High-potential pluripotent stem cells

Abstract

High-potential pluripotent stem cells according to the present invention have: the properties of conventional pluripotent stem cells, that is, the ability to be differentiated into an embryonic tissue which is all cells making up the body; and the ability to be differentiated into a germ line and / or cells of an extraembryonic tissue such as the placenta, that is, a differentiation ability similar to totipotency. Accordingly, since the high-potential pluripotent stem cells have the ability to be differentiated into cells of an embryonic tissue such as the placenta, in addition to conventional regenerative medicine for various diseases with all injuries in the body, a damaged site of the extraembryonic tissue can be reconstituted to improve or recover the functions of the damaged site, and the high-potential pluripotent stem cells can be applied to new fields of regenerative medicine, such as being able to be used for a treatment pertaining to reproduction such as an infertility treatment.

Description

[Technical Field]

[0001] The present invention relates to a cell preparation for regenerative medicine, more specifically to high-potential pluripotent stem cells isolated from extraembryonic tissues and the like, and a cell preparation containing the high-potential pluripotent stem cells. [Background technology]

[0002] Mesenchymal stem cells, a type of tissue stem cell, are present in mesenchymal tissues such as bone marrow, skin, and adipose tissue and are composed of a population containing various cells. However, unlike other tissue stem cells, they have been reported to differentiate not only into mesodermal cells such as bone, fat, and cartilage, which belong to the same developmental lineage, but also into ecto- and endodermal cells beyond the germ layers. This ability to differentiate into the three germ layers of mesoderm, endoderm, and ectoderm suggests pluripotency, but due to the low differentiation rate, it was assumed that not all mesenchymal stem cells are pluripotent, but rather that only some cells are. Based on these findings, the inventors conducted extensive research to discover pluripotent stem cells that are thought to exist among mesenchymal stem cells, and discovered new pluripotent stem cells (multilineage-differentiating stress enduring cells: Muse cells) (Patent Document 1, Non-Patent Documents 1-3).

[0003] Muse cells are pluripotent cells that can differentiate into various cells of the three germ layers across germ layers, but are not tumorigenic.They have been found to home to and engraft in damaged tissue simply by administering them intravenously, and to bring about tissue repair and functional recovery by spontaneously differentiating into tissue-specific cell types according to field theory.Application of these cells to regenerative medical therapies for various diseases is being investigated (Patent Documents 2 to 4).

[0004] Meanwhile, Muse cells can be obtained from bone marrow fluid, adipose tissue (Non-Patent Document 4), dermal connective tissue of the skin, etc., and are also known to exist widely in the connective tissue of organs and peripheral blood.

[0005] Previous research into mesenchymal stem cells has often used as raw materials cells collected from bone marrow fluid, adipose tissue, etc. by invasive methods. However, recently, research has also been conducted into the use of cells collected by non-invasive methods from medical waste such as placental tissue (including umbilical cord, umbilical cord blood, placenta, chorion, amniotic membrane, and amniotic fluid) (Patent Document 5). [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5185443 [Patent Document 2] International Publication No. WO2014 / 027684 [Patent Document 3] Japanese Patent Application Laid-Open No. 2015-159895 [Patent Document 4] International Publication No. WO2018 / 235834 [Patent Document 5] Special Publication No. 2020-503375 [Non-patent literature]

[0007] [Non-Patent Document 1] Kuroda Y et al. Proc Natl Acad Sci USA,2010: 107: 8639-8643. [Non-patent document 2] Wakao S et al. Proc Natl Acad Sci USA,2011: 108: 9875-9880. [Non-patent document 3] Kuroda Y et al. Nat Protc, 2013: 8: 1391-1415. [Non-patent document 4] Ogura F. et al., Stem Cells Dev., 23, 717-728, doi:10.1089 / scd.2013.0473 (2014) Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide high-potential pluripotent stem cells that have the properties of conventional Muse cells, i.e., the ability to differentiate into embryonic tissues, which are all cells that make up the body, as well as the ability to differentiate into cells of extraembryonic tissues such as the placenta and / or germ cell lineages, i.e., a differentiation ability close to totipotency, and a cell preparation containing said high-potential pluripotent stem cells. [Means for solving the problem]

[0009] The inventors attempted to isolate Muse cells from cells that can be collected non-invasively, such as extraembryonic tissues (umbilical cord, umbilical cord blood, placenta, placental blood, decidua, chorion, amnion, amniotic fluid, etc.), which are biological tissues that cease to function as organs and become waste after pregnancy, childbirth, and delivery. After detailed examination of the SSEA-3-positive cells (so-called Muse cells), the inventors discovered that among the SSEA-3-positive cells obtained, there exist high-potential pluripotent stem cells distinct from Muse cells. These high-potential pluripotent stem cells possess, in addition to the useful properties of Muse cells conventionally obtained from bone marrow, etc., the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, and thus possess differentiation potential approaching totipotency, leading to the completion of the present invention.

[0010] That is, the present invention is as follows. [1] SSEA-3 positive high potential pluripotent stem cells. [2] The SSEA-3-positive high-potential pluripotent stem cells according to [1] above, wherein the pluripotent stem cells are derived from extraembryonic tissue. [3] The SSEA-3-positive high-potential pluripotent stem cells according to [2] above, wherein the extraembryonic tissue is selected from the group consisting of umbilical cord, umbilical cord blood, placenta, placental blood, decidua, chorion, amniotic membrane, amniotic fluid, etc. [4] The SSEA-3-positive high-potential pluripotent stem cells according to any one of [1] to [3] above, wherein the pluripotent stem cells are CD133-positive. [5] SSEA-3-positive high-potential pluripotent stem cells according to any one of [1] to [4] above, which have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages. [6] The SSEA-3-positive high-potential pluripotent stem cells according to any one of [1] to [5] above, which are pluripotent stem cells having at least one of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability. [7] The SSEA-3-positive high-potential pluripotent stem cells according to any one of [1] to [6] above, which are pluripotent stem cells having all of the following properties: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability. [8] A regenerative medicine drug comprising the SSEA-3-positive high-potential pluripotent stem cells described in [1] to [7] above. [Effects of the Invention]

[0011] The high-potential pluripotent stem cells of the present invention possess the properties of conventional Muse cells, i.e., the ability to differentiate into embryonic tissues, which comprise all cells in the body, as well as the ability to differentiate into cells of extraembryonic tissues such as the placenta and / or germ cell lineages, i.e., a differentiation potential close to totipotency. Furthermore, they possess properties such as being positive for CD133, a cell surface marker that is negative for conventional Muse cells obtained from bone marrow, peripheral blood, adipose tissue, skin, etc. Therefore, in addition to being used in regenerative medicine for various diseases involving damaged tissues in the body, their ability to differentiate into cells of extraembryonic tissues such as the placenta can also reconstitute damaged sites in these extraembryonic tissues, improving or restoring the function of the damaged sites. Furthermore, their ability to differentiate into germ cell lineages can also be used to treat reproductive disorders, such as infertility treatment. Therefore, the high-potential pluripotent stem cells of the present invention and cell preparations containing the high-potential pluripotent stem cells can be applied to new fields of regenerative medicine.

[0012] Like conventional Muse cells, the high-potential pluripotent stem cells of the present invention can selectively migrate to, accumulate in, and engraft at sites of injury. They differentiate into cells that constitute tissue at the engrafted site and reconstruct tissue by replacing damaged and dead cells. Therefore, differentiation into target cells is not required prior to transplantation (administration). Furthermore, they are non-tumorigenic and have excellent safety. Furthermore, like conventional Muse cells, the high-potential pluripotent stem cells of the present invention are not subject to immune rejection, enabling treatment with allogeneic preparations produced from donors without the need for HLA matching or long-term administration of immunosuppressants. Therefore, the high-potential pluripotent stem cells of the present invention, possessing the above-described excellent properties, can provide a viable means for treating patients with damage to tissues formed from embryonic and / or extraembryonic tissues, as well as for reproductive treatments. Furthermore, the high-potential pluripotent stem cells of the present invention highly express HLA-G, suggesting their high immunoregulatory function. This suggests their usefulness, for example, in reducing the risk of rejection during transplantation of donor preparations. Furthermore, the high-potential pluripotent stem cells of the present invention highly express desmosome-related molecules, particularly desmoglein 2, desmocollin 2, 3, plakoglobin, etc., which are also expressed in early embryos and pluripotent stem cells during normal development and are thought to be essential molecules for maintaining pluripotency and self-renewal in human pluripotent stem cells, suggesting that the high-potential pluripotent stem cells of the present invention have excellent pluripotency maintenance and self-renewal functions, and that they may bring about better improvement or recovery of the function of the damaged site in various diseases involving tissue damage, and furthermore, due to their ability to differentiate into germ cell lineages, they may be used as useful pharmaceuticals for regenerative medicine. [Brief explanation of the drawings]

[0013] [Figure 1] Diagram (including some photographs) showing the isolation process of SSEA-3-positive cells (UC-HP-PSCs) from human umbilical cord. [Figure 2] FIG. 1 shows the results of examining the expression of various markers in human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs). [Figure 3] Figure (photo) showing embryoid body-like cell clusters (top) obtained by suspension culture of human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs), and the results of immunostaining of the embryoid body-like cell clusters (bottom). [Figure 4] Embryoid body-like cell clusters of human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSC) were cultured on gelatin-coated dishes and allowed to spontaneously differentiate. The expression of three germ layer markers was examined by immunostaining or RT-PCR. (Photo) [Figure 5] Diagram (photo) showing the results of differentiation of human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) into triploid germ layer cells. [Figure 6]Photographs of a pluripotent cell cluster (1st cluster) obtained by suspension culture of human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs), a pluripotent cell cluster (2nd cluster) obtained by breaking this pluripotent cell cluster into single cells, subjecting them to adherent culture, and then subjecting them to suspension culture, and a pluripotent cell cluster (3rd cluster) obtained by repeating the adherent culture-suspension culture cycle. Figure 1 also shows the results of RT-PCR analysis of the expression of endodermal, mesodermal, and ectodermal markers after spontaneous differentiation of each of these clusters through adherent culture on gelatin-coated dishes. [Figure 7] Figure (photo) shows the results of examining the presence or absence of teratoma formation in mice transplanted with human umbilical cord-derived SSEA-3 positive cells (UC-HP-PSC). [Figure 8-1] Photograph showing the morphology of cells when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and various Muse cells were induced to differentiate into trophoblast lineage cells (conditions: 10 ng / mL BMP4, 1 μM A83-01, 0.1 μM PD173074). [Figure 8-2] Photograph showing the morphology of cells when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and various Muse cells were induced to differentiate into trophoblast lineage cells (conditions: 10 ng / mL BMP4, 20 μM SB431542, 20 μM SU5402). [Figure 8-3] Diagram (photograph) showing the morphology of cells when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and various Muse cells were induced to differentiate into trophoblast lineage cells. [Figure 9] Figure (photo) shows the results of immunostaining to examine the expression of trophoblast markers when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and various Muse cells were induced to differentiate into trophoblast cell lineages. [Figure 10-1] This figure shows the results of quantitative PCR analysis of the expression of trophoblast markers when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and various Muse cells were induced to differentiate into trophoblast lineage cells (conditions: 10 ng / mL BMP4, 1 μM A83-01, 0.1 μM PD173074). [Figure 10-2] This figure shows the results of quantitative PCR analysis of the expression of trophoblast markers when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and various Muse cells were induced to differentiate into trophoblast lineages (conditions: 10 ng / mL BMP4, 20 μM SB431542, 20 μM SU5402). [Figure 11-1] FIG. 1 shows the results of examining CD133 expression in NTERA-2 cells, a human pluripotent embryonal carcinoma cell line. [Figure 11-2] FIG. 1 shows the results of examining CD133 expression in human bone marrow-derived Muse cells (BM-Muse). [Figure 11-3] FIG. 1 shows the results of examining CD133 expression in human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs). [Figure 12-1] This figure shows the results of quantitative PCR to examine the expression of germ cell lineage markers when human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) were induced to differentiate under the same conditions as those for inducing iPS cell differentiation into primordial germ cells. [Figure 12-2] FIG. 1 shows the results of quantitative PCR analysis of the expression of germ cell lineage markers when human bone marrow-derived Muse cells were induced to differentiate under the same conditions as those for inducing differentiation of iPS cells into primordial germ cells. [Figure 13] Human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) and human bone marrow-derived Muse cells (BM-Muse) were fixed with 4% PFA / 0.1M phosphate buffer on days 4 and 6 after differentiation induction under conditions consistent with the differentiation induction conditions for iPS cells into primordial germ cells. The figure (photo) shows the results of immunostaining for the germ cell lineage marker Blimp1. [Figure 14-1] Differentiation induction method using male mouse primordial germ cells <1> <2> and <3> Figure 1 shows the results of quantitative PCR analysis of the expression of germline markers Prdm14, Blimp1, Dppa3, Daz1, Nanos3, SSEA-1, Stra8, and Sycp3 3, 5, 7, and 14 days after differentiation of human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) using IFN-γ. [Figure 14-2] Differentiation induction method using female mouse primordial germ cells <1> <2> and <3> Figure 1 shows the results of quantitative PCR analysis of the expression of germline markers Prdm14, Blimp1, Dppa3, Daz1, Nanos3, SSEA-1, Stra8, and Sycp3 3, 5, 7, and 14 days after differentiation of human umbilical cord-derived SSEA-3-positive cells (UC-HP-PSCs) using IFN-γ. [Figure 15] Differentiation induction method <3> SSEA-3 positive cells derived from human umbilical cord (UC-HP-PSC) were induced to differentiate by IFN-γ-γ (Fcγ-γ), and the cells were fixed in 4% PFA / 0.1M phosphate buffer on day 5. The cells were then immunostained for mitochondria and the germ cell lineage marker Blimp1 (photo). [Figure 16] A diagram (including some photographs) showing human amniotic mesenchymal stem cells (AM-HP-PSCs), SSEA-3-positive cells derived from them, and embryoid body-like cell masses obtained by suspension culture. [Figure 17] Embryoid body-like cell clusters of human amnion-derived SSEA-3-positive cells (AM-HP-PSCs) were cultured in an adherent state on a gelatin-coated dish, and the results of observation and immunostaining for endodermal, mesodermal, and ectodermal markers are shown in the photograph. [Figure 18] FIG. 1 shows the results of flow cytometry analysis of CD105 or CD90 expression in human amniotic membrane and human bone marrow mesenchymal stem cells and SSEA-3-positive cells derived from them (AM-HP-PSCs and BM Muse cells). [Figure 19] FIG. 1 shows the results of flow cytometry analysis of CD44 or CD133 expression in human amniotic membrane and human bone marrow mesenchymal stem cells and the SSEA-3-positive cells (AM-HP-PSCs and BM Muse cells) derived from them, respectively. [Figure 20] FIG. 1 shows the results of flow cytometry analysis of the expression of CD34 or CD45 in human amniotic membrane and human bone marrow mesenchymal stem cells. [Figure 21]Figure 1 shows the results of a comparative quantitative PCR analysis of germline-specific gene expression in human amnion-derived SSEA-3-positive cells (AM-HP-PSCs) and human bone marrow-derived Muse cells (BM Muse cells). [Figure 22] Figure 1 shows the results of a comparative quantitative PCR analysis of the expression of various pluripotency markers in human amnion-derived SSEA-3-positive cells (AM-HP-PSCs) and human bone marrow-derived Muse cells (BM Muse cells). [Figure 23] Diagram (partial photograph) showing the method for preparing high-potential pluripotent stem cells (P-HP-PSCs) derived from human placental tissue. [Figure 24] FIG. 1 shows the results of flow cytometry analysis of CD133-positive expression in SSEA-3-positive cells derived from human placenta. [Figure 25-1] Human placenta-derived high-potential pluripotent stem cells (P-HP-PSCs) were induced to phagocytose male mouse primordial germ cells and differentiate into germ cell lineages. The expression of early and late germ cell lineage markers was evaluated by quantitative PCR. [Figure 25-2] Human placenta-derived high-potential pluripotent stem cells (P-HP-PSCs) were induced to phagocytose female mouse primordial germ cells and differentiate into germ cell lineages. The expression of early and late germ cell lineage markers was evaluated by quantitative PCR. [Figure 26-1] FIG. 1 shows the results of flow cytometry analysis of the percentage of cells expressing HLA-G in human umbilical cord-derived high-potential pluripotent stem cells (UC-HP-PSCs). [Figure 26-2] FIG. 1 shows the results of flow cytometry analysis of the percentage of cells expressing HLA-G in human placenta-derived high-potential pluripotent stem cells (P-HP-PSCs). [Figure 26-3] FIG. 1 shows the results of flow cytometry analysis of the percentage of cells expressing HLA-G in human bone marrow-derived Muse cells (BM-Muse). [Figure 26-4]FIG. 1 shows the results of flow cytometry analysis of the percentage of cells expressing HLA-G in human adipose-derived Muse cells (ADSC-Muse). [Figure 26-5] FIG. 1 shows the results of flow cytometry analysis of the percentage of cells expressing HLA-G in human skin-derived Muse cells (NHDF-Muse). [Figure 27-1] Figure showing the ejection fraction (EF) when vehicle alone (Control), human bone marrow-derived Muse cells (BM-Muse), human umbilical cord-derived high-potential pluripotent stem cell fraction (UC-HP-PSC), or human bone marrow-derived SSEA-3-negative MSCs (BM non-Muse) were administered to a rat myocardial infarction model. [Figure 27-2] Figure 1 shows the change in ejection fraction (ΔEF) when vehicle alone (Control), human bone marrow-derived Muse cells (BM-Muse), human umbilical cord-derived high-potential pluripotent stem cell fraction (UC-HP-PSC), or human bone marrow-derived SSEA-3-negative MSCs (BM non-Muse) were administered to a rat myocardial infarction model. [Figure 27-3] Figure 1 shows the left ventricular fractional shortening (%FS) when vehicle alone (Control), human bone marrow-derived Muse cells (BM-Muse), human umbilical cord-derived high-potential pluripotent stem cell fraction (UC-HP-PSC), or human bone marrow-derived SSEA-3-negative MSCs (BM non-Muse) were administered to a rat myocardial infarction model. [Figure 27-4] Figure 1 shows the change in left ventricular fractional shortening (Δ%FS) when vehicle alone (Control), human bone marrow-derived Muse cells (BM-Muse), human umbilical cord-derived high-potential pluripotent stem cell fraction (UC-HP-PSC), or human bone marrow-derived SSEA-3-negative MSCs (BM non-Muse) were administered to a rat myocardial infarction model. [Figure 28]This figure shows the results of RNA-seq gene expression analysis of desmosome-related molecules in human umbilical cord-derived high-potential pluripotent stem cells (UC-HP-PSCs), human bone marrow-derived Muse cells (BM-Muse), human adipose-derived Muse cells (ADSC-Muse), and human skin-derived Muse cells (NHDF-Muse). The vertical axis shows fragments per kilobase of exon per million mapped reads (FPKM). DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention relates to high-potential pluripotent stem cells isolated from extraembryonic tissues and the like, and to cell preparations containing the high-potential pluripotent stem cells. The present invention is described in detail below.

[0015] 1. Cell Preparation (1) High-potential pluripotent stem cells The high-potential pluripotent stem cells used in the cell preparations of the present invention are high-potential pluripotent stem cells isolated from extraembryonic tissues, etc., and have the same useful properties of pluripotent stem cells as Muse cells described below. In addition, they have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, have differentiation potential close to totipotency, and are positive for CD133, a cell surface marker that is negative in Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc. In particular, as shown below, the human umbilical cord-derived SSEA-3+ cells obtained in Example 1 have useful properties of pluripotent stem cells similar to those of Muse cells conventionally obtained from bone marrow, peripheral blood, fat, skin, etc., as determined by other analyses in Example 1, and in addition have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, have differentiation potential close to totipotency, and are 99.9% or more positive for the cell surface marker CD133. For these reasons, the human umbilical cord-derived SSEA-3+ cells obtained in Example 1 are referred to as "human umbilical cord-derived high potential pluripotent stem cells," sometimes abbreviated as UC-HP-PSC (Umbilical cord High Potential Pluripotent stem cells). Furthermore, other analyses have shown that the human amnion-derived SSEA-3-positive cells obtained in Example 2 have useful properties of pluripotent stem cells similar to those of Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc., and in addition have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, have differentiation potential close to totipotency, and are approximately 70% or more positive for the cell surface marker CD133. For these reasons, the human amnion-derived SSEA-3-positive cells obtained in Example 2 are referred to as "human amnion-derived high-potential pluripotent stem cells" or "human amnion-derived high-potential pluripotent stem cell fraction," and are sometimes abbreviated as AM-HP-PSC (Amniotic membrane High Potential Pluripotent stem cells). Furthermore, other analyses have shown that the human placenta-derived SSE-3-positive cells obtained in Example 3 have useful properties of pluripotent stem cells similar to those of Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc., and in addition have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, have differentiation potential close to totipotency, and are approximately 80% or more positive for the surface marker CD133. For these reasons, the human placenta-derived SSEA-3-positive cells obtained in Example 3 are referred to as "human placenta-derived high-potential pluripotent stem cells" or "human placenta-derived high-potential pluripotent stem cell fraction," and may be abbreviated as P-HP-PSC (Placenta High Potential Pluripotent stem cells). Furthermore, it has been confirmed that the high-potential pluripotent stem cells of the present invention exist among conventional bone marrow-derived SSEA-3-positive cells, albeit in very small numbers (up to about a few percent). Therefore, the high-potential pluripotent stem cells of the present invention can be obtained not only from extraembryonic tissues, but also from conventional bone marrow, peripheral blood, fat, skin, etc.

[0016] Therefore, the high-potential pluripotent stem cells of the present invention can be considered to be primarily a subset of specialized Muse cells isolated from extraembryonic tissues. That is, the high-potential pluripotent stem cells of the present invention have useful properties of pluripotent stem cells similar to those of Muse cells, which means the properties of Muse cells described below.

[0017] Muse cells can be obtained from bone marrow fluid, adipose tissue (Non-Patent Document 4), and dermal connective tissue of the skin, and are also known to be present in connective tissues of organs and peripheral blood. These cells possess properties of both pluripotent stem cells and mesenchymal stem cells, and are identified, for example, as cells positive for the cell surface marker "SSEA-3," preferably as double-positive cells positive for SSEA-3 and CD105. Therefore, Muse cells or cell populations containing Muse cells can be isolated from biological tissues using, for example, the expression of SSEA-3 alone or the expression of SSEA-3 and CD105 as indicators. Details of the isolation, identification, and characteristics of Muse cells are disclosed in International Publication No. WO 2011 / 007900. Furthermore, taking advantage of the high resistance of Muse cells to various external stresses, Muse cells can be selectively enriched by treatment with proteolytic enzymes or by culturing under various external stress conditions, such as hypoxia, low phosphate, low serum concentration, low nutrition, exposure to heat shock, the presence of harmful substances, the presence of reactive oxygen species, mechanical stimulation, and pressure treatment. Herein, pluripotent stem cells (Muse cells) or cell populations containing Muse cells prepared from in vivo mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an indicator may be referred to simply as "SSEA-3-positive cells."

[0018] Muse cells or cell populations containing Muse cells can be prepared from biological tissues (e.g., mesenchymal tissues) using the cell surface markers SSEA-3 or SSEA-3 and CD105 as indicators. Here, "biological organism" refers to a mammalian organism. In the present invention, "biological organism" does not include fertilized eggs or embryos at developmental stages earlier than the blastula stage, but does include embryos at developmental stages after the blastula stage, including fetuses and blastulas. Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparations of the present invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are directly isolated from biological tissues and possess markers. Furthermore, "mesenchymal tissue" refers to tissues such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, umbilical cord, umbilical cord blood, and amniotic membrane, as well as tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, adipose tissue, blood, dental pulp, umbilical cord, umbilical cord blood, and amniotic membrane. For example, it is preferable to collect mesenchymal tissue from a living organism and prepare and use Muse cells from this tissue. Alternatively, Muse cells may be prepared from cultured mesenchymal cells such as fibroblasts or bone marrow mesenchymal stem cells using the above-mentioned preparation methods.

[0019] Alternatively, a cell population containing Muse cells can be prepared by a method comprising applying an external stress stimulus to mesenchymal tissue of a living body or cultured mesenchymal cells, selectively proliferating cells that are resistant to the external stress, and recovering cells with an increased abundance. The external stress may be any one or a combination of protease treatment, culture at a low oxygen concentration, culture under low phosphate conditions, culture at a low serum concentration, culture under low nutrient conditions, culture under exposure to heat shock, culture at low temperature, freezing treatment, culture in the presence of a harmful substance, culture in the presence of active oxygen, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical impact. The protease treatment time is preferably 0.5 to 36 hours in total to apply external stress to the cells. The protease concentration may be any concentration that is used when detaching cells adhered to a culture vessel, disaggregating cell clumps into single cells, or recovering single cells from tissue. The protease is preferably a serine protease, an aspartic acid protease, a cysteine ​​protease, a metalloprotease, a glutamic acid protease, or an N-terminal threonine protease, and more preferably trypsin, collagenase, or dispase.

[0020] The Muse cells may be autologous or allogeneic to the recipient of the cell transplant.

[0021] As described above, Muse cells or a cell population containing Muse cells can be prepared from biological tissue using, for example, SSEA-3 positivity or double positivity for SSEA-3 and CD105 as an indicator. Adult human skin is known to contain various types of stem and progenitor cells, including skin-derived progenitor cells (SKPs), neural crest stem cells (NCSCs), melanoblasts (MBs), pericytes (PCs), endothelial progenitor cells (EPs), and adipose-derived stem cells (ADSCs). Because Muse cells are not identical to these cells, they can be prepared using the "non-expression" of markers specific to these cells as an indicator. More specifically, Muse cells can be separated using the non-expression of at least one, for example, two, three, four, five, six, seven, eight, nine, ten, or eleven, of eleven markers selected from the group consisting of CD34 (a marker for EPs and ADSCs), CD117 (c-kit) (a marker for MBs), CD146 (a marker for PCs and ADSCs), CD271 (NGFR) (a marker for NCSCs), NG2 (a marker for PCs), vWF factor (von Willebrand factor) (a marker for EPs), Sox10 (a marker for NCSCs), Snail (a marker for SKPs), Slug (a marker for SKPs), Tyrp1 (a marker for MBs), and Dct (a marker for MBs). For example, but not limited to, it can be prepared using the non-expression of CD117 and CD146 as an indicator, and further it can be prepared using the non-expression of CD117, CD146, NG2, CD34, vWF, and CD271 as an indicator, and further it can be prepared using the non-expression of the above 11 markers as an indicator.

[0022] Furthermore, Muse cells having the above characteristics are as follows: (i) low or absent telomerase activity; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) Self-renewing ability Preferably, the Muse cells used in the present invention have all of the above properties. Here, with regard to (i) above, "low or no telomerase activity" means that the telomerase activity is low or undetectable when detected using, for example, a TRAPEZE XL telomerase detection kit (Millipore). "Low" telomerase activity means, for example, that the telomerase activity is at the same level as that of human fibroblasts, which are somatic cells, or that the telomerase activity is 1 / 5 or less, preferably 1 / 10 or less, of that of HeLa cells. Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endodermal, mesodermal, and ectodermal) in vitro and in vivo. For example, by in vitro induction culture, they can differentiate into hepatocytes (including hepatoblasts or cells expressing hepatocyte markers), neurons, skeletal muscle cells, smooth muscle cells, osteocytes, adipocytes, etc. In addition, when transplanted into the testis in vivo, they may also exhibit the ability to differentiate into three germ layers. Furthermore, when transplanted into a living body by intravenous injection, they have the ability to migrate and engraft in injured organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells appropriate for the tissue. Regarding (iii) above, Muse cells proliferate at a rate of approximately 1.3 days. In suspension culture, they proliferate from a single cell, forming embryoid-like cell clusters. Once they reach a certain size, proliferation ceases after approximately 14 days. However, when these embryoid-like cell clusters are transferred to adherent culture, cell proliferation resumes, and cells proliferate from the cell clusters and spread at a rate of approximately 1.3 days. Furthermore, when transplanted into the testis, they do not become cancerous for at least six months. Furthermore, with regard to (iv) above, Muse cells have the ability to self-renew (self-replicate). Here, "self-renewal" refers to the fact that when cells contained in an embryoid-like cell mass obtained by culturing a single Muse cell in suspension culture are transferred to adherent culture, differentiation into three germ layers can be confirmed; and when cells from the embryoid-like cell mass are transferred to suspension culture as a single cell to form a next-generation embryoid-like cell mass, which is then transferred to adherent culture, differentiation into three germ layers can again be confirmed; and when an embryoid-like cell mass is again formed by culturing a single cell in suspension culture, differentiation into three germ layers can again be confirmed. Self-renewal can be achieved by repeating one or more cycles.

[0023] The high-potential pluripotent stem cells used in the cell preparation of the present invention are high-potential pluripotent stem cells isolated from extraembryonic tissues, etc., and have the same useful properties of pluripotent stem cells as Muse cells described above. In addition, they have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, and have differentiation potential close to totipotency. Furthermore, they are positive for CD133, a cell surface marker that is negative in Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc. CD133 is a glycoprotein and a cell surface marker also known as Prominin 1. Examples of human CD133 include proteins registered in the NCBI (National Center for Biotechnology Information) Protein Database under accession numbers AER93377 and AER93376. CD133 positive refers to cells that are stained when cells are stained using a CD133 antibody or cells that are selected with a CD133 antibody by flow cytometry. The high-potential pluripotent stem cells of the present invention can be a cell population containing CD133-positive cells, and preferably contain 50% or more CD133-positive cells, more preferably 60% or more, 70% or more, or 80% or more, and even more preferably 90% or more. The positivity of the cell surface marker CD133 can be confirmed by a conventional identification method using an antibody or the like.

[0024] The high-potential pluripotent stem cells of the present invention can be obtained from extraembryonic tissues, etc. Here, extraembryonic tissues, etc. can be obtained from biological tissues that have completed their role as organs and become waste after pregnancy, birth, and delivery, such as umbilical cord, umbilical cord blood, placenta, placental blood, decidua, chorion, amniotic membrane, and amniotic fluid.

[0025] Furthermore, the high-potential pluripotent stem cells of the present invention are capable of differentiating into all germ layers that constitute an individual (the three germ layers of ectoderm, mesoderm, and endoderm), and further have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, and have differentiation potential close to totipotency. Here, "having the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, and having differentiation ability close to totipotency" means the ability to differentiate into various cells, including extraembryonic tissues such as the placenta, gametes (sperm or eggs) of the germ cell lineage, and the primordial germ cells (PGCs) that form these. Specifically, the differentiation potential of extraembryonic tissues into cells and / or germ cell lineages can be confirmed by, for example, differentiation into various cells in each extraembryonic tissue, differentiation into trophoblast cell lineages, cell multinucleation, expression of trophoblast markers (e.g., ERVW-1, human chorionic gonadotropin alpha chain (hCGA), etc.; early markers such as CDX2, TP63, and ID2; late markers such as GCM1, PGF, and ERVFRD-1), expression of genes involved in germ cell lineage differentiation (e.g., Blimp1, Dappa3, ITGA, Sycp3, TBX3, TFAP2c, TP63, Nanos3, PRDM14, SSEA-1, Daz1, Stra8, etc.), and expression of genes suggesting pluripotency (e.g., Oct3 / 4, Nanog, Sox2, KLF2, KLF4, REX1, TERT, etc.).

[0026] In particular, the high-potential pluripotent stem cells of the present invention can be obtained by a conventional method for isolating Muse cells from extraembryonic tissues and the like (International Publication No. WO2011 / 007900). For example, the high-potential pluripotent stem cells of the present invention can be isolated by mincing extraembryonic tissue, culturing the tissue fragments, culturing mesenchymal cells derived from the extraembryonic tissue, and increasing the number of cells until an effective cell mass is reached, and then using the following antigen markers as indicators: (i) SSEA-3 alone, (ii) a double SSEA-3 and CD105, (iii) a double SSEA-3 and CD133, or (iv) a triple SSEA-3, CD105, and CD133. Furthermore, the high-potential pluripotent stem cells of the present invention can be obtained from embryonic tissues such as bone marrow, peripheral blood, fat, and skin, in accordance with the methods for isolating them from the above-mentioned extraembryonic tissues.

[0027] Furthermore, the high-potential pluripotent stem cells of the present invention can be confirmed to be cells with differentiation ability close to totipotency, for example, by examining the characteristics of naive or primed pluripotent stem cells shown in Table 1 below. [Table 1] <References: 1) L. Weinberger, M. Ayyash, N. Novershtern, JH Hanna, Dynamic stem cell states: naive to primed pluripotency in rodents and humans. Nat. Rev. Mol. Cell Biol. 17, 155-169 (2016). 2)M. Ueda, Y. Takashima, History of Pluripotent Stem Cells and Human Naive Pluripotent Stem Cells. Cytometry Research 27, 19-24 (2017). 3) Kiichiro Tomoda, Biochemistry, Vol. 90, No. 2, pp. 187-191 (2018)

[0028] Furthermore, the high-potential pluripotent stem cells of the present invention can be confirmed by the expression of desmosome-related molecules, particularly desmoglein 2, desmocollin 2, 3, plakoglobin, etc., which are also expressed in early embryos and pluripotent stem cells during normal development and are thought to be essential molecules for maintaining pluripotency and self-renewal in human pluripotent stem cells, etc.

[0029] (2) Preparation and use of cell preparations The cell preparation of the present invention can be obtained by suspending the high-potential pluripotent stem cells of the present invention obtained in (1) above or a cell population containing such cells in physiological saline or an appropriate buffer solution (e.g., phosphate-buffered saline). In this case, if the high-potential pluripotent stem cells of the present invention isolated from autologous or allogeneic tissue are scarce, the cells may be cultured and expanded until a predetermined cell number is obtained before cell transplantation. Because the high-potential pluripotent stem cells of the present invention are non-tumorigenic, even if undifferentiated cells recovered from biological tissue are contained, the possibility of canceration is low and safe. Furthermore, the recovered high-potential pluripotent stem cells of the present invention can be cultured in a conventional growth medium (e.g., α-minimal essential medium (α-MEM) containing 10% fetal bovine serum), without particular limitation. For more details, with reference to the pamphlet of International Publication No. WO 2011 / 007900, appropriate medium and additives (e.g., antibiotics, serum), etc., can be selected to prepare a solution containing the high-potential pluripotent stem cells of the present invention at a predetermined concentration during the culture and expansion of the high-potential pluripotent stem cells of the present invention. When the cell preparation of the present invention is administered to a human subject, human extraembryonic tissue is collected, and for example, umbilical cord tissue-derived mesenchymal stem cells are cultured as adherent cells from umbilical cord tissue until a cell quantity sufficient to obtain an effective therapeutic amount of the high-potential pluripotent stem cells of the present invention is reached, and the high-potential pluripotent stem cells of the present invention are then isolated using the SSEA-3 antigen marker as an indicator, and autologous or allogeneic high-potential pluripotent stem cells of the present invention can be prepared as a cell preparation. Alternatively, for example, extraembryonic tissue-derived mesenchymal stem cells obtained from human extraembryonic tissue can be cultured under external stress conditions to proliferate and concentrate the high-potential pluripotent stem cells of the present invention until an effective therapeutic amount is reached, and then autologous or allogeneic high-potential pluripotent stem cells of the present invention can be prepared as a cell preparation.

[0030] Furthermore, when using the high-potential pluripotent stem cells of the present invention in a cell preparation, the cell preparation may contain dimethyl sulfoxide (DMSO) or serum albumin to protect the cells, or antibiotics to prevent bacterial contamination and proliferation. Furthermore, the cell preparation may contain other pharmaceutical components (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.). Those skilled in the art can add these factors and drugs to the cell preparation at appropriate concentrations. Thus, the high-potential pluripotent stem cells of the present invention can also be used as a pharmaceutical composition containing various additives. The dosage form of the cell preparation is not particularly limited, but is preferably a parenteral administration formulation, and more preferably an injectable formulation.

[0031] The number of high-potential pluripotent stem cells of the present invention contained in the cell preparation prepared as described above can be adjusted appropriately, taking into consideration the subject's gender, age, weight, condition of the affected area, and the condition of the cells used, so as to achieve the desired effect on the disease being treated. Target individuals include, but are not limited to, mammals such as humans. The cell preparation of the present invention may be administered once, or multiple times (e.g., 2 to 10 times) at appropriate intervals (e.g., twice a day, once a day, twice a week, once a week, once every two weeks, once a month, once every two months, once every three months, or once every six months) until the desired therapeutic effect is achieved. The administration time may be acute, subacute, or chronic, or may be two or more times selected from these, as long as the therapeutic effect is achieved. The therapeutically effective amount varies depending on the condition of the subject, but is, for example, 1 × 10 per individual per administration. 3 cells ~1×10 10 The preferred dose is 1 to 10 times per cell. The total dose per individual is not limited, but is preferably 1 x 10 3 cells ~1×10 11 cells, preferably 1 x 10 4 cells ~1×10 10 cells, more preferably 1 x 10 5 cells ~1×10 9 Cells, etc.

[0032] The high-potential pluripotent stem cells of the present invention used in the cell preparation of the present invention have the property of migrating to and engrafting at the site of damage caused by the disease to be treated. Therefore, the administration site and method of the cell preparation are not limited, and the cell preparation may be administered locally to the affected area or intravenously.

[0033] The cell preparation of the present invention can reconstruct tissue at the damaged site in a patient suffering from a disease to be treated, and can improve or restore lost function at the damaged site in the patient.

[0034] The present invention will be explained in more detail with reference to the following examples, but the present invention is not limited to these examples in any way. [Example]

[0035] Example 1-1: Isolation of high-potential pluripotent stem cells from human umbilical cord High-potential pluripotent stem cells can be obtained from human umbilical cords according to the method described in International Publication No. WO 2011 / 007900 regarding the isolation and identification of human Muse cells. More specifically, high-potential pluripotent stem cells derived from human umbilical cords were obtained by FACS separation using SSEA-3 as an indicator, according to the steps shown in Figure 1. Specifically, a 35-week-old human umbilical cord was minced into 0.5 mm cubes and placed at 1 cm intervals in a 10 cm dish. αMEM, 10% FBS, 1 ng / mL bFGF, and 2 mM GlutaMAX were added and cultured for approximately 10 days using the explant method. The established human umbilical cord-derived mesenchymal stem cells were expanded and subcultured, and then SSEA-3-positive cells derived from the human umbilical cord tissue (i.e., the human umbilical cord-derived high-potential pluripotent stem cells of the present invention: UC-HP-PSC) were isolated using an anti-SSEA-3 antibody. Approximately 3% of the established human umbilical cord-derived mesenchymal stem cells contained SSEA-3-positive cells.

[0036] Example 1-2: High-potential pluripotent stem cells derived from human umbilical cord co-express mesenchymal cell surface markers The cell surface markers of the human umbilical cord-derived SSEA-3-positive cells obtained in Example 1-1 were analyzed by FACS. As a result, as shown in Figure 2, the human umbilical cord-derived SSEA-3-positive cells co-expressed the mesenchymal stem cell markers CD105, CD90, CD44, CD73, and CD166. However, they were negative for the hematopoietic markers CD34 and CD45, the neural crest-derived stem cell marker CD271, and the endothelial progenitor marker von Willebrand factor (vWF).

[0037] Example 1-3: Human umbilical cord-derived high-potential pluripotent stem cells have the following properties similar to conventional human bone marrow-derived Muse cells: (1) High-potential pluripotent stem cells derived from human umbilical cords form pluripotent cell clusters Single-cell suspension culture was performed using human umbilical cord-derived SSEA-3-positive cells obtained by FACS in Example 1-1. On day 7 of culture, 44.2% of the SSEA-3-positive cells formed embryoid body-like cell clusters (Figure 3, top). These cell clusters were cryo-embedded and thin-sectioned, and immunostained for the pluripotency markers Nanog, Oct3 / 4, Sox2, PAR4, and Tra-1-81. As a result, as shown in Figure 3, bottom, the embryoid body-like cell clusters expressed all of the pluripotency markers, confirming that the embryoid body-like cell clusters formed from human umbilical cord-derived SSEA-3-positive cells were pluripotent cell clusters.

[0038] (2) High-potential pluripotent stem cells derived from human umbilical cord spontaneously differentiate into three germ layers To confirm the differentiation potential of SSEA-3+ cells derived from a single human umbilical cord, we transferred pluripotent cell clumps to gelatin-coated dishes and cultured them as adherent cells. After approximately 10 days, immunohistochemistry revealed the expression of cytokeratin 7 (CK7, an endodermal marker), α-smooth muscle actin (SMA, a mesodermal marker), and neurofilament-M (an ectodermal marker), as shown in Figure 4. RT-PCR also confirmed the expression of α-fetoprotein (α-FP, an endodermal marker), Nkx2.5 (a mesodermal marker), and microtubule-associated protein-2 (MAP-2, an ectodermal marker), as shown in Figure 4. These results demonstrate that SSEA-3+ cells derived from human umbilical cord have the spontaneous differentiation potential to differentiate into three germ layers.

[0039] (3) High-potential pluripotent stem cells derived from human umbilical cord differentiate into three germ layers upon differentiation induction For neural induction, 1 × 10 human umbilical cord-derived SSEA-3 positive cells were cultured in poly-HEMA-coated dishes. 5 The cells were cultured at a density of 1000 cells / mL in Neurobasal medium (Gibco) containing B-27 supplement for 7 days to form spheres (spherical cell masses). For osteocyte induction, 4 × 10 human umbilical cord-derived SSEA-3 positive cells were used. 3 cells / cm 2 The cells were cultured at a density of 1000 for 16 days using the osteocyte induction medium from the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems). For adipocyte induction, 1 × 10 human umbilical cord-derived SSEA-3 positive cells were used. 4 cells / cm 2 The cells were cultured at a density of 1000 for 16 days using the adipocyte induction medium from the Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems). For hepatocyte induction, 1 × 10 human umbilical cord-derived SSEA-3 positive cells were used. 4 cells / cm 2The cells were seeded onto collagen-coated plates at a density of 100 μg / well. After seeding, they were cultured in DMEM-low glucose, 20 ng / mL EGF, and 10 ng / mL bFGF for 2 days. They were then cultured in DMEM-low glucose, 0.5 mg / mL BSA, 1× insulin-transferrin-selenium, 5 mM nicotinamide, 10 nM dexamethasone, 150 ng / mL HGF, and 50 ng / mL FGF4 for 28 days. For endothelial cell induction, 5 x 10 cells 3 cells / cm 2 The cells were cultured at a density of 1000 for 8 days in DMEM-low-glucose, 2% FBS, and 20 ng / mL bFGF.

[0040] The results are shown in Figure 5. Neural induction led to the formation of spheres, which were confirmed to be positive for neural stem cell markers NeuroD1, Nestin, and Musashi-1. Expression of osteocalcin was observed in bone cell induction, FABP-4 in adipocyte induction, and CD141 in vascular endothelial cell induction. Expression of α-fetoprotein, albumin, and cytokeratin 19 (CK19) was confirmed by hepatocyte induction. These results indicate that human umbilical cord-derived SSEA-3 positive cells have the ability to differentiate into three germ layer cells even when induced to differentiate.

[0041] (4) High-potential pluripotent stem cells derived from human umbilical cords have the ability to self-renew The self-renewal potential of human umbilical cord-derived SSEA-3+ cells was assessed using their differentiation potential into three germ layers. Pluripotent cell clusters were formed from single human umbilical cord-derived SSEA-3+ cells, and the single cell clusters were then cultured on uncoated or gelatin-coated dishes. The cell clusters cultured on uncoated dishes were cultured for 7 days, then single cells were treated with trypsin for 5 minutes and recultured in suspension to form pluripotent cell clusters (second cluster). Meanwhile, cell clusters cultured on gelatin-coated dishes were cultured for approximately 10 days, after which RNA was extracted. This suspension-adherent culture cycle was repeated three times, and each generation confirmed the expression of α-fetoprotein (α-FP, an endoderm marker), Nkx2.5 (a mesoderm marker), and microtubule-associated protein (MAP-2, an ectoderm marker) (Figure 6). These results demonstrate that human umbilical cord-derived SSEA-3+ cells possess self-renewal potential and undergo clonally expanding growth.

[0042] (5) High-potential pluripotent stem cells derived from human umbilical cords do not form tumors when transplanted into living organisms To evaluate the tumorigenicity of human umbilical cord-derived SSEA-3-positive cells in vivo, we used immunodeficient mice, CB17 / IcrJcl-Prkdc scid 1 × 10 testis of a male (2 months old) 5 Human umbilical cord-derived SSEA-3 positive cells were transplanted using glass microtubes. 1 × 10 cells were used as a control. 5 Mouse ES cells (positive control), 1 x 10 5 Mitomycin C-treated MEF (mouse embryonic fibroblast) cells and PBS (negative control) were transplanted. As a result, as shown in Figure 7, mouse ES cells formed teratomas 3 months after transplantation, whereas human umbilical cord-derived SSEA-3-positive cells did not form teratomas even 6 months after transplantation. This indicates that human umbilical cord-derived SSEA-3-positive cells do not have tumorigenic potential.

[0043] Example 1-4: High-potential pluripotent stem cells derived from human umbilical cord tissue have the following properties not found in conventional bone marrow, fat, or skin-derived Muse cells: (1) Induction of differentiation into trophoblast lineage The differentiation of SSEA-3 positive cells derived from the human umbilical cord obtained in Example 1-1 into trophoblast lineage cells was confirmed by the following two induction methods, according to the differentiation induction conditions below for inducing differentiation of ES cells and iPS cells into trophoblast lineage cells. As a control group, Muse cells derived from human bone marrow (BM-Muse), human adipose tissue (ADSC-Muse), and human skin (NHDF-Muse) were used. 1 × 10 cells were cultured at 1 × 10 4 cells / cm 2 The cells were seeded onto dishes at a density of 100 μg / cm2 and cultured for 3 weeks in [1] DMEM-low-glucose, 2% FBS, 10 ng / mL BMP4, 1 μM A83-01, 0.1 μM PD173074, or [2] DMEM-low-glucose, 2% FBS, 10 ng / mL BMP4, 20 μM SB431542, and 20 μM SU5402 (Yabe, S. et al. Proc. Natl. Acad. Sci. USA 113, E2598-2607(2016); Sudheer, S. et al. Stem Cells Dev 21, 2987-3000(2012)).

[0044] (2) High-potential pluripotent stem cells derived from human umbilical cords were differentiated into multinucleated cells by the differentiation induction method described above [1][2]. Placental syncytiotrophoblast-like cells are multinucleated cells. As shown in Figure 8, in SSEA-3-positive cells derived from human umbilical cord, multinucleated cells appeared after 2 weeks using induction method [1] and after 3 weeks using induction method [2]. The morphology of these cells is very similar to that of syncytiotrophoblast-like cells derived from human ES cells. However, no multinucleated cells were observed in Muse cells derived from bone marrow (BM), adipose tissue (ADSC), or skin (NHDF) using any of the induction methods.

[0045] (3) High-potential pluripotent stem cells derived from human umbilical cords expressed trophoblast markers using the differentiation induction methods described above [1][2]. Three weeks after induction, the cells were fixed in 4% PFA / 0.1M phosphate buffer and immunostained for the trophoblast markers ERVW-1 and human chorionic gonadotropin alpha chain (hCGA). As shown in Figure 9, ERVW-1 and hCGA were detected in SSEA-3-positive cells derived from human umbilical cord using both induction methods [1] and [2], but expression was not observed in Muse cells derived from bone marrow, adipose tissue, or skin. We also evaluated the expression of trophoblast markers CDX2, GCM1, and ERVFRD-1 at 3 and 5 days, and 1, 2, and 3 weeks after induction using quantitative PCR. As shown in Figures 10-1 and 10-2, the early marker CDX2 was detected in SSEA-3-positive cells derived from human umbilical cord after 3 days of induction, while the late markers GCM1 and ERVFRD-1 were expressed after 2 weeks. Furthermore, compared with induction method [1], induction method [2] showed higher expression of all trophoblast markers, suggesting it is a more efficient induction method. On the other hand, no expression of any markers was observed in Muse cells derived from bone marrow (BM), adipose tissue (ADSC), or skin (NHDF). This indicates that, unlike Muse cells derived from bone marrow, adipose tissue, and skin, SSEA-3-positive cells derived from the human umbilical cord have the ability to differentiate into trophoblast cells, an extraembryonic tissue.

[0046] Example 1-5: High-potential pluripotent stem cells derived from human umbilical cord tissue are positive for the cell surface marker CD133 CD133 is a glycoprotein and a cell surface marker also known as Prominin 1. Although its function remains unknown, it has been revealed that it is expressed on some stem / progenitor cells, such as hematopoietic stem cells and neural stem cells. The expression of CD133 in the human umbilical cord-derived SSEA-3-positive cells obtained in Example 1-1 was confirmed by FACS. Bone marrow-derived Muse cells were used as a comparison group, and the pluripotent human embryonal carcinoma cell line NTERA-2 was used as a positive control. As shown in Figures 11-1, 11-2, and 11-3, 84.9% of SSEA-3-positive cells in NTERA-2 were CD133-positive, while 99.9% of SSEA-3-positive cells in human umbilical cord-derived SSEA-3-positive cells were CD133-positive. On the other hand, only 2.0% of SSEA-3-positive cells in bone marrow-derived Muse cells were CD133-positive, a significantly lower level than that of human umbilical cord-derived SSEA-3-positive cells.

[0047] Example 1-6: High-potential pluripotent stem cells derived from human umbilical cord tissue have the ability to differentiate into germline cells (1) Induction of differentiation into primordial germ cells The differentiation of the human umbilical cord-derived SSEA-3 positive cells obtained in Example 1-1 into primordial germ cells was confirmed by the following induction method, according to the differentiation induction conditions below for inducing differentiation of iPS cells into primordial germ cells. Human bone marrow-derived Muse cells were used as a control group, and primordial germ cells induced from iPS cells (iPS-PGCs) were used as a positive control group. 5 × 10 human umbilical cord-derived SSEA-3 positive cells or human bone marrow-derived Muse cells were used. 4 cells / cm 2 The cells were seeded at a density of 3 × 10 cells / well in a dish, and then cultured in DMEM-low-glucose, 10% FBS, 50 ng / mL Activin A, 3 μM CHIR99021, and 10 μM Y-27632 for 2 days. 3 Cells were seeded at 100 cells / well, and cultured in suspension for 4 days in DMEM-low-glucose, 10% FBS, 200 ng / mL BMP-4, 1000 U / mL LIF, 100 ng / mL SCF, 50 ng / mL EGF, and 10 μM Y-27632. The expression of germline markers Blimp1, Nanos3, Dppa3, TFAP2C, PRDM14, and SSEA-1 was assessed by quantitative PCR on days 4 and 6 after induction. The results are shown in Figures 12-1 and 12-2. Blimp1 and Nanos3 were detected in human umbilical cord-derived SSEA-3+ cells from day 4 onward. Furthermore, Dppa3 expression was observed on day 6 after induction, and SSEA-1 expression also increased. In contrast, bone marrow-derived Muse cells expressed some markers, including Nanos3 and Dppa3, but Blimp1 and SSEA-1 expression was lower than in human umbilical cord-derived SSEA-3+ cells. Immunostaining for the germline marker Blimp1 was also performed on days 4 and 6 after induction. The results are shown in Figure 13. Blimp1 was detected in human umbilical cord-derived SSEA-3+ cells, but was barely expressed in bone marrow-derived Muse cells. These results suggest that human umbilical cord-derived SSEA-3+ cells have the ability to differentiate more efficiently into germline cells than bone marrow-derived Muse cells.

[0048] (2) High-potential pluripotent stem cells derived from human umbilical cord cells can be induced to differentiate into germline cells more efficiently by phagocytosing apoptotic primordial germ cells. Commercially available human umbilical cord-derived SSEA-3 positive cells were used to confirm the induction of differentiation into primordial germ cells using the following induction method. 5 × 10 human umbilical cord-derived SSEA-3 positive cells were used. 3 cells / cm 2 After seeding at a density of 1 × 10 cells, male or female mouse primordial germ cells were killed with 100 μM rotenone and 100 μM oligomycin A. 4 cells / cm 2 The cells were added at a density of 100 μg / ml and allowed to phagocytose in DMEM-low-glucose, 10% FBS for 3 days. <1> After removing the apoptotic mouse primordial germ cells, they were cultured in DMEM-low-glucose, 10% FBS as adherent cells for 11 days. <2> 5 × 10 SSEA-3 positive cells derived from human umbilical cord 3 cells / cm 2After seeding in a dish at a density of 5 × 10 cells, mitomycin C-treated somatic cells in the genital ridges other than primordial germ cells were added. 3 cells / cm 2 The cells were added to a density of 100 μg / cm2 and cultured in DMEM-low-glucose, 10% FBS for 11 days as adherent cells. <3> 5 × 10 human umbilical cord-derived SSEA-3 positive cells were cultured in a low-cell binding V-bottom 96-well plate. 3 cells / well, 5 × 10 somatic cells in the genital ridge other than primordial germ cells treated with mitomycin C 4 The cells were suspended at 100 cells / well and cultured in suspension in DMEM-low-glucose, 10% FBS for 11 days. Quantitative PCR was used to evaluate the expression of germline markers PRDM14, Blimp1, Dppa3, Dazl, Nanos3, SSEA-1, Stra8, and Sycp3 on days 3, 5, 7, and 14 after induction. Primordial germ cell-like cells (PGCLCs) induced from human iPS cells were used as a positive control. The results are shown in Figures 14-1 and 14-2. Induction method <1> In this study, increased expression of markers such as Blimp1, Dppa3, and Nanos3 was observed regardless of whether male or female primordial germ cells were phagocytosed. <2> So, the induction method <1> Although some markers showed higher levels than in the control group, most markers were expressed at the same level. <3> In this method, the expression of all markers was high five days after induction, demonstrating that this method is a more efficient method of inducing differentiation into germline cells than other induction methods. However, after seven days, the expression levels decreased due to an increase in cell death caused by the effects of suspension culture. <3> On day 5, the cells were fixed with 4% PFA / 0.1M phosphate buffer and immunostained for human mitochondria and the germline marker Blimp1. The results are shown in Figure 15. Blimp1 expression was confirmed in human mitochondria-positive cells regardless of whether the primordial germ cells were phagocytosed. This indicates that human umbilical cord-derived SSEA-3-positive cells efficiently differentiate into germline cells by phagocytosing apoptotic primordial germ cells. The commercially available human umbilical cord-derived SSEA-3-positive cells used in this example were a human umbilical cord-derived high-potential pluripotent stem cell fraction containing 91.2% CD133-positive cells.

[0049] Based on other analyses in Example 1, the human umbilical cord-derived SSEA-3+ cells obtained in Example 1 above have useful properties of pluripotent stem cells similar to those of Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc., and in addition have properties such as the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, having differentiation potential close to totipotency, and being positive for the cell surface marker CD133. For these reasons, CD133-positive pluripotent stem cells capable of differentiating into cells of extraembryonic tissues and / or germ cell lineages, as exemplified by the human umbilical cord-derived SSEA-3+ cells obtained in Example 1 above, are referred to as novel SSEA-3+ pluripotent stem cells and are referred to as "high-potential pluripotent stem cells" in the present invention, and those derived from human umbilical cord in particular are referred to as "human umbilical cord-derived high-potential pluripotent stem cells."

[0050] Example 2-1: Isolation of high-potential pluripotent stem cells from human amniotic membrane High potential pluripotent stem cells were obtained from human amniotic membrane according to the method described in International Publication No. WO2011 / 007900 regarding the isolation and identification of human Muse cells. After expanding and subculturing the obtained human amnion-derived mesenchymal stem cells, we isolated human amnion-derived SSEA-3-positive cells (i.e., human amnion-derived high-potential pluripotent stem cells: AM-HP-PSCs) using an anti-SSEA-3 antibody. As a result, as shown in Figure 16, approximately 1% of the human amnion-derived mesenchymal stem cells contained SSEA-3-positive cells. Furthermore, when the obtained human amnion-derived SSEA-3-positive cells were cultured in single cell suspension, approximately 25% of the SSEA-3-positive cells formed embryoid body-like cell clusters on day 6 of culture (Figure 16).

[0051] Example 2-2: Examination of the properties of high-potential pluripotent stem cells derived from human amniotic membrane (1) Clustering and differentiation of high-potential pluripotent stem cells derived from human amniotic membrane into three germ layers in adherent culture To confirm the differentiation potential of human amnion-derived SSEA-3+ cells into tripartite germ layers, pluripotent cell clumps were transferred to gelatin-coated dishes and cultured as adherents. After approximately 10 days, immunohistochemistry detected cytokeratin 7 (CK7, an endodermal marker), alpha-smooth muscle actin (SMA, a mesodermal marker), and neurofilament-M (an ectodermal marker) (Figure 17). These results demonstrate that human amnion-derived SSEA-3+ cells have the potential to spontaneously differentiate into tripartite germ layers.

[0052] (2) Analysis of cell surface markers of human amniotic mesenchymal stem cells (MSCs) and high-potential pluripotent stem cells derived from human amniotic membrane Among human amnion-derived SSEA-3-positive cells, the double positive rates for SSEA-3 and CD105 were 81.1%, for SSEA-3 and CD90 were 84.0%, for SSEA-3 and CD44 were 74.2%, and for SSEA-3 and CD133 were 71.5% (Figures 18 and 19). Human amnion-derived MSCs and human amnion-derived SSEA-3-positive cells, like bone marrow-derived MSCs and Muse cells, barely express hematopoietic cell surface markers such as CD34 and CD45 (Figure 20).

[0053] (3) Gene expression analysis of high-potential pluripotent stem cells derived from human amniotic membrane and conventional human bone marrow Muse cells 1) Expression analysis of germline-specific genes (Blimp1 / Dppa3 / ITGA / Sycp3 / TFAP2c / TP63) We performed quantitative PCR to compare germline-specific gene expression in human amnion-derived SSEA-3+ cells and human bone marrow-derived Muse cells. As shown in Figure 21, human amnion-derived SSEA-3+ cells expressed significantly higher levels of germline markers, including Blimp1, Dppa3, ITGA, and TFAP2c, than bone marrow-derived Muse cells. These results suggest that human amnion-derived SSEA-3+ cells may be capable of differentiating into germline cells and, furthermore, may be capable of inducing differentiation into cells of extraembryonic tissues beyond the three germ layers.

[0054] 2) Genes suggesting pluripotency (a) Primed pluripotency markers (Oct3 / 4 / Nanog / Sox2) Quantitative PCR was used to compare the expression of primed pluripotency markers, Oct3 / 4, Nanog, and Sox2, in human amnion-derived SSEA-3+ cells, human amnion-derived SSEA-3-negative cells (AM-non-Muse), and human bone marrow-derived Muse cells. Human amnion-derived SSEA-3+ cells showed higher expression of these primed pluripotency markers than bone marrow Muse cells (Figure 22). These results suggest that human amnion-derived SSEA-3+ cells are more pluripotent than bone marrow Muse and human amnion-derived SSEA-3-negative cells.

[0055] (b) Naive pluripotency markers (KLF2 / KLF4 / REX1) Quantitative PCR was used to compare the expression of naive pluripotency markers KLF2, KLF4, and REX1 in human amnion-derived SSEA-3-positive cells, human amnion-derived SSEA-3-negative cells (AM-non-Muse), and human bone marrow-derived Muse cells. There was no difference in expression between human amnion-derived SSEA-3-positive cells and bone marrow Muse cells (Figure 22). However, human amnion-derived SSEA-3-positive cells showed higher expression than human amnion-derived SSEA-3-negative cells.

[0056] (c) Telomerase reverse transcriptase gene (TERT) We performed quantitative PCR to compare the expression of the telomerase reverse transcriptase gene (TERT) in human amnion-derived SSEA-3-positive cells and human bone marrow-derived Muse cells. TERT was not expressed in either human amnion-derived SSEA-3-positive cells or bone marrow Muse cells (Figure 22). This suggests that this property is a common property (non-tumorigenicity) of SSEA-3-positive pluripotent stem cells regardless of their tissue of origin.

[0057] Other analyses have shown that the human amnion-derived SSEA-3-positive cells obtained in Example 2 above possess useful properties of pluripotent stem cells similar to those of Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc. In addition, they have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, have differentiation potential close to totipotency, and are approximately 70% or more positive for the cell surface marker CD133. Thus, Example 2 above demonstrates that the "high-potential pluripotent stem cells" of the present invention also exist in human amnion, an extraembryonic tissue. In particular, the human amnion-derived SSEA-3-positive cells obtained in Example 2 above can be referred to as a human amnion-derived high-potential pluripotent stem cell fraction, which contains 70% or more of human amnion-derived SSEA-3 and CD133 double-positive high-potential pluripotent stem cells.

[0058] Example 3-1: Isolation of high-potential pluripotent stem cells from human placental tissue High-potential pluripotent stem cells (HPS) can be obtained from human placental tissue using the method described in International Publication No. WO 2011 / 007900 for the isolation and identification of human Muse cells. More specifically, HPS derived from human placental tissue were obtained by FACS separation using SSEA-3 as an indicator, as shown in Figure 23. Specifically, human placenta was minced into 0.5 mm cubes and the tissue pieces were placed at 1 cm intervals on a 10 cm dish. The cells were cultured for approximately 10 days using the explant method in a medium containing αMEM, 15% FBS, 1 ng / mL bFGF, and 2 mM GlutaMAX. After expansion and subculture, the established human placental tissue-derived mesenchymal stem cells were isolated using an anti-SSEA-3 antibody. Approximately 1% of the established human placenta-derived mesenchymal stem cells contained SSEA-3-positive cells. When SSEA-3 positive cells derived from human placenta isolated by FACS were cultured in single cell suspension, 10.3% of the SSEA-3 positive cells formed embryoid body-like cell clusters on the 7th day of culture.

[0059] Example 3-2: High-potential pluripotent stem cells derived from human placenta tissue are positive for the cell surface marker CD133 The expression of CD133 in the human placenta-derived SSEA-3-positive cells obtained in Example 3-1 was confirmed by FACS. As a result, as shown in Figure 24, 85.2% of the SSEA-3-positive cells derived from the human placenta were CD133-positive. Therefore, the SSEA-3-positive cells derived from human placenta tissue obtained in Example 3 can be referred to as a human placenta-derived high-potential pluripotent stem cell fraction, which contains 80% or more high-potential pluripotent stem cells derived from human placenta (P-HP-PSCs).

[0060] Example 3-3: High-potential pluripotent stem cells derived from human placenta efficiently induce differentiation into germ cell lineages by phagocytosing apoptotic primordial germ cells The human placenta-derived high-potential pluripotent stem cell fraction obtained in Example 3-1 was used to confirm the induction of differentiation into primordial germ cells by the following induction method. 5 × 10 SSEA-3 positive cells of the human placenta-derived high-potential pluripotent stem cell fraction were cultured in a medium containing 5 × 10 3 cells / cm 2 After seeding at a density of 1 × 10 cells, male or female mouse primordial germ cells were killed with 100 μM rotenone and 100 μM oligomycin A. 4 cells / cm 2 The cells were added at a density of 5 × 10 cells per well and phagocytosed in DMEM-low-glucose, 10% FBS for 3 days. After removing the dead mouse primordial germ cells, they were cultured in DMEM-low-glucose, 10% FBS, 20 μM 5-azacytidine, and 10 μM retinoic acid for 3 days as adherent cells. After that, they were cultured in a low-cell binding V-bottom 96-well plate at a density of 5 × 10 cells per well. 3 The cells were suspended at 100 cells / well and cultured in suspension in DMEM-low-glucose, 10% FBS for 2 days. Quantitative PCR was used to assess the expression of early germline markers PRDM14, Blimp1, Sox17, Tfap2c, Dppa3, Nanos3, and SSEA-1, late germline markers Dazl and Ddx4, and meiotic markers Stra8 and Sycp3 at 8 days postinduction. Primordial germ cell-like cells (PGCLCs) derived from human iPS cells or the human gestational choriocarcinoma cell line JEG3 were used as positive controls. The results are shown in Figures 25-1 and 25-2. Phagocytosis of both male and female primordial germ cells resulted in an increase in early germline markers such as Blimp1 and Nanos3. The expression of the late germline marker Dazl and the meiotic marker Stra8 was particularly significant. These results demonstrate that, like the human umbilical cord-derived high-potential pluripotent stem cell fraction, the human placenta-derived high-potential pluripotent stem cell fraction can efficiently differentiate into germline lines by phagocytosing apoptotic primordial germ cells. Therefore, it is suggested that the high-potential pluripotent stem cells of the present invention have the ability to differentiate into germ cell lineages and have a differentiation potential close to totipotency.

[0061] Example 4: High-potential pluripotent stem cells derived from human umbilical cord tissue and placenta express HLA-G at higher levels than Muse cells derived from bone marrow, adipose tissue, and skin. HLA-G is expressed in extravillous trophoblast cells, which form the outermost layer of the placenta through which fetal tissue invades maternal tissue, and has an immunosuppressive function that allows the fetus to escape from the maternal immune system. HLA-G expression was confirmed by FACS using the human umbilical cord-derived high-potential pluripotent stem cell fraction obtained in Example 1-1 and the human placenta-derived high-potential pluripotent stem cell fraction obtained in Example 3-1. Bone marrow-, adipose-, and skin-derived Muse cells were used as comparison groups. As shown in Figures 26-1, 26-2, 26-3, 26-4, and 26-5 and Table 2, the percentages of HLA-G-positive cells among SSEA-3-positive cells in the human umbilical cord (UC)- and human placenta-derived high-potential pluripotent stem cell fractions were 66.6% and 52.6%, respectively. On the other hand, the percentages of HLA-G-positive cells among SSEA-3-positive cells in bone marrow-, adipose-derived (ADSC), and skin-derived (NHDF) Muse cells were 16.5%, 9.5%, and 1.2%, respectively, significantly lower than the high-potential pluripotent stem cell fractions derived from human umbilical cord and human placenta. Therefore, the high-potential pluripotent stem cells of the present invention have higher HLA-G expression and are suggested to have stronger immunoregulatory functions than Muse cells, suggesting usefulness such as a reduced risk of rejection when administering donor preparations.

[0062] [Table 2]

[0063] Example 5: Effect of human umbilical cord-derived high-potential pluripotent stem cells in a rat myocardial infarction model (1) Creation of a rat myocardial infarction model The experimental animal protocol used in this study was reviewed and approved by the Animal Experimentation Committee of the Shiga Research Institute of Nissei Baylis Co., Ltd. The day before administration of the test substance, a myocardial infarction model was created. Specifically, rats were anesthetized by subcutaneous administration of a triple anesthetic mixture at a volume of 2.5 mL / kg, fixed in a dorsal position, and an endotracheal tube was inserted orally into the airway. Artificial ventilation was performed using a small animal ventilator. The left lateral thoracic wall was opened to expose the heart. The left anterior descending coronary artery (LAD) was occluded for 30 minutes using a suture needle (Elb suture needle M10-50B2, Akiyama Seisakusho Co., Ltd.). During this time, electrocardiograms (lead II) were recorded using a LabChart-Pro (AD Instruments), and ST potential elevation and myocardial whitening were visually observed to confirm the presence or absence of occlusion. If ventricular fibrillation (VF) occurred, resuscitation measures were performed. A myocardial infarction model (ischemia-reperfusion) was created by reperfusion after 30 minutes of occlusion. After reperfusion, the chest was closed and the incision was sutured. To prevent infection, an appropriate amount of 2 mg / mL ampicillin sodium (Viccillin Injection 0.5 g, Meiji Seika Pharma Co., Ltd.) was dripped into the incision. After suturing, the incision was disinfected with a povidone-iodine preparation (Povidone Solution 10%, Yoshida Pharmaceutical Co., Ltd.). Atipamezole 0.15 mg / kg (Atipame Injection, Kyoritsu Pharmaceutical Co., Ltd.) was then administered subcutaneously. After awakening, the animal was returned to its cage. The day after the myocardial infarction model was designated as day 0. The analgesic meloxicam (Metacam 0.5% Injection, Boehringer Ingelheim Animal Health Japan Inc.) was administered subcutaneously for two days starting the day after surgery.

[0064] (2) Evaluation in a rat myocardial infarction model (echocardiographic measurements) The day after the myocardial infarction model rats prepared above (day 0), human bone marrow-derived Muse cells (BM-Muse), human umbilical cord-derived high-potential pluripotent stem cell fraction (UC-HP-PSC) obtained in Example 1-1, and human bone marrow-derived SSEA-3-negative MSCs (BM non-Muse) were injected as test substances into the tail vein of the model rats at a dose of 3 × 10 4The control group received 1 mL of physiological saline as a vehicle. Echocardiography was performed on days 14 and 28 after administration of the test substance to evaluate the effect on myocardial infarction. For echocardiographic evaluation, rats were anesthetized with 0.5–3.0% isoflurane (isoflurane inhalation anesthetic solution "Pfizer," Mylan Pharmaceuticals, Inc.) and placed in a supine or lateral position. A linear or sector probe (5–13 MHz) was applied to the chest using a general-purpose ultrasound imaging system (Vivid S6, GE Medical Systems) or an ultrasound imaging system (Nemio SSA-550A, Toshiba Medistems Corporation). Left ventricles were visualized in short-axis sections and measured in M-mode. Left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), end-diastolic left ventricular anterior wall thickness (LVAWd), end-diastolic left ventricular posterior wall thickness (LVPWd), left ventricular fractional shortening (%FS), and ejection fraction (EF). These measurements were taken over three heartbeats, and the average was used as the measurement value. The results for EF and %FS are shown in Figures 27-1 to 27-4, respectively. At 14 days after administration, EF and %FS were significantly higher in the BM-Muse and UC-HP-PSC groups compared with the control group. At 28 days after administration, %FS was significantly higher in the UC-HP-PSC group compared with the control group. On the other hand, no significant differences were observed between the BM non-Muse group and the control group at either 14 or 28 days after administration. No significant differences were observed between the groups in LVIDs, LVAWd, or LVPWd.

[0065] These findings indicate that both BM-Muse and UC-HP-PSCs were effective in a rat myocardial infarction model, but UC-HP-PSCs showed a significant effect on %FS even 28 days after administration, suggesting that they have a superior effect compared to BM-Muse.

[0066] Example 6: High-potential pluripotent stem cells derived from human umbilical cord tissue express desmosome-associated molecules Desmosomes are intercellular adhesion mechanisms composed of cell membrane proteins such as desmoglein and desmocollin, and intracellular components such as plakoglobin. Desmosomes are known to be expressed primarily in epithelial cells and cardiomyocytes, but are also expressed in early embryos and pluripotent stem cells. In human pluripotent stem cells, desmosome-associated molecules (especially desmoglein 2) have been reported to be essential for maintaining pluripotency and self-renewal (Jongjin Park et al., DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells. Stem Cell Reports, 11, 115-127 (2018)). Comprehensive gene expression analysis was performed using RNA-seq to examine the expression of desmosome-associated molecules in the high-potential pluripotent stem cells derived from human umbilical cord obtained in Example 1-1. Muse cells derived from bone marrow, adipose tissue, and skin were used as comparison groups. As shown in Figure 28, the high-potential pluripotent stem cells derived from human umbilical cord showed higher expression of the desmosome-associated molecules desmoglein 2, desmocollin 2 and 3, and plakoglobin than Muse cells derived from bone marrow (BM), adipose tissue (ADSC), and skin (NHDF).

[0067] These findings suggest that the high-potential pluripotent stem cells of the present invention have superior maintenance of pluripotency and self-renewal capabilities compared to conventional bone marrow-, adipose-, and skin-derived Muse cells, and may bring about better improvement or recovery of the function of the damaged site in various diseases involving tissue damage. Furthermore, due to their ability to differentiate into germ cell lineages, they may be used as useful regenerative medicines. [Industrial Applicability]

[0068] The high-potential pluripotent stem cells of the present invention possess the properties of conventional pluripotent stem cells, i.e., the ability to differentiate into embryonic tissues, which are all cells that make up the body, as well as the ability to differentiate into cells of extraembryonic tissues such as the placenta and / or germ cell lineages, i.e., a differentiation potential close to totipotency. Therefore, in addition to conventional regenerative medicine for various diseases resulting from damage to the body's structure, the ability to differentiate into cells of extraembryonic tissues such as the placenta and uterus can reconstitute damaged areas of the extraembryonic tissues, thereby improving or restoring the function of the damaged areas. Furthermore, due to their ability to differentiate into germ cell lineages, they can also be used in treatments related to reproduction, such as infertility treatments. Therefore, the high-potential pluripotent stem cells of the present invention and cell preparations containing the high-potential pluripotent stem cells can be applied to new fields of regenerative medicine.

Claims

1. A method for producing SSEA-3 positive high potential pluripotent stem cells, comprising: isolating SSEA-3-positive high-potential pluripotent stem cells from human tissue (excluding embryos) and confirming the expression of CD133; The method for producing SSEA-3-positive high-potential pluripotent stem cells has all of the following properties: (i) telomerase activity is absent, or is at the same level as that of human fibroblasts, which are somatic cells, or is 1 / 5 or less compared to HeLa cells; (ii) have the ability to differentiate into cells of any of the three germ layers; (iii) does not exhibit neoplastic growth; and (iv) It has self-renewal ability.

2. The method of claim 1 , wherein the human tissue is an extraembryonic tissue.

3. The method of claim 2, wherein the extraembryonic tissue is selected from the group consisting of umbilical cord, umbilical cord blood, placenta, placental blood, decidua, chorion, amniotic membrane, and amniotic fluid.

4. The production method according to any one of claims 1 to 3, wherein the SSEA-3-positive high-potential pluripotent stem cells are CD133-positive.

5. The method according to any one of claims 1 to 4, wherein the SSEA-3-positive high-potential pluripotent stem cells have the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages.

6. A method for producing a regenerative medicine containing SSEA-3-positive high-potential pluripotent stem cells as an active ingredient, A step of producing SSEA-3-positive high-potential pluripotent stem cells by the production method according to any one of claims 1 to 5; and A manufacturing method comprising the step of manufacturing a pharmaceutical for regenerative medicine by incorporating the SSEA-3-positive high-potential pluripotent stem cells as an active ingredient.

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