WO2003038074A1

WO2003038074A1 - Method of inducing differentiation of mesoblast stem cells or es cells into nerve system cells

Info

Publication number: WO2003038074A1
Application number: PCT/JP2001/009510
Authority: WO
Inventors: Osamu Honmou
Original assignee: Renomedix Institute Inc.; Teikoku Hormone Mfg.Co.,Ltd.; Mitsui Sumitomo Insurance Care Network Company, Limited; Hitachi,Ltd.
Priority date: 2001-10-30
Filing date: 2001-10-30
Publication date: 2003-05-08
Also published as: JP4118236B2; JPWO2003038075A1; CN1610738A; WO2003038090A1; KR101022349B1; KR20040062610A

Abstract

It is found out that when mesoblast stem cells or ES cells prepared from a mononuclear fraction separated from bone marrow fluid or cord blood are cultured in a fundamental liquid culture medium, the differentiation of these mesoblast stem cells or ES cells into nerve system cells or glial cells is induced. It is further found out that the induction of the differentiation of mesoblast stem cells or ES cells into nerve system cells is promoted by adding an ischemic brain extract to the above-described fundamental liquid culture medium. It is clarified that the nerve system cells obtained by the above method are capable of regenerating nerve in a cerebral infarction model, a dementia model, a spinal injury model and a demyelination model.

Description

Description Method for inducing differentiation from mesoderm stem cells or ES cells to nervous system cells

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

Oligodendrocyte (Archer, DR. Et al., Exp Neurol, 1994, 125, 268-77., Blakemore, WF. And Crang, AJ., Dev Neurosci, 1988, 10, , G 、 el, M. et al., Ann New York Acad Sci, 1987, 495, 71-85.), Or Schwann cells (Blak emore, WF., Nature, 1977, 266, 68—9. , Blakemore, WF. And Crang, AJ., Dev Neurosci, 1988, 10, 1-11, Honmou, 0. et al., J Neurosc i, 1996, 16, 3199-208. Olfactory ensheating cells (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.) Transplanted myelinating cells can induce remyelination and restore electrophysiological function in animal models (Utzschneider, DA. Et al., Proc Natl Acad Sci USA, 1994, 91, 53-7., Honmou, 0. et al., J Neur osci, 1996, 16, 3199-208.). It is not impossible to prepare such cells from patients or others and use them in cell therapy, but 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 capable of self-renewal, It is 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., Lois, C. and Alvarez- Buyl la, A., Proc Natl Acad Sci USA, 1993, 90, 2074-7., Morshead, CM. Et al., Neuron, 1994, 13, 1071—82., Re ynolds, 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., Neurosc i, 1997, 76, 1121-8 > Moyer, MP. Et al., Transplant Proc, 1997, 29, 2040-1, Svendsen, CN. Et al., Exp Neurol, 1997, 148, 135-4 6.), and remyelinated (Flax, JD. Et al., Nat Biot echnol, 1998, 16, 1033- 9.). 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.).

These studies are of great interest as they suggest that the cells may be used in the repair of nervous system diseases. (Akiyam a, Y. et a to Exp Neurol, 2001., Chalmers-Redman, RM. Et a to Neurosci, 1997, 76, 1121-8., Moyer, MP. Et al., Transplant Proc, 1 997, 29, 2040—1, Svendsen, CN. Et al., Exp Neurol, 1997, 148, 1 35-46, Yandava, BD. Et al., Proc Natl Acad Sci USA, 1999, 96, 7 029-34 .)

Already, the present inventor has extracted human adult-derived neurons from the brain, cultured them, made cell lines, examined their functions, and found that these neurons have a self-replicating function and a multi-differentiation function. It was. In other words, neuronal progenitor cells obtained from the brain were expanded into single cells, and cell lines were cloned in vitro. However, self-renewal ability (ie, proliferative ability) and multipotency [ie, neuron (astrolog tes), oligodendrocytes (oligodendrocytes; oli differentiation into godendr ocy tes) was confirmed, confirming that this cell has the properties of neural stem cells.

Actually, when transplantation experiments were performed on these cells using a rat ischemia model and a trauma model, they showed very good engraftment, migration, and differentiation. 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 and electrophysiologically Ifc 5 shinobied in behavioral science.

Judging from the above facts, collecting a small amount of nerve tissue from the own cerebrum, extracting and culturing neural stem cells, and transplanting the obtained neural stem cells to the damaged site of the own spinal cord 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, although neurological deficits do not occur during collection. Therefore, establishing safer and simpler autotransplantation therapy was extremely important in terms of establishing treatments for today's complex diseases. In contrast, the present inventor has 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 to obtain donor cells. Developed (patent application

2001 -1 60579). That is, the present inventor has found that the mononuclear cell fraction prepared from bone marrow cells has the ability to differentiate into neural cells. Further, the present inventor further provides a cell fraction containing mesoderm stem cells isolated from the mononuclear cell fraction, a cell fraction containing stromal cells, and a cell fraction containing AC 133-positive cells. Has been found to have the ability to differentiate into neural cells. Disclosure of the invention

The present invention has been made in view of such a situation, and an object of the present invention is to provide a method for inducing differentiation from mesoderm stem cells or ES cells to nervous system cells, a nervous system cell obtained by the method, and the nervous system. It is an object of the present invention to provide a composition for treating a nervous system disease including cells, and a method for treating a nervous system disease using the composition.

The inventor newly found that mesodermal 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 were added to culture medium 1 (DMEM (Dulbecco's modi fied essential medium) 50%, F -12 50%, FSC 1%, bFGF (Basib fibrobl as t growth f ac tor) 10 ng / ml continuously added caro, EGF (Epidermal growth fact or) 10 ng / ml added daily), or culture solution 2 (NPBM (Neural Progenito r Basal Medium), 2% Neural survival factors (Clonetics)> 0.2% EGF (human Epidermal growth f ac tor) 0.2% Gentami cine-ampho ter ici nB, 0.2% FGF (human fibroblast growth factor), added daily to bFGF 10 ng / ml, in daily addition) the EGF 10 ng / ml, in a state in which floating, 5% C0 _2, 37 ° and cultured in C, and mesodermal stem cells or the ES cells Was found to induce differentiation into neural stem cells.

In addition, mesoderm stem cells or ES cells contained in the mononuclear cell fraction isolated 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: Clon etics), 2% Neural survival factors (Clonetics), 0.2% hEGF (human Epidermal growth factor), 0.2% Gentamicine-amphotericinB, 0.2% FGF (human fibroblast growth factor)), and found that mesoderm stem cells or ES cells induce differentiation into neurons or Darya cells It was.

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

In addition, the nerve regeneration ability of the nervous system cells obtained by the above differentiation induction method was examined in a cerebral infarction model, a dementia model, a spinal cord injury model, and a demyelination model. It turns out that it has the same reproducibility.

From the above results, it has become 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 / regenerative 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 mesoderm stem cells or ES cells into 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” to promote regeneration of nerve tissue will be possible.

That is, the present invention relates to a method for inducing differentiation from mesoderm stem cells or ES cells to neural cells, a nervous system cell obtained by the method, a composition for treating a nervous system disease comprising the neural cell, the composition To treat neurological diseases More specifically about the law,

[1] Mesodermal stem cells or ES cells contained in the mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from vertebrate animals, in a basic culture medium at 33 to 38 ° C. A method of inducing into nervous system cells by culturing with

[2] The fraction of mononuclear cells is bone marrow fluid or umbilical cord blood collected from vertebrate animals, and is subjected to density gradient centrifugation in solution for fc at a speed sufficient for the specific gravity at 2000 rpm. After centrifugation, the specific gravity is 1.07 The method according to [1], which is a cell fraction that can be prepared by collecting a cell fraction in the range of g / ml to 1. Ig / ml,

[3] Mesodermal stem cells have the characteristics of SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD (-), CD34 (-), CD45 (-) The method according to [1] or [2], which is a cell,

[4] The method according to any one of [1] to [3], wherein the neural cell is selected from the group consisting of a neural stem cell, a neural progenitor cell, a neural cell, and a glial cell.

[5] The method according to any one of [1] to [4], wherein bFGF, EGF, or ischemic brain extract is added to the basic culture solution.

[6] A cell obtained by the method according to any one of [1] to [5],

[7] A composition for treating a nervous system disease, comprising the cell according to [6],

[8] Nervous system diseases include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, stroke, brain tumors, higher dysfunction, mental illness, cancer, traumatic nervous system diseases, and The composition according to [7], which is selected from the group consisting of spinal cord infarction,

[9] A method for treating a nervous system disease, comprising transplanting the cell according to [6] or the composition according to [7] to a recipient,

[1 0] Nervous system diseases are central and peripheral demyelinating diseases, The treatment according to [9], which is selected from the group consisting of peripheral degenerative diseases, stroke, brain tumor, higher dysfunction, mental illness, epilepsy, traumatic nervous system disease, and spinal cord infarction Method,

[1 1] The therapeutic method according to [9] or [10], wherein the cells to be transplanted are derived from a recipient.

In the present invention, first, mesoderm stem cells or ES cells contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from a vertebrate animal are used in a basic culture solution at 33 ° (: to 38 °). Provided is a method for inducing to nervous system cells by culturing under the condition of ° C.

In the present invention, the vertebrate is preferably a mammal (eg, mouse, rat, rabbit, buyu, inu, 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 is an established technique to puncture the umbilical cord directly at birth and suck it with a syringe to collect and store the umbilical cord blood.

In the present invention, the 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 the specific gravity at 2000 rpm, and after centrifugation, the specific gravity is 1 It can be prepared by collecting cell fractions in the range of 07g / ml to 1.lg / ml. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for a cell to occupy a position according to its specific gravity in a solution for density gradient centrifugation. Usually, it is about 10 to 30 minutes. The specific gravity of the recovered cell fraction is preferably in the range of 1.07 g / m 1 to 1.08 g / ml (eg, 1.077 g / ml). Density gradient centrifugation As a solution for this, Ficol solution or Percol solution can be used, but is not limited thereto.

For example, bone marrow fluid (5-10 zl) collected from a vertebrate is first mixed with a solution (2 ml of 1L-15, 3 ml of Ficol), and centrifuged (2000 rpm for 15 minutes). Extract the nuclear cell fraction (approx. Lml). This mononuclear cell fraction is mixed with a culture solution (2 ml of NPBM) for cell washing and centrifuged again (2000 rpm 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 medium containing the mononuclear cell fraction, the mononuclear cell fraction is, for example, culture medium 1 (DMEM (Dulbecco's Modified Eagles Medium-Low Glucose), 10¾ FBS (fetal bovine serum), 1% ant i -biot c-ant imycot ic solution), Medium 2 (MSCBM (Mesenchymal Stem Cel 1 Basal Medium)), 10% MCGS (Mesenchymal Cel 1 Growth Suppl ement), 4 mM L-Glutamine, 1% Penici 11 in-Streptomycin 37) in culture medium 3 (DMEM (sigma), 10% FBS (gibco), 1% Penici 11 in-St reptomycin, 2 mM L-Glutamine (gibco)). It can be prepared by culturing under the conditions of C, 5¾ C0 ₂ in air.

In the present invention, mesoderm stem cells are prepared from the above mononuclear cell fraction (including the state of the culture solution). “Mesodermal stem cells” refer to cells that constitute a tissue that is developmentally classified as mesoderm, including blood cells. Mesodermal stem cells are cells that have the ability to copy (divide and proliferate) cells that have the same ability as themselves and 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.

Mesodermal stem cells are, for example, bone marrow fluid or umbilical cord blood collected from vertebrates Centrifugation at 900 g for a time sufficient for separation according to the specific gravity, followed by density gradient centrifugation in the solution, and after centrifugation, the fraction of cells with a specific gravity in the specific gravity range of 1.07 g / ml to 1. lg / ml It is also possible to prepare by recovering. Here, “a sufficient time for separation according to specific gravity” means a time sufficient for a cell to occupy a position according to its specific gravity in a solution for density gradient centrifugation, usually 10 to 30 About a minute. 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 a solution for density gradient centrifugation, Ficol solution or Percol solution can be used, but is not limited thereto.

Specifically, first, bone marrow fluid (25 ml) or umbilical cord blood collected from a vertebrate is 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 4X10 ⁷ cells / ml) to remove blood components. After that, mix 5 ml with Percol solution (1.073 g / ml), and centrifuge (900 g for 30 minutes) to extract the mononuclear cell fraction. For cell washing, the extracted mononuclear cell fraction is mixed with, for example, culture solution 1 (D MEM, 10¾ FBS, anti-biotic-antimycotic solution), culture solution 2 or culture solution 3, and centrifuged ( For 15 minutes at 2000 rpm). Then, the supernatant was removed after centrifugation, the precipitated cells were collected and cultured (37 ° C, 5% CO 2 in air) 0

In addition, mesoderm stem cells are, for example, from the above mononuclear cell fraction, the above SH 2 (1), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CDH (-) , CD34 (−), CD45 (−) and other cells having cell surface markers can be obtained by selecting using an antibody. The selection method is not particularly limited, and examples thereof include a method using magnet beads or a method using a normal cell sorter (FACS or the like).

The method for preparing ES cells in the present invention is well known to those skilled in the art. It can be prepared by the method (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, the mesoderm stem cell or the ES cell is cultured in a basic culture solution under the conditions of 33 to 38 ^.

The basic culture medium in the present invention is not particularly limited as long as it is a normal culture medium used for cell culture, but preferably DMEM (Dulbecco's modi fied essential medi 籠) or NPBM (Neural progenitor cell). basal m edium: CI one tics). The other components of the basic culture solution are not particularly limited, but preferred embodiments include F-12, FSC, Neural survival factors (Clonetics) and the like contained in the culture solution described in Example 2. It is The concentration in these culture media is not particularly limited, but preferably F-12 is 50% and FSC is 1%. Also, C0 ₂ concentration in the culture solution is good Mashiku is 5%, is not particularly limited.

In a preferred embodiment of the present invention, bFGF (Basib fibroblast growth factor) or EGF (Epidermal growth factor) is added to the basic culture solution for the mesoderm stem cells or the ES cells. In this case, these may be added alone or both may be added. Examples of the concentration of bFGF or EGF include lng / ml to 100 ng / ml, and preferably 10 ng / ml. There are no particular restrictions on the timing of addition or the method of addition, but a method of adding the mesodermal stem cells or the ES cells every day while culturing the mesoderm stem cells or the ES cells in the basic culture solution is preferable. In addition, when mesoderm stem cells are induced to differentiate into neural stem cells, it is preferable to add bFGF and EFG to the basic culture solution. 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 (Neuroscope state) or attached to the culture vessel. Examples of the culture container include a non-coating dish (non-coating dish) and the like.

In the present invention, by adding an ischemic brain extract to the basic culture solution containing the basic culture solution and other components, the mesodermal stem cell or the ES cell to the nervous system cell is added. It is possible to promote the induction of In the present invention, such a method for promoting induction into neural cells is also provided.

The ischemic brain extract in the present invention can be prepared, for example, by centrifuging a crushed liquid of ischemic brain of a vertebrate. Specifically, the whole brain is removed from an animal model of whole cerebral ischemia (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 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 cerebral 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 particular limitation on the addition time.

In the present invention, the mesoderm stem cells or the ES cells are cultured under the above conditions to induce to the nervous system cells. Specific examples of the nervous system cells include neural stem cells, neural progenitor cells, neural cells, glial cells, and the like. The present invention provides a cell obtained by the above method. The cell is a neuronal cell, and examples thereof include, but are not limited to, neural stem cells, neural progenitor cells, neural cells, and dahlia cells.

The present invention also provides a composition for treating a nervous system disease 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 have been added or genes have been introduced.

In the preparation of the composition of the present invention, for example, (1) the 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 the introduction of a gene having such an effect (2) Addition of a substance that improves the survival rate of the cells of the present invention in damaged nerve tissue, or introduction of a gene having such an effect (3) Blocks adverse effects of the cells of the present invention from damaged nerve tissue Addition of substances 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 Introduction of substances that regulate the cell cycle Addition or introduction of genes with such effects, ⑥ Addition of substances aiming to suppress immune reactions, or introduction of genes with such effects, ⑦Energy -Addition of a substance that activates metabolism, or introduction of a gene having such an effect, ⑧ Introduction of a substance that improves the migration ability of a donor cell in the host tissue, or introduction of a gene having such an effect, ⑨ Examples include, but are not limited to, introducing a substance that improves blood flow, or introducing a gene having such an effect (including induction of angiogenesis).

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 For example, 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 Diseases, epilepsy, traumatic neurological diseases (including head trauma, brain contusion, spinal cord injury), and spinal cord infarction, but are not limited to these.

According to the present invention, cells obtained by separation from recipient-derived bone marrow fluid or umbilical cord blood can be transplanted as donor cells (autologous transplantation 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. If autotransplantation therapy is difficult, cells from other people or other animals can be used. The cells may be stored frozen.

For example, cells transplanted to a patient are stored in a syringe in a state where the cells to be transplanted are suspended using artificial cerebrospinal fluid or physiological saline, and the damaged nerve tissue is exposed by surgery. This can be done by direct injection with a needle. Since the cells of the present invention have high migration ability, they can move in nervous system tissues. 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. In addition, it can be expected to be injected into arteries and veins. Therefore, transplantation can be performed in the manner of normal blood transfusion, which is preferable in that transplantation operation in a ward is possible.

In addition, the cells of the present invention may be used as a gene carrier because of their high migration ability. For example, it is expected to be used as a vector therapy vector for various neurological diseases such as brain tumors. Brief Description of Drawings

Figure 1 is a photograph showing cultured mesoderm stem cells. It expresses SH3, a marker for mesodermal cells (A), but is negative for nestin, a marker for neural stem cells (B). After differentiation induction under culture conditions, morphologically changed to neural stem cell-like, and the marker of mesoderm cells, SH3, became negative (C) and the neural stem cell marker nestin was positive (D). The scale bar indicates Ιθ ΐη for A and B, and 200 ^ m for C and D. Figure 2 shows that when mesoderm stem cells are induced to differentiate into neural stem cells under culture conditions, and subsequently induced to differentiate, neurons (A, D), astrocytes (B, E), oligodendrocytes (C, F) Photograph showing differentiation into D is a photograph immunostained with NSE (neuron-specific enolase), E ¾ GFAP (glial fibrillary a cidic protein), and F is GalC (gal actocerebros ide). The scale bar indicates 25 mm.

Figure 3 is a photograph showing that the transplanted cells repaired the cerebral infarction. 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 (expresses E. coli galactose seed) and reacts when treated with the substrate X-gal blue Color. Therefore, it is possible to track donor cells in the host brain tissue. Rat brain infarction model (middle cerebral artery temporary occlusion model, basal ganglia, temporal lobe, hippocampus etc. fall into cerebral infarction) transplanted with donor cells marked with LacZ gene, resulting in cerebral infarction The tissue was repaired by engraftment in the basal ganglia, temporal lobe, hippocampus, etc.

Figure 4 is a photograph showing that the transplanted cells repaired the spinal cord injury site. As a result of transplanting donor cells marked with the LacZ gene into a rat spinal cord injury model (a model traversed at the level of the first thoracic spinal cord), the donor cell is not only the damaged site, but also the brain (A), myelin ( B) also migrated to the lumbar spine (C) Repair was done. D, E, and F are photographs of A, B, and C observed at high magnification. The scale bars in A, B, C, D, E and F are 200 m for A, B and C, and 10 xm for D, E and F.

Figure 5 is a photograph showing that the transplanted cells repaired the spinal cord demyelination site. A is a photograph showing remyelination after transplantation of neural stem cells derived from adult mesoderm stem cells to a mature rat spinal cord demyelination region. B is a photograph of remyelinated axons observed at high magnification. The scale parameters for A, B, C, D, E and F are 250 m for A, B and C, and 10 m for D, E and F. Fig. 6 is a photograph of a nestin-positive neurosphere, which is a neural stem cell derived from ES cells. BEST MODE FOR CARRYING OUT THE INVENTION

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] Mononuclear cell fraction and mesoderm stem cell preparation

(1) Mononuclear cell fraction and culture solution of the mononuclear cell fraction

Cell samples collected from mice (and humans) were diluted in L-15 medium (2 ml) containing 3 ml of Ficoll and centrifuged (2, OOOrpnu for 15 minutes). Cells are collected from the mononuclear cell fraction, suspended in 2 ml of serum-free medium (Neural Progenitor cell Maintenance Medium: 腦 M), and then centrifuged (2,000 rpm, 15 minutes). The supernatant was removed and the sedimented cells were recovered. The cells were suspended again in NP 匪 to prepare a mononuclear cell fraction.

In addition, the mononuclear cell fraction was added to culture medium 1 (DMEM (Dulbecco's Modified Eagles Medium-Low Glucose) 10¾ FBS (fetal bovine serum), 1¾ anti-biotic-antimycotic solution), culture medium 2, among the liquid 3, and cultured in a 37 ° C, 5% C0 ₂ conditions, 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 (+), CD29 (+), CD44 Cells having characteristics such as (+), CD14 (-), CD34 (-), CD45 (-) were extracted using antibodies. As a sorting method, a method using a magnetic bead or a method using a normal cellosphere (such as FACS) was used.

[Example 2] Differentiation induction from mesoderm stem cells to nervous system cells

(1) Induction from mesoderm stem cells to neural stem cells

First, wash, then peel the cells from the culture solution by enzyme treatment (reagent (0.05% trypsin, 0.02¾ EDTA) ', room temperature, 5 minutes), add an equal volume of culture solution, and several times Pipette to break up to a single cell. Next, it was centrifuged at 600 rpm for 5 minutes. The precipitated cells were sucked up with a pipette. Next, add fresh medium 1 (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC 1¾, Basib fibroblast growth factor (bFG F) 10 ng / ml 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), 0.2% hE GF (human Epidermal growth factor ), 0.2% Gentamicine-amphotericin B, 0.2% hFGF (human fibroblast growth factor) Basib fibroblast growth factor (bFGF) 10 ng / ml, and epidermal growth factor (EGF) 10 ng / ml added daily) In the culture vessel (on-treated polystyrene dish), the culture was continued at 5% CO ₂ and 37 in a floating state.

(2) Induction from mesoderm stem cells to neurons and dahlia cells

The culture medium of cultured mesoderm stem cells is used as a new culture medium (DMEM (Dulbecco's modified essential medium) 50%, F-12 50%, FSC \%) or Change to nutrient solution 2 (NPBM (Neural progenitor cell basal medium: Clonetics), 2% Neural survival factors (Clonetics), 0.2% hEGF (human Epidermal growth factor), 0.2% Gentamicine-amphoter icinB, 0.2% hFGF) By culturing for about 4 weeks, we also succeeded in inducing differentiation from mesoderm stem cells directly into neural cells and Darya cells (without conversion to neural stem cells).

Confirmation of differentiation induction from mesoderm stem cells to nervous system cells by the methods (1) and (2) above shows that SH2 and SH3, which are the best of mesodermal cells, disappear (marker of neural stem cells) Instead, nestin was negative), but instead, the neural stem cell marker nestin was positive (Figure 1).

Induced differentiation from mesoderm stem cells to neural cells, the cultured cells also changed morphologically. Specifically, cells that were single cells formed a neurosphere (Fig. 1). It was also confirmed that neural cells (NSE positive) and glial cells (GFAP positive) differentiated 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

By applying ischemic stress to the brain and adding the ischemic brain extract to the cultured mesoderm stem cells, we 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 could 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 opened through the anterior abdominal wall to expose the heart and ascending aorta. An incision is made in the apex and a tube is inserted from the left ventricle into the ascending artery. Next, make an incision in the right atrial appendage, Saline was perfused for 3 minutes to perform full 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 whole cerebral 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 NP 匪 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 ^ m membrane filter to obtain an ischemic brain extract.

(2) Induction of differentiation of cultured mesodermal stem cells (MSC) into neural stem cells

The MSC cells 100 mm non-coat ing dish ( IWAKI), conditioning mediu m (3 types: otherwise), incubation was continued at 37 ° C 5% C0 ₂ conditions. At 90% confluent, the medium was aspirated with a Pasteur pipette and rinsed 3 times with Dulbecco's PBS. 2 ml of 0.05% Trypsin, 0.02% EDTA in PBS was added and incubated at 37 ° C for 2-5 minutes until the cells detached. 2ml of conditioning medium was added to stop Tryps in reaction. The supernatant and detached cells were collected in a centrifuge tube with a Pasteur pipette, pipetted several times, and then centrifuged at 100 rpm for 5 minutes. The supernatant was discarded and NPMM was added to resuspend. The cells were seeded in 50% NP 匪 and 50% ischemic brain extract medium that had been warmed at 37, and cultured in suspension in 100 ° non-coating dish (IWAKI) at 37 ° C and 5% C02. , 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), demyelination model (Fig. 5) As a result of the study in (1), it was found that the cells had the same regenerative ability as neural stem cells extracted and cultured from the brain. [Example 5] Differentiation induction from ES cells to neural cells

Mouse ES cells in 100 mm gelatin-coating dish (IWAKI), conditioning medium 20 ml (DMEM, 10% FCS, 100 ^ M 2-mercaptoethanol, 1000 UNITs / ml ESGRO (CHEMICON)), 37 ° C 5% and the culture was continued at C0 ₂ of the conditions. At 90¾ confluent, blot the culture with a Pasteur pipette and rinse three 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. The FCS was 400 1 calories and the Trypsin reaction was stopped. 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. Plated on 50% NPMM, 50% ischemic brain extract medium which had been warmed to 37 ° C, 37 ° C, 5 C0 under ₂ conditions, and cultured in suspension 100 mm non-coating di sh ( IWAKI). bFGF 10 ng / ml and EGF 10 ng / ml were added every day. Industrial applicability

The present invention greatly contributes to the treatment of cerebral infarction, dementia, spinal cord injury, demyelinating disease and the like. In addition, the present invention is more general and can be applied to nerve transplantation / regenerative therapy for cranial nerve injury in a wide area. In other words, 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 and peripheral nervous systems.

Furthermore, the differentiation inducing method of the present invention provides a clue to unravel the mechanism of differentiation from mesoderm stem cells or ES cells into 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, “gene therapy” to promote regeneration of nerve tissue It is highly expected that it will be possible.

Claims

The scope of the claims

1. Mesodermal stem cells or ES cells contained in a mononuclear cell fraction isolated from bone marrow fluid or umbilical cord blood collected from a vertebrate animal in a basic culture medium at a temperature of 33 ° C to 38 ° C Induction to nervous system cells by culturing in

2. 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 2 000 rotations and sufficient time for separation according to the specific gravity. The method according to claim 1, which is a cell fraction that can be prepared by collecting a cell fraction contained in the range of g / nil to 1.lg / ml.

3. Mesodermal stem cells have the characteristics of SH2 (+), SH3 (+), SH4 (+), CD29 (+), CD44 (+), CD1 4 (-), CD34 (-), CD45 (-) The method according to claim 1 or 2, which is a cell.

4. The method according to claims 1 to 3, wherein the neural cells are selected from the group consisting of neural stem cells, neural progenitor cells, neural cells, and Daria cells.

5. The method according to claim 1, wherein bFGF, EGF or ischemic brain extract is added to the basic culture solution.

6. A cell obtained by the method according to any one of claims 1 to 5.

7. A composition for treating a nervous system disease comprising the cell according to claim 6.

8. Nervous system diseases include central and peripheral demyelinating diseases, central and terminal degenerative diseases, stroke, brain tumors, higher dysfunction, mental illness, epilepsy, traumatic nervous system diseases, and spinal cord The composition according to claim 7, wherein the composition is selected from the group consisting of infarctions.

9. A method for treating a nervous system disease, comprising transplanting the cell according to claim 6 or the composition according to claim 7 into a recipient.

1 0. Nervous system diseases include central and peripheral demyelinating diseases, central and peripheral degenerative diseases, strokes, brain tumors, higher dysfunction, mental illness, cancer, traumatic nervous system diseases, and 10. The treatment method according to claim 9, which is selected from the group consisting of spinal cord infarction.

1 1. The treatment method according to claim 9 or 10, wherein the cells to be transplanted are derived from a recipient.