TWI307717B - Placental stem cell and methods thereof - Google Patents

Description

1307717 发明Invention Description: [Technical Field] The present invention relates to a stem cell derived from a postpartum placenta and a method for obtaining and cultivating the same. The invention further relates to compositions comprising placental stem cells and methods of using placental stem cells. [Prior Art]

Stem cells have the potential to differentiate into various cells in the body. In theory, stem cells can divide and proliferate without restriction to supplement the number of their cells. When stem cells divide, each new cell maintains the state of the stem cells or becomes a seed cell having a specialized function, such as muscle cells, red blood cells, or brain cells. Stem cells are usually classified into totipotent, pluripotent, and multipotent. Almighty stem cells have the ability to grow into all the different types of cells in the body, including germ cells. For example, a fertilized egg is a versatile stem cell. The pluripotent stem cells grow into any cell in the body other than the cell type required to develop the fetus. Limited-potential stem cells can only grow into two or more different cell types in a given organ or a designated tissue. Compared to stem cells, progenitor cells (progenitwceH, or mother cells) are unable to self-renew and can only grow into a few cells. Embryonic stem cells ((1) and adult stem cells are the main source of stem cells. Embryonic stem cells are derived from embryos. For the purpose of research, it can be carried out in a clinical clinic such as Body 3 1307717 for donor consent. In vitro fertilization to make the egg develop into an embryo' and obtain embryonic stem cells from it for research. Usually the embryo can be obtained on the 4th or 5th day after fertilization. At this time, the embryo is like a micro-organ composed of cells. The globule, called blastocyst. The blastocyst contains three structures 'separate' 疋: trophoblast, which is a layer of cells surrounding the blastocyst; blastocoel, which is a blastocyst The inner cavity; and the inner cell mass, which is a group of cells concentrated at one end of the blastocyst and composed of about 3 cells.

These inner cell populations are isolated and cultured in vitro to obtain embryonic stem cells. The inner cell population is typically grown on a layer of feeder cells, typically mouse embryonic fibroblasts, which serve as an attachment layer and source of nutrients for the inner cell mass. Embryonic stem cells are pluripotent stem cells that can be made into all kinds of cells in the body. An adult stem cell or somatic stem cell is an undifferentiated cell that can be found in differentiated cells of an organ or tissue. Adult stem cells can self-replicate and renew (cell self-renew) and can differentiate into specific organs or tissues. Cell Types The scientific community believes that having a specific region in each tissue allows adult stem cells to remain inactive for many years (ie, not splitting) until they are activated by factors such as disease or tissue injury. . The number of adult stem cells present in each tissue is extremely small, and adult stem cells have been found in tissues and organs including brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, umbilical cord, adipose tissue, alpaca, and liver. 4 1307717

Since stem cells provide a source of new cells or tissues, stem cells play an important role in various diseases, physical abnormalities, and disability treatments. Hematopoietic stem cells (HSCs) used to form blood cells in the bone marrow are currently the only stem cells that have been commonly used. Hematopoietic stem cells are used to treat leukemia, lymphoma, and several transmissible blood diseases. However, other stem cells also have the potential to treat other diseases. Many reports have suggested that certain adult stem cells have the ability to differentiate into several types of cells. For example, hematopoietic stem cells may differentiate into brain cells (neural cells, oligodendrocytes, and astrocytes) (Haul et al., H. Hematother. Stem Cell Res. 12:23-32, 2003; Zhao et al., PNAS 100: 2426-2431, 2003; Bonilla et al., Eur. J. Neurosci. 1 5 : 575-5 82, 2002), skeletal muscle cells (Ferrari et al., Science 279: 1528) -1530, 1998;

Gussoni et al., Nature 401: 390-394, 1999), cardiomyocytes (Jackson et al., J. Clin. Invest. 107: 1395-1402, 2001) and hepatocytes (Lagasse et al., Nat. Med·) 6:1229-1234, 2000). Bone marrow stromal cells may differentiate into cardiomyocytes and bone myocytes (Galmiche et al., Blood 82: 66-76, 1993; Wakitani et al., Muscle Nerve 1 8: 1417-1426, 1 995), while brain stem cells may differentiate into hematocrit and bone path muscle cells rwsoM ei al., Science 283:534-547, 1999; Galli et al., Nat.

Neurosci. 3: 986-99 1,2000). Embryonic stem cells and adult stem cells have their own advantages and disadvantages in the application of cell regeneration therapy. One of the advantages of adult stem cells is that they can culture the cells of 5 1307717 themselves, enlarge the number of cells, and then replant them back into the patient's body. The use of the patient's own adult stem cells avoids the situation where the immune system rejects foreign cells without the use of immunosuppressive drugs. Conversely, if the embryonic stem cells provided by the donor are implanted into the patient, it may cause transplant rejection.

Conversely, embryonic stem cells can differentiate into all cell types in the body, although there is evidence in the above that there is evidence that adult stem cells may differentiate into other cell types, but usually adult stem cells can only differentiate into cell types of their own source tissues. . In addition, embryonic stem cells are relatively easy to culture to amplify the number, and the number of adult stem cells in adult tissues is scarce, and it is difficult to amplify the number of cells by cell culture. For the above reasons, embryonic stem cells are therefore advantageous for replacing stem cells with a large number of cells. Embryonic stem cells are usually taken from embryos that are artificially fertilized or donated in gamete. However, because embryonic stem cells are closely related to life, they are highly controversial in their application. In contrast, adult stem cells do not cause moral controversy, but their ability to proliferate and differentiate is lower than that of embryonic stem cells. In addition, invasive surgery is usually required to obtain adult stem cells. Furthermore, embryonic stem cells may produce teratomas (a benign tumor composed of three layers of germ layers), and adult stem cells do not have this problem. Neural stem cells (NSCs) are one of adult stem cells. Such stem cells have the ability to differentiate into three cell types in the nervous system: nerve cells, stellate cells, and oligosaccharide 6 1307717 cells. Neural stem cells can be isolated from the mesenteric nervous system of the adult or fetus (e a c 〇 e e ί a /., E X p . N e u r ο 1., 1 4 9: 2 8 - 4 1,

\99Z\ Reynolds & Weiss, Dev. Biol., 175:1-13, 1996' Gage, Science, 287:1433-1438. 200 0). Transplanted neural stem cells can successfully survive and differentiate into neural cells and glial cells (glia), increasing their potential for therapeutic potential α/·, Cell 66:713-729, 1 9 91 ; Gage et Al., Proc. Natl. Acad. Sci. USA, 92:1 1 879-1 1 883, 1995; Lundberg et al., Brain Res., 737:295-300, 1 9 9 6 ! Sv ends en et al ., Dev. Brain. Res., 99:253-258, 1997). However, many issues such as invasive intracranial sampling from adult brains, ethical considerations for obtaining cells from the fetus, and immunocompatibility limit the clinical application of neural stem cells (NSCs). SUMMARY OF THE INVENTION The present invention provides a stem cell obtained from a postpartum placenta. One aspect of the present invention provides a method for obtaining placental stem cells, the method comprising: obtaining a postpartum placenta; preparing a single-cell suspension of placental cells; culturing the placental cells; and obtaining a Placental stem cells. The placental stem cells may be finite-potential or pluripotent stem cells' and the placenta may be a human placenta. Another aspect of the present invention provides a method for culturing placental stem cells, the method comprising: obtaining a postpartum placenta; preparing a single cell suspension of placental cells; culturing the placental cells; obtaining a placental stem cell; and culturing the placental stem cells . The placental stem cells may be of limited potential or 7

1307717 pluripotent stem cells, and the placenta can be a human placenta. Yet another aspect of the present invention is to provide an isolated placental stem cell having certain characteristics such as a cell marker. It is a further aspect of the present invention to provide a homogenous population of isolated finite-potential or pluripotent placental stem cells having certain features including, for example, cell markers. Accordingly, one aspect of the present invention also provides a method of obtaining a placental stem cell based on certain cell marker characteristics. Furthermore, the present invention has a further orientation to provide cryopreserved placental stem cells from a postpartum placenta. The invention also provides a method of differentiation of placental stem cells. The placental stem cells can be obtained by the method of the present invention. An aspect of the present invention provides a method for making placental stem cells into OdiP〇cytic differentiation, and another aspect of the present invention provides a method for performing osteogenic differentiation of placental stem cells. Another aspect of the present invention provides a method of cartilage differentiation' and the present invention further provides a method of neural differentiation. A further aspect of the invention provides a composition comprising a placental stem cell and/or a differentiated placental stem cell, and a pharmaceutical composition comprising a placental stem cell and/or a differentiated placental stem cell. The invention also provides a method of treating a patient, the method comprising administering to the patient an effective amount of placental stem cells and/or differentiated placental stem cells. 8

1307717 [Embodiment] The present invention provides a stem cell derived from a postpartum placenta. Due to the origin of the fetus and a part of the embryo, fetal cells and growth factors provide a rich source. To this end, placental dry energy has higher cell proliferation and differentiation than other adult stem cells, and the placenta is a temporary organ used in the uterus to maintain fetal survival and production. Therefore, the placenta obtained after childbirth does not cause ethical competition for invasive surgery to obtain cells. The embodiments described herein and the terms used are for the purpose of describing the examples only and are not intended to limit the invention. Other embodiments that are not specifically described herein are also covered by the scope of the present invention, but the present invention can be readily understood and implemented after reading the present disclosure. The present invention relates to stem cells taken from a postpartum placenta. In the text, the term "stem cell" refers to a dominant cell, which can replicate indefinitely to form specific cells of an organ or tissue. Stem cells can divide to produce two progeny stem cells, one progeny stem cell and one progenitor cell (or transition cell: cell), which then proliferate into mature and developing cells in the tissue. The term "stem cell" as used herein includes both finite-potential and pluripotent stem cells. The term "pluripotent cell" as used herein refers to cells with a variety of fully differentiated abilities, that is, cells that can grow in the body of an animal other than the cells needed to develop the fetus. Multipotent cells can be self-renewed, and the thin placenta is a disk that can be used as a stem cell. Subsequent discards are also used in the implementation of the text (master function or production > transit complete stem cell system refers to a breastfeeding any seed can remain dormant or inactive in the 9 1307717 organization.

Here, the term "multip〇tent cel1" means a cell having the ability to grow into two or more different types of cells in a given organ or tissue. However, 'limited potential cells have the ability to become pluripotent cells. For example, hematopoietic stem cells were originally thought to be a finite-potential plastic cell, a stem cell that can only develop into several types of blood cells but cannot develop into brain cells. However, as mentioned above, there is recent evidence that hematopoietic stem cells may be pluripotent cells because they may differentiate into other cell types including brain cells. Here, the term "progenitor cell" Refers to a cell that is specifically used to differentiate into a particular cell type or to form a particular species of tissue. Here, the term "post-partum placenta" refers to a placenta that is excreted from the uterus after birth and does not contain an umbilical cord. Therefore, the method of obtaining placental stem cells of the present invention is different from the conventional method of using umbilical cord or cord blood (Migliaccio et al., Blood 96: 2717-2722, 2000; Rubinstein et al., New England J Medicine 3 3 9: 1565-1 577 1 998; Far/W ei fl/·, US Patent Publication No. 20030 i 80269) 产 can obtain postpartum placentas from any mammalian species, including rodents, humans, non-human primates, horses, Canine, feline, bovine subfamily, sylvestris, ovis, rabbits and other animal species. The placenta system used in an embodiment of the present invention is obtained from a human. Placental stem cells can be characterized according to their cellular markers. A variety of different cell markers are currently known. These known cell markers can be referred to the following resources 10 1307717

Cooked Stem Cells: Scientific Progress and Future Research Directions·, Department of Health and Human Services. June 2001; http://www.nih.gov/news/stemcell/scireport.htm» Available such as immunochemistry (immunochemistry) Or conventional methods such as flow cytometry to detect cell markers. Flow cytometry allows rapid measurement of light scattering and fluorescence produced by luminescent cells or particles. A signal is generated when a cell or particle passes through a beam of light. The signal of each cell or particle is measured separately, and the output can represent the cytometric characteristics of individual accumulation of cells or particles. The fluorescent dye can be detected by flow cytometry by calibrating the fluorescent dye on an antibody specific for the cell marker. For the above methods, please refer to the following information: βί αΛ, Rev. Sci. Instrum 43:404-409,1972; Herzenberg et al., Immunol. Today 21 :383-390, 2000 ; Julius et al., PNAS 69:1934-1938 , 19 7 2 ; Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press, 1997 ; Jaroszeski et al. (eds.), Flow Cytometry Protocols in Methods in Molecular Biology No. 9 1, Humana Press, 1997; Practical F / ow ί σ τη eir , 3 rded ·, W i 1 ey - L iss, 1 9 9 5. In an embodiment of the invention, a human placental stem cell can exhibit at least one of the following cell markers: CD9, CD13, CD29, CD44, CD90/Thy-1, CD105/SH-2/endoglin, CD166, SH-3, SH-4, vimentin, HLA-ABC, SSEA-4, TRA-1-60 and TRA-1-81. In yet another embodiment, a human placental stem 11 1307717 cell is negative for at least one of the following cell marker tests: c D 1 4, CD 3 4, CD45, AC or CD 1 3 3/2, cytokeratin 7 (Cyt 〇keratin 7), von Willebrand factor, HLA-DR, HLA G,

Glycophorin A, piacentai alkaline phosphatase and β-human chorionic gonadotropin. In another embodiment of the invention, a placental stem cell is positive for at least one of the following cell marker tests: CD9, CD13 'CD29, CD44, CD90/Thy-1, CD 105/SH-2/endothelin, CD166, SH- 3. SH-4, melanin, HLA-ABC, SSEA-4, TRA-1-6 0 and TRA-1-81, and tested negative for at least one of the following cell labeling tests: CD14, CD34 'CD45, AC or CD133/2, cytokeratin 7, Wenger's factor, HLA-DR, HLA G, glycophorin A, placental alkaline phosphatase and β-human chorionic gonadotropin. The present invention also provides a homogeneous cell population of placental stem cells. In the text, the term "population of cells" refers to a cluster containing at least two cells. As used herein, the term "homogeneous population" refers to a group of cells that exhibit substantially the same phenotype, for example, using cell markers to determine whether a cell is of the same phenotype. At least about 7 of a homogeneous population of cells 0% of the cells are substantially identical, or at least about 80%, 90%, 92%, 96% or 99% of the cells are substantially identical. The invention therefore provides a method for isolating a placental cell having certain cellular characteristics to obtain a Method for placental stem cells. A fetal cell having the above-described cellular characteristics can be isolated from a single cell suspension of placental cells obtained from the above-mentioned postpartum placenta, or from placental cells isolated and cultured from the placenta.

1307717 Plate cells. The cells are separated by flow cytometry as described above and based on cell characteristics. In one embodiment of the invention, a placental stem cell is obtained by isolating placental cells having at least one of the following characteristics: the cells are labeled with CD9, CD1 3, CD29, CD44, CD90/Thy-1, CD105 /SH-2/endothelin, CD166, SH-3, SH-4, melanin, HLA-ABC, SSEA-4, TRA-1-60 and TR A-1 - 8 1 tested positive; Test of cell markers CD14, CD34, CD45, AC or CD133/2, cytokeratin 7, Wilberg's factor, HLA-DR, HLA G, glycophorin A, placental alkaline phosphatase and β-human chorionic gonadotropin Negative reaction; or cell markers CD9, CD13, CD29, CD44, CD9 0/Thy-1, CD105/SH-2/endothelin, CD166, SH-3, SH-4, melanin, HLA-ABC , SSEA-4, TRA-1-60 and TRA-1-81 were positive, and the cells were labeled with CD14, CD34, CD45, AC or CD133/2, cytokeratin 7, Wenger's factor, HLA-DR 'HLA G, blood glycoprotein A, placental alkaline phosphatase and β-human chorionic gonadotropin were negative. The invention also provides a method of obtaining placental stem cells. The method comprises at least the steps of: obtaining a postpartum placenta, preparing a single cell suspension of the placental cells, culturing the placental cells, and obtaining a placental stem cell. The method of obtaining a postpartum placenta can be obtained from a caesarean section or a normal delivery process after consultation with the donor. The placenta can be mechanically cut into small pieces using instruments such as scissors. Recombination of the placenta with enzymes such as trypsin 13 1307717 (trypsin), chym〇trypsin, lysozyme, arnylase or κ protein degrading enzyme (protease Κ) A single cell suspension. The culture medium containing standard medium such as DMEM (Gibco) and 10% calf serum (designated Hyclone product) is used to culture the placental cells obtained in the above process and can supplement the culture with appropriate glucose and / or antibiotics. Placental stem cells can be obtained from placental cells that are continuously cultured in the culture medium.

Placental stem cells that occur during culture can be detected based on their ability to differentiate into different cell types. For example, the ability of cells in culture to differentiate into adipocytes, osteoblasts (or osteoblasts) and/or nerve cells can be tested. The term "differentiation" as used herein refers to the occurrence or alteration of one or more characteristics characteristic of a particular type of cell. Such characteristics may include, but are not limited to, the expression of a characteristic cell marker of a particular type of cell. Other characteristics may include the appearance or alteration of a cell type, or the expression of a specific protein of a particular type of cell. Thus, for example, adipogenic differentiation refers to the appearance of one or more fat cell-like properties in a cell. Similarly, osteoblast differentiation and neural cell differentiation refer to the appearance of one or more characteristics similar to osteoblasts or nerve cells. Fat cells are the connective tissue cells responsible for the synthesis and storage of fat. Osteoblasts are the main cells responsible for bone formation and are thought to be derived from the skeletal progenitors in skeletal tissues. Osteoprogenitor cell. In the text, "neurai cells" - 14 1307717 refers to neurons or glial cells. Glial cells can be further divided into stellate cells and oligodendrocytes. Placental stem cells can be differentiated by induction of an effective dose of a differentiation agent. The term "effective amount" refers to an amount of a differentiation agent that induces differentiation of a cell. A "differentiation agent" may be any chemical agent, cytokine, protein, peptide or other substance that induces differentiation of a cell.

The cells can be cultured in a cell containing 20% rabbit serum to induce adipocyte differentiation, wherein the rabbit serum is a known inducing factor for adipogenesis of medullary bone-derived primary cells ei al., J. Bone Miner. Res. 13:96-106, 1988). The expression of the fat cell transcription factor ΡΡΑΙΙγ2 and/or CEBPot is detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) to determine the differentiation of adipocytes, wherein ΡΡΑΙΙγ2 The full name is peroxisome proliferator activated receptor gamma and the full name of CEBPa is CCAAT/enhancer binding protein alpha. Alternatively, after the cells are cultured in an adipocyte-inducing differentiation culture medium, oil red 0 (0il Red 0) is added for staining to show the accumulation of lipids in the cells to detect the differentiation of fat cells {Conget and Minguell, J. Cellular Physiology 181: 67-73, 199 9). Other methods of inducing and detecting adipocyte differentiation can also be used (see Pittenger et al., Science 284: 143-147, 1999; rcAoM^a/ova ei α/., Obesity Research 8: 664-672, 2000). . 15 1307717 Bacteria, parasites and virions. The nucleic acid vector or the biological load can be introduced into the cells by an ex vivo gene therapy protocol, the method comprising: excising cells or tissues from a patient's body, introducing the nucleic acid vector or the biological vector into the cell The cut cells or tissues, and the cut out cells are replanted back into the patient (please refer to the ruler "〇e//α/., Am. J.

Health Syst. Pharm. 55: 899-904, 1998; Raymon et al., Exp. Neurol. 144:82-91, 1997; Culver et al., Hum. Gene Ther. 1:399-410, 1990; Kasid et Al., Proc. Natl. Acad. Sci. USA 87: 473-477, 1990). A nucleic acid vector or a biological vector can be introduced into the excised cell or tissue using, for example, calcium phosphate-mediated transfection (扒gier ei α/., Cell 14: 725, 1 978; Corsaro and Pearson) , Somatic Cell

Genetics 7: 603, 1 9 8 1; Graham and Van der Eb, Virology 52: 456, 1973). Other methods can also be used to introduce the nucleic acid vector into a host cell, such as electroporation (WeMW flwn et al., EMBO J. 1: 841-845, 1982). The cells of the present invention may also be used in combination with other agents such as other types of cells, growth factors and antibiotics. The type of other agent can be determined by a person skilled in the art. The treatment of the cytoplasmic treatment of the medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal medicinal It is divided. The material has been made to the genus of the genus or the sacral body of the sacral body. The method of the sputum is the same as the sputum of the sputum. The treatment of the nucleus is the same as that of the package.

Use together. The differentiated placental stem cells of the present invention can be used in any tissue that requires replacement or regeneration. For example, the placental stem cells of the present invention for performing neural cell differentiation can be used to treat neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Pik c's s d i s e a s e, and amyotrophic lateral sclerosis. The differentiated cells can also be used to treat brain or spinal cord injuries. In another embodiment, placental stem cells that undergo adipocyte differentiation can be used to treat conditions such as diabetes. In yet another embodiment, placental stem cells for chondrocyte differentiation can be used to treat degenerative joint diseases such as osteoarthritis or rheumatoid arthritis, as well as other types of cartilage defects, such as cartilage defects caused by traumatic fractures. . The placental stem cells of the present invention may be used to treat genetic hereditary joint diseases such as hereditary femoral head necrosis. In still another embodiment, placental stem cells exhibiting osteoblast differentiation can be used to treat bone defects caused by traumatic fractures, age-related diseases such as osteoporosis, and genetic hereditary diseases such as osteopetrosis. It is to be understood that the number of ingredients, the reaction conditions, and the like, or the numbers used in the description of the present application, in the examples or elsewhere, are approximate. Accordingly, the numerical parameters set forth in the specification are approximations, and may vary depending upon the results of the invention as desired. Moreover, these parameters are not intended to limit the principles equivalent to the scope of the patent application of the present invention, but rather the preferred data obtained using conventional conventional techniques. Although the above-mentioned data ranges and parameters for indicating the maximum range of the present invention are approximate values, the data values described in the above specific embodiments have been 22

1307717 As precise as possible. However, each measurement experiment has its standard deviation, and any data value must have some error value. It should be understood that specific terms such as (a), "and" and "the" are used in the context of the description and the scope of the appended claims. Contains number patterns. Unless otherwise defined, all the disciplines used in the text have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be used to practice the methods of the invention. The methods and materials described are for illustrative purposes only. All references mentioned in this application are incorporated by reference in their entirety to disclose and describe the relevant methods and/or materials described in this document. In addition, the document discussed herein only discloses the prior application of the present invention. And there is no literature showing that the present invention has been disclosed in the prior art. The actual data obtained in the context of the present invention will differ from the data disclosed in the present disclosure by the individual implementations. Various methods of practicing the techniques and/or experimental procedures (collectively) of the present invention include any conventional method for achieving the same objectives, and are not intended to be merely specific examples of the methods described in the specification. In addition, although some methods have been described in the specific content of the book, which are for illustrative purposes, the use of the methods in the present invention is not limited to the description. The invention is illustrated by the following examples, which are not intended to be limiting. Therefore, r is a reference to its reciprocal and content texts. The method disclosed by the Science and Technology Institute and the "Description of Limitation" in this publication 23 2307717 Example 1 Test _ The stem cell of the stem cell is first taken from the healthy mother who was born by caesarean section or spontaneously delivered and attached with The Human Body Test Committee certifies that the placenta is only used for the purpose of the study for the full-term placenta (about 3-4 weeks to 4 weeks of placenta). Carefully cut the placenta and use a dish buffer solution (ph〇) Sphate buffered saline, PB S) Wash several times. Cut the postpartum placental tissue pieces into smaller pieces with scissors' and use the 0.25% trypsin_EDTA (Gibco) solution to make the tissue fragments at 37 C. Decompose for 丨〇min. After centrifugation to pellet the cells' and wash the cells once with the acidate buffer solution, the cells were suspended in penicillin (Gibco) containing 10% calf serum (Hycl〇ne), 1 〇〇 unit/ml. And 100 units/ml of streptomycin (Gibc) in dmeM medium (Gibco). The cells were cultured in a 37 t and saturated atmosphere containing 5% carbon dioxide' and the culture medium was updated twice a week. The number covers 80% For the culture dish area, the cells were collected with 〇.25% trypsin-pentamethoxine (0113 (?〇) solution, and the cells were re-distributed in a ratio of 1:2 to 1:3. Then the cells were plated. After at least 9 days of growth, an analytical test can be performed. Example 2 Adipocyte differentiation of placental stem cells The squamous cell differentiation is cultured in an adipogenic medium or an osteogenic medium according to the examples. 1 obtained cells, wherein the fat cell culture medium contains 0.5 mM isobutylmethyl yellow 24 1307717 嘌呤, 1 μ Μ adrenocortic ketone, 10 μ Μ insulin and 60 μΜ indomethacin (Z) eMnz\s et al. J. Bone and Mineral Research 1 4: 700-709, 1 999), while the osteoblast cell culture medium contains Ο.ΙμΜ-upper corticosterone, 10 mM β-citric acid glycerin (p_giyCer〇i phosphate) And 50 μπι ascorbate (ie, vitamin CHJabwa/ ei α/., J Cell Biochem. 64:295-312, 1997). The intracellular accumulated neutral fat vacuoles were subsequently stained by Oil Red staining to assess the fat cells. Formation situation (Conge i and J. Cellular

Physiology 1 81:67-73, 1999)· and using von Kossa staining or alizarin red staining to detect calcium accumulation in cells to assess the differentiation of osteoblasts (Jakwa/ ei a/·, J Cell Biochem. 64:295-312, 1997 and ΡΓαη e"/·, Chin. J. Traumatol. 5:374-379, 2002). Figure 1 shows the results of osteoid mesenchymal stem cells detecting their adipocyte differentiation by Oil Red staining (Fig. 1A) and the results of von Kossa staining for detecting osteoblast differentiation (Fig. 1B). For the control group. Figure 2 shows the differentiation potential of the placental stem cells of the present invention. Figure 2A shows placental stem cells that have undergone adipocyte differentiation but have not been stained. Figure 2B shows placental stem cells stained with adipocytes and stained with Oil Red to detect accumulation of fat in the cells. Figure 2B shows that placental stem cells obtained according to the method of the present invention can differentiate into adipocytes. As shown in Figures 2C and 2D, the placental stem cells of the present invention can also differentiate into osteoblasts. Both alizarin red staining (Fig. 2C) and von Kossa staining (Fig. 2D) can stain osteoblasts in cultured placental stem cells. Therefore, the placental stem cells obtained according to Example 1 have the ability to form a variety of cells in 25 1307717. Example 3

Chondroblast is a cell that secretes the cartilage matrix and becomes a chondrocyte when it is surrounded by a matrix. The microculture technique was used to induce the placental stem cells obtained in the above Example 1 for chondroblast differentiation (Barry ef fli-, Experimental Cell Research 2 6 9:1 8 9 - 2 0 0, 2 Ο Ο 1 and Z m A : e ί a / ·, T issue E ngineering 7:211-28, 2001). Quickly place approximately 200,000 cells in a 15 ml polypropylene circular graduated test tube containing 1 ng/g of transforming growth factor (TGF)-pl or TGF-P3 (both purchased from R&D systems) ), 0.1 mM ascorbic acid-2-phosphate (Sigma), 1M07 Μcorticosterone (Sigma) and 1% insulin-transferrin-sodium·selenite medium supplement (Sigma) The culture solution is cultured for 3 to 6 hours. The cells were transferred to a 24-well plate and cultured for another 2 to 3 weeks. These cells are then stained with Alci an Blue (pH 1) to detect proteoglycans present in cartilage and other connective tissues. In addition, anti-human second type knee proprotein The antibody (Santa Cruz Biotechnology, Inc.) was diluted at a ratio of 1 ··100, and the cells were reacted with the antibody at 4 ° C for one night by immunocytochemistry to detect the type 2 collagen in the cells. Subsequently, biotinylated anti-gat IgG secondary antibodies (ABC kit, Vector Labs) were diluted 1:500 times 26 1307717 with the above cells at room temperature The reaction was carried out for 45 minutes for secondary anti-staining. Finally, the cells were observed using a Leica DM IRB inverted microscope (Leica, Germany).

It can be confirmed from Figs. 3A and 3B that the placental stem cells of the present invention have the potential to form cartilage. Figure 3A clearly shows a placental stem cell-derived cell stained blue by Alfalfa blue, which indicates that the cell produces proteoglycans. Figure 3B also shows a placental stem cell-derived cell that is recognized by an anti-type 2 collagen antibody, which means that the cell produces type II collagen, while type II collagen mostly appears in cartilage and for bone Or other connective tissue is essential for the normal development process. Example 4 Phenotypic characteristics of placental stem cells The cell markers of the placental stem cells obtained in Example 1 were analyzed by flow cytometry and/or immunochemical staining. Bone marrow cells obtained according to the method of Pittenger et al. (Science 284: 143-147, 1999), Reyes et al. (Blood 98:2615-162 5, 2001), and according to the method of Eric es et al. Umbilical cord blood (Br. J. Haematol. 109: 235-242, 2000) and placental stem cells (MPSC) obtained in Preferred Example 1 were subjected to cell marker analysis' and the cell marker comparison results of each cell are listed in Table 1. in. Table 1 27 1307717 Cell marker Pittenger et al (spinal) Reyes et al (spinal) Erices et al (umbilical cord blood) MPSC CD9 (+) (+) CD 13 (+ ) (+) (1) CD14 (-) (―) ( -) CD29 (+) (+) (+) CD34 (-) (-) (-) (-) CD44 (+) (+) (+) CD45 (-) (-) (-) (-) CD90/ Thv-1 (+) (+) (+) CD105/SH2/endothelial factor (+) (+) CD117/c-kit (-) (-) AC or CD133/2 (-) CD166 (+) (+) SH3 (+) (+) (+) SH4 (+) (+) (+) Interstitial protein (+) Cytokeratin 7 (-) Wenwei Burr factor (-) (-) (-) HLA-ABC (+ (-) (+) HLA-DR (-) (-) (-) HLA-G (-) Glycophorin A (-) (-) SSEA-4 (+) T RA -1 -60 (+) TRA-1 -8 1 (+) β-human gonadotropin 1 (+) placental alkaline phosphatase (-)

Example 5 Placental Stem Cells_<Following Further Characterizations The placental stem cells obtained according to Example 1 were analyzed again using flow cytometry, immunochemical analysis, immunofluorescence, and/or reverse transcriptase polymerase chain reaction. . Flow cytometry was performed using a FACSCalibur flow cytometer purchased from BD Biosciences, Inc. (San Jusem USA) to perform flow cytometry. Using trypsin to treat fifth-generation fifth passages of placental dry fines 28 1307717 cells, and the following antibodies were used to label the placental stem cells, including: anti-CD14 antibody or anti-CD90 antibody bound to fluorescent isothiocyanate (FITC); combined with phycoerythrin (phycoerythrin, PE) anti-CD45 antibody; or, without any fluorescently labeled anti-CD34 antibody (BD Biosciences), anti-STRO-1 antibody (Hybridoma bank, U. of Iowa, 10, USA) or anti-CD105 /SH2 antibody (HybΓidoπlabank). A rat anti-mouse IgG antibody (BD Biosciences) combined with FITC was used as the secondary antibody.

Fluorescence-assisted cell screening assay (FAS C) was used to confirm that these undifferentiated placental stem cells were negative for CD14, CD45 and CD34, and CD14, CD45 and CD34 were both hematopoietic cell markers. These placental stem cells also responded negatively to the bone marrow stromal progenitor cell marker STR0-1. Therefore, it is indicated that these placental stem cells are not of hematopoietic origin or are not contaminated by cord blood cells. It was further confirmed that these placental stem cells exhibited CD90 (a surface marker which can be observed on mesenchymal stem cells) and CD105. The morphology of these placental stem cells is similar to that of mesenchymal stem cells (MSCs), which exhibit a bell-shaped shape and adhere to the surface of a plastic culture with cell-like cell colonies. _Free _ cytochemical analysis, immunofluorescence and anti-Korean recording of 厶 厶 enzyme chain and 廒 固定 该 该 该 该 该 该 该 该 该 该 该 该 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定 固定The cells are treated for 2 minutes to penetrate the cell membrane of the cells. The slides with the cells were then allowed to react with the primary antibody at 4 ° C for one night. Followed by immunocytochemical analysis, 29 1307717

A primary antibody recognizing a neuron-specific enolase (NSE) is used to react with the above cells at a dilution ratio of 1:25, followed by an anti-rabbit antibody labeled with biotin and a horseradish that binds avidin-biotin. The oxidase was reacted, wherein the avidin-biotin conjugated horseradish peroxidase was purchased from Vector Laboratories, Burlingame, CA, USA. And use primary antibody against NeuN (1:100, Chemicon International Inc., Temecula» CA, USA), primary antibody against nestin (1:100, BD Biosciences), primary antibody against glial fibrillary acidic protein (GFAP) Immunofluorescence staining was performed (1:100, Chemiconlnentimentalnc.), primary antibody against microtubule-associated protein 2 (1:200, Chemicon Intemationallnc.), and primary antibody against NG2 (1:50, Chemicon Incorporated allnc.). The cells were then reacted with a secondary antibody labeled with Cy3 or luciferin (€1〇1^63〇6丨11) for 1 hour at 37 °C, and 4',6-diamino-2- Phenylhydrazine is stained to identify the nucleus, of which 4',6-diamino-2-phenylindole (DAPI) is from KPL Inc. Gaithersburg, MA, USA. Finally, cells were observed using a co-focusing fluorescence microscope (Zeiss, LSM510).

Anti-Korean Polymerase Chain Reaction The total RNA in these cultured cells was isolated using the Purescript reagent kit purchased from Gentra System (Minneapolis, MN, USA), and 2 μg (/zg) of total RNA was purchased. Reverse transcription was performed from Superscript II of Invitrogen (Carlasbad, CA, USA). The cDNA was synthesized using T a q P1 a t i n u m (I n v i t r 〇 g e η ) with 0.0 2 5 μg/ml of oligo(d T) 30 1307717. Each sample was taken out in equal volumes and amplified to replicate human genes with the following primers from Μ'. These primers include: Human Nestin Gene: 5'-CCC TGA CCA CTC CAG ΤΤΤ Α-3', and 5 *-GAG TCC TGG ATT TCC TTC-3* Human Oct-4 gene: 5'-GTG AAG CTG GAG AAG GAG AAG CTG-3', and 5'-CAA GGG CCG CAG CTT ACA CAT G-3'

Nerve fiber light chain gene (NF-L): 5,-CCC GAC TCA GTT TCA CC-3', and 5'-CAG CCT TAG ACG CCT C-3' purchased from (:1 elbow (:11 company (? &1 into 11〇, €人, 1; 38) Human glyceraldehyde-3-can acid dehydrogenase (GAPDH) primer as the internal control group of polymerase bond-locking reaction. 94 ° C 1 The sample was cycled 30 times in minutes, 65 ° C for 1 minute, and 72 ° C for 1 minute to replicate the cDNA of each sample. Then electrophoresis was performed with 1.5% agarose gel to separate the PCR product and auto-sequence loading. The sequence of the PCR products was confirmed. The reverse transcription polymerase chain reaction and immunofluorescence staining of the undifferentiated placental stem cells showed that the placental stem cells labeled "nestin" for nerve cells (Fig. 5A and 5C). And the embryonic stem cell marker "〇ct4" (Fig. 5B and 5D) shows a positive reaction plant, CeU, 60:585-595, 1990, Reubinoff et α/ } nature Biotech.

1 8: 399_4 〇 4, 2000). These undifferentiated placental stem cells also showed a weak positive reaction to the early neural cell markers "Tujil", "gfAP" and "NeuN 31 1307717 (see Figure 6). Placental stem cells of the present invention Unlike other stem cells, it has been shown to have the ability to differentiate into neural cells. For example, stem cell lines derived from human adipose tissue exhibit a STRO-1 positive response (Zm^· ei a/·, M〇l.

Niol Cell, 13: 4279-4295, 2002), and the placental stem cells of the present invention

Then no. In addition, the placental stem cells of the present invention have a strong positive reaction to the surface marker "CD9", while the finite-potential stem cells derived from cord blood have no surface CD90 (Go〇<iiW/j 〇/., Bio· Bl〇dd Marrow Transplant., 7:58 1 -588, 2001; Lee et al., Blood, 103: 1669-1675, 2004). The placental stem cells of the present invention are also positive for 〇ct_4 and several other embryonic stem cell markers, suggesting that the placental stem cells are equivalent to a group of excellent and primitive finite-potential stem cell populations. Example 6 Differentiation of neural cells of placental stem cells The placental stem cells obtained according to Example 1 were colonized to be uncoated or coated with fibronectin, gelatin or poly-L-lysine. In a 35 micron culture dish. The cell lines were cultured in serum-free DMEM-LG medium (Gibco) at different cell densities and exposed to 10 ng/ml of alkaline fibroblast growth factor (b- FGF)/20 ng/ml epidermal growth factor (EGF) (purchased from R&D), 10-6 M retinoic acid (RA, purchased from Sigma), /3 - thiolethanol / dimethyl sulfoxide / Butylated hydroxymethyl ether (1χ1〇_5Μ β -ΜΕ/2% DMSO/100/z Μ BHA) (Sigma) or 0.5 mM 1-methyl-3-isobutylxanthine 32

Incubate for 3 to 6 days in a drug such as 1307717 (IBMX, Sigma). These cells were then collected and subjected to & transcription & synthase-locked reaction immunoassay. The cells differentiated into neuron-like cells were quantified to know how many percentages of the stem cells in the total number of cells were in the process of differentiation. The cells were stained to detect the expression of NSE, and stained and analyzed under inverted microscope. Five low-power photos with no overlap were taken for each sample. The number of cells showing a tight cell body and NSE staining positive was calculated as positive neural cells. And use 4',6 diamino-2-phenylindole (DAPI) or anti-human nuclear antibody (1:5〇, Chemic(>n ΐη^咖心心)

Inc.) was stained to calculate the total number of cells in which DApi stained only the nuclei (nuclear specificity). Morphological changes in some placental stem cells were observed after 3 to 6 days of culture in culture medium containing retinoid (10-6 M) or lBMX (0.5 mM). In particular, 'placental stem cells in DmEm medium (Gibco) supplemented with 1% fetal calf serum (FBS), 1 〇〇 unit/ml penicillin and 1 gram/ml streptomycin (Section 7a), the cells cultured in the culture solution containing retinoid or IBMX were observed to have a cytoplasmic state of contraction and have outwardly extending projections (see Fig. 7E). In addition to the morphological changes, the cells cultured in the culture medium containing retinoid or sputum showed more neuronal markers "N S Ε" than the control group, see Figures 7, 7D and 7F, respectively. The cells in the control group and the cells cultured in b-FGF/EGF or /9 ME/DMSO/BHA were not observed to have a cell-like change as described in the above 33 1307717 (data not shown). After six days of culture, about 40% to 60% of the cells in the IB MX-treated cell population differentiate into neuron-like cells, and about 10% of the retinoid-treated cells. The cells were differentiated (data not shown). In addition, those cells treated with IBMX and showing a strong positive response to NSE staining exhibited bipolar or multipolar morphology with long branches. Sometimes, several neuron-like cells can be observed under the microscope to form a network-like structure.

When the cells were treated with IB MX for 2 hours, a change in the morphology of some of the placental stem cells was observed (data not shown). The longer the culture time, the greater the number of differentiated cells (Fig. 8A-8C). According to the results of three independent experiments, about 7% of placental stem cells differentiated after 24 hours of treatment with IBMX, and after 5 days, the number of differentiated cells increased to over 40%. In addition, after 5 days of treatment with placental stem cells by IBMX, the expression of the mature neuronal marker "Neural Fiber Light Chain (NF-L)" was detected by reverse transcription polymerase chain reaction. The placental stem cells treated with IBMX contain cells expressing the neural marker MAP2 (Fig. 9A), the stellate cell marker GFAP (Fig. 9B) or the oligodendrocyte marker NG2 (Fig. 9C), suggesting that Placental stem cells can differentiate into these three types of nerve cells (neurons, stellate cells, and dendritic cells). In addition, these MAP2-positive cells exhibit a neuronal morphology with a tight cell body and a number of outwardly extending axons (Fig. 9A). The number of these neurons, stellate cells and oligodendrocytes in the total number of cells is about 16% '1% and 17% respectively, and this result is related to the human bone marrow treated by IBMX reported by Deng et al. The recommended method of matrix fine 34 1307717 is to label these undifferentiated placental stem cells to distinguish between these undifferentiated placental stem cells and brain cells. After 24 hours, the undifferentiated placental stem cells labeled with CM-Dil were added to the culture well plate containing the brain cells and the cell density was 2×10 4 cells/cm 2 and in DMEM-LG medium. Cultivate. After the cells were co-cultured for 3 to 5 days, immunofluorescence staining was used to detect the expression of MAP2 and GFAP.

The placental stem cell line is cultured on a single layer of neonatal rat primary brain cells' and the fluorescence emitted by the Cell Tracker CM-Dil label on the cells can be observed under a fluorescence microscope to distinguish the two cells (i 〇B and 10E map). Cells labeled with CM-Dil are shown to exhibit both MAP2 and GFAP protein markers (see Figure 10D). Figures 1C and 10F show the nucleus of these MAP2-positive and GFAP-positive CM-Dil labeled cells after DAPI staining. The white arrows in Fig. 10C indicate the placental stem cells that have been differentiated into neuronal phenotypes, and the hollow arrows in the first OF diagram indicate the placental stem cells that have differentiated into stellate cell phenotypes. The contents of this specification can be fully understood from the teachings of the referenced materials described in the specification, and the entire contents of each of the entire disclosures. The examples in the specification are merely illustrative of preferred embodiments of the invention and are not intended to limit the scope of the invention. It is to be understood by those skilled in the art that the present invention is not limited by the scope of the invention, and the scope of the invention is defined by the scope of the appended claims. [Simplified illustration] 36 1307717 Figure 1 shows the staining results of the control group of mesenchymal stem cells, used as a positive control group, if oil red (Oil Red) staining method to detect fat cell differentiation, then The staining result of the positive control group was red (such as the dark area of Figure 1A). If the vonKossa staining method was used to invert the bone cell differentiation phenomenon, the staining result of the positive control group was brown (as in Figure 1B). Dark dark area).

Fig. 2 shows the results of staining of placental stem cells of the present invention to demonstrate their ability to differentiate into other species of cells. Figure 2A shows unstained placental stem cells, and Figure 2B shows staining of cells with Oil Red to detect fat cells (dark areas in the figure). Figure 2C shows cells stained with alizarin red, and 2D shows cells stained with vonKossa staining. Both 2C and 2D showed osteoblasts detected in cultured placental stem cells (respectively It is red and brown, and appears as a dark dark area in the figure). Figure 3 shows the potential of the placental stem cells of the present invention to differentiate into chondrocytes. Figure 3A shows that placental stem cell-derived cells are stained blue by the Alcian Blue dye (a darker region in the cell), indicating that the cell will exhibit proteoglycans, or proteoglycans. . Figure 3B shows a placental stem cell-derived cell (more dark region) that binds to an anti-type 2 collagen antibody. Figure 4 shows a histogram of the presentation of CD 14, CD45, CD34, CD90, CD105 and STRO-1 on undifferentiated placental stem cells. 37 1307717 Figure 5 shows the appearance of nestin (Nestin) and Oct-4 in placental stem cells. Figure 5A shows the amount of nestin expression detected by the reverse transcription polymerase chain reaction. Figure 5B shows the amount of 0 c t -4 detected by the reverse transcription polymerase chain reaction. Glyceraldehyde-3-acid dehydrogenase (GAPDH) is used as an internal control of the reverse transcription polymerase chain reaction. Figures 5C and 5D show the expression of nestin and Oct-4 detected by immunofluorescence, respectively. Figure 6 shows the performance of Tujil (泠-tubulin III), GFAP and NeuN in placental stem cells detected by immunofluorescence and conjugated focus microscopy. The cell lines were stained with DAPI and magnified at a magnification of 400 times to take a photograph.

Figure 7 shows photographs of placental stem cells after three days of culture in different culture broths. Figures 7A and 7B show cells cultured in expansion medium as a negative control group. The 7C and 7D lines show cells cultured in a culture solution containing retinoic acid (RA, or A acid or vitamin A acid). Figures 7D and 7F show cells cultured in a culture solution containing isobutylphosphonium xanthine (IBMX). Figures 7A, 7C and 7E show phase difference plots for Figures 7B, 7D and 7F, which show the detection of neuron specific enolase (NSE) by immunocytochemical staining. Figure 8 shows the placental stem cells treated with isobutylmethylxanthine and the reverse transcription polymerase chain reaction to detect the expression of the nerve filament light chain (NF-L). Figures 8A, 8B and 8C show photographs of phase difference between treatment with isobutylmethylxanthine for 24 hours, 72 hours 38 1307717 and 1 08 hours, respectively, and the photographs were taken at a magnification of 200 times. Figure 8D shows the expression of the nerve fiber light chain detected by reverse transcription polymerase chain reaction after treatment with isobutylphosphonium xanthine for 0 hours, 6 hours, 3 days, and 5 days, and glycerol Aldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control group for reverse transcription polymerase chain reaction.

Fig. 9 shows the results of immunofluorescence tests of placental stem cells after three days of culture in a culture solution containing isobutylphosphonium xanthine with various labels. Fig. 9A shows the results of staining with MAP2, Fig. 9B shows the results of staining with GFAP, and Fig. 9C shows the results of staining with NG2. Fig. 10 shows photographs of cells after CM-Dil-labeled placental stem cells were co-cultured with rat primary brain cells for 5 days, and the photographs were taken at a magnification of 400 times. Figures 10A-10C and 10D-1 0F show cells in the same field of view, respectively. Figure 10A shows the results of immunostaining with anti-Μ AP2 antibody. Figure 10B shows the results of CM-Dil staining with the same cells. Figure 10C shows the nuclei in the same cells after DAPI staining. Fig. 10D shows the results of immunostaining with an anti-GFAP antibody. Figure 10E shows the results of CM-Dil staining with the same cells. Figure 10F shows the nuclei in the same cells after DAPI staining. [Simplified description of component symbol] 39

Claims (1)

1307717 Shore 7.! ❼ ❼ Ά ) ) 正 正 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I · · · · · The placental stem cells are cultured in a medium containing an effective dose of !-mercapto-3_isobutylxanthine to obtain cells having a neuronal characteristic. The nerve cells are characterized by neuron-specific enolase. (neuron-specific enolase, NSE) 'Nu-specific nuclear protein (NeuN), microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein 'GFAP' or oligodendrocyte labeled NG2. 2. The method according to claim 1, wherein the placental stem cell has a negative reaction to the cell marker CD 1 1 7 / ckit, the assay disk liquid enzyme and HLA-DR, and the cell marker η LA - ABC was positive. 3. The method of claim 2, wherein the placental stem cells have at least one of the following characteristics: a. labeling cells with CD9, CD 13, CD29, CD44, CD90/Thy-1, CD105/SH-2 Endothelin (endoglin), CD 1 66 'SH-3, SH-4, vimentin, SSEA-4 'TRA-1-60 and TRA-1-81 tested positive; 1?. Mark the cells with 0014. 034, 0045, 八 (3 or 00133/2, cytokeratin 7 , 〇 伯 BER TF (v〇n 40 1307717 Willebrand factor), HLA G, glycophorin A and β-human fluff The test for gonadotropin (β-human chorionic g ο nadotropi η) was negative; or c. For cell markers CD9, CD13, CD29, CD44, CD90/Thy-1, CD105/SH-2/endothelin, CD166, SH-3, SH-4, melanin, SSEA-4, TRA-1 - 60 is positive for the test with TRA-1 -8 1 and labeled with cells such as CD14, CD34, CD45, AC or CD133/2, cytokeratin 7, Wenger's factor, HLA G, glycophorin A and β-human The test for chorionic gonadotropin was negative. 4. A cell population obtained by the method of claim 1, 2 or 3. 5. A method of preparing a population of cells having a neuronal cell characteristic, comprising: culturing the human placental stem cells in a medium containing an effective amount of 1-mercapto-3-isobutylxanthine to obtain A neuron-characterized cell characterized by neuron-specific enolase (NSE), neuron-specific nuclear protein (NeuN), microtubule-associated protein 2 (MAP2), and glial fibrillary acid A glial fibrillary acidic protein (GFAP) or an oligodendrocyte labeled NG2; and the cell population is isolated. The method of claim 5, wherein the placental stem 'cell has a negative reaction to the cell marker CD1 17/ckit, placental alkaline phosphatase and HLA-DR, and the cell is labeled with HLA-ABC. The method of claim 6, wherein the placental stem cell has at least one of the following characteristics: a· for cell markers CD 9 , CD 13 , CD 2 9 , CD44 , CD90/Thy-1 , CD105/SH-2 / endoglin (endoglin), CD166, SH-3, SH-4, melanin (vimentin) ), SSEA-4, TRA-1-60 and TRA-1 - 8 1 test positive; 1?. For cells labeled € 014, 匚 034, 0045, human (3 or € 0133/2, cytokeratin 7 (cytokeratin 7), von W i 11 ebrandfact 〇r, HLAG, glucagon A (g 1 yc 〇ph 〇ri η A) and β-human chorionic hormone (β-human chorionic g ο nadotropi η) tested negatively; or c. labeled cells for CD9, CD 13 , CD29, CD44, CD90/Thy-1, CD105/SH-2/endothelin, CD166, SH-3, SH-4 , melanin, SSEA-4, TRA-1-60 and TRA-1-81 were tested positively, and the cells were labeled with CD14, CD34, CD45, AC or CD133/2, cytokeratin 7, and Wenwei's factor. , HLA G, blood group enzyme protein A and β-human chorionic gonadotropin test were negative. 8. A kind of 42 1307717 as described in claim 5, 6 or 7 The method of obtaining a population of cells. 9. Application of the cell population of patentable scope of item 8, which has a system for treating a neurodegenerative disease, brain injury or spinal cord injured patients. The cell population according to claim 9, wherein the neurodegenerative disease is selected from the group consisting of Parkinsin's disease, Alzheimer's disease, and Alzheimer's disease. , in the group consisting of Pick's disease, Huntington's disease, and amyotrophic lateral sclerosis. 1 1. A placental stem cell such as the Food Industry Development Institute's accession number BCRC960221. 43