DESCRIPTION PLURIPOTENT STEM CELL FOR TREATING DIABETIC SKIN ULCER TECHNICAL FIELD [0001] The present invention relates to a cell preparation for use in regenerative medicine. More particularly, the present invention relates to a cell preparation 10 comprising pluripotent stem cells effective for repair and regeneration of skin tissue in skin ulcers including skin ulcers caused by diabetes, and to a method for treating skin ulcers that uses these pluripotent stem cells. 15 BACKGROUND ART [0002] Diabetes is a disease that is associated with persistent hyperglycemic state and is said to occur as a 20 result of the actions of a diverse range of environmental and genetic factors. The main regulatory factor of blood sugar is insulin, and hyperglycemia is known to occur due to insulin deficiency or an excess of factors that inhibit the action thereof (such as genetic 25 predisposition, lack of exercise, obesity or stress). Diabetes is classified as type 1 diabetes, which occurs primarily due to a decrease in pancreatic insulin secretory function attributable to such factors as autoimmune diseases, and type 2 diabetes, 1-1hich is caused 30 by a decrease in pancreatic insulin secretory function or insulin resistance caused by pancreatic exhaustion associated with persistently high levels of insulin secretion. In Japan, diabetes has become a modern-day national affliction and more than 95% of diabetes 35 patients (which is estimated to exceed 20 million people when including persons at risk to the onset of diabetes) have been diagnosed 1-1ith non-insulin-dependent diabetes - 2 - mellitus, and increases in the number of patients is becoming a problem accompanying lifestyle changes. The number of sufferers of diabetes around the world has been estimated at roughly 200 million persons (Non-Patent 5 Document 1), and the global market for antidiabetic drugs is on the order of roughly 1 trillion yen. This makes diabetes the top-ranked disease both in terms of market size and population. 10 [0003] l'lany serious complica'cions of· diabetes such· as heart disease, kidney failure or blindness have an effect on individuals together with diabetes, and complications involving the lower extremities resul in the greatest damage. As much as 40% to 70% of all lower extremity 15 amputations are related to diabetes mellitus, and in actuality, 85% of all diabetes-related lower extremity amputations occur following ulcers of the leg or foot. Patients afflicted with diabetes mellitus are at greater risk to the onset of chronic skin ulcers such as ulcers 20 of the leg and foot accompanying long-term complications. Ulcers occur as a result of ischemia and/or nerve disorders. Local tissue ischemia is the major cause of diabetic ulcers. Similar to large vessel disease, patients ~lith diabetes are at greater risk to skin 25 perfusion associated with non-conductive arteries during the course of impairment of the microcirculation control mechanism referred to as atherosclerosis or microvascular disease. Under normal circumstances, blood flow increases in order to promote healing in response to 30 injury. However, in cases in which microvascular disease (or ischemia) is present, this response is significantly impaired, and this is considered to most likely be important in the etiology of ulcers together with a tendency towards the occurrence of thrombosis in the 35 microcirculation during reduced blood flow. On the other hand, nerve disorders lack an adequately established treatment method 1.zith respect to both the symptomatic - -------------- - 3 - treatment thereof and the prevention of the progressive decline of nerve function, and is one of the major complications of diabetes mellitus. The effects of peripheralnerve disorders are particularly complex. 5 Although the mechanism leading to nerve damage associated with diabetes has yet to be fully understood, it is said to involve multiple factors, such as genetic predisposition, metabolic and vascular abnormalities or a lack of perturbation of related growth factors. 10 [0004] Attention is being· focused on regenerative medicine using pluripotent stem cells for treatment of the aforementioned refractory diseases. Adipose tissuederived stromal cells (ASC) are knmm to be one type of 15 such cells, and are thought ·to have the ability to differentiate into not only adipocytes and blood vessels, but also various other tissue cells and tissue lines (Non-Patent Documents 2 to 4). An attempt to treat ischemic injury by producing diabetic mice has been 20 reported as an example of the use of ASC (Non-Patent Document 5). In addition, an example of applying adipose-derived regenerative cells to the clinical treatment of diabetic ulcers of the lower extremities of patients afflicted 1vith peripheral arterial diseases has 25 also been reported (Non-Patent Document 6). However, these attempts have not led to complete recovery at the injured site. [0005] In addition, although mesenchymal stem cells (MSC) 30 having the ability to differentiate into bone, cartilage, adipocytes, nerve cells or skeletal muscle and the like are knmm to be examples of cells obtained from the adult body that have. the ability to differentiate (Non-Patent Documents 7 and 8), these constitute a cell population 35 that includes various cells, the actual state of that differentiation ability is not fully understood, and there have been considerable variations in the - 4 - therapeutic effects thereof. In addition, although iPS cells have been reported to be adult-derived pluripotent stem cells (as reported in Patent Document 1, for example), in addition to the establishment of iPS cells 5 requiring an extremely complex procedure consisting of introducing a specific gene into mesenchymal cells in the form of skin fibroblasts or introducing a specific compound into somatic cells, iPS cells are also highly tumorigenic, thereby resulting in the presence of 10 extremely difficult obstacles to th~ir:clinical application. [0006] According to research conducted by Dezawa, one of the inventors of the present invention, pluripotent stem 15 cells present in· a· mesenchymal cell fraction that-· express a surface antigen in the form of stage-specific embryonic antigen-3 (SSEA-3) (referred to as multilineagedifferentiating stress enduring cells (Muse cells)) are responsible for the pluripotency of that mesenchymal cell 20 fraction, and were determined to have the potential to be applied to disease treatment targeted at tissue regeneration (Patent Document 2, Non-Patent Documents 9 to 12). However, there have yet to be any examples describing the use of Muse cells for the prevention 25 and/or treatment of skin ulcer that clearly demonstrate the obtaining of anticipated therapeutic effects. [Prior Art Documents] [Patent Documents] 30 [0007] Patent Document 1: Japanese Patent No. 4183742 Patent Document 2: International Publication No. WO 2011/007900 [Non-Patent Documents] 35 [0008] Non-Patent Document 1: Stumvoll, M., et al., Lancet, Vol. 365, p. 1333-1346 (2005) 5 - 5 - Non-Patent Document 2: Rehman, J., et al., Circulation, Vol. 109, p. 1292-1298 (2004) · Non-Patent Document 3: Planat-Benard,·v., et al., Circulation, Vol. 109, p. 656-663 (2004) Non-Patent Document 4: Miranvile, A., et al., Circulation, Vol. 110, p. 349-355 (2004) Non-Patent Document 5: Kim, E. K., et al., Plast. Reconstr. Surg., Vol. 128, p. 387-394 (2011) Non-Patent Document 6: Marino, G., et al., J. Surg. 10 Res., Vol. 185, p. 36-44 (2013) 15 Non-Patent Document 7: Deza1va, M., et al., J. Clin. Invest., Vol. 113, p. 1701-1710 (2004) Non-Patent Document 8: Dezawa, M., et al., Science, Vol. 309, p. 314-317 (2005) Non-·Patent Document 9: Li, S .·; et al·., Cancer· Gene Therapy, Vol. 12, p. 600-607 (2005) Non-Patent Document 10: Kuroda, Y., et al., Proc. Natl. Acad. Sci. USA, Vol. 107, p. 8639-8643 (2010) Non-Patent Document 11: Wakao, S., et al., Proc. 20 Natl. Acad. Sci. USA, Vol. 108, p. 9875-9880 (2011) 25 Non-Patent Document 12: Kuroda, Y., et al., Nat. Protoco., Vol. 8, p. 1391-1415 (2013) DISCLOSURE OF THE INVENTION [Problems to be Solved by the Invention] [0009] An object of the present invention is to provide a novel medical application using pluripotent stem cells 30 (Muse cells). More specifically, an object of the present invention is to provide a cell preparation for the prevention and/or treatment of skin ulcers that contains Muse cells. 35 [Means for Solving the Problems] [0010] The inventors of the present invention found that, - 6 - by administering Muse cells to mice with diabetic skin ulcer, the Muse cells are able to reconstruct and repair skin tissue that results in healing of the ulcer, thereby leading to completion of the present invention. 5 [0011] Namely, the present invention is as described below. [1] A cell preparation for preventing and/or treating skin ulcer, comprising pluripotent stem cells positive for SSEA-3 isolated from biological mesenchymal 10 tissue or cultured mesenchymal cells. 15 [2] The cell preparation described in [1] above, comprising a cell fraction wherein pluripotent stem cells positive for SSEA-3 have been concentrated by-external stress stimulation. [3]The cell preparation described-in [1] or [2] above, wherein the skin ulcer is selected from the group consisting of diabetic skin ulcer, decubitus ulcer, venous stasis ulcer, arterial ulcer, radiation ulcer, necrotizing fasciitis and third degree burns. 20 [4] The cell preparation described in [1] to [3] above, wherein the pluripotent stern cells are CD105- positive. [5] The cell preparation described in [1] to [4] above, wherein the pluripotent stern cells are CD117- 25 negative and CD146-negative. [6] The cell preparation described in [1] to [5] above, 1vherein the pluripotent stern cells are CD117- negative, CD146-negative, NG2-negative, CD34-negative, vWF-negative and CD271-negative. 30 [7] The cell preparation described in [1] to [6] above, wherein the pluripotent stern cells are CD34- negative, CD117-negative, CD146-negative, CD271-negative, NG2-negative, vWF-negative, Sox10-negative, Snailnegative, Slug-negative, Tryp1-negative and Oct-negative. 35 [8] The cell preparation described in [1] to [7] above, wherein the pluripotent stern cells are pluripotent stern cells having all of the following properties: - 7 - (i) low or absent telomerase activity; (ii) ability to differentiate into cells of any of the three germ layers; (iii) absence of demonstration of neoplastic 5 proliferation; and (iv) self-renewal ability. [9] The cell preparation described in [1] to [8] above, wherein the pluripotent stem cells have the ability to differentiate into one or more cells selected 10 from the group consisting of epidermal·keratinocytes, vascular endothelial cells, vascular per-icytes, - adipocytes, preadipocytes, skin fibroblasts and nerve sheath cells. 15 [Effects of the Invention] [0012] The present invention is able to inhibit the progression of skin ulcer and repair skin tissue by a skin tissue regeneration mechanism by which Muse cells 20 differentiate into cells that constitute skin tissue at the site of a skin ulcer follovling administration of the Muse cells to that site in a subject afflicted 1vith skin ulcer. 25 BRIEF DESCRIPTION OF THE DRAWINGS [0013] [Figure 1] A. Since mice typically have an inherently high level of wound healing ability that enables wounds to heal rapidly, it is difficult to clearly differentiate 30 differences in I'IOUnd healing Hhen evaluating the Hound healing effect of a drug. Consequently, immunodeficient mice afflicted with diabetes, which is characterized by impaired progression of 1wund healing, were used to evaluate 1wund healing effects. In order to induce type 35 1 diabetes, 5-Heek-old female SCID mice were intraperitoneally injected with streptozotocin (STZ) after fasting for 24 hours. The mice Here investigated - 8 - for hyperglycemia (blood glucose > 300 mg/dl) 3 days after administration of STZ (150 mg/kg). Administration of STZ (150 mg/kg) was repeated in the case hyperglycemia was not observed. Skin defects were produced on the 5 backs of 9-week-old DM-SCID mice. FIG. 1B indicates typical changes in blood sugar levels. Nearly all of the mice became hyperglycemic after one or two injections of STZ. [Figure 2] MSC are known secrete a growth factor required 10 during the inflarrunatory phase and cell. grm·1th phase of wound healing. Therefore, the relat·i ve values of growth factor production in Muse cell fractions and mesenchymal cell fractions (MSC) cultured for 48 hours under hypoxic (1% 02 ) and normoxic conditions were measured by ELISA. 15 The measured cytokines consisted of HGF; SDF-1 ;- PDGF-BB, VEGF, EGF, TGF-~, NGF-~, bFGF and TNF-a. Absorbance at 450 nm is plotted on the Y axis. Values are shoHn as the mean± SO (n~3). Asterisks (*) indicate Pvay 10 by these examples. [Examples] [0028] Example 1: Preparation of Human Muse Cells 15 ( 1) Sampling of Human Tissue and Cell Pr.ep.ara.tion Lipoaspirates Here acquired by liposuction surgery from the abdomens and/or thighs of five non-obese women (age: 26.6±8.7 years, BMI: 21.5±2.0) from whom consent Has obtained Hith the approval of the ethics committee of the 20 Graduate School of Medicine and Faculty of Medicine of the University of Tokyo. Stromal vascular fractions (SVF) containing adipose-derived stromal/stem cells (ASC) Here isolated from the aspirated adipose tissue as previously described (see Yoshimura, K. et al., J. Cell 25 Physiol., Vol. 208, p. 64-76 (2006)). Simply put, aspirated adipose tissue was Hashed Hith PBS and digested in PBS containing 0.075% collagenase for 30 minutes at 37°C on a shaker. Mature adipocytes and connective tissue v1ere separated from the pellet by centrifugal separation. 30 The cell pellet >vas then re-suspended and lysed by passing through 100 ~m, 70 ~m and 40 ~m screens. The cell pellet containing adipose-derived stromal/stem cells (ASC) (equivalent to SVF) was cultured in a culture dish containing Dulbecco's Modified Eagle's Medium (DMEM, 35 Nissui, Tokyo, Japan) enriched with 10% fetal bovine serum (FBS) . The ASC that proliferated about 2 weeks - 20 - after culturing were sub-cultured using the same medium. The second generation of sub-cultured hASC 1vere recovered over the course of 5 minutes at 37°C using 0.25% trypsin containing 2 mM EDTA and then used to isolate Muse cells. 5 [0029] (2) Isolation of Muse Cells A magnetic-activated cell sorter (MACS, autoMACS, Miltenyl Biotec, Bergisch Gladbach, Germany) was used to recover SSEA-3-positive Muse cells. Since Muse cells 10 expressed SSEA-3 on the surface thereof, anti-SSEA-3 antibody coupled to phycoerythrin (PE, 1:3 dilution, Miltenyl Biotec) and anti-PE microbeads (1:2 dilution, Miltenyl Biotec) were used for MACS isolation of the Muse cells. Target cells labeled with microbeads were trapped 15 in the magnetic field followed by recovery in the form of a positive fraction. The cell solution that did not bind to the magnetic column was recovered in the form of a negative fraction. A MACS program was used in which the cell solution v1as applied t1vice to the magnetic column at 20 an extremely slow speed in order to more favorably purify the Muse cells. The resulting positive cell fraction was used in the following examples as a Muse cell population, while the negative cell fraction was used as mesenchymal cell fraction (MSC) . 25 [0030] Example 2: Production of Immunosuppressed Diabetic Mice Model Five-week-old severe combined immunodeficient (SCID) mice (C. Bl 7 /Icr-scid scid/ sci d) 1vere purchased from Clea 30 Japan, Inc. (Tokyo, Japan). All animal experiments v1ere carried out with the approval of the Institutional Animal Care and Use Committee of the University of Tokyo. After allowing the SCID mice to fast for 24 hours, the animals were intraperitoneally injected with freshly prepared 35 citrate-buffered saline (pH 4.5) containing streptozotocin (STZ, 150 mg/kg, Sigma-Aldrich, St. Louis MO) . Blood glucose levels were measured using a - 21 - g1ucometer and test strips (Glucose Pilot, Aventir Biotech LLC, Carlsbad, CA) on the third day after injection of STZ. Mice were considered to have diabetes mellitus (OM) if blood glucose level exceeded 300 mg/dl. 5 Those mice that did not exhibit hyperglycemia (blood glucose level in excess of 300 mg/dl) were subjected to a second round of STZ injection (150 mg/kg). foll01ved by monitoring blood glucose levels three days later. 10 [0031] Skin defects v1ere produced on the backs of the mice as previously described (see Galiano, R. D., et al., Wound Repair Regen., Vol. 12, p. 485-492 (2004) and Tepper, 0. M., Diabetes, Vol. 10, 2337/db09-0185) in order to evaluate healing of skin wounds. More 15 specifically, each mouse was anesthetized by intraperitoneal injection of pentobarbital (65 mg/kg). After shaving the backs of the animals with an electric trimmer and depilatory cream, two full-thickness skin wounds (diameter: 6 mm) penetrating to the dermomuscular 20 layer were produced on the backs of the mice using a sterilized circular biopsy punch (Kai Industries Co., Tokyo, Japan) . A doughnut-shaped silicon splint (silicon rubber sheet having an inner diameter of 9 mm, outer diameter of 15 mm and thickness of 1.0 mm, Kyowa 25 Industries, Saitama, Japan) \'las placed on the wounds and fixed in position using 6-0 nylon suture to avoid contraction of the wound (FIG. 1A). An occlusive bandage (Perme-roll, Nitta Medical, Osaka, Japan) was used to prevent the wound from drying and forming a scab. 30 [0032] Five experimental groups were prepared consisting of lvild-type mice, non-DM-SCID mice, OM-induced SCID mice, OM-induced SCID mice treated with the Muse cell population, and OM-induced SCID mice treated with the 35 mesenchymal cell fraction (MSC) . Six mice were used in each group. Muse cells (1.0 x 105 cells/mouse) were mixed with 0.1 ml of crosslinked hyaluronic acid (Restylane, Q- _I - 22 - MED, Uppsala, S1veden) followed by injecting subcutaneously.around the wounds. The amount of time until wound closure (number of days until complete epidermal regeneration) was investigated macr:oscopically; 5 The wounds were sequentially photographed on days 0, 3, 7, 10 and 14 using an ordinary digital camera (IXY Digital 90, Canon, Tokyo, Japan). The photographs 1vere evaluated using image analysis soft1vare (Photoshop CS6, Adobe Systems, San Jose, CA) foll01ved by measurement of wound 10 area. [0033] Example 3: Cytokine Production Assay (ELISA) MSC are known to secrete growth factors (such as PDGF, bFGF, TGF-P or EGF) required in the inflammatory 15 phase and cell growth phase of wound healing (Maxson, s., et al., Stem Cells Transl. Med., Vol. 1, p. 142-149 (2012)). Therefore, the Muse cell population and MSC were cultured in vitro under hypoxic and normoxic conditions to examine cytokines secreted into the culture 20 broth. The experiment was carried out in the manner indicated below. 4.0 x 105 cells of the Muse cell population and MSC were disseminated in a 60 mm culture dish follov1ed by culturing in serum-free DMEM under hypoxic (1% 02 ) and normoxic (6% 02 ) conditions. The 25 culture medium was recovered 48 hours later and filtered using a 0.22 ~m filter (Millex-GV Filter, Millipore, Billerica, MA) . The amounts of cytokines secreted into the culture broth v1ere compared and examined using an ELISA kit for hepatocyte growth factor (HGF) and stromal 30 cell-derived factor 1 (SDF-1) (both available from R&D Systems, Minneapolis, MN), and using a cytokine array kit for vascular endothelial growth factor (VEGF), epidermal groHth factor (EGF), platelet-derived growth factor {PDGF-BB), nerve growth factor-p (NGF-p), steam cell 35 factor (SCF), tumor necrosis factor-a (TNF-a), basic fibroblast growth factor (bFGF) and transforming grm1th - 23 - factor-13 (TGF-13) (Signosis #EA-1101, Santa Clara, CA). Absorbance was measured at 450 nm by spectrophotometry using an Infinite microplate reader (MlOOO, Tecan Group, Mannedorf, Switzerland). 5 [0034] The results of adherent culturing of the Muse cell population and MSC under normoxic (6% 0 2 ) and hypoxic (1% 0 2 ) conditions and comparing the concentrations of cytokines present in the culture medium 48 hours later 10 are shoi-m in FIG. 2. The Larger amounts of EGF, POGF··BB, NGF-13, SCF, TNF-a, bFGF and TGF-13 were detected in the Muse cell population in comparison \vith MSC cultured at the same oxygen pressure. Moreover, the concentrations of VEGF, EGF, POGF,-BB, NGF-13, SCF, TNF-a, bFGF and TGF-(3 15 were higher in the Muse cell population under hypoxic conditions than the concentrations thereof under normoxic conditions. 20 [0035] Example 4: Wound Healing in OM-SCID Mice Although streptozotocin (STZ) induced type 1 OM by damaging pancreas 13 cells, the admini~tration dosage and method of STZ differed from those of previous reports (see Schmidt, R. E., et al., Am. J. Pathol., Vol. 163, p. 2077-2091 (2003); Lee, R. H., et al., Proc. Natl. Acad. 25 Sci. USA, Vol. 103, p. 17438-17443 (2006); and, Schmidt, R. E., et al., Exp. Neural., Vol. 209, p. 161-170 (2008)). When STZ was administered at 200 mg/kg, many of the SCIO mice died within 1 week after administration due to severe \veight loss and metabolic abnormalities. However, 30 when STZ was injected into SCIO mice 24 hours after the start of fasting at 150 mg/kg, hyperglycemia was able to be induced comparatively consistently and a state of OM (blood glucose level exceeding 300 mg/dl) continued for more than 30 days (FIG. lB). Those SCIO mice in which OM 35 was successfully induced by injection of STZ in a single administration ( 9 of 29 mice: 31. 0%) or after two 5 - 24 - administrations (13 of 29 mice: 44.8%) were used in a wound healing experiment for 30 days after the final injection of STZ. [0036] Wound healing was significantly delayed in DM-SCID mice in the case of comparing v1ith wild-type (WT) mice (n=6) or non-DM-SCID mice (n=6) (FIG. 3). Although WT mice and non-DM-SCID mice demonstrated wound sizes of 56.9±12.0% and 67.5±6.5%, respectively, on day 7, DM-SCID 10 mice (n=6) demonstrated wound size·s of 95. 4±3 .1% (WT vs. DM-SCID: P