and SV40T gene, which are known as genes related to cell carcinogenesis, were also introduced into stromal cells.
3. PBABE-hygro-hTERT (provided by Dr. Robert A Weinberg) and pBABE-hygro-hTERT are available in Proc. Natl. Acad. Sci. USA vol95, p14723-14728. The hTERT EcoRV-SalI fragment obtained by PCR from pCI-Neo-hTERT-HA was cloned into pBABE-hygro as described in 1. pBABE-puro-rasV12 (provided by Dr. Scott W Lowe) was prepared by the method described in Cell, 88, 593-602, 1997. pMFG-tsT-IRES-neo is an IRES to MFG-tsT (pZIPtsU19 {obtained from Dr. R. McKay} and incorporated into MFG vector {Lab.Invest. 78, 1467-1468,1998}) A BamHI fragment of -neo (extracted from pRx-hCD25-ires-neo {Human gene therapy 9, 1983-1993, 1998}) was cloned and prepared.
4). The production of retrovirus-producing cells and the resulting infection of the virus were carried out in accordance with the “Experimental Method for Gene Transfer & Expression Analysis by Separate Experimental Medicine The Protocol Series (edited by Izumi Saito, Juno Yodosha)” (P58-62).

Specifically, using BOSC23 packaging cells (Proc. Natl. Acad. Sci. USA, 90: 8392-8396, 1993), ΨCRIP packaging cells (Proc. Natl. Acad. Sci. USA, 90: 3539-3543, 1993).
4-1. Preparation of recombinant retrovirus vector-producing cells (i) 5.5 × 10 ⁶ BOSC23 cells were seeded in a 10 cm dish 18-24 hours before transfection.
(Ii) 800 μl of OPTI-MEM (Gibco / BRL) was gently added to 15 μg of DNA (retroviral vector) and stirred to prepare solution A.
(Iii) 750 μl of OPTI-MEM was taken into a sterilized tube, 50 μl of LIPOFECTAMINE (2 mg / ml Gibco / BRL) was added and mixed slowly to prepare a solution B.
(Iv) Liquid A was gently mixed with liquid B to prepare liquid C, and left at room temperature for 30 to 45 minutes.
(V) BOSC23 cells were washed once in a medium at 37 ° C. excluding antibiotics and FBS.
(Vi) C solution (1.6 ml) was gently added to BOSC23 cells.
(Vii) Further, 2.4 ml of OPTI-MEM was added.
(Viii) 5 hours, and incubated under 5% CO _2.
(Ix) 4 ml of DMEM containing 20% fetal bovine serum was added and incubated overnight.
(X) The medium was changed to 37 ° C. containing 10% fetal bovine serum, and at the same time, 1 to 2 × 10 ⁶ ΨCRIP as packaging cells were seeded in a 10 cm dish.
(Xi) After 24 hours, the medium of BOSC23 cells was filtered with a 0.45 or 0.20 μm syringe filter, and the medium of ΨCRIP was replaced with 5 ml of filtered medium. At the same time, polybrene (Hexadimethrine Bromide, SIGMA H-9268) was added to 8 μg / ml.
(Xii) After culturing for 4 to 24 hours, 5 ml of a medium was added and further cultured overnight.
(Xiii) Drug selection is performed to produce ΨCRIP cells that produce retroviruses.

Next, the above three types of vectors were separately grown on retrovirus-producing cells (ψCRIP / P131), and stromal cells were transfected as follows (FIG. 8).
First, the day before transfection, stromal cells were reseeded to a 5 × 10 ⁴ cell / 10 cm dish, and ψCRIP / P131 medium producing retrovirus was changed from DMEM containing 10% bovine serum to 12.5% inactivated horse serum 12.5%. Incubate instead of α-MEM medium containing% inactivated fetal bovine serum / 2-Mercaptoethanol / hydrocortisone. On the day, the culture supernatant was filtered through a 0.20 μm filter, and polybrene was added to a final concentration of 8 μg / ml. Next, stromal cells were infected with the recombinant retroviral vector produced in the supernatant. After 4 hours, the culture supernatant was changed to a new medium and cultured for another 2 days. After that, pBABE-hygro-hTERT is hygromycin 100 μg / ml for 5 days, pBABE-puro-rasV12 is puromycin 1 μg / ml for 5 days, and pMFG-tsT-IRES-neo is G418 1 mg / ml for 5 days. Went.

For infection, three types of retroviral vectors were used: (1) control, (2) pBABE-hygro-hTERT vector only, (3) pMFG-tsT-IRES-neo vector only, (4) pBABE-puro-ras- V12 vector only, (5) pMFG-tsT-IRES-neo / pBABE-hygro-hTERT 2 types of vectors, (6) pBABE-puro-ras-V12 / pBABE-hygro-hTERT 2 types of vectors, (7) pMFG-tsT -IRES-neo / pBABE-puro-ras-V12 2 vectors, (8) pBABE-puro-ras-V12 / pMFG-tsT-IRES-neo / pBABE-hygro-hTERT 3 vectors I let you.
These viruses and cells are stored by the inventors and can be distributed at any time after the patent.

[Example 7] Differentiation ability of mesenchymal stem cells The ability to differentiate into bone, cartilage, muscle and the like, and the differentiation ability of mesenchymal stem cells (mesenchymal stem cells) having both self-replication ability and the support ability of blood stem cells were examined. .
Bone marrow was punctured from the iliac bones of healthy adults, mononuclear cells were collected by specific gravity centrifugation, and cultured overnight in DMEM containing 10% inactivated fetal bovine serum. The adherent cells are cultured from the next day. Two weeks later, the cells collected with TE (trypsin-EDTA) were cryopreserved as primary mesenchymal stem cells. Thereafter, the hTERT gene was introduced in the same manner as in Example 1. Next, the growth curves of primary mesenchymal stem cells and immortalized mesenchymal stem cells introduced with the hTERT gene were compared. The results are shown in FIG. As is clear from FIG. 9, the primary mesenchymal stem cells had a 42-day generation 17 (PD = 17) and cell division was stopped (crisis), but the immortalized mesenchymal stem cells had a 270-day generation 54 ( Even after PD = 54), it was considered that a stable cell line was established since subculture was possible without slowing the division rate.

Next, it was examined whether the prepared mesenchymal stem cells retain pluripotency.
(1) First, differentiation into adipocytes was induced using 1 μM dexamethazone, 60 μM indomethacine, 0.5 μM 3-isobutyl-1-methylxanthine, 5 μg / ml insulin. . After about 1 week of culture, it was stained with Oil Red O staining (FIG. 10; red ones are fat droplets).
(2) Next, 1 μM dexamethazone, 50 μg / ml ascorbate-2-phosphate, 6.25 μg / ml insulin, 6.25 μg / ml transferrin, 5.35 μg Cartilage differentiation was induced using / ml selenic acid, 1.25 mg / ml linoleic acid, 10 ng / ml TGF-β. After 2-3 weeks of culture, it was stained with Alcian blue. Staining (frozen section) Chondroitin in the cartilage matrix is stained blue (FIG. 11).
(3) Furthermore, bone differentiation was induced with 1 μM dexamethazone, 50 μM ascorbate-2-phosphate, and 10 mM β-glycerophosphate. It was stained with von Kossa staining in 2-3 weeks of culture (mineral deposition). The results are shown in FIG.

[Example 8] Differentiation induction from mesoderm stem cells to neural cells (1) Induction from mesoderm stem cells to neural stem cells Immortalized mesoderm stem cells by introducing the hTERT gene by the same method as in the above example The following mesenchymal stem cells (MSC) were used for the following experiment.
First, the immortalized cells are washed, then the cells are removed from the culture solution by enzymatic treatment (reagent (0.05% trypsin, 0.02% EDTA), room temperature, 5 minutes), an equal volume of culture solution is added, and several Pipet up and down to single cells. Then it was centrifuged at 900g for 5 minutes. The precipitated cells were sucked up with a pipette. Next, fresh culture medium 1 (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FCS 1%, Basic fibroblast growth factor (bFGF) 10 ng / ml added daily, Epidermal growth factor (EGF) 10 ng / ml added daily) or culture 2 (NPBM (Neural progenitor cell basal medium: Clonetics), 2% Neural survival factors (Clonetics), 10 ng / ml hEGF (human Epidermal growth factor), 0.2% Gentamycine-amphotericinB, 10 ng / ml hFGF (human fibroblast growth factor), basic fibroblast growth factor (bFGF) 10 ng / ml added daily, Epidermal growth factor (EGF) 10 ng / ml added daily), culture vessel (on-treated polystyrene dish) Then, the culture was continued at 37 ° C. with 5% CO ₂ in a floating state.

Confirmation of differentiation induction from mesoderm stem cells to neural stem cells by this method disappears from markers of mesoderm cells, SH2 and SH3 (neural stem cell markers nestin are originally negative), instead, markers of neural stem cells This was done by nestin being positive (FIG. 13).
The cultured cells were also morphologically changed by induction of differentiation from mesoderm stem cells to neural stem cells. Specifically, cells that were single cells formed neurospheres (FIG. 7). It was also confirmed that nerve cells (NSE positive) and glial cells (GFAP positive) were differentiated from these neural stem cells (FIG. 14).

(2) Induction from mesoderm stem cells to neurons and glial cells [1]
The culture medium of the above-mentioned mesodermal stem cells being cultured is a new culture medium (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC 1%) or culture medium 2 (NPBM (Neural progenitor cell basal medium) : Clonetics), 2% Neural survival factors (Clonetics), 10 ng / ml hEGF (human Epidermal growth factor), 0.2% Gentamycine-amphotericinB, 10 ng / ml hFGF) We succeeded in inducing differentiation from mesoderm stem cells directly into neural cells and glial cells (without conversion to neural stem cells).

(3) Induction from mesoderm stem cells to neurons and glial cells [2]
Differentiation induction from mesoderm stem cells to neural cells can also be performed as follows. The nerve induction was performed according to the past literature (journal of neuroscience research 61: 364-370, 2000). First, the number of cells of MSC KY hTERT was prepared. Specifically, the cells were cultured in a 10 cm dish in advance, and at the time of 70-80% confluence, the supernatant was aspirated and washed with 5 ml of PBS, and then 1 ml of Trypsin-EDTA was added to collect the cells. Cells were pelleted by centrifugation at 210 g for 10 minutes, and the cell concentration was adjusted to 2 × 10 ⁴ cells / 1 ml 10% fetal bovine serum-containing DMEM. Seed 2 ml (4 x 10 ⁴ cells) in a 6-well plate, remove DMEM containing 10% fetal bovine serum 24 hours later, and change the medium to 2 ml of DMEM containing 10% fetal bovine serum containing 1 mM 2-ME. (Pre-guidance). After 24 hours, serum was removed and the following three types of nerve induction were performed. (1) DMEM 2 ml containing 1 mM 2-ME, (2) DMEM 2 ml containing 10 mM 2-ME, (3) 200 μM butylated hydroxyanisole (BHA, Sigma) and 2% dimethylsulfoxide (DMSO, NACALAI ) Containing 2 ml of DMEM. Morphological observation was performed after induction for 24 hours in the above medium.

  As a result, (1) MSC KY hTERT with a flat cytoplasm showed a form in which the cytoplasm regressed and communicated with adjacent cells while having two or multiple poles. (2) (3) More cells with almost the same morphology were seen compared to (1). Such morphological changes showed the same findings as in the past literature, and as a further analysis, immunostaining of neural markers was performed. Three types of antibodies, Glial fibrillary acidic protein (GFAP, Cappel), Myelin basic protein (MBP, Chemicon), and Neuron specific enolase (NSE, ARP), were used by diluting 50 times, 200 times, and 200 times the stock solution, respectively. In addition, Vectastain Elite ABC kit (VECTOR laboratories) was used for immunostaining, and DAB substrate kit (VECTOR laboratories) was used as a substrate, and fixation was performed with 2% paraformaldehyde and 0.3% hydrogen peroxide as an endogenous peroxidase inhibitor. The nerve induction was performed as described above, and immunostaining was also performed 24 hours after the induction.

[Example 9] Evaluation of nerve regeneration ability
Regarding the nerve regeneration ability of the nervous system cells obtained by the above differentiation induction method, as a result of examination in the cerebral infarction model, dementia model, spinal cord injury model, demyelination model, it is the same level as the neural stem cells extracted and cultured from the brain It was found that there was a reproducibility (FIG. 15).

[Example 10] Neuronal differentiation of neurosphere-like cells Mesenchymal stem cells (MSC-hTERT), which are mesoderm stem cells immortalized by introducing the hTERT gene by the same method as in the above example Similarly, stromal cells into which hTERT gene was introduced (Stroma-hTERT), PDF cells, Hela cells, and HepG2 cells were suspended and cultured in Neural Progeniter basal medium / Non-treated Dish. When cell morphology was observed 48 hours later, MSC-hTERT cells, Stroma-hTERT cells, and PDF cells were sphere-like (FIG. 16).

In addition, after inducing differentiation of each of the above cells into neural stem cells by the method of the present invention, that is, in a state of being suspended in Neural Progeniter basal medium / Non-treated Dish, cultured for 1 day, 2 days, 5 days, and then total RNA Preparation and nestin expression were observed by RT-PCR.
A culture was continued as it was with DMEM containing 10% FBS as a control. Confirmation of differentiation induction from mesoderm stem cells to stem cells by this method was performed as described in Example 8 when nestin, a marker for neural stem cells, was positive.
As a result, the expression of nestin was observed in the lanes of the cultured cells of 2 days and 5 days of MSC-hTERT, MSC, Stroma-hTERT, PDF (FIG. 17).

[Example 11] Differentiation into neurons, formation of neurospheres
Each cell used in Example 10 was seeded in DMEM 6 wells containing 10% FBS so as to be 5 × 10 ⁴ cells / well. After overnight culture, the medium was exchanged with 10% FBS-containing DMEM supplemented with 1 mM β-ME, and further cultured overnight. Subsequently, induction was performed by exchanging with DMEM medium containing 2% DMSO / 200 μM butylated hydroxyanisole (BHA). Four days later, the cells were collected, total RNA was extracted, and RT-PCR was performed.

In addition, each of the above cells is seeded in a non-coated dish container using NPBM (FGF (20 ng / ml) and EGF (20 ng / ml)) medium so as to form 5 × 10 ⁵ cells / 10 cm dish. Formed. Four days later, the cells were collected, and each cell was seeded in 1 × 10 ⁵ cells / well (poly-D-lysine / laminine coated 6 wells) NPBM (FGF, EGF (−)). After 10 days, cells were collected, total RNA was prepared, and RT-PCR was performed.
In order to correct the difference in the cDNA concentration in the sample, the same analysis was performed for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as an internal standard for correction.
As a result, nerve cells derived from MSC-hTERT (FIG. 18 lane 6; BHA, DMSO) and cells induced to differentiate from MSC-hTERT via neural stem cells into neurons (FIG. 18 lane 13; NPBM ( -) PDL / laminin) NF-M expression was particularly strong.

[Example 12] Expression of Nestin
When Nestin became positive for MSC-hTERT, a vector expressing EGFP was introduced into a cell line. When differentiation was induced into neural stem cells in vitro and the cells were observed with a confocal laser microscope, it was confirmed that Nestin-positive MSC-hTERT emits green fluorescence (FIG. 19).
Furthermore, Nestin expression was also confirmed by RT-PCR (FIG. 20).

  According to the present invention, a method for inducing differentiation from mesoderm stem cells or ES cells to neural cells, a sufficient amount of cells are obtained by growing mesoderm stem cells, and further differentiation induction into neural stem cells and nervous system cells efficiently There are provided a novel method, a nervous system cell obtained by the method, a composition for treating a nervous system disease containing the nervous system cell, and a method of treating a nervous system disease using the composition.

  While normal human cultured mesoderm stem cells are restricted to a certain extent under culture conditions, mesoderm stem cells introduced with the hTERT gene in the present invention can be stably increased in a very large amount. Moreover, the transformation of the cell itself, which is seen when destabilizing by introducing an oncogene or the like, is not recognized, and immortalization can be realized while retaining the properties of the original cell. It is also possible to remove the hTERT gene after sufficient growth has been obtained.

  The methods of the present invention include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumors, higher dysfunctions including dementia, For treating neurological disorders such as mental illness, epilepsy, traumatic nervous system diseases (including head trauma, brain contusion, spinal cord injury), inflammatory diseases, infectious diseases (eg Jacob disease), and spinal cord infarction It contributes greatly. In addition, the present invention is more general and is considered to be applicable to nerve transplantation / regeneration therapy for wide-area cranial nerve injury. Central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke (including cerebral infarction, cerebral hemorrhage, subarachnoid hemorrhage), brain tumor, higher dysfunction including dementia, mental illness, epilepsy It can be applied to traumatic nervous system diseases (including head injury, brain contusion, spinal cord injury), inflammatory diseases, infectious diseases (eg Jacob disease), and autotransplantation therapy for spinal cord infarction.

  Furthermore, the differentiation induction method of the present invention provides a clue to unravel the mechanism of differentiation from mesoderm stem cells to neural cells. If genes that regulate such differentiation are identified and analyzed, mesoderm stem cells can be efficiently and sufficiently transformed into neural cells using these genes. Therefore, it is highly expected that “gene therapy” for promoting regeneration of nerve tissue will be possible.

  In addition, the method of the present invention is expected to be applied to functional tests for drug discovery, toxicity tests, and cDNA cloning. There are usually many cell-based assay systems, such as functional and toxicological tests for the discovery of therapeutic agents that work on nerves, and when searching for new cDNAs that modify the function of human-derived neural cells. However, it has been extremely difficult to supply sufficient human neurons. In addition, it has been relatively easy to obtain an immortal cell line from cells such as mice, but the species specificity between non-human animal cells and human neurons is often high. In experimental results using cells and individual animals, it was often difficult to apply to humans as an alternative to human cells. By using the method and cells of the present invention, it is possible to easily prepare a large amount of human nervous system cells sufficient to carry out all experiments, and also to maintain the properties and functions of the original human neurons. It can be prepared in a held state. In application to functional testing for drug discovery, toxicity testing, and cDNA cloning, dramatic technological innovation can be expected for methods that were impossible in the past because of quantitative limitations.

  For example, until now, functional tests and toxicity tests of compounds (drugs) have been conducted by administering them to experimental animals such as rats, but the results of experiments in rats often do not apply to humans. Experiments with samples have been anxious. In particular, since it is very difficult to obtain human nervous system cells, if a sufficient amount of human nervous system cells can be obtained, functional tests and toxicity tests using human nervous system cells can be facilitated. Very useful. By obtaining a large amount of cells with our immortalized human MCS and inducing them into neural cells by the method of the present invention, a large amount of human neural cells can be obtained.

  Furthermore, in the present invention, a technique for inducing from mesoderm cells to neural cells is presented. At this time, the genes that control proliferation and differentiation can be expected by cloning the changing genes. In addition, this research requires a large amount of cells. However, since human MCS immortalized by the method of the present invention can be obtained in large quantities, there are advantages such as easy gene cloning.

It is a photograph which shows a cultured mesoderm stem cell. Although it expresses SH3 which is a marker of mesodermal cells (A), nestin which is a marker of neural stem cells is negative (B). After differentiation induction under culture conditions, morphologically changes to a neural stem cell-like state, and the mesodermal cell marker SH3 becomes negative (C), and the neural stem cell marker nestin becomes positive. (D). The scale bar is 10 μm for A and B, and 200 μm for C and D. When mesoderm stem cells are induced to differentiate into neural stem cells under culture conditions and then further induced to differentiate, they differentiate into neural cells (A, D), astrocytes (B, E), and oligodendrocytes (C, F). It is a photograph showing this. D is a photograph immunostained with NSE (neuron-specific enolase), E is GFAP (glial fibrillary acidic protein), and F is GalC (galactocerebroside). The scale bar indicates 25 μm. It is a photograph which shows that the transplanted cell repaired the cerebral infarction focus. Donor cells that have been induced to differentiate mesoderm stem cells into neural stem cells under culture conditions are genetically marked with the LacZ gene (expressed in E. coli β-galacto and expressed), reacting with the substrate X-gal and reacting blue Color develops. Thus, donor cells can be tracked within the host brain tissue. The rat cerebral infarction model (the middle cerebral artery temporary occlusion model in which the basal ganglia, temporal lobe, hippocampus, etc. falls into cerebral infarction) is transplanted with donor cells marked with the LacZ gene, resulting in cerebral basal ganglia They were engrafted in temporal lobes, hippocampus, etc., and tissue repair was performed. It is a photograph which shows that the transplanted cell repaired the spinal cord injury site. As a result of transplanting a donor cell marked with the LacZ gene into a rat spinal cord injury model (a model traversed at the first thoracic spinal cord level), the donor cell is not only the damaged site, but also the brain (A), cervical spinal cord (B), It also migrated to the lumbar spine (C) to perform tissue repair. D, E, and F are photographs of A, B, and C observed at a high magnification. The scale bars in A, B, C, D, E, and F are 200 μm for A, B, and C, and 10 μm for D, E, and F. It is a photograph which shows that the transplanted cell repaired the spinal cord demyelination site. A is a photograph showing remyelination after transplantation of a neural stem cell derived from an adult mesoderm stem cell to an adult rat spinal cord demyelination region. B is a photograph of remyelinated axons observed at high magnification. The scale bars in A, B, C, D, E, and F indicate 250 μm in A, B, and C, and 10 μm in D, E, and F, respectively. It is a photograph of a neurosphere that is a neural stem cell derived from ES cells and is nestin positive. It is a figure which shows the structure of the vector used for the gene introduction | transduction to a stromal cell. It is a figure which shows typically the transfection to a stromal cell. It is a figure which shows the cell division generation number of the mesoderm stem cell (primary culture: primary culture) and the mesoderm stem cell immortalized by hTERT introduction | transduction. It is a photograph which shows the mesodermal stem cell (A) immortalized by hTERT introduction | transduction, and the fat cell (B: Oil RedO dyeing | staining) differentiation-induced from the immortalized mesodermal stem cell. It is a photograph which shows the mesoderm stem cell (A) immortalized by hTERT introduction | transduction, and the chondroblast cell (B: Alcian blue dyeing | staining) differentiation-induced from the immortalized mesoderm stem cell. Staining (frozen section) Chondroitin in the cartilage matrix is stained blue. It is a photograph which shows the mesoderm stem cell (A) immortalized by hTERT introduction | transduction, and the osteoblast (B: von Kossa staining; mineral deposition) induced differentiation from the immortalized mesoderm stem cell. It is a photograph showing nestin positive neural stem cells induced to differentiate from mesoderm stem cells immortalized by introduction of hTERT. The scale bar indicates 100 μm. It is a photograph which shows the NSE positive nerve cell (A) and the GFAP positive glial cell (B) which further induced differentiation from the neural stem cell which induced differentiation from the mesoderm stem cell immortalized by hTERT introduction. The scale bar is 20 μm for A and 10 μm for B. It is a photograph which shows that the spinal cord demyelination site | part was repaired by transplanting the mesoderm stem cell immortalized by hTERT introduction | transduction. A is a photograph showing that the mesoderm stem cells into which the hTERT gene has been introduced have been remyelinated after transplantation to the demyelinating region of the mature rat spinal cord. B is a photograph of remyelinated axons observed at high magnification. The scale bar is 250 μm for A and 10 μm for B. It is a photograph which shows the result of having tried differentiation induction of each cell to the neural stem cell by the method of this invention. Each cell was suspended, cultured in Neural Progeniter Basal medium / Non-treated Dish, and observed 48 hours later.Each cell was able to survive in suspension, but MSC-hTERT, Stroma-hTERT, PDF Only had a neurosphere-like appearance. FIG. 4 is a photograph in which after each cell was induced to induce differentiation into neural stem cells by the method of the present invention, total RNA was prepared and Nestin expression was confirmed by RT-PCR. Lane A is DMEM-10% FBS: culture dish, B is NPBM: Non-treated Dish / 1day, C is NPBM: Non-treated Dish / 2day, D is NPBM: Non-treated Dish / 5day. NPBM refers to Neural Progeniter basal medium. It is the photograph which confirmed that the neural stem cell induced | guided | derived from MSC differentiated into the nerve cell by RT-PCR. The upper row shows the result of correction by GAPDH (GAPDH is used as an internal control), and the lower row shows the result by NF-M, which is a subunit of neurofilament (NF). Lane 1 is DDW, 2 is neural stem cell, 3 is MSC-hTERT, 4 is Stroma-hTERT, 5 is PDF-hTERT, 6 is MSC-hTERT BHA, DMSO, 7 is Stroma-hTERT BHA, DMSO , 8 is PDF-hTERT BHA, DMSO, 9 is MSC-hTERT NPBM (-), 10 is Stroma-hTERT NPBM (-), 11 is PDF-hTERT NPBM (-), 12 is NSC NPBM (-) PDL / laminin , 13 is MSC-hTERT NPBM (-) PDL / laminin, 14 is Stroma-hTERT NPBM (-) PDL / laminin, and 15 is PDF-hTERT NPBM (-) PDL / laminin. When Nestin becomes Positive in MSC-hTERT, it is a photograph showing the result of introducing a gene expressing EGFP and inducing differentiation into neural stem cells by the method of the present invention. Observation with a confocal laser microscope showed that EGFG, which was not expressed before induction of differentiation into neural stem cells, became strongly expressed after induction. It is the photograph which confirmed the Nestin expression of MSC-hTERT by RT-PCR. Lane 1; cells in which Nestin enhancer / promoter EGFP has been introduced into MSC-hTERT, lane 2; cells in which Nestin enhancer / promoter EGFP has been introduced into MSC-hTERT and induced to differentiate into neural stem cells.

Claims (21)

  By culturing mesoderm stem cells or ES cells contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from vertebrates in a basic culture solution at a temperature of 33 ° C. to 38 ° C., A method of induction into nervous system cells. A method for inducing mesoderm stem cells into neural cells, comprising the following steps (a) and (b):
(A) providing an immortalized mesoderm stem cell by highly expressing or activating the immortalizing gene;
(B) A step of inducing the mesoderm stem cell to a nervous system cell by culturing the immortalized mesoderm stem cell in the step (a) in a basic culture medium.   The method according to claim 2, wherein the immortalized gene is highly expressed or activated by introducing the immortalized gene into a mesoderm stem cell.   The method according to claim 3, wherein the mesoderm stem cell is immortalized by introducing the immortalization gene into the mesoderm stem cell using a retroviral vector.   The method of claim 4, wherein the retroviral vector is pBabe.   The method according to any one of claims 2 to 5, wherein the immortalized gene can be removed or removed from the nervous system cell by gene removal treatment.   The method according to claim 6, wherein the gene removal treatment of the immortalized gene is performed by sandwiching the immortalized gene with a loxP sequence or a loxP-like sequence and then treating with a recombinase.   The method according to any one of claims 2 to 7, wherein the immortalizing gene is any one of a telomerase gene, a gene derived from telomerase, or a gene that regulates the expression or activity of telomerase.   The mesoderm stem cells are derived from any of bone marrow fluid, umbilical cord blood, peripheral blood, skin, skin hair root cells, muscle tissue, ES cells, or cells derived from ES cells. the method of.   The method according to any one of claims 2 to 8, wherein the mesoderm stem cell is a mesoderm stem cell contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from a vertebrate.   Mononuclear cell fraction is obtained by subjecting bone marrow fluid or umbilical cord blood collected from vertebrates to density gradient centrifugation in the solution for a time sufficient for separation according to specific gravity at 2000 rpm, and after centrifugation, specific gravity 1.07 g / ml The method according to claim 1 or 10, which is a cell fraction that can be prepared by recovering a cell fraction contained in the range of 1 to 1.1 g / ml.   Mesodermal stem cells have the characteristics of SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD14 (-), CD34 (-), CD45 (-) The method according to claim 1.   The method in any one of Claims 2-12 which culture | cultivates on the conditions of 33 to 38 degreeC.   The method according to claim 1, wherein bFGF, EGF, or ischemic brain extract is added to the basic culture solution.   The method according to claim 1, wherein the neural cells are selected from the group consisting of neural stem cells, neural progenitor cells, neural cells, and glial cells.   The cell obtained by the method in any one of Claims 1-15.   A composition for the treatment of a nervous system disease comprising the cell of claim 16.   Nervous system diseases include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke, brain tumor, higher dysfunction, mental illness, epilepsy, traumatic nervous system disease, inflammatory disease, infection The composition according to claim 17, which is selected from the group consisting of sexually transmitted diseases and spinal cord infarction.   A method for treating a nervous system disease, comprising transplanting the cell according to claim 16 or the composition according to claim 17 to a recipient.   Nervous system diseases include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke, brain tumor, higher dysfunction, mental illness, epilepsy, traumatic nervous system disease, inflammatory disease, infection The treatment method according to claim 19, which is selected from the group consisting of sexually transmitted diseases and spinal cord infarction.   The treatment method according to claim 19 or 20, wherein the cells to be transplanted are derived from a recipient.