WO2010110602A2 - Method for regenerating dopaminergic neurons in the substantia nigra pars compacta using human adult stem cells or precursor cells - Google Patents

Abstract

The present invention relates to a method for regenerating or restoring dopaminergic neurons from human adult stem cells or neural precursor cells obtained by the ex vivo culturing of the human adult stem cells. More particularly, the present invention relates to a method for regenerating or restoring dopaminergic neurons in the substantia nigra pars compacta using human adult stem cells or neural precursor cells differentiated therefrom. The method of the present invention can serve as an efficient source for cell replacement therapy for neurological diseases such as Parkinson's disease.

Description

Regeneration of Dopamine Neurons in Black Density Using Human Adult Stem Cells or Progenitor Cells

The present invention relates to a method for regenerating or restoring dopamine neurons in dentin of black matter using human adult stem cells or progenitor cells.

Of the various organs of the human body, the brain is particularly vulnerable to ischemia. When the blood flow to the brain is completely blocked for even five minutes, the death of vulnerable neurons begins. In part, the marked vulnerability of brain tissue to ischemic injury is due to the high metabolic rate of the brain and the limitation of energy substrates that rely entirely on glucose, which has limited brain accumulation.

In addition, it has been demonstrated that exposure to moderate hypoxia early in birth does not cause neurological damage but selectively disrupts synaptic function, and permanent hypoxic changes occur when exposed to hypoxia during development. It is known that dopamine plays an important role with excitatory amino acids, but the mechanism of brain damage of dopamine is not fully understood, but there are two possibilities.

First, catecholamine neurotransmitters are characterized as oxidative promoters. Oxygen free radicals produced by dopamine through autoxidation are directly toxic to neurons. Second, when hypoxic-ischemic dopamine is associated with neuronal receptors It binds and acts on brain cell death, which has been shown to have neuroprotective effects when administered dopamine receptor antagonists. However, changes in monoamines and their metabolites in the immature brains of neonatal rats have been studied in the context of transient hypoxia or anaerobics, and very few studies of relatively severe brain injury with ischemia.

In addition, the deficiency of dopamine in the brains of animals including humans causes various serious disorders. Among them, the movement disorder is particularly serious, which is well known in patients with Parkinson's disease. Parkinson's disease usually occurs after the 50s, and the dopamine deficiency in the striatum is caused by the death of dopaminergic neurons in the melanoma of the middle brain. This dyskinesia is almost the same in experimental animals treated with 6-hydroxydopamine (OHDA).

Dopamine neurons are present in the ventral and ventral side of the middle brain and regulate postural reflection, locomotion, and reward-related behavior. These neurons stimulate many structures in the entire brain, and brain damage, Parkinson's disease, schizophrenia, and drug addiction due to hypoxia and ischemia are associated with their degeneration or dysfunction (Hynes et al., 1995, Cell 80: 95-101). Among them, Parkinson's disease is a neurodegenerative disease caused by the death of dopaminergic neurons in the brain regions that control movement. Regression causes a lack of brain signaling chemistry (neurotransmitter) known as dopamine and causes motor disorders specifically observed in the disease, such as bradykinesia, akinesis, anxious posture, and stiffness.

Therapies that can replace neurons lost in these neurodegenerative disorders and neurological diseases are gene therapy or cell transplantation.

In particular, in connection with the latter method of cell transplantation, it has been shown that transplantation of dopamine neurons in human fetuses has a beneficial effect (Freed et al., 2001, N. Engl. J. Med. 344: 710). -719). However, fetal niger transplantation therapy requires human fetal tissue obtained from at least 5-10 weeks fetus to have a clinically effective effect in the patient. Therefore, in order to treat degenerative neurological disorders and neurological diseases, alternative sources of neurons such as dopamine neurons are urgently required.

In addition, there is still a problem that differentiated dopamine neurons from human embryonic stem cells are obtained in low yield compared to the total cell ratio in the population, and problems with the formation of teratoma by undifferentiated cells at the time of transplantation. Still remains undecided. Therefore, cell replacement therapy that requires a large number of dopamine neurons is not effectively used.

The present inventors have obtained nestin-positive neural progenitor cells up to 100% of the adult stem cells from human placenta-derived pluripotent adult stem cells, and transplanted them into the striatum of hypoxic ischemic (HI insult) mice. It was confirmed that dopamine neurons were regenerated and restored in the substantia nigra pars compacta (SNc) to improve the motility of the rats, and completed the present invention.

Summary of the Invention

Disclosure of Invention An object of the present invention is to provide a cell population for regenerating or restoring dopamine neurons in the densely packed region, a method for producing the cell population, and a method for regenerating and restoring dopamine neurons using the cell population.

It is another object of the present invention to provide a composition for regenerating and restoring dopamine neurons in the densely packed region of black matter and for restoring (restoring) the composition of the neural liner circuit.

In order to achieve the above object, the present invention provides a method for generating a dopamine neuron regeneration and cell population for dendritic cells in black dense, including the differentiation of primate adult stem cells or neural progenitor cells.

At this time, the method for differentiating the adult stem cells into nestin-positive neural progenitor cells may be characterized in that only adult stem cells are aggregated without specific ectoderm differentiation-inducing substance. The differentiated neural progenitor cells are characterized by expressing one or more genes selected from the group consisting of Nestin , Dcx, Sox1 and HuD . Transplantation of the neural progenitor cells into the striatum is characterized by the effect of regeneration and restoration of dopamine neurons in the dense cortex.

In addition, the present invention provides a cell population for regeneration of dopamine neurons in the dense part of the black matter, containing primate adult stem cells or neural progenitor cells differentiated from the same, wherein the cell population is dense black matter. Dopamine neurons are regenerated and reconstructed in the dendritic region of the vaginal region by inducing differentiation or maturation into dopamine neurons in the region.

The present invention also provides a method for regenerating and restoring dopamine neurons in the dendritic region of black matter using primate adult stem cells or cell populations containing the neural progenitor cells that differentiated them.

Accordingly, the present invention provides the use of "regeneration and restoration of dopamine neurons in black densities" of a population of cells containing adult stem cells and / or neural progenitor cells for regenerating dopamine neurons in the dentin.

Preferably, the composition for regenerating and restoring dopamine neurons in the dendritic region of the black matter, containing primate adult stem cells or a population of nestin-positive neural progenitor cells differentiated from the adult stem cells, and for restoring the black ancestral circuit (for repair) ) Provides a composition.

Other features and embodiments of the present invention will become more apparent from the following detailed description and the appended claims.

1 is a result of karyotype analysis by proliferating cell number and Kimja staining of hPLC.

Figure 2 is a result of flow cytometry (FACS analysis) showing the surface expression of hPLC.

3 shows the results of RT-PCR analysis of hPLC.

Figure 4 is a micrograph according to the differentiation of hPLC into neural progenitor cells, after the formation of the ellipsoid (B) by the undifferentiated hPLC (A), hPLC aggregation (B), after the ellipsoid culture (C) and when the cells are filled in the culture dish The case of proliferation (D) is shown.

5 is a result of Western blot analysis according to the differentiation step [Nv: undifferentiated hPLC; Sph: Ellipsoid Formation; + P1: passage 1; + P3: passage 3].

Figure 6 shows the results of RT-PCR analysis of markers of neural progenitor cells according to differentiation stage [Nv: undifferentiated hPLC; Sph: Ellipsoid Formation; + P1: passage 1; + P3: passage 3].

FIG. 7 is a micrograph showing damage of dopamine neurons (A) and disruption of the melanoma progenitor circuit (B) in VTA and SNc following hypoxic ischemic injury.

Figure 8 shows the results of the rotarod test for the motor coordination ability of the rat after implanting the neural progenitor cells of the present invention in the striatum (black bars: control group (n = 14); Gray bar: hP + transplant group (n = 22); White bar: normal rat group (n = 16)].

FIG. 9 is a photograph related to differentiation in the striatum after neural progenitor cells transplanted using specific antibodies (green) and HN antibodies (red), showing NeuN-positive cells (A), GFAP + showing differentiation into mature neurons. / HN + double positive cells (B) and TH positive cells (C) showing differentiation into dopamine neurons.

10 is a photograph showing the development of TH-immunoreactive fiber structure in striatum [A: experimental group 2 weeks after transplantation; B: experimental group 8 weeks after transplantation; C: control group.

11 is a photograph showing regeneration of dopamine neurons in SN and VTA over time after transplantation [A: 2 weeks after transplantation; B: 4 weeks after transplantation; C: 8 weeks after transplantation].

12 is a photograph showing dopamine neuron regeneration in SNc using TH (red) and BrdU (green) antibodies.

Figure 13 is a photograph showing the recovery of the coronary ancestral circuit by regenerated dopamine neurons, when A is labeled with FG (Fluorogold), B is labeled with FG and TH, C is all TH + cells of SNc and VTA When half of the TH + cells of FF were labeled with FG, D represents the case of FG (white), TH (red) and BrdU (green).

14 is a micrograph of the SVZ cross section immunostained with an antibody against Dcx [a: control, b: experimental group].

15 is a photograph confirming the presence of nestin-positive cells in normal SNc (FIG. 15 a), SNc implanted with control after injury (FIG. 15 b), and SNc implanted with hP + after injury (FIG. 15 c).

Detailed Description of the Invention and Specific Embodiments

In one aspect, the present invention relates to a cell population for regenerating and restoring dopamine neurons in a dense black region containing primate adult stem cells or neural progenitor cells differentiated therefrom and a method for producing such cell populations.

In another aspect, the present invention relates to the use of such cell populations, specifically, primate adult stem cells or differentiated nestin-positive neural progenitor cells, for example, which regenerates or restores dopamine neurons in dense black matter. Use in the form of a composition; And use in the form of a composition for restoring the choroid circulatory circuit by striatum targeting of the dopamine neurons regenerated (restored) in the choroidal region.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated in detail.

Isolation of Human Adult Stem Cells

Stem cells used in the present invention are adult stem cells. Preferably, they are adult stem cells of primates, more preferably humans.

Stem cells (stem cells) refers to the undifferentiated cells of the stage before the differentiation (differentiation) to each cell constituting the tissue, and refers to the differentiation to specific cells by a specific differentiation stimulation (environment). Stem cells, unlike differentiated cells that have ceased cell division, are capable of self-renewal by cell division and thus have proliferation (expansion) characteristics. Differentiation into cells can be differentiated into other cells by different environment or differentiation stimulus, so it has plasticity in differentiation. These stem cells can be divided into embryonic and adult stem cells according to their origin. In the present invention, adult stem cells are used instead of embryonic stem cells, which have many biological, ethical and legal problems, and thus have many limitations in clinical applications.

The "adult stem cell" is a stem cell extracted from grown body tissue, and is a primitive cell just before being differentiated into cells of a specific organ. Adult stem cells are difficult to proliferate and have a strong tendency to differentiate, but instead can differentiate into tissue-specific progenitor cells inherent in the human body. Adult stem cells can be differentiated to become cells with a variety of properties, and have the potential to generate replacement cells for a wide range of tissues and organs such as the heart, pancreas, nerve tissue, muscle, and cartilage. The present invention relates to differentiation into cells that make up neural tissue. More specifically, it relates to the differentiation of human adult stem cells or neural progenitor cells differentiated from dopamine neurons.

Such adult stem cells can be obtained from most tissues such as primates, preferably human bone marrow, fat, umbilical cord blood, blood, liver, skin, gastrointestinal tract, placenta, uterus, brain, pancreas, liver, eyes and fetal tissue. have. In one embodiment, placental stem cells that are capable of differentiating to a variety of tissues, are easily manipulated in vitro, and have immunomodulatory capacity, can be used, for example, in the first trimester of pregnancy. Placenta can be used for 5 to 7 weeks.

As a method for separating adult stem cells from various tissues of humans, methods conventional in the art, which are appropriate for each tissue, can be used. For example, the obtained specific tissues are separated into single cell bodies by treatment with trypsin solution and / or collagenase, and then cultured in a suitable medium in which growth factors such as bFGF and EGF are added in an appropriate amount. Then, it can be separated by FACS or the like can be used to isolate adult stem cells according to the growth rate.

Generation of neural progenitor cells

The present invention utilizes the obtained primate adult stem cells or neural progenitor cells differentiated from the adult stem cells.

The obtained primate adult stem cells can be differentiated into neural progenitor cells by conventional methods known in the art. Specific examples of the differentiation method include the following methods.

Adult stem cells can be carried out by culturing in a medium containing biochemical differentiation inducers such as soluble factors and growth factors to differentiate into ectoderm cells such as neural progenitor cells.

At this time, the "growth factor" refers to a protein that binds to the receptor on the cell surface as a primary result of cell proliferation and differentiation activity, and as soluble factors, for example, neurotrophin, mitogen, stem cell factor , Growth factors, differentiation factors (eg, TGF-β phase), TGF-β phase and agonists, neurotrophic factors, oxidants, neurotransmitters and survival factors. Many soluble factors stimulate cell division in many different cell types, while others are specific for certain cell types. Common differentiating agents that specifically promote differentiation of neuronal cell types include progesterone, putrescine, laminin, insulin, sodium selenite, transferrin, neuroturin, sonic hedgehog (SHH), nogin, follistatin, epithelium Growth factor (EGF), fibroblast growth factor (e.g., FGF-4, FGF-8, basic fibroblast growth factor (bFGF), growth and differentiation factor 5 (GDF-5), neurotrophin 3 (NT-3) ), Neurotrophin 4 (NT-4), brain induced neurotrophic factor (BDNF)), transforming growth factor α (TGF-α), transforming growth factor beta-3 (TGF β3), platelet induced growth factor ( PDGF-AA), insulin-like growth factor (IGF-1), bone morphogenic proteins (BMP-2, BMP-4), glial cell induced neuronal factor (GDNF), retinoic acid (RA), midkine, ascorb Ligands for receptors complexing with acids, dibutyryl cAMP, dopamine and gp130 (eg, LIF, CNTF, SCF, IL-11 and IL-6) and the like.

As a method for differentiating adult stem cells to obtain neural progenitor cells, the above conventional differentiation method may be used, but may be differentiated by aggregation without using a biochemical derivative for ectoderm tissue induction.

The method uses a method of aggregating human adult stem cells to form an ellipsoid to differentiate into nestin-positive neural progenitor cells only by enhanced 'cell interaction' between the stem cells.

Aggregation of adult stem cells increases cellular interactions between the aggregated stem cells and differentiates them into ectoderm tissues, particularly neural tissue-producing precursor cells, without inducing other ectoderm differentiation-inducing substances. Or poly-D-lysine may be aggregated in a culture plate coated with an ellipsoid.

For example, when using placental-derived adult stem cells, polyethyleneimine or poly-D-lysine exerts a positive charge on the surface of the culture dish, enhancing the ability of the stem cells to adhere to the surface, but at a high concentration. When cultured, stem cells do not adhere to the surface of the culture plate and aggregate to form an ellipsoid. The preferred number of stem cells to culture is 0.2 x 10 ⁵ to 2.5 x 10 ⁵ cells / cm 2, more preferably 1.0 x 10 ⁵ to 1.5 x 10 ⁵ cells / cm 2. In the case of culturing more stem cells than 2.5 x 10 ⁵ cells / ㎠, the ellipsoid formed was too large to maintain its shape, and in the case of culturing less than 0.2 x 10 ⁵ stem cells, most of them were cultured. It will adhere to the surface of the dish.

It is preferable that the ellipsoid formed by aggregation of pluripotent adult stem cells has a diameter of 50 to 400 µm, and more preferably, a diameter of 100 to 200 µm.

When the ellipsoid formed by the above method is cultured in a conventional medium, it is differentiated into ectoderm tissue, particularly neural progenitor cells for neurogenesis. This can be confirmed by the expression of most of the cells of the ellipsoid Nestin (B of Figure 3). Nestin is a cell marker unique to neural stem cells and neural progenitor cells, and the expression of nestin is a strong signal for ectoderm differentiation by enhancing the interaction between cells that have been aggregated into ellipsoids. Hints.

When the nestin-expressing cells are transplanted into a conventional culture dish, the nestin-expressing cells are grown outside the ellipsoid and form a rosette-like spread, and the method of the present invention All of the cells, ie 100% of the cells become nestin + (FIG. 4C).

As described above, the method of differentiation by the aggregation of adult stem cells not only exerts an excellent effect of 100% adult stem cells into neural progenitor cells, but also the process is very simple, which significantly shortens the time required for differentiation and differentiated cells. It has the advantage of reducing the risk of contamination by other substances by increasing the viability of, and by eliminating the process of differentiation-inducing drugs or gene conversion.

In the present invention, all neural progenitor cells obtained by differentiating adult stem cells by an appropriate method by use of a biochemical derivative for ectoderm tissue induction or by aggregation of stem cells can be used, but more preferably, biochemical for ectoderm tissue induction. Without using derivatives, ellipsoids are formed and cultured by aggregation to obtain and use neural progenitor cells.

Meanwhile, the progenitor cells obtained by differentiating adult stem cells in the present invention are characterized by expressing at least one or more of genes such as Nestin , Dcx, Sox1, and HuD . In one embodiment of the present invention, all of the nestin , Dcx, Sox1, and HuD genes may be expressed. The Dcx, Sox1 and HuD genes are expressed in early neuronal progenitor cells.

Regeneration and Restoration of Dopamine Neurons in Black Density

The present invention relates to a method for regenerating and restoring dopamine neurons in the dendritic region of black matter using human adult stem cells or neural progenitor cells obtained by differentiating them.

As used herein, the term "differentiation" means that an undifferentiated pluripotent stem cell or progenitor cell obtains a more specialized ending, wherein the differentiated cell has a phenotype that is characteristic of a particular cell type or tissue.

The term "dopamine neuron" as used herein refers to a neuron expressing tyrosine hydroxylase (TH). Dopamine neurons are specifically located in the middle brain melanoma and stimulate the striatum, limbic system and neocortex in vivo to regulate postural reflection, locomotion, and compensation-related behavior.

As used herein, the term "regeneration" refers to a phenomenon in which a portion of a formed organ or individual is lost when it is replaced, and "reconstruction" may also be referred to as "reconstitution". It means the construction and reconstruction of a tissue or organ from a cell or tissue dissociated once.

The present invention relates to a method of replenishing (regenerating) or reconstructing (recovering) damaged dopamine neurons in the 'black dense' region.

In one embodiment of the present invention, when the adult stem cells or neural progenitor cells obtained above are implanted into the striatum of an animal that artificially killed dopamine neurons with hypoxia and ischemia, it is a marker of dopamine neurons in the striatum. Not only did we show expression of tyrosine hydroxylase (TH) and GFAP, a marker protein of astrocytes, but also induces differentiation and maturation of dopamine neurons in the densely packed region of the vaginal region to regenerate and restore damaged dopamine neurons. Confirmed.

However, in general, neurogenesis in adults is known to occur only in two places: the subventricular zone (SVZ) of the entire brain and the subgranular zone (SGZ) of the hippocampus. The cells in the SVZ are arranged in a relatively small and dense form, not as a single cell but as a mosaic population of immature cells with slightly differentiation. The neural stem cells present in the SVZ are differentiated into neuroblasts, move through the RMS (rostral migration stream) to the olfactory bulb, and finally into the intermediate neurons (granule cell layer). ). The neural stem cells of SGZ are differentiated by traveling relatively close distances and finally function as projection neurons in the granule layer of the hippocampus dentate gyrus.

That is, since adult neurogenesis is known to occur only in the above two regions, the fact that "dopamine neurons can be regenerated in the black dense part" has great significance.

Substantia nigra is a nucleus that extends from the upper end of the cerebrospinal grey to the location of the subthalamic nucleus, almost the entire midbrain, between the crus cerebri and the mesothelial epithelium. Is divided into two parts: the pars compacta located on the inverted side and the pars reticulata located on the base of the cerebral base. In the dense part, large neurons are concentrated in the cytoplasm of melanin pigments, and these cells contain dopamine as a neurotransmitter. Cells of dense parts are large multipolar cells, nucleolus is large and distinct, and nistle bodies are large and darkly stained and distinct. The dopamine-containing neurons in the dense part of the cortex are mainly centrifugal fibers (striatum, caudate nucleus and conch nucleus putamen) that send centrifugal fibers (nigrostriatal fibers). The dopaminergic fibers, which are projected by the ancestors, are also called the ancestor related dopaminergic system.

As such, since the dopamine neurons are densely packed, the dense cortex is closely related to diseases characterized by damage to the dopamine neurons in the mesenchymal medulla, such as Parkinson's disease. It can be predicted that a replaceable treatment method will have a very large effect on the disease and the like.

The present invention is useful for such a cell replacement therapy because it uses the cells to promote the regeneration and restoration of dopamine neurons in the dentin of black matter.

At this time, the dopamine neurons regenerated in the dense parts of the black matter differentiate into dopamine neurons from the stem cells or precursors inherent in the dense parts of the black matter, not the differentiation of transplanted stem cells or neural progenitor cells that have been migrated from the striatum. Or by maturity. In other words, the dopamine neurons regenerated in the dense part of the vagina are not stem cells or neural progenitor cells migrated from the implanted striatum, nor dopamine neurons migrated from the SVZ, but are stem cells present in the dense part itself. Or matured and / or differentiated from precursors.

This means that by transplantation of adult stem cells or neural progenitor cells into the striatum, stem cells or precursors in the dentin are stimulated or induced to mature and / or differentiate into dopamine neurons, resulting in regeneration of dopamine neurons. do.

The present invention also provides the use of adult stem cells or neural progenitor cells to restore central nervous system function in another aspect. In particular, it provides a therapeutic use for neurological diseases caused by damage to dopamine neurons.

For example, these cells can be used therapeutically by implanting them directly into sites such as striatum, depending on the disease or condition to be treated. In addition, primate adult stem cells, preferably human adult stem cells or neural progenitor cells derived therefrom, may be used therapeutically by administration or implantation in the form of a composition containing a therapeutically effective amount. The present invention also includes a method for treating neurodegenerative disorders or neuropathies characterized by degeneration or modification of dopamine neurons.

The therapeutic use of the present invention may be advantageously carried out by direct implantation into the lesion site in the form of the neural progenitor cells or differentiated neurons of the present invention or compositions containing them. Methods of neurotransplantation and cell culture can use methods known to those skilled in the art.

In one embodiment, adult stem cells or neural progenitor cells of the invention may be directly implanted into the striatum of the patient. About eight weeks after transplantation, dopamine neurons are almost completely regenerated (restored) in the dense cortex, replacing the damaged dopamine neurons, resulting in animals with experimental lesions of the striatum dopamine functional system. Can restore motor function.

In addition, as the restored dopamine neurons are effectively targeted to the striatum in order to effectively perform the original neurotransmission role, they re-establish the destroyed black tissue circuits as in hypoxia and ischemia.

In another embodiment, the adult stem cells or neural progenitor cells for regenerating dopamine neurons may be provided in the form of a composition comprising PBS and an antioxidant (such as ascorbic acid).

Therefore, the present invention relates to a composition for the production of dopamine neurons in the dense part of the black matter, containing human adult stem cells or neural progenitor cells derived from the adult stem cells. In addition, since it reestablishes the neural crest circuit destroyed by targeting of the regenerated dopamine neurons to the striatum, in another aspect of the present invention, it contains primate adult stem cells or neural progenitor cells derived from the adult stem cells. The present invention relates to a composition for restoring (recovering) a black matter liner circuit.

The term "treatment" or "treat" as used herein is a therapeutic treatment and a means of prevention or prevention. Therefore, those in need of treatment include those already with neurodegenerative disorders or neuropathies, as well as those who wish to prevent neurodegenerative disorders or neuropathies. The method of the invention is not limited to treating humans, primates and any mammals in need of treatment, including livestock, breeding, pet or sport animals, such as dogs, horses, cats, sheep, pigs, cattle, and the like. Can be used. A "disorder" is any condition that benefits from treatment with two types of neuronal primitive cells, differentiated neurons or cells of the present invention. Examples of disorders that may benefit from dopamine neurons that are regenerated and restored by transplanting adult stem cells or neural progenitor cells include improper postural reflexes, migration and reward-related behaviors, such as those associated with Parkinson's disease, schizophrenia, and drug addiction. to be.

As used herein, a “therapeutically effective amount” of cells is used to stop or alleviate the physiological effects of a patient caused by the loss, damage, or degeneration of differentiated neurons, such as dopamine neurons, astroglia, and oligodendrocytes. That's enough.

The therapeutically effective amount of cells used will depend on the patient's need, the patient's age, physiological condition and health, the desired therapeutic effect, the size and area of tissue to be targeted for treatment, the extent of the lesion and the route of delivery chosen. For example, treatment of a disorder affecting a larger area of the brain may require more cells to achieve a therapeutic effect when compared to smaller target areas. In addition, the cells may be administered at one or more sites within a given target tissue in multiple small grafts of low cell dose. The cells of the invention can be completely isolated before transplantation, for example to form a suspension of single cells, or almost completely isolated before transplantation, for example to form small aggregates of cells. The cells can be administered by transplanting or migrating them to a predetermined tissue site and reconstructing or regenerating functionally deficient regions.

Appropriate ranges of cells that can be administered to achieve a therapeutically effective amount can be suitably used in accordance with the patient within the ordinary knowledge of those skilled in the art. For example, it may be about 1,000 to 10,000,000 cells, or more, but 2,000,000 to 4,000,000 may be preferable since a high dose may not completely exclude the possibility of turning into cancer cells.

The ability to regenerate dopamine neurons in black matter dense from adult stem cells as disclosed herein is of clinical relevance for transplantation treatment for various neurodegenerative disorders and neuropathies. For example, dopamine neurons are neurodegenerative disorders and neuropathies characterized by abnormalities in postural reflexes, movement and compensation-related behavioral regulation, such as hypoxic ischemic injury, Parkinson's disease, schizophrenia and drug addiction, as well as trauma, or It can be used to treat lesions caused by Parkinson-like conditions, such as other diseases that result in ataxia and postural abnormalities.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1 Isolation of Human Adult Stem Cells

(1) Isolation and Culture of Human Adult Stem Cells

The placenta, which is 5-7 weeks old at the beginning of the first trimester of pregnancy, was sterilely isolated from Maria Fertility Hospital, washed with Dulbecco's Phosphate Buffered Saline (DPBS), chopped, and then chopped at 37 ° C. for 5-20 minutes. Treated with trypsin solution (0/25% trypsin / 0.5 nM EDTA) to separate into single cell bodies. The isolated cells were recovered using 100 μm nylon mesh and then washed with DPBS.

After centrifugation, 10% fetal bovine serum (FBS), 4 ng / ml fibroblast growth factor, 0.1 mM beta-mercaptoethanol, and 1% non-essential amino acids Incubated in a Dulbecco's Modified Eagle's Media (DMEM) medium containing nonessential amino acids and antibiotics in a humidified chamber at 37 ° C. and 5% CO ₂ .

After 5 days, the adherent tissue debris was removed while changing medium. When cells proliferated and became dense (passfluent ¹ ) on the culture surface (75 cm ² ). Through this process, a total of 11 independent human placenta stem cells (hPLCs) were established.

(2) RT-PCR Analysis

Total RNA was extracted using Trizol reagent (Invitrogen), and cDNA was reacted for 15 minutes at 72 ° C for 15 hours using Superscript II (Invitrogen) and ₂₀ oligo-d (T) primers. Synthesized. Target sequences from cDNA were pre-denatured for 3 minutes at 95 ° C, denatured for 30 seconds at 95 ° C, annealed for 45 seconds at 53-60 ° C and 72 ° C using premix kits (Invitrogen, Bioneer) The process of amplification (extension) for 45 seconds was repeated 35 times. The amplified cDNA was isolated by 2% agarose gel and confirmed by staining with ethidium bromide.

As a result, it was confirmed that hPLC expresses Oct4, Nanog, Tbn, Klf4, and activin (FIG. 3). This is the same as that commonly found in omnipotent embryonic stem cells or undifferentiated embryonic cells prior to the onset of cystogenesis.

(3) FACS analysis

To analyze the surface antigens of the cells, the cells were harvested and rinsed with PBS added with 4 mM EDTA / 5% FBS, followed by 2% FBS / at 4 ° C. containing antibody bound to mouse IgG or FITC or PE (phycoerythrin). Treated at 4 ° C. and 30 min in PBS buffer. Cells were then washed and analyzed using FACS Vantage SE (Becton & Dickinson). 5 μg / ml of propidium iodide was added prior to FACS analysis to remove dead cells and debris. The results obtained were analyzed using the CellQuest program (Becton & Dickinson).

Flow cytometry was positive for CD13, CD9, CD29, CD44, CD90, CD105 and HLA-ABC; Negatives were shown for CD10, CD14, CD24, CD34, CD45, CD50, CD38, and HLA-DR (FIG. 2). These results are similar to those expressed in mesenchymal stem cells (MSCs) of umbilical cord blood. Expression of CD13, CD9, CD90 and CD105 and lack of CD45, CD10 and HLA-DR were common to both cells. In the hPLC of the present invention, the expression of CD34, which is an identification marker of MSCs, did not appear.

(4) Karyotype Analysis

About 70% of the cells grown on a 75 cm ² culture surface were exposed to 0.1 μg / ml of colcemid solution for 2 hours, collected and washed, and then washed for 20 minutes in a 75 mM KCL stock solution at 37 ° C. Treated. After incubation, the stock solution was washed, centrifuged and fixed with 5 ml of methanol / acetic acid (3: 1) fixative.

Fixed cells were harvested and resuspended in 0.5 ml of fixative. A drop of suspension was dropped onto a frozen slide at a height of 30 cm. Slides dried in air were exposed to 0.025% trypsin for 30 seconds and washed with PBS. The chromosomes were stained with 10% Giemsa solution and karyotype was analyzed by Cytovision (Applied Imaging).

As a result, there was no change in the number of chromosomes or G-banding in isolated hPLCs and showed the same degree of homeostasis even when continuously cultured for 90 to 150 days (FIG. 1).

(5) Histochemical analysis of the obtained hPLC

The cell lines incubated on a cover slip were fixed with 4% paraformaldehyde solution at room temperature for 15 minutes and then washed with PBS. The immobilized cells were permeabilized with 0.1% Triton X-100 / PBS containing 10% normal goat serum, blocking nonspecific antigens, and then blocking primary antibodies at room temperature. Treated and left for 1 hour. After washing, the secondary antibody was again treated at room temperature and left for 1 hour. Nuclei of cells were labeled with Hoeshst 33342 (10 μg / ml, Sigma) for 10 minutes and observed by fluorescence microscopy. The antibody used was anti-Nestin (1: 500, Santa Cruz).

As a result, about 30% of undifferentiated hPLC expressed Nestin (A of FIG. 4). This fact, together with the expression of Nanog, Oct4, Klf, etc., the RT-PCR analysis results, suggests that undifferentiated hPLCs have potential for differentiation into ectoderm tissues, and yet, specifically to specific tissues Shows that this has not been done.

Example 2 Formation of Neural Progenitor Cells

Example for a hPLC obtained in 1 to induce differentiation into neural progenitor cells of the 0.2 x 10 ^{5 cells / cm 2 0.01 ~ 1} % polyethylenimine-coated culture dishes and poly -D- lysine (0.1 ~ 100 ㎍ / ml) were each incubated in a coated dish. On day 2, stem cells aggregated together to form an ellipsoid. The stem cell ellipsoids were examined for response to nestin antibodies.

As a result, as shown in Fig. 4, the expression of nestin was increased at the time of ellipsoid formation (Fig. 4B) and rosette-like spread was obtained when the ellipsoid was transferred to a general culture dish and cultured. Formed and most of the cells, ie 100% of the cells, expressed nestin (FIG. 4C). In addition, even when passaged for more than one month it was confirmed that the expression of the nestin is still maintained (D in Fig. 4). Nestin induction at each step was confirmed by Western blot (FIG. 5). And the induced phenotype was maintained even if cryopreserved for more than 2 months (data not shown).

In addition, RT-PCR extraction of RNA from differentiated neural progenitor cells resulted in the loss of expression of genes associated with pluripotency and marked expression of Sox1 , Dcx , and HuD mRNA , which are characteristic of neural progenitor cells. It was confirmed that the increase (Fig. 6).

These results suggest that enhanced cell interaction is a potent and efficient inducer of ectoderm differentiation in hPLC.

Example 3 Regeneration of Dopamine Neurons in the Gray Density

(1) HI-induced dopamine deficiency in neonatal rat brain

Electrical paralysis of both carotid arteries in young rats at 5 days of age resulted in several brain damages due to ischemic and hypoxic insult, but the ventral overtegmental area (VTA) and SNc Hypoxic insults resulted in a gradual killing of tyrosine hydroxylase-positive (TH +) cells in hypoxic insult (FIG. 7A). Regression of dopamine neurons in SNc disrupted the cortical circuit and subsequently resulted in the loss of TH-immunoreactivity in the striatum (FIG. 7B).

(2) transplantation into striatum

Two weeks after hypoxic ischemic injury, mice were anesthetized with Equithesin and mounted on a stereotaxic frame (David Kopf Instruments) for transplantation. The skull was opened and 2 x 10 ⁵ hPLC in 4 μl 0.5% glucose / PBS were implanted into the four sites as follows, based on the bregma in the bilateral striata.

AP +0.5 mm, ML +1.2 mm, DV -4.5 mm;

AP +0.5 mm, ML -1.2 mm, and DV -4.5 mm;

AP +0.5 mm, ML +1.2 mm, and DV -3.3 mm;

AP +0.5 mm, ML -1.2 mm, and DV -3.3 mm relative to bregma ⁵⁰

(Where AP stands for anterior-posterior, which means +0.5 mm forward from the bregma of the brain, ML stands for medial-laterla, left and right, +1.2 mm stands for 1.2 mm to the right, and DV stands for Abbreviation for dorso-ventral, dorsal-dorsal, and -4.5 mm means 4.5 mm downward from the surface of the brain.)

1 μl of cell suspension was injected at each site for 5 minutes, and then the needle was slowly removed. Cyclosporin A (10 mg / kg, Novartis) was intraperitoneally administered daily throughout the survival period.

(3) Behavioral test

Two weeks after transplantation, a rotarod test was performed on mice transplanted with progenitor cells differentiated from hPLC. As a control, glucose / PBS was transplanted.

Accelerated Rotarod (Ugo Basile) can measure the motor coordination ability of the rat. First, a 10-minute session was trained to stay on a spinning rod at a low speed of 5 rpm three or more times, and then the fall latency of the animals was measured on the rod accelerated to 5 rpm at 5 rpm for 600 seconds or longer.

As a result, it was confirmed that the transplanted animals showed a significant improvement in motor activity compared to the control group (FIG. 8). In addition, in the group of animals transplanted with adult stem cells or neuronal precursor cells (hP +) derived therefrom, the time for the animals to stay on the rotating rods is gradually increased, so that at 8 weeks of transplantation, the performance of the animals is almost similar to that of normal animals. Showed.

Animals were anesthetized by overdose with Equithesin, heparin solution (1 U / ml PBS) was perfused through the heart to remove blood, and then tissues were fixed by perfusion in 4% paraformaldehyde dissolved in PBS. The brains were harvested from fixed animals and the tissues equilibrated to 30% sucrose at 4 ° C. for 2 days until they sank to the bottom. Once equilibrated, the tissues were mounted with OCT compounds (Sakura Finetech) and frozen sections (-20 ° C.) to a thickness of 10-40 μm, followed by histological analysis. Using the method of Example 5 (5), the antibody is anti-TH (1: 500, Santa Cruz); anti-BrdU (1: 200, Roche); anti-HN (1: 500, Chemicon); anti-NeuN (1: 500, Santa Cruz); And anti-GFAP (1: 500, Sigma) were used.

As a result, 8 weeks after transplantation, the presence of cells having a human nuclei in the striatum of an animal transplanted with hP + was confirmed. Some of the transplanted neural progenitor cells differentiated into mature neurons (FIG. 9A) and some were dopamine cells expressing TH protein (FIG. 9C). In addition, the presence of GFAP-expressing astrocytes was also partially confirmed. This suggests that hP + can differentiate into other cell types in the host brain (FIG. 9B).

These results show that the final differentiation and / or maturation of transplanted neural progenitor cells can be effected by the host microenvironment and partially relieve host brain disease by production of dopamine neurons in the striatum.

(4) Dopamine Neuron Regeneration and Restoration in Black Density

Although transplanted hP + was observed to differentiate into TH + cells, the number of TH + cells in the striatum did not increase proportionally with the gradual improvement of motor activity until 8 weeks after transplantation. Following this discrepancy, we tested whether recovery of normal activity was due to dopamine neuronal regeneration in SNc.

Fluorochrome LLC was diluted with 0.9% saline to make a 2% working solution. According to the following six experimental designs, the diluted solution (0.2 μl) was injected from the bregma to the following coordinates using a stereotaxic frame.

AP +1.1 mm, ML +2.0 mm, DV -3.5 mm;

AP +1.1 mm, ML +2.0 mm, DV -2.5 mm;

AP +0.0 mm, ML +2.0 mm, DV -3.75 mm;

AP +0.0 mm, ML +2.0 mm, DV -2.75 mm;

AP -0.8 mm, ML +2.75 mm, DV -3.75 mm; And

AP -0.8 mm, ML +2.75 mm, DV -2.5 mm relative to bregma ⁵⁰ .

By this method all TH + cells in the striatum were successfully labeled (FIG. 10).

As a result, a surprising increase in the number of TH + cells was clearly observed in both SNc and VTA 2 two weeks after transplantation (FIG. 11A). Thereafter, the number of TH + cells increased markedly on both sides, at 4 weeks the overall morphology of VTA was close to normal (FIG. 11B), and regeneration of dopamine neurons at SNc was nearly complete at 8 weeks after transplantation. (FIG. 11C).

Bromodeoxyuridine (BrdU, Sigma) was used to more closely observe the regeneration of dopamine neurons in the dentin. BrdU (Sigma) was diluted in 0.9% saline and administered intraperitoneally at 50 mg / kg daily until rat death. BrdU is an analog of thymine and can be infiltrated into the DNA of cells when the DNA is replicated in large quantities. Therefore, this is the most efficient way to specifically label only new cells that are in the process of cell division during injection. Brain sections (30 mm thick) of animals injected with BrdU were treated in 2 N HCl for 1 hour at room temperature, then neutralized twice with 0.1 M borate buffer (pH 8.5) for 10 minutes and then washed with PBS. In order to ensure the cell membrane permeability of the tissues were treated for 1 hour in PBS containing 0.3% Triton X-100 and 3% BSA in PBS, and then exposed to antibodies against BrdU overnight at 4 ℃. Thereafter, washings and secondary antibodies were treated.

As a result of the administration of BrdU, it was shown that many neurons were newly generated in or near SNc, and they were surrounded by TH + cytoplasm (FIG. 12A). Observation of BrdU / TH double positive cells in both directions with three-dimensional confocal microscopy revealed that both signals were localized within a single cell representing newly generated TH + neurons. Consecutive microscopic observation of cells through z-axis at 0.5-mm intervals revealed that the BrdU and TH proteins were in the same cell in a single cell (FIG. 12B). That is, the regeneration of dopamine neurons in the SNc region was clearly confirmed.

On the other hand, an increase in many TH + cells was clearly observed in both SNc and VTA 2, and because dopamine neurons produced in SVZ can travel long distances from the SVZ via the rostral migratory stream (RMS), Dopamine neuron migration from the SVZ was to determine whether the regeneration in the SNc region.

To this end, the SVZ cross section by immunostaining with antibodies to Dcx, it was confirmed the movement of Dopaminergic Neurons immature cells that can contribute to reproduction in the SNc.

Compared with the SVZ of the control transplant group (FIG. 14 a) and the hP + transplant group (FIG. 14 b), there was no significant difference in the amount of Dcx + cells. New dopamine neurons have been identified as unlikely to migrate.

Thus, hP + implantation into the striatum induced adult stem cells or precursors in the SNc region to induce maturation and / or differentiation into dopamine neurons, indicating that damaged dopamine neurons were regenerated and restored within the SNc itself. have.

For further confirmation, the presence of nestin positive cells in normal SNc (FIG. 15 a), SNc with control implanted after injury (FIG. 15 b), and SNc with hP + implanted after injury (FIG. 15 c) were examined.

As a result of immunohistochemical analysis, nestin-positive progenitor cells were present in SNc of normal animals, and it was confirmed that neural progenitor cells involved in SNc regeneration were intrinsic in the SNc region. And the hypoxic ischemic injury (HI insult) through the fact that the expression of the nestin protein in the SNc significantly increased, it was confirmed that the damage activates the precursor inherent in the SNc. In other words, it can be seen that the precursors inherent in the SNc itself are activated by hypoxic ischemic injury, which tends to promote the regeneration of nerve cells.

Moreover, in histological studies with human nuclei (HN) antibodies, there is no evidence indicating that the nestin-positive cells in these SNc are hP + cells that have migrated from the implanted site (the striatum) to the SNc region, and furthermore, real-time RT- PCR results indicate that 3.5-fold induction of Nestin mRNA occurs in hypoxic ischemic-damaged SNc (not shown). These findings suggest that dopamine neurons regenerated in SNc have been transferred from striatum or neural progenitors. The differentiation or maturation of dopamine neurons from stem cells or precursors inherent in SNc, not the differentiation of cells.

That is, dopamine neurons regenerated in SNc are not caused by stem cells or neural progenitor cells migrated from the striatum, nor dopamine neurons migrated from SVZ, and are matured from stem cells or precursors present in SNc itself. And / or differentiated.

However, when adult stem cells or neural progenitor cells are transplanted into the striatum, they do not differentiate and move directly to SNc. However, the transplantation stimulates the stem cells or precursors embedded in SNc to mature and / or differentiate into dopamine neurons. Or induces the regeneration of dopamine neurons in the SNc.

Transplantation of adult stem cells or neural progenitor cells into the striatum may function to induce maturation and / or differentiation from stem cells or precursors present in the SNc.

(5) Restoration of the black ancestral circuit

At 4 weeks after transplantation, TH + fibers developed in the striatum. At 8 weeks, the degree was not different from normal animals. With the advent of abundant TH + fibers in these striatum and the gradual improvement of motor activity, the dopamine neurons regenerated in SNc predict the neural pathways to the corresponding striatum in their original roles, and in fact the restoration of the stromal ancestral circuit was achieved. Whether or not was observed.

To this end, retrograde tracer fluorogold was injected into the striatum three days before the rats were killed (FIG. 13A), and brain sections were immunostained to confirm the presence of TH protein.

As a result, as expected, many cells appeared pink by double staining of TH and fluorogold (B, C, D of FIG. 13). This indicates that TH + cells took up fluorogold from their ends in the striatum.

In addition, in order to determine whether double positive (fluorogold + / TH +) cells were also newly formed cells, the brain sections of animals injected with BrdU and fluorogold showed that some of the fluorogold + / TH + cells were clearly BrdU positive. (FIG. 13D).

These results show that the cells of fluorogold + / TH + / BrdU + are dopaminergic neurons regenerated in the dentin of the vaginal cells, and play a role in transmitting nerves to the corresponding striatum, and further show that the stromal ancestral circuit has been restored.

Transplanting human adult stem cells or neural progenitor cell populations differentiated from the same according to the present invention may induce differentiation or maturation of dopamine neurons in the dense part of the cortex, thereby regenerating and restoring dopamine neurons in the region. Since the dopaminergic neurons are directly replaced in the dentin region of the vaginal region, they may be used as more effective cell replacement therapy for neurological diseases related to ischemic hypoxia damage.

Having described the specific part of the present invention in detail, it is obvious to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (10)

1. A cell population for regeneration of dopamine neurons in black matter densities containing neural progenitor cells differentiated from adult stem cells, which express at least one gene selected from the group consisting of nestin , Dcx, Sox1 and HuD genes.
2. The cell population according to claim 1, wherein the differentiated neural progenitor cells are obtained by culturing an ellipsoid formed by aggregation of adult stem cells.
3. The cell population according to claim 2, wherein the ellipsoid has a diameter of 100 to 200 µm.
4. A method of producing a population of dopamine neurons in the dendritic cells of black matter, comprising aggregating and culturing an ellipsoid by aggregating primate adult stem cells.
5. The method of claim 4, wherein the aggregation of the adult stem cells is performed in a culture vessel coated with polyethyleneimine or poly-D-lysine.
6. 6. The method of claim 5, wherein the aggregation of the adult stem cells is carried out in a medium containing no biochemicals for ectoderm differentiation.
7. A composition for regenerating dopamine neurons in black matter dense parts containing primate adult stem cells or neural progenitor cells differentiated from the adult stem cells.
8. The composition of claim 7, wherein the neural progenitor cells express one or more genes selected from the group consisting of nestin , Dcx, Sox1 and HuD .
9. Primate adult stem cells or neural progenitor cells containing neural progenitor cells differentiated from the adult stem cells, the composition for restoring (recovery).
10. The composition of claim 9, wherein the neural progenitor cells express one or more genes selected from the group consisting of nestin , Dcx, Sox1, and HuD .

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