Umbilical cord-derived pluripotent stem cells of extremely immature infant

Abstract

Ultra-high potential pluripotent stem cells according to the present invention have ability to differentiate into cells of extraembryonic tissues of placenta and the like and / or a germ cell line, that is, ability to differentiate which is close to totipotency, in addition to properties of conventional pluripotent stem cells, that is, ability to differentiate into embryonic tissues which are all cells constituting a body. Thus, having the ability to differentiate into embryonic tissue cells of placenta or the like, the ultra-high potential pluripotent stem cells can reconstruct a damaged site of the extraembryonic tissue and improve or recover the function of the damaged site and also can be used for reproductive treatment such as infertility treatment, in addition to conventional regenerative medicine to various diseases involving damage to any of all parts that constitute a body. Further, the ultra-high potential pluripotent stem cells may be more effectively applied to a new regenerative medicine field, having excellent ability to proliferate and excellent ability to differentiate.

Description

Pluripotent stem cells derived from the umbilical cord of extremely preterm infants

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

[0002] Mesenchymal stem cells (MSCs), 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, MSCs have been reported to differentiate not only into mesodermal cells such as bone, adipose, and cartilage, which belong to the same developmental lineage, but also into ectodermal and endodermal cells across germ layers. While their ability to differentiate into the three germ layers of mesoderm, endoderm, and ectoderm suggests pluripotency, their low differentiation rate suggests 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 identify pluripotent stem cells thought to exist within mesenchymal stem cells, and discovered a new pluripotent stem cell (multilineage-differentiating stress-enduring cell: Muse cell) (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. Their application in 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 on 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).

[0006] The present inventors previously discovered that high-potential pluripotent stem cells, distinct from conventional Muse cells obtained from bone marrow, exist among SSEA-3-positive cells obtained from umbilical cords, and that these high-potential pluripotent stem cells, in addition to the useful properties of conventional Muse cells, also possess the ability to differentiate into cells of extraembryonic tissues and / or germ cell lineages, and have differentiation potential approaching totipotency (Patent Document 6). However, due to increasing demand for regenerative medicine, further investigation of pluripotent stem cells has been required.

[0007] Patent No. 5185443 International Publication No. WO2014 / 027684 JP 2015-159895 International Publication No. WO2018 / 235834 JP 2020-503375 International Publication No. WO2021 / 201286

[0008] Kuroda Y et al. Proc Natl Acad Sci USA, 2010: 107: 8639-8643.Wakao S et al. Proc Natl Acad Sci USA, 2011: 108: 9875-9880.Kuroda Y et al. Nat Protc, 2013: 8: 1391-1415.Ogura F. et al., Stem Cells Dev., 23, 717-728, doi:10.1089 / scd.2013.0473 (2014)

[0009] The present invention aims to provide 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 potential close to totipotency, and that also have additional functions, and a cell preparation containing such stem cells.

[0010] The inventors isolated Muse cells from extraembryonic tissues (particularly the umbilical cord), which are biological tissues that cease functioning as organs and become waste after pregnancy, childbirth, and delivery, using cells that can be collected non-invasively. They then conducted detailed studies on the properties and therapeutic effects of Muse cells collected from the umbilical cords of preterm and full-term infants. They discovered that Muse cells obtained from the umbilical cords of extremely preterm infants (<28 weeks gestation) possess useful properties different from those of Muse cells obtained from bone marrow, etc., and Muse cells obtained from the umbilical cords of full-term infants (37 to 42 weeks gestation), leading to the completion of the present invention.

[0011] That is, the gist of the present invention is as follows: [1] SSEA-3-positive ultra-high potential pluripotent stem cells derived from the extraembryonic tissue of an extremely preterm infant. [2] The ultra-high potential pluripotent stem cells, wherein the extraembryonic tissue is selected from the group consisting of umbilical cord, umbilical cord blood, placenta, placental blood, decidua, chorion, amnion, and amniotic fluid. [3] The ultra-high potential pluripotent stem cells, wherein the extraembryonic tissue is umbilical cord. [4] The ultra-high potential pluripotent stem cells, wherein the extremely preterm infant is a baby born at less than 28 weeks gestation. [5] The ultra-high potential pluripotent stem cells are characterized in that the expression of at least one gene selected from the group consisting of HAND1, HOXD13, NOG, TFAP2A, EFS, LCP1, MYO1D, TPPP3, ANGPTL4, GATA3, HOXA13, PODXL, EPCAM, ICAM5, JAM2, L1CAM, BARX1, EREG, and KRT81 is increased compared to the expression in SSEA-3-positive pluripotent stem cells derived from the extraembryonic tissue of a full-term infant. [6] The ultra-high potential pluripotent stem cells are pluripotent stem cells that have at least one of the following properties: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew. [7] The ultra-high potential pluripotent stem cells are pluripotent stem cells having all of the following properties: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) no neoplastic growth; and (iv) the ability to self-renew. [8] A regenerative medicine drug comprising the ultra-high potential pluripotent stem cells. [9] The regenerative medicine drug is for treating a lung disease.

[10] The regenerative medicine drug is for treating a lung disease selected from the group consisting of pediatric chronic lung disorder, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

[11] SSEA-3-positive pluripotent stem cells derived from the extraembryonic tissue of an extremely preterm infant, characterized in that the expression of at least one gene selected from the group consisting of HAND1, HOXD13, NOG, TFAP2A, EFS, LCP1, MYO1D, TPPP3, ANGPTL4, GATA3, HOXA13, PODXL, EPCAM, ICAM5, JAM2, L1CAM, BARX1, EREG, and KRT81 is increased compared to the expression in SSEA-3-positive pluripotent stem cells derived from the extraembryonic tissue of a full-term infant.

[0012] The previously reported high-potential pluripotent stem cells 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. These stem cells possess differentiation potential close to totipotency. Furthermore, they express 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 reconstitute damaged extraembryonic tissues and improve or restore the function of those damaged tissues. Furthermore, their ability to differentiate into germ cell lineages can be used to treat reproductive disorders, such as infertility. Therefore, the high-potential pluripotent stem cells and cell preparations containing the high-potential pluripotent stem cells can be applied to new fields of regenerative medicine, and the ultra-high-potential pluripotent stem cells of the present invention are characterized by superior proliferation and differentiation capabilities compared to high-potential pluripotent stem cells, and therefore may be even more useful as pharmaceuticals for regenerative medicine.

[0013] Like conventional Muse cells and high-potential pluripotent stem cells, the ultra-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 and high-potential pluripotent stem cells, the ultra-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 ultra-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 ultra-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 ultra high potential pluripotent stem cells of the present invention, like high potential pluripotent stem cells, are also expressed in early embryos and pluripotent stem cells during normal development, and highly express desmosome-associated molecules, particularly desmoglein 2, desmocollin 2, 3, plakoglobin, etc., which are thought to be essential molecules for maintaining pluripotency and self-renewal in human pluripotent stem cells, suggesting that they have excellent pluripotency 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.Furthermore, due to their ability to differentiate into germ lineage cells, they may be useful as a pharmaceutical for regenerative medicine.Furthermore, the ultra high potential pluripotent stem cells of the present invention are characterized by a higher migration ability to lung tissue than high potential pluripotent stem cells, and therefore may be even more useful as a pharmaceutical for treating lung diseases.

[0014] Body weight changes in a rat model of BLM-induced lung injury. (A) Schematic diagram of the experimental design. BLM: bleomycin; IT: intratracheal administration; IV: intravenous administration; SpO2: oxygen saturation; HR: heart rate. (B) Body weight changes measured on days 0, 1, 2, 3, 4, 5, 7, 10, 14, 17, and 21 (n = 5 per group). The value on day 0 was set as 100%. BLM(-) = uninjured, untreated group; PBS = BLM-injured, PBS-treated group; Non-Muse = BLM-injured, non-Muse cell-treated group; BM-Muse = BLM-injured, BM-Muse cell-treated group; Term UC-Muse = BLM-injured, Term UC-Muse cell-treated group; Preterm UC-Muse = BLM-injured, Preterm UC-Muse cell-treated group. (C) Body weight on day 21. *p<0.05, **p<0.01, and ***p<0.001. Serum SP-D levels on day 14 are shown. Five animals were used in each group. *p<0.05 and **p<0.01. Pulmonary function measurements are shown. (A) SpO2 and heart rate (HR) were recorded on day 9. A representative 30-second recording is shown for each group. (B) SpO2 was measured for 60 minutes on day 9 (n=3 per group). *p<0.05, **p<0.01, ***p<0.001. (C) HR was measured for 60 minutes on day 9 (n=3 per group). Histopathological evaluation results are shown (some photographs). (A) Representative H&E images of lungs at low (x12.5), medium (x40), and high (x400) magnifications. Scale bars: x12.5 = 2 mm, x40 = 500 μm, x400 = 50 μm. (B) Assessment of pulmonary fibrosis. H&E sections (n ​​= 5 per group) were evaluated according to the Ashcroft scale. *p<0.05, **p<0.01, and ***p<0.001. (C) Assessment of pulmonary inflammation and fibrosis. H&E sections (n ​​= 5 per group) were evaluated according to the modified ATS document scale. *p<0.05, **p<0.01, and ***p<0.001. Results of confirmation of lung engraftment of injectable cells are shown (some photographs). GFP-expressing cells were introduced in all groups. (A) Representative images of anti-GFP immunostaining in the lung, heart, intestine, kidney, spleen, and liver from the Non-Muse, BM-Muse, Term-UC-Muse, and Preterm-UC-Muse groups. Scale bar = 50 μm.(B) Quantification of GFP-positive cells detected in the lungs (n = 3 per group). The area percentage was defined as (GFP-positive cell area / total lung cell area) × 100. *p<0.05 and **p<0.01. Results confirming marker expression in GFP-positive Muse cells in the lungs are shown (selected photographs). (A) Immunofluorescence staining for podoplanin (red), GFP (green), and DAPI (blue) in preterm-derived UC-Muse, term-derived UC-Muse, and bone marrow-derived Muse cell groups. (B) Percentage of podoplanin relative to total GFP-positive cells. (C) Immunofluorescence staining for pro-SPC (red), GFP (green), and DAPI (blue) in preterm-derived UC-Muse, term-derived UC-Muse, and BM-Muse groups. (D) Percentage of proSP-C-positive cells among GFP-positive cells. (E) Immunofluorescence staining for CD31 (red), GFP (green), and DAPI (blue) in preterm-derived UC-Muse, term-derived UC-Muse, and BM-Muse groups. (F) Percentage of CD31-positive cells relative to the total GFP-positive cell population. Scale bar = 50 μm. *p < 0.05 and ***p < 0.001. Differential gene expression and pathway analysis are shown. (A) Comparison of preterm-derived UC-Muse, term-derived UC-Muse, and BM-Muse cells by RNA-seq. Dendrograms and unsupervised hierarchical clustering heatmaps of preterm-derived UC-Muse (1, 2, 3), term-derived UC-Muse (1, 2, 3), and BM-Muse cells (1, 2, 3). Similarity between gene expression profiles is represented by the vertical distance on the dendrogram. Highest correlation is indicated by a shorter vertical distance. (B) Gene ontology analysis: Genes upregulated in preterm (red) and term (blue) UC-Muse cells compared to BM-Muse cells are listed. Genes upregulated in preterm UC-Muse (orange) and BM-Muse (light green) cells compared to term UC-Muse cells are listed. Genes upregulated in term UC-Muse (green) and BM-Muse (light blue) cells compared to preterm UC-Muse cells are listed. (C) Heatmap of genes related to lung development, vascular development, cell migration, and cell adhesion.(D) Evaluation of the migration ability of preterm-derived UC-Muse cells, term-derived UC-Muse cells, BM-Muse cells, and BM-non-Muse cells toward injured lung tissue. For each sample, the number of migrated cells was counted at 20x magnification in four randomly selected fields, and the average value of three samples was used for comparison. ***p<0.001. Analysis of differentially expressed genes is shown. (A) Gene ontology (GO) analysis identified GO terms from a group of genes with more than two-fold higher expression in Preterm-UC-Muse than in Term-UC-Muse. (B) Expression of genes with the GO terms detected in (A) is displayed as a heatmap. All genes shown on the right are genes with more than two-fold higher expression in Preterm-UC-Muse than in Term-UC-Muse. Evaluation of pulmonary fibrosis is shown (partial photographs). (A) Representative Masson's Trichrome stained images of lungs. Scale bar = 500 μm. (B) Quantification of collagen-positive cells detected in the lungs (n = 3 per group). The area percentage was defined as (collagen-positive cell area / total lung cell area) x 100. *p<0.05, **p<0.01, and ***p<0.001. The results of confirming engraftment of injected cells into the lungs are shown (photos). GFP-expressing cells were introduced in all groups. Low-magnification images of anti-GFP immunostaining of lungs in the Non-Muse, BM-Muse, Term-UC-Muse, and Preterm-UC-Muse groups. Scale bar = 100 μm.

[0015] The present invention is described in detail below. One aspect of the present invention relates to ultra-high potential pluripotent stem cells isolated from extraembryonic tissues of extremely preterm infants (hereinafter, sometimes referred to as "ultra-high potential pluripotent stem cells of the present invention"). Another aspect of the present invention relates to a cell preparation containing the ultra-high potential pluripotent stem cells (hereinafter, sometimes referred to as "cell preparation of the present invention").

[0016] 1. Cell Preparation (1) Ultra High Potential Pluripotent Stem Cells The ultra high potential pluripotent stem cells used in the cell preparation of the present invention are pluripotent stem cells isolated from extraembryonic tissues of extremely preterm infants, etc., and possess the same useful properties of pluripotent stem cells as conventional Muse cells obtained from bone marrow, peripheral blood, adipose tissue, skin, etc., as described below. In addition, they have the same differentiation potential into extraembryonic tissue cells and / or germ cell lineages as Muse cells derived from extraembryonic tissues of full-term infants, possess differentiation potential close to totipotency, and are positive for CD133, a cell surface marker that is negative in conventional Muse cells derived from bone marrow, peripheral blood, adipose tissue, skin, etc. Furthermore, compared to Muse cells derived from extraembryonic tissues of full-term infants, they have superior proliferation and differentiation potential, and have a high migration ability to lung tissue. In particular, as shown below, the human extremely preterm umbilical cord-derived SSEA-3+ cells obtained in the Examples and other analyses have been shown to possess useful pluripotent stem cell properties similar to those of Muse cells conventionally obtained from bone marrow, peripheral blood, adipose tissue, skin, etc. Additionally, similar to Muse cells collected from full-term infant umbilical cords, they have the potential to differentiate into extraembryonic tissue cells and / or germ cell lineages, and possess differentiation potential approaching totipotency. Furthermore, compared to Muse cells collected from full-term infant umbilical cords, they possess superior proliferation and differentiation potential, enhanced migration ability to lung tissue, and other properties. For these reasons, the human extremely preterm umbilical cord-derived SSEA-3+ cells obtained in the Examples are referred to as "human umbilical cord-derived ultra-high potential pluripotent stem cells." These cells are also referred to herein as "UC-Muse cells (preterm umbilical cord Muse cells) derived from preterm infants."

[0017] Furthermore, even among conventional bone marrow-derived SSEA-3-positive cells, the ultra-high potential pluripotent stem cells of the present invention may exist, albeit in very small numbers (up to approximately several percent). Therefore, the ultra-high potential pluripotent stem cells of the present invention can be obtained not only from extraembryonic tissues, but also from conventional sources such as bone marrow, peripheral blood, fat, and skin. Therefore, the ultra-high potential pluripotent stem cells of the present invention can be considered to be primarily a subset of special Muse cells isolated from the extraembryonic tissues of extremely preterm infants. In other words, the ultra-high potential pluripotent stem cells of the present invention possess useful properties of pluripotent stem cells similar to those of Muse cells, which means the properties of Muse cells described below.

[0018] 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 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 hypoxic conditions, low phosphate conditions, low serum concentration, low nutritional conditions, 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."

[0019] 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.

[0020] Alternatively, a cell population containing Muse cells can be prepared by a method comprising applying an external stress stimulus to mesenchymal tissue or cultured mesenchymal cells in vivo, selectively expanding cells resistant to the external stress, and recovering cells with an increased abundance of such cells. The external stress may be protease treatment, culture under low oxygen concentration, culture under low phosphate conditions, culture under low serum concentration, culture under low nutrient conditions, culture under heat shock, culture at low temperature, freezing, culture in the presence of harmful substances, culture in the presence of reactive oxygen species, culture under mechanical stimulation, culture under shaking, culture under pressure, or physical impact, or a combination of these. The protease treatment is preferably carried out for a total of 0.5 to 36 hours to impart external stress to the cells. The protease concentration may be any concentration 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.

[0021] The Muse cells may be autologous or allogeneic to the recipient of the cell transplant.

[0022] As described above, Muse cells or cell populations containing Muse cells can be prepared from biological tissues using, for example, SSEA-3 positivity or double positivity for SSEA-3 and CD105. 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 absence of markers specific to these cells as an indicator. More specifically, Muse cells can be separated based on 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 (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.

[0023] Furthermore, Muse cells having the above characteristics may have at least one property selected from the group consisting of: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew. Preferably, Muse cells used in the present invention have all of the above properties. Regarding (i), "low or no telomerase activity" refers to low or undetectable telomerase activity when detected using, for example, a TRAPEZE XL telomerase detection kit (Millipore). "Low" telomerase activity refers to, for example, telomerase activity equivalent to that of human fibroblasts, which are somatic cells, or telomerase activity at or below 1 / 5, preferably 1 / 10, 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. However, in suspension culture, they proliferate from a single cell, form embryoid-like cell clusters, and 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 proliferating from the cell clusters 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. Regarding (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 adhesive culture, differentiation into three germ layers can be confirmed, and at the same time, when cells from the embryoid-like cell mass are again transferred to suspension culture as a single cell, an embryoid-like cell mass of the next generation is formed, and when this is transferred to adhesive culture, differentiation into three germ layers can again be confirmed, and at the same time, an embryoid-like cell mass can again be confirmed by culturing a single cell in suspension culture. Self-renewal can be achieved by repeating one or more cycles.

[0024] The ultra-high potential pluripotent stem cells used in the cell preparations of the present invention are isolated from extraembryonic tissues and the like. They possess the same useful properties of pluripotent stem cells as Muse cells, as well as the potential to differentiate into extraembryonic tissue cells and / or germ cell lineages, similar to Muse cells derived from extraembryonic tissues of full-term infants, and possess differentiation potential approaching totipotency. Furthermore, they are positive for CD133, a cell surface marker that is negative for Muse cells derived from bone marrow, peripheral blood, adipose tissue, skin, etc. Furthermore, compared to Muse cells derived from extraembryonic tissues of full-term infants, they possess superior proliferation and differentiation potential, enhanced migration ability to lung tissue, and other properties. 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 by flow cytometry with a CD133 antibody. The ultra-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. Positivity for the cell surface marker CD133 can be determined by an identification method using a conventional antibody or the like.

[0025] The ultra-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 cease functioning as organs and become waste after pregnancy, birth, and delivery, such as the umbilical cord, umbilical cord blood, placenta, placental blood, decidua, chorion, amniotic membrane, and amniotic fluid. Among these, the umbilical cord is preferred. The ultra-high potential pluripotent stem cells of the present invention can be obtained from extraembryonic tissues of very preterm infants. In the present invention, "very preterm birth" specifically refers to birth at less than 28 weeks of gestation, for example. Furthermore, "full-term birth" specifically refers to birth at 37 to 41 weeks and 6 days of gestation, for example.

[0026] Furthermore, the ultra-high potential pluripotent stem cells of the present invention are capable of differentiating into all germ layers constituting 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, exhibiting differentiation potential approaching totipotency. Furthermore, the ultra-high potential pluripotent stem cells of the present invention have properties such as superior proliferation and differentiation ability and a high migration ability to lung tissue, compared to Muse cells derived from the extraembryonic tissues of full-term infants. Furthermore, the ultra-high potential pluripotent stem cells of the present invention have the property of increased expression of at least one specific gene associated with tissue morphogenesis, actin filament-based process, tube morphogenesis, cell-cell adhesion, and epithelial cell differentiation, as defined by Gene Ontology, compared to Muse cells derived from the extraembryonic tissues of full-term infants. Specifically, specific genes associated with tissue morphogenesis include HAND1, HOXD13, NOG, and TFAP2A; specific genes associated with actin filament-based processes include EFS, LCP1, MYO1D, and TPPP3; specific genes associated with tube morphogenesis include ANGPTL4, GATA3, HOXA13, and PODXL; specific genes associated with cell-cell adhesion include EPCAM, ICAM5, JAM2, and L1CAM; and specific genes associated with epithelial cell differentiation include BARX1, EREG, and KRT81.

[0027] 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, germ cell lineages (gametes (sperm or eggs)), and the primordial germ cells (PGCs) that form the basis of gametes. Specifically, the differentiation potential of extraembryonic tissues into cells and / or germ cell lineages can be confirmed by, for example, differentiation into various cells that make up 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, and Stra8), and expression of genes suggesting pluripotency (e.g., Oct3 / 4, Nanog, Sox2, KLF2, KLF4, REX1, and TERT). Here, whether or not the cells have "superior proliferation and differentiation capabilities compared to Muse cells derived from the extraembryonic tissues of full-term infants" can be confirmed, for example, by measuring and comparing the proliferation capabilities of the cells using methods known in the art, and by measuring and comparing the expression levels of the above-mentioned differentiation-related markers of the cells using methods known in the art. Whether or not the cells have "higher migration capabilities to lung tissue compared to Muse cells derived from the extraembryonic tissues of full-term infants" can be confirmed, for example, by measuring and comparing the migration capabilities of the cells using methods known in the art (e.g., the migration assay described in the Examples below).Whether or not "expression of at least one specific gene associated with tissue morphogenesis, actin filament-based process, tube morphogenesis, cell-cell adhesion, and epithelial cell differentiation, as defined in Gene Ontology, is increased compared to Muse cells derived from the extraembryonic tissue of a full-term infant" can be confirmed, for example, by measuring and comparing the expression levels of the above genes in cells using methods known in the art. "Increased gene expression" preferably means that the gene expression is increased by at least 1.5-fold, and more preferably at least 2-fold, compared to that in Muse cells derived from the extraembryonic tissue of a full-term infant.

[0028] In particular, the ultra-high potential pluripotent stem cells of the present invention can be obtained by conventional methods for isolating Muse cells from extraembryonic tissues, etc. (International Publication No. WO 2011 / 007900). For example, the ultra-high potential pluripotent stem cells of the present invention can be obtained by mincing extraembryonic tissues, culturing the resulting tissue fragments, culturing mesenchymal cells derived from the extraembryonic tissues, and expanding them until an effective cell mass is reached. Then, the ultra-high potential pluripotent stem cells can be isolated using the following antigen markers as indicators: (i) SSEA-3 alone, (ii) a double SSEA-3 and CD105 marker, (iii) a double SSEA-3 and CD133 marker, or (iv) a triple SSEA-3, CD105, and CD133 marker. Furthermore, the ultra-high potential pluripotent stem cells of the present invention can be obtained from embryonic tissues, such as bone marrow, peripheral blood, adipose tissue, and skin, using methods similar to those for isolating extraembryonic tissues, etc.

[0029] Furthermore, the ultra-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.

[0030] References: 1) L. Weinberger, M. Ayyash, N. Novershtern, J.H. 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).

[0031] Furthermore, the ultra-high potential pluripotent stem cells of the present invention are also expressed in early embryos and pluripotent stem cells during normal development, and can be confirmed by the expression of desmosome-related molecules, particularly desmoglein 2, desmocollin 2, 3, plakoglobin, etc., which are thought to be essential molecules for maintaining pluripotency and self-renewal in human pluripotent stem cells, etc.

[0032] (2) Preparation and Use of Cell Preparations The cell preparations of the present invention can be obtained by suspending the ultra-high potential pluripotent stem cells of the present invention obtained in (1) above or a cell population containing said cells in physiological saline or an appropriate buffer (e.g., phosphate-buffered saline). In this case, if the ultra-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. Furthermore, because the ultra-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 cancer development is low and the preparation is safe. Furthermore, the recovered ultra-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, etc.), without particular limitation. More specifically, with reference to the above-mentioned International Publication No. WO2011 / 007900, a solution containing a predetermined concentration of the ultra-high potential pluripotent stem cells of the present invention can be prepared by appropriately selecting a medium, additives (e.g., antibiotics, serum), etc., for the culture and proliferation of the ultra-high potential pluripotent stem cells of the present invention. When the cell preparation of the present invention is administered to a human subject, extraembryonic tissue from a human extremely preterm infant is collected, and, for example, umbilical cord tissue-derived mesenchymal stem cells are cultured as adherent cells from the umbilical cord tissue and expanded to a cell quantity sufficient to obtain an effective therapeutic amount of the ultra-high potential pluripotent stem cells of the present invention. The ultra-high potential pluripotent stem cells of the present invention can then be isolated using the SSEA-3 antigen marker as an indicator, and autologous or allogeneic ultra-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 ultra-high potential pluripotent stem cells of the present invention until an effective therapeutic amount is reached, and then autologous or allogeneic ultra-high potential pluripotent stem cells of the present invention can be prepared as a cell preparation.

[0033] Furthermore, when using the ultra-high potential pluripotent stem cells of the present invention in cell preparations, 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 ultra-high potential pluripotent stem cells of the present invention can also be used as pharmaceutical compositions 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.

[0034] The number of ultra-high-potential pluripotent stem cells of the present invention contained in the cell preparation prepared as described above can be appropriately adjusted to achieve the desired effect on the disease being treated, taking into consideration the subject's gender, age, weight, condition of the affected area, and the condition of the cells used. The target individual includes, but is 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 may be performed during the acute, subacute, or chronic phase, or at two or more of these phases, as long as the therapeutic effect is achieved. The therapeutically effective amount depends on the condition of the subject, but may be, for example, 1 x 10 per administration per individual. 3 Cell ~1×10 10 The preferred dose is 1 to 10 times the amount of cells. The total dose per individual is not limited to, but may be 1 x 10 3 Cell ~1×10 11 cells, preferably 1 x 10 4 Cell ~1×10 10 cells, more preferably 1 x 10 5 Cell ~1×10 9 Examples include cells.

[0035] The ultra-high potential pluripotent stem cells of the present invention used in the cell preparation of the present invention have the ability to migrate to and engraft at the site of injury caused by the disease being 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. The ultra-high potential pluripotent stem cells of the present invention used in the cell preparation of the present invention are characterized by their high ability to migrate to the lungs and can therefore be preferably used to treat or prevent any lung injury, disease, or condition, including, but not limited to, pediatric chronic lung disorder (also known as bronchopulmonary dysplasia (BPD)), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), emphysema, cystic fibrosis (CF), pulmonary hypoplasia, and pulmonary hypertension. Exemplary diseases or conditions include, but are not limited to, pediatric chronic lung disorder (also known as bronchopulmonary dysplasia (BPD)), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), acute respiratory distress syndrome (ARDS), emphysema, cystic fibrosis (CF), pulmonary hypoplasia, and pulmonary hypertension.

[0036] The cell preparation of the present invention can reconstruct tissue at the damaged site of a patient suffering from a disease to be treated, and can improve or restore lost function at the damaged site of the patient, i.e., can be used as a pharmaceutical for regenerative medicine.

[0037] 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.

[0038] Muse cells derived from the umbilical cords of extremely preterm human infants, i.e., ultra-high potential pluripotent stem cells, can be obtained according to the method described in International Publication No. WO 2011 / 007900, which relates to the isolation and identification of human Muse cells. Specifically, the ultra-high potential pluripotent stem cells of the present invention were obtained as follows. Their functions were also confirmed.

[0039] <Methods> (Human umbilical cord (UC) samples) Human UC samples were collected from six infants born at 23 weeks of gestation (preterm birth, one boy and two girls) or 38 weeks of gestation (term birth, three girls) after obtaining written parental consent (Table 2).

[0040]

[0041] (Preparation of human UC-MSCs and BM-MSCs) Human UC-MSCs from preterm and term infants were isolated and cultured according to a previously reported method. Briefly, umbilical cords (wet weight 2-3 g) from preterm and term infants were collected and cut into 2-3 mm pieces. 3 The cells were cut into pieces of 100 μm diameter, enzymatically dispersed with Liberase DH Research Grade (Roche, Mannheim, Germany), and filtered through a 100 μm cell strainer (BD Bioscience, Bedford, MA, USA). The harvested cells were cultured in α-minimum essential medium (Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (Millipore Sigma, St. Louis, MO, USA) and 1% antibiotic-antimycotic solution (Invitrogen, Carlsbad, CA) at 37°C (5% CO2, 95% air) until confluent and then passaged. Human BM-MSCs purchased from Lonza (Tokyo, Japan) were cultured in low-glucose Dulbecco's modified Eagle's medium (Life Technologies, Carlsbad, CA, USA) containing 10% fetal bovine serum and 0.1 mg / mL kanamycin (Invitrogen) at 37°C (5% CO and 95% air) until confluence was reached, and then passaged as previously described.

[0042] (Preparation of human Muse and non-Muse cells) Human mesenchymal stem cells (passages 5–8) were labeled with green fluorescent protein (GFP)-lentivirus according to a previously reported method. GFP(+) / SSEA-3(+) and GFP(+) / SSEA-3(-) cells were isolated from the GFP-labeled mesenchymal stem cells using a fluorescence-activated cell sorter (FACSAria II, Becton Dickson, Franklin Lakes, NJ) as Muse and non-Muse cells, respectively. Human preterm-derived UC-Muse, term-derived UC-Muse, bone marrow-derived Muse, and non-Muse cells were prepared from preterm-derived UC-MSCs, term-derived UC-MSCs, bone marrow-derived MSCs, and bone marrow-derived MSCs, respectively. In this study, all human Muse cells were used at passages 6–9.

[0043] (BLM-Induced Rat Lung Injury Model) Male 6-week-old Lewis rats were obtained from SLC (Shizuoka) and maintained under standard conditions with free access to water and laboratory rodent chow. At the start of the study (day 0), Lewis rats weighing 180-200 g were anesthetized with isoflurane (Wako Pure Chemical Industries, Ltd.) in a closed chamber. The anesthetized rats were intubated using an aerosol spray (Natsume Seisakusho, Tokyo, Japan). Rats received a single dose of BLM (Nippon Kayaku Co., Ltd., Tokyo, Japan, 12 mg / kg dissolved in 300 μl of dH2O) or an equal volume of phosphate-buffered saline (PBS). On day 3, 1 × 10 UC-Muse cells derived from preterm infants, UC-Muse cells derived from term infants, BM-Muse cells, and BM-non-Muse cells (non-Muse cells) were added to the rats. 5 Cells / 1.0 ml PBS or the same volume of PBS without immunosuppressant treatment were injected into the tail vein. On day 21, all rats were euthanized under deep anesthesia with an overdose of isoflurane and subjected to histopathological analysis. Body weights were measured and recorded on days 0, 1, 2, 3, 4, 5, 7, 10, 14, 17, and 21.

[0044] (Measurement of surfactant protein D (SP-D)) Peripheral blood was collected from the tail vein without anesthesia on day 14, and serum was separated and stored at -80°C until use. SP-D levels were measured using a Rat / Mouse SP-D ELISA kit (Yamasa, Tokyo, Japan) according to the manufacturer's instructions.

[0045] Cardiopulmonary function assessment was performed on freely moving rats fitted with a collar-type sensor to detect vital signs from the carotid artery using a mouse / rat pulse oximeter, MouseOX Plus (Starr Life Sciences, Oakmont, PA, USA). On day 7, the hair around the neck was removed under isoflurane anesthesia. On day 8, rats were allowed to acclimate to the sensor for 2 hours to minimize stress during pulmonary function measurements. On day 9, oxygen saturation (SpO2) and heart rate were recorded for 60 minutes.

[0046] (Tissue section preparation) On day 21, all rats were euthanized by isoflurane overdose and cervical dislocation, and the lungs, heart, intestine, kidneys, spleen, and liver were removed. For paraffin sections, the left and right lungs were inflated through the trachea with 10% buffered neutral formalin solution (Muto Chemicals, Tokyo, Japan) before fixation. All tissues were fixed in 10% buffered neutral formalin solution (Muto Chemicals) at room temperature for 24 hours, embedded in paraffin, and cut into 4-μm-thick sections. For frozen sections, the right and left lungs were inflated through the trachea with 4% paraformaldehyde (PFA) in 0.1 M PBS, fixed in 4% PFA in 0.1 M PBS at 4°C for 24 hours, embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetech Japan, Tokyo, Japan), and cut into 6-μm-thick frozen sections.

[0047] Paraffin sections were deparaffinized with xylene, hydrated with alcohol, and stained with hematoxylin and eosin (H&E). To assess the severity of lung injury on day 21, H&E-stained sections were analyzed and scored according to the Ashcroft scale and the modified American Thoracic Society (ATS) documentation scale. For the Ashcroft scale, a pathologist blinded to treatment group scored pulmonary fibrosis on a scale ranging from 0 to 8 in 20 randomly selected fields. For the modified ATS documentation scale, a pathologist blinded to treatment group scored eight pathological categories (areas of atypical proliferation, granulomas, necrosis, cysts, alveolar septum thickening, neutrophils, lymphocytes, and nuclear fragments) on a scale ranging from 0 to 23 in 45 randomly selected fields.

[0048] Immunohistochemistry. To detect injected human Muse cells, paraffin sections were deparaffinized with xylene, hydrated with alcohol, blocked with 3% H2O2 and Blocking One (Nacalai Tesque, Kyoto, Japan), and incubated with anti-GFP rabbit polyclonal antibody (MBL, Nagoya, Japan). Then, sections were treated with horseradish peroxidase-conjugated anti-rabbit IgG goat polyclonal antibody (dilution 1:1,000, Nichirei Biosciences, Tokyo, Japan), visualized with 3,3'-diaminobenzidine tetrahydrochloride, and counterstained with hematoxylin. Images were taken using a BX53 microscope (Ebiden, Tokyo, Japan). To count GFP-positive cells, 15 randomly captured images per group (three non-overlapping random fields per paraffin section; n = 5 per group) were processed using the BZ-X800 Analyzer software (Keyence, Osaka, Japan).

[0049] To evaluate differentiation of injected cells in rat lungs, cryosections were washed with PBS and incubated in 20% Block-Ace / 5% bovine serum albumin / 0.3% Triton X-100 in PBS for blocking. Then, they were incubated with rabbit anti-GFP antibody (1:100; Abcam, ab6556) or goat anti-GFP antibody (1:200; Abcam, ab6673), followed by incubation with either Alexa Fluor 488-conjugated donkey anti-rabbit IgG antibody (1:200; Jackson ImmunoResearch, West Grove, PA, USA, 711-546-152) or Alexa Fluor 488-conjugated donkey anti-goat IgG antibody (1:200; Jackson ImmunoResearch, 705-545-003). After washing with PBS, the sections were stained with mouse anti-podoplanin antibody (a marker for type I alveolar epithelial cells; 1:200; Abcam, ab10288), rabbit anti-prosurfactant protein C (proSP-C; a marker for type II alveolar epithelial cells; 1:100; MilliporeSigma, AB3786), or goat anti-CD31 antibody (a marker for endothelial cells; 1:50; Santa Cruz Biotechnology, Dallas, TX, USA, sc-1506), followed by Alexa Fluor 594-conjugated donkey anti-mouse IgG antibody (1:200; Jackson ImmunoResearch, 715-586-150), Alexa Fluor 594-conjugated donkey anti-rabbit IgG antibody (1:200; Jackson ImmunoResearch, 711-586-152), or Alexa Fluor 594-conjugated donkey anti-goat IgG antibody (1:200; Jackson ImmunoResearch, Sections were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 1:500; Thermo Fisher Scientific, D1306). Images were acquired with a confocal laser scanning microscope (A1; Nikon). GFP and podoplanin, pro-SPC, or CD31 double-positive cells were quantified using ImageJ.1.53t and calculated from five randomly acquired images in each section.

[0050] (RNA sequencing analysis) Total RNA was isolated using NucleoSpin RNA (Macherey-Nagel, Düsseldorf, Germany, 740902.50). The purity and concentration of the extracted RNA were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Libraries were constructed using the NEB Next Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Hitchin, UK, E7490) and the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB, E7530). Sequencing was performed by Rhelixa (Tokyo, Japan) using a NovaSeq 6000 (Illumina). Quality control and adapter trimming of the sequencing data were performed using fastp version 0.20.0. DNA reads were mapped to human reference genomic DNA (GRCh38.94) using default settings in HISAT2 version 2.2.1. Gene counts from the mapped HISAT2 output for downstream gene expression analysis were generated using featureCounts version 2.0.1. Read counts for each gene were normalized per million transcripts. Hierarchical clustering with heatmaps was generated using iDEP version 0.96. To compare biological processes in preterm UC-Muse, term UC-Muse, and BM-Muse cells, pathway and process enrichment analysis was performed using Metascape with default parameters. For marker genes of lung and vascular development and cell migration, genes annotated with Gene Ontology terms related to "lung development," "vascular development," "cell migration," and "cell adhesion" were selected.

[0051] Migration Assay: This experiment was performed using Matrigel invasion chambers (BD, 354480) according to the manufacturer's protocol. Lung tissue slices from intact Wistar rats fed serum-free medium alone or α-minimal essential medium were placed in the lower chamber. Human preterm UC-Muse, full-term UC-Muse, BM-Muse, or BM-non-Muse cells (2.5 × 10 4 ) were placed in the upper chamber and cultured at 37°C and 5% CO2 for 24 hours. Migrated cells were fixed with 4% PFA in 0.1 M phosphate buffer for 15 minutes and stained with Mayer's hematoxylin (Fujifilm, Tokyo, Japan, 131-09665). For each sample, the number of migrated cells in four fields was counted using a 20x objective lens, and the average of three samples was calculated using ImageJ.1.53t.

[0052] Statistical Analysis: All data are presented as mean ± standard error of the mean (SEM). Intergroup comparisons were performed using one-way ANOVA with Holm-Sidak multiple comparison test for two or more groups, or Student's t-test for two groups. A p value of less than 0.05 was considered statistically significant. Statistical analysis was performed using GraphPad Prism (version 10.2, GraphPad Software, Boston, MA).

[0053] <Results> (Body weight in BLM-induced lung injury model) To evaluate the therapeutic effect of UC-Muse cells on BLM-induced lung injury, male Lewis rats weighing 180-200 g were intratracheally administered BLM (12 mg / kg) on ​​day 0. On day 3, 1x10 5Preterm-UC-Muse, term-UC-Muse, BM-Muse, or non-Muse cells were intravenously injected into rats and sacrificed on day 21 (Figure 1A). Control rats received the same volume of PBS intravenously. Body weight loss was observed in all groups except the BLM(-) intact group until day 3 after BLM administration. Unlike the PBS group, the preterm-UC-Muse, term-UC-Muse, and BM-Muse groups showed recovery by day 5 (Figure 1B). The non-Muse group showed only a small recovery compared to the three Muse groups (Figure 1B). On day 21, the preterm UC-Muse group showed the greatest weight recovery compared to the other groups, being significantly higher than the BM-Muse group (p<0.01) and non-Muse groups (p<0.01), but not the full-term UC-Muse group (p=0.370) (Figure 1C). The non-Muse group had the lowest weight recovery on day 21. The mean weight was not significantly different from that of the PBS group (p = 0.351; Figure 1C). The weights of the term-UC-Muse and BM-Muse groups on day 21 were not significantly different from each other (p = 0.335; Figure 1C).

[0054] Serum SP-D levels were measured on day 14 because SP-D is more closely associated with the severity of BLM-induced lung injury than other conventional biomarkers, such as lactate dehydrogenase, monocyte chemoattractant protein-1, aspartate aminotransferase, alanine aminotransferase, high-mobility group box 1, and C-reactive protein (Fig. 1A). SP-D concentrations were significantly higher in the PBS-treated group (1285.2 ± 129.6 ng / mL) than in the non-BLM-treated group (122.2 ± 4.2 ng / mL; p < 0.01) and decreased after Muse cell treatment, suggesting that BLM treatment induced lung injury. SP-D in the preterm-UC-Muse group was the lowest among all treatment groups, and significant differences were observed between the term-UC-Muse, BM-Muse, and non-Muse groups (all p < 0.05). On the other hand, no significant difference was observed in SP-D concentrations among the full-term UC-Muse, BM-Muse, and non-Muse groups (Figure 2), suggesting that the preterm UC-Muse group showed the highest recovery from lung injury on day 14 compared with the other three groups.

[0055] (Cardiopulmonary function evaluation) Next, on day 9, cardiopulmonary function was evaluated using a mouse / rat pulse oximeter (Figure 1A). SpO2 and heart rate were continuously recorded using a collar-type sensor attached to freely moving, awake rats. Baseline SpO2 in the BLM(-) intact group was 02 The SpO2 recovery rate (95.9 ± 0.1%) was significantly lower than that of the PBS group (67.4 ± 0.7%, p < 0.001), suggesting that BLM administration worsened pulmonary function (Figures 3A and 3B). Although the difference in SpO2 between the BM-Muse and non-Muse groups was not statistically significant, the preterm UC-Muse group had the highest SpO2 recovery rate compared to the full-term UC-Muse group (p < 0.05), BM-Muse group (p < 0.01), and non-Muse group (p < 0.01) (Figure 3B). Furthermore, the term UC-Muse group also showed a significantly higher SpO2 recovery rate compared to the BM-Muse and non-Muse groups (both p < 0.05). 02 was high.

[0056] Sp 02In contrast, changes in basal heart rate were less consistent: no statistically significant differences were observed among the BLM(-) intact (97.3 ± 8.1 / min), PBS (70.9 ± 7.9 / min), non-Muse (78.7 ± 2.7 / min), BM-Muse (78.2 ± 7.4 / min), term-UC-Muse (92.0 ± 3.8 / min), and preterm-UC-Muse (92.4 ± 17.6 / min) groups (Figure 3C).

[0057] Histopathological analysis of the lungs was performed on day 21 (Figure 1A). As shown in Figures 4A and 9, structural changes were evident in the PBS group compared to the BLM(-) intact group. Because BLM induces an acute inflammatory response, which leads to a fibrotic response in the early stage and fibrosis in the later stage, two scales were used to quantify the effect of cell therapy on lung histopathology.

[0058] The Ashcroft scale assesses fibrosis and is the most widely used measure of lung injury in animals. This scale assigns a score of 9, with 0 representing a normal lung and 8 representing complete fibrotic obstruction of the lung. The Ashcroft scale score for the BLM(-) intact group was 0.22 ± 0.10, while that for the PBS group was 3.37 ± 0.10 (p < 0.001). The Muse cell group had a lower Ashcroft scale score than the PBS group. Although the difference between the BM-Muse and full-term UC-Muse groups was not statistically significant, the preterm UC-Muse group (1.53 ± 0.13) had a lower score than the full-term UC-Muse (1.96 ± 0.06; p < 0.05), BM-Muse (2.15 ± 0.10; p < 0.05), and non-Muse groups (3.15 ± 0.12; p < 0.01; Figure 4B).

[0059] Because the Ashcroft scale only assesses fibrosis, we also used a modified ATS documented scale to assess the degree of lung inflammation and fibrosis. This scale assigns a score out of 25, with 0 representing healthy lungs and 24 representing completely abnormal lungs. The BLM(-) intact group had a score of 1.07 ± 0.30, while the PBS group had a score of 15.18 ± 0.17 (p < 0.001). Consistent with the Ashcroft scale, the preterm UC-Muse group (7.84 ± 0.65) had the lowest score compared with the full-term UC-Muse (9.91 ± 0.15; p < 0.05), BM-Muse (10.75 ± 0.46; p < 0.05), and non-Muse groups (12.91 ± 0.28; p < 0.01). Of the three groups, the full-term UC-Muse, BM-Muse, and non-Muse groups were significantly different from each other, but no significant difference was observed between the full-term UC-Muse and BM-Muse groups (Figure 4C).

[0060] Engraftment of GFP-labeled injectable cells in the BLM-induced lung injury model. Engraftment of GFP-labeled injectable cells was further evaluated in the lung, heart, intestine, kidney, spleen, and liver using anti-GFP immunostaining on day 21. GFP signals were detected in the lung but were barely detectable in other tissues in the preterm UC-Muse, term UC-Muse, BM-Muse, and non-Muse groups (Figures 5A and 10). The percentage of GFP-positive cells relative to the total lung cell area was quantified, and the preterm UC-Muse group (18.54 ± 1.43%) showed the highest value compared with the term UC-Muse group (11.08 ± 0.56%; p < 0.05), BM-Muse group (7.11 ± 1.62%; p < 0.01), and non-Muse group (2.77 ± 0.49%; p < 0.01) (Figure 5B). However, although there was no significant difference in the percentage of GFP-positive cell area between the adult UC-Muse and BM-Muse groups, the adult UC-Muse and BM-Muse groups showed higher engraftment rates than the non-Muse group (p<0.05; Figure 5B).

[0061] Immunohistochemical analysis of marker expression. Marker expression in GFP-labeled cells engrafted into the lungs on day 21 was examined in the preterm-UC-Muse, term-UC-Muse, BM-Muse, and non-Muse groups. Not only did the non-Muse group have the lowest number of GFP-positive cells, but the cells also showed almost undetectable expression of the type I pneumocyte marker podoplanin, the type II pneumocyte marker proSP-C, and the vascular endothelial cell marker CD31 (data not shown)

[58] . Therefore, this group was excluded from the following experiments.

[0062] In the preterm UC-Muse group, 40.5 ± 0.8% of GFP-positive cells expressed podoplanin, compared with 35.9 ± 2.2% in the term UC-Muse group and 31.0 ± 2.2% in the BM-Muse group (p < 0.05 for preterm UC-Muse vs. term UC-Muse, p < 0.001 for preterm UC-Muse vs. BM-Muse, p < 0.05 for term UC-Muse vs. BM-Muse), suggesting that the proportion of podoplanin-positive / GFP-positive cells was highest in the preterm UC-Muse group (Figures 6A and 6B). The percentage of proSP-C(+) cells was higher in the preterm-derived UC-Muse (45.1±3.1%; p<0.05) and term-derived UC-Muse (43.9±2.6%; p<0.05) groups compared with the BM-Muse (35.8±2.8%) group. However, there was no statistically significant difference between the preterm-derived and term-derived UC-Muse groups (Figures 6C and 6D).

[0063] The endothelial cell marker CD31 was expressed in 15.5 ± 0.8% of GFP-positive cells in the preterm UC-Muse group, comparable to the full-term UC-Muse group (15.8 ± 1.2%) (Fig. 6E, 6F). Of note, the proportion of CD31-positive cells among GFP-positive cells was highest in the BM-Muse group (29.0 ± 0.8%), significantly higher than in the preterm UC-Muse group (p < 0.001) and full-term UC-Muse group (p < 0.001) (Fig. 6E, 6F).

[0064] Differential Gene Expression and Pathway Analysis. Three replicates of cultured preterm-derived UC-Muse, term-derived UC-Muse, and BM-Muse cells were subjected to RNA-seq. Genes showing ≥2-fold differential expression were extracted and compared between groups for pathway and process enrichment analysis. Genes with ≥4-fold higher expression in BM-Muse cells compared to preterm and term-derived UC-Muse cells were also identified (Figure 7A).

[0065] Furthermore, genes with over 2-fold higher expression in preterm and term Muse cells than in BM-Muse cells were associated with cell migration (actin filament-based processes in preterm and term UC-Muse cells, positive regulation of cell migration in preterm UC-Muse cells, and amoeboid cell migration in term UC-Muse cells) and differentiation (tissue and cell morphogenesis in preterm and term UC-Muse cells, and cell-cell adhesion in term UC-Muse cells; Figure 7B). Genes with over 2-fold higher expression in early UC-Muse cells compared with late UC-Muse cells were also associated with vascular and epithelial differentiation (tissue morphogenesis, ductal morphogenesis, epithelial cell differentiation) and migration (actin filament-based processes, cell-cell adhesion). On the other hand, genes whose expression levels were more than two-fold higher in full-term UC-Muse cells compared to preterm UC-Muse cells were associated with embryonic skeletal development, cell population proliferation, and tissue remodeling.

[0066] Genes that were overexpressed in BM-Muse cells at least four times higher than in preterm and term UC-Muse cells were associated with skeletal development, renal development, vascular differentiation (vasculature development compared to preterm UC-Muse cells and ductal morphogenesis compared to term UC-Muse cells), oral development (preterm UC-Muse cells), and cardiac development (term UC-Muse cells; Figure 7B).

[0067] We further compared gene expression related to lung and vascular development, cell migration, and cell adhesion. UC-Muse cells derived from preterm and term infants showed higher expression of genes related to lung development (e.g., forkhead box F1, forkhead box P4), cell migration (e.g., coagulation factor III, S1P receptor 1), and cell adhesion (e.g., intercellular adhesion molecules 1 and 5) compared with BM-Muse cells (Figure 7C). On the other hand, BM-Muse cells showed higher expression of genes related to vascular development (e.g., early growth response 3 and vascular endothelial growth factor A) than UC-Muse cells derived from preterm and term infants (Figure 7C). Furthermore, gene ontology (GO) analysis identified GO terms from a group of genes with more than two-fold higher expression in Preterm-UC-Muse compared to Term-UC-Muse (Figure 8A). The expression of genes containing the detected GO terms was displayed as a heatmap. The genes shown on the right side of Figure 8B (HAND1, HOXD13, NOG, TFAP2A, EFS, LCP1, MYO1D, TPPP3, ANGPTL4, GATA3, HOXA13, PODXL, EPCAM, ICAM5, JAM2, L1CAM, BARX1, EREG, and KRT81) were shown to be more than twice as expressed in Preterm-UC-Muse as in Term-UC-Muse.

[0068] In vitro migration assay. In various animal models of injury, Muse cells have been reported to selectively migrate to injury sites via the S1P and S1PR2 axis. As described in the Methods section, we examined the in vitro migration ability of preterm-derived UC-Muse cells, term-derived UC-Muse cells, bone marrow-derived Muse cells, and bone marrow-derived non-Muse cells toward injured lung tissue specimens from intact Wistar rats. Preterm-derived UC-Muse cells (60.2 ± 1.5 cells / field), term-derived UC-Muse cells (50.6 ± 1.6 cells / field), and BM-Muse cells (40.9 ± 1.2 cells / field) exhibited significantly higher migration potential than BM-non-Muse cells (all p < 0.001; Figure 7D). Furthermore, UC-Muse cells from preterm and term infants exhibited higher migratory potential compared with BM-Muse cells (both p<0.001), and UC-Muse cells from preterm infants exhibited higher migratory potential than UC-Muse cells from term infants (p<0.001; Figure 7D).

[0069] Discussion: BLM-induced lung injury displays a series of events, including acute inflammatory responses, fibrotic changes, extracellular matrix deposition, and alterations in smooth muscle and microvasculature. Because human lung disease is fundamentally characterized by a combination of inflammation and fibrosis, BLM-induced lung injury is widely used as an animal model for BPD (Bronchopulmonary Pathology), a pulmonary complication associated with preterm infants, and age-related lung diseases such as IPF and COPD. While endogenous lung stem cells are depleted and / or dysfunctional in preterm infants with BPD and elderly COPD patients, MSCs are unable to colonize the injured lung and differentiate into lung components, limiting their preventive or therapeutic effects.

[0070] Muse cells have several unique advantages. Surgical treatment is not required; noninvasive intravenous administration is effective enough to deliver Muse cells to target damaged tissues by sensing S1P produced by damaged or apoptotic cells. Muse cells already possess pluripotent cell-like properties, capable of migrating to damaged tissues and replacing damaged cells through a phagocytic differentiation mechanism, eliminating the need for gene transfer or differentiation induction. Furthermore, expression of HLA-G, associated with immune tolerance in the placenta, obviates the need for HLA matching or immunosuppression. These advantageous properties have been demonstrated in various animal models, and the safety and efficacy of clinical-grade bone marrow-derived Muse cells have been reported in several diseases, including acute myocardial infarction, subacute cerebral infarction, epidermolysis bullosa, and amyotrophic lateral sclerosis.

[0071] Although SSEA-3-positive Muse cells have been isolated from human UC tissues in previous studies, the differences in their basic characteristics and therapeutic effects between preterm and term UC-Muse cells had not been evaluated until this study. In this study, human Muse cells were intravenously administered without immunosuppressants to a rat BLM-induced lung injury model and the therapeutic effects of preterm and term UC-Muse cells were compared with bone marrow-derived Muse cells (standard Muse cells). Although MSCs have been extensively tested in clinical trials, Muse cells account for only a small percentage of the total MSC population. To rigorously evaluate the therapeutic effects of MSCs, we used Muse-free MSCs, which account for 97–99% of the total cell population, as a control.

[0072] The results of this study suggest that UC-Muse cells derived from preterm / newborn infants are superior to BM-Muse cells and non-Muse cells in terms of weight loss, serum SP-D levels, SpO2, Ashcroft score, and modified ATS document score in terms of recovery. Furthermore, UC-Muse cells from preterm / term infants exhibited significantly superior homing ability to the injured lung and significantly higher expression of type 1 and type 2 pneumocyte markers compared with BM-Muse cells and non-Muse cells. When comparing UC-Muse cells derived from preterm infants with those derived from term infants, UC-Muse cells derived from preterm infants showed significantly superior recovery in terms of serum SP-D levels, SpO2, Ashcroft score, and modified ATS score, and also exhibited superior lung homing and expression of type 1 pneumocyte markers compared with UC-Muse cells derived from term infants.

[0073] The basis for these differences between UC-Muse cells and BM-Muse cells from preterm and term infants was investigated in terms of gene expression patterns and in vitro migration into injured lung tissue. Expression of genes related to cell migration, cell adhesion, and differentiation, particularly those related to lung differentiation, was higher in UC-Muse cells from preterm / term infants than in BM-Muse cells. Meanwhile, BM-Muse cells expressed higher levels of genes related to skeletal, renal, vascular, and cardiac differentiation factors than preterm / term UC-Muse cells. The higher proportion of lung-homing cells positive for type 1 and type 2 pneumocytes may be related to the higher expression of forkhead box F1 and forkhead box P4, which are associated with lung differentiation, in UC-Muse cells from preterm / term infants than in BM-Muse cells. Expression of factors related to vascular and epithelial differentiation, as well as migration, was higher in UC-Muse cells from preterm infants than in UC-Muse cells from term infants.

[0074] In vitro cell migration assays demonstrated that UC-Muse cells derived from preterm and term infants migrated to lung tissue sections more efficiently than bone marrow-derived Muse cells. Notably, preterm-derived UC-Muse cells exhibited even higher migration capacity than term-derived UC-Muse cells. This was consistent with the aforementioned differential gene expression results. Taken together, these results suggest that human preterm-derived UC-Muse cells may have superior therapeutic potential for lung diseases such as BPD, IPF, and COPD compared with term-derived UC-Muse cells and bone marrow-derived Muse cells.

[0075] UC-Muse cells derived from preterm infants have several advantages over other Muse cells. First, UC cells can be collected noninvasively at the time of neonatal delivery, unlike BM-Muse cells, which require invasive sampling from the donor's bone marrow. Second, UC-MSCs have previously been reported to have a higher proliferation capacity than BM-MSCs, so collection of UC-Muse cells on a clinical scale may be easier than BM-Muse cells. Furthermore, with regard to gestational age, preterm UC-MSCs have superior proliferation and differentiation potential compared to full-term UC-MSCs, so the number of MSCs and Muse cells collected from preterm UC may be higher than that from full-term UC. Although umbilical cord blood (UCB) is present in umbilical cord blood, collection of umbilical cord blood is difficult due to the recommended delayed cord sectioning method for preterm infants. Therefore, UC-MSCs from preterm infants may be a more suitable source for Muse cells than preterm cord blood. Based on these findings, preterm UC-Muse cells from preterm infants are expected to be an ideal source for stem cell therapy in human patients with BPD, IPF, and COPD.

[0076] As described above, the results of this study demonstrated that preterm-derived UC-Muse cells have a stronger therapeutic effect than full-term-derived or adult bone marrow-derived UC-Muse cells in treating BLM-induced lung injury in a rat model.

[0077] The ultra-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, their ability to differentiate into cells of extraembryonic tissues such as the placenta and uterus can reconstruct damaged areas of the extraembryonic tissues and improve or restore the function of the damaged areas. Furthermore, their ability to differentiate into germ cell lineages allows them to be used in reproductive treatments such as infertility treatments. Furthermore, compared to high-potential pluripotent stem cells derived from extraembryonic tissues of full-term infants, they have additional properties such as superior proliferation and differentiation potential and a high migration ability to lung tissue. Therefore, the ultra-high potential pluripotent stem cells of the present invention and cell preparations containing these ultra-high potential pluripotent stem cells can be more effectively applied in the new field of regenerative medicine.

Claims

1. SSEA-3 positive ultra-high potential pluripotent stem cells derived from extraembryonic tissues of extremely preterm infants.

2. The ultra-high potential pluripotent stem cells of claim 1, 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.

3. The ultra-high potential pluripotent stem cells according to claim 2, wherein the extraembryonic tissue is an umbilical cord.

4. The ultra-high potential pluripotent stem cells according to claim 1, wherein the extremely preterm infant is a baby born at less than 28 weeks gestation.

5. The ultra-high potential pluripotent stem cells of claim 1, characterized in that the expression of at least one gene selected from the group consisting of HAND1, HOXD13, NOG, TFAP2A, EFS, LCP1, MYO1D, TPPP3, ANGPTL4, GATA3, HOXA13, PODXL, EPCAM, ICAM5, JAM2, L1CAM, BARX1, EREG, and KRT81 is increased compared to the expression in SSEA-3-positive pluripotent stem cells derived from the extraembryonic tissue of a full-term infant.

6. The ultra-high potential pluripotent stem cells of claim 1, wherein the pluripotent stem cells are pluripotent stem cells having at least one of the following properties: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew.

7. The ultra-high potential pluripotent stem cells of claim 1, which are pluripotent stem cells having all of the following properties: (i) low or no telomerase activity; (ii) the ability to differentiate into cells of any of the three germ layers; (iii) the lack of neoplastic growth; and (iv) the ability to self-renew.

8. A regenerative medicine comprising the ultra-high potential pluripotent stem cells of claim 1.

9. The regenerative medicine drug according to claim 7, which is for treating pulmonary diseases.

10. The regenerative medicine drug according to claim 9, wherein the pulmonary disease is selected from the group consisting of pediatric chronic lung disorder, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis.

11. SSEA-3-positive pluripotent stem cells derived from the extraembryonic tissue of an extremely preterm infant, characterized in that the expression of at least one gene selected from the group consisting of HAND1, HOXD13, NOG, TFAP2A, EFS, LCP1, MYO1D, TPPP3, ANGPTL4, GATA3, HOXA13, PODXL, EPCAM, ICAM5, JAM2, L1CAM, BARX1, EREG, and KRT81 is increased compared to the expression in SSEA-3-positive pluripotent stem cells derived from the extraembryonic tissue of a full-term infant.

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