Technology and Innovation, Vol. 21, pp. 143-152, 2020 ISSN 1949-8241 • E-ISSN 1949-825X Printed in the USA. All rights reserved. http://dx.doi.org/10.21300/21.2.2020.143 Copyright © 2020 National Academy of Inventors. www.technologyandinnovation.org

REMOVING BARRIERS TO REGENERATIVE MEDICINE AND PROMOTING INNOVATIVE APPLICATIONS: THE DISCOVERY OF HUMAN ENDOGENOUS PLURIPOTENT MUSE CELLS

Mari Dezawa

Department of Biology and Histology, Tohoku University Graduate School of Medicine, Sendai, Japan

Multilineage-differentiating stress-enduring (Muse) cells are naturally existing, non-tumorigenic reparative endogenous stem cells identified by SSEA-3 expression. Muse cells are able to differ- entiate into nearly all cell types in the body, mobilize from the bone marrow to the peripheral blood and distribute to the connective tissue of organs, and contribute to daily minute repair of damaged/lost cells by spontaneous differentiation into tissue-compatible cells. Endogenous Muse cells express receptors for damage signaling by sphingosine-1-phosphate and are thus able to specifically home to sites of damage to regenerate healthy tissue by simultaneous dif- ferentiation into multiple tissue-constituent cells. When the number of endogenous Muse cells is not sufficient, administration of exogenous Muse cells delivers robust functional recovery. Muse cells do not need to be “induced” or genetically manipulated to exhibit pluripotency or to differentiate into various cell types for clinical use. Intravenous drip is the main method of administration, making surgery unnecessary. Furthermore, because Muse cells have an immunomodulatory system similar to the placenta, donor-derived Muse cells can be directly administered to patients without human leukocyte antigen-matching or immunosuppression therapy. Clinical trials for the treatment of myocardial infarction, stroke, and epidermolysis bullosa by intravenous delivery of donor-derived Muse cells are currently being conducted by the Life Science Institute Inc., a member of the Mitsubishi Chemical Holdings Corporation. Overall, Muse cells may safely provide clinically relevant regenerative effects compatible with the ‘body’s natural repair systems’ by a simple, cost-effective strategy—collection of Muse cells, large-scale expansion, and intravenous administration.

Key words: Muse cells; Repair; Intravenous injection; Sphingosine-1-phosphate; Differentia- tion; Immunomodulation

INTRODUCTION stage-specific embryonic antigen-3 (SSEA-3) as well Multilineage-differentiating stress enduring as pluripotent master genes, including Oct3/4, Nanog, (Muse) cells are endogenous pluripotent reparative and , and are able to differentiate into cells of all stem cells considered to contribute to tissue homeo- three germ layers from a single cell and to self-renew stasis through daily minute tissue repair and to be (2). They are able to differentiate into mesodermal distinct from other known stem cells (Figure 1) (1). (cardiomyocytes, glomerular cells, osteocytes, adi- Muse cells express the pluripotent surface marker pocytes, and vascular endothelial cells), endodermal

______

Accepted: November 1, 2019. Address correspondence to Mari Dezawa, Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, 2-1, Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. [email protected] +81-22-717-8025

143 144 DEZ AWA

(hepatocytes and cholangiocytes), and ectodermal (MSCs) and as several percent of the total (neural cells, melanocytes, and keratinocytes) cells in cells (1). While Muse cells exhibit pluripotent prop- vitro, either spontaneously or by cytokine induction erties similar to embryonic stem (ES) and induced (3-5). They also exhibit triploblastic-lineage differen- pluripotent stem (iPS) cells, they are non-tumori- tiation in vivo with unique characteristics. Without genic, consistent with the fact that they exist in vivo. prior cytokine induction, Muse cells home to the In fact, gene expression levels of factors relevant to damaged tissue and differentiate into the multiple cell cycle progression are similar between Muse cells cell types comprising the damaged tissue with a rapid and somatic cells, and Muse cell activity, time-course. For example, in a liver damage model, an indicator of tumorigenesis, is low compared with engrafted Muse cells spontaneously differentiate into that of iPS cells and tumorigenic HeLa cells. They hepatocytes, cholangiocytes, sinus endothelial cells, do not form teratomas for up to six months when and Kupffer cells, while in damaged glomeruli, they transplanted into the testis of immunodeficient mice differentiate into podocytes, mesangial cells, and cap- (2,9). On the other hand, their proliferation speed is illary endothelial cells (6,7). ~1.3 days/cell division, similar to or slightly slower than that of fibroblasts, and are thus expandable to Figure 1. Schematic diagram for Muse cell dynamics in the steady state. Muse cells mobilize from the bone marrow to clinical scale (2). the peripheral blood and are distributed to connective tissue Muse cells are characterized as endogenous repar- of organs, where they participate in tissue repair to maintain ative stem cells (Figure 1). Reparative effects of Muse tissue homeostasis. This figure was reproduced from Kushida, cells have been demonstrated following their intrave- Wakao, and Dezawa’s “Muse Cells Are Endogenous Reparative Stem Cells” in Advances in Experimental Medicine and Biology nous or local injection into animal models of acute with permission of Springer, copyright 2018. myocardial infarction, stroke, liver damage, chronic kidney disease, aortic aneurism, and skin ulcers of diabetes mellitus (6,7,10-15). In these models, Muse cells specifically home to the site of damage after administration, replenish the lost/damaged cells by spontaneous differentiation into new tissue-specific cells, and repair the damaged tissue. Clinical data of patients with acute myocardial infarction support this concept (16).

Muse Cells Tolerate Extensive Genotoxic Stimuli and Quickly Repair DNA Muse cells have a robust, effective DNA damage checkpoint and DNA repair system for the complete recovery of cells to maintain their reparative func- Muse cells are identified as SSEA-3(+) in tis- tion. Compared with general somatic cells, Muse cells sues. In vivo, they are found in the bone marrow, are more resistant to chemical genotoxic stress, such peripheral blood, and connective tissue of organs as hydrogen peroxide, and physical genotoxic stress, (1). Importantly, they are also found in extra-em- such as UV irradiation (17). The levels of senescence bryonic tissue such as the umbilical cord, which is and apoptosis are lower in Muse cells than in somatic rich in connective tissue (8). The proportion of Muse cells under both types of genotoxic stress because, in cells to mononucleated cells in the bone marrow Muse cells, the DNA damage repair system is prop- is approximately 1:3000 (~0.03%). Muse cells are erly activated following injury, and the damage is also found in the peripheral blood at a proportion of quickly repaired within six hours. This is partly due to approximately 0.01%~0.2% of the mononuclear frac- the prompt increase in non-homologous end joining tion (1). Muse cells are also found in commercially (NHEJ) enzymatic activity in Muse cells. Activation available cultured mesenchymal stem/stromal cells of RAD51 and DNA-PK, which are involved in the MUSE CELLS AS ENDOGENOUS REPARATIVE STEM CELLS 145 homologous recombination and NHEJ repair sys- 2) Muse cells are stress-tolerant and able to sur- tems, respectively, occurs soon after DNA damage vive in the hostile microenvironment of damaged and declines to basal levels within six hours after the tissue. The immunomodulatory effect of Muse cells induction of stress in Muse cells, suggesting that Muse allows intravenously administered allograft cells to cells have a powerful NHEJ system that allows them escape from host immunologic attack, enabling them to survive strong genotoxic stress. to efficiently home to the site of damage and repair the damaged tissue. Therefore, donor-derived Muse Muse Cells Function as Reparative Stem Cells cells can be directly administered to patients with- A series of studies, in which exogenous Muse cells out human leukocyte antigen (HLA)-matching or were administered, unveiled many mechanisms immunosuppression therapy. Allogeneic Muse cells underlying the tissue repair effects of endogenous and remain in the host tissue as functional cells for over exogenous Muse cells. The innate reparative functions six months without immunosuppression. of Muse cells substantiate the feasibility of utilizing 3) After homing, Muse cells replenish new func- Muse cells for clinical applications. tional cells by spontaneous differentiation into tissue-compatible cells (Figure 3). Figure 2. Schematic diagram for Muse cell dynamics and their 4) Exogenously administered Muse cells sur- mechanism under critical situations such as acute myocar- dial infarction. Damaged tissue produces S1P. Following an vive and remain integrated in the host tissue for an increase in the serum S1P level, large numbers of Muse cells extended period as functional cells. Therefore, their are mobilized from the bone marrow to the peripheral blood. pleiotropic effects, including anti-fibrotic, anti-in- Circulating Muse cells accumulate in the damaged tissue via flammatory, anti-apoptotic, and paracrine effects, the S1P-S1PR2 system. When the number of endogenous Muse cells is insufficient for tissue repair, intravenous administration are long-lasting and effective. of exogenous Muse cells may reinforce their reparative effects. 5) Muse cells can be supplied to patients in three This figure was reproduced from Kushida, Wakao, and Dezawa’s simple steps: collection from tissue sources, expan- “Muse Cells Are Endogenous Reparative Stem Cells” in Advanc- sion, and intravenous administration (Figure 4). es in Experimental Medicine and Biology with permission of Springer, copyright 2018. 6) Unlike ES and iPS cells, Muse cells require no gene introduction or in vitro cytokine induction prior to treatment, providing feasible regenerative therapy through an intravenous drip, which is an expedient approach for patients. Based on these characteristics, Life Science Institute Inc, a group company of Mitsubishi Chemical Holdings Corporation, initiated clinical trials for acute myocardial infarction (http://www.lsii.co.jp/ en/pdf/20180115-1.pdf), stroke (http://www.lsii. co.jp/en/pdf/20180903-1.pdf), and epidermolysis bullosa (http://www.lsii.co.jp/en/pdf/20181220-1. pdf) in 2018 under the approval of the regulatory organization Pharmaceuticals and Medical Devices Agency in Japan. The characteristics of the reparative functions of Muse cells are as follows: 1) Both endogenous circulating and exogenously administered Muse cells sense the location of the site of damage and migrate to and home to the site by the sphingosine-1-phosphate (S1P, actively produced by damaged cells)-S1P receptor 2 (S1PR2, expressed on the Muse cell surface) system (Figure 2). 146 DEZ AWA

Figure 3. Spontaneous differentiation of homed Muse cells into tissue-constituent cells. After homing to the damaged tissue, Muse cells spontaneously and simultaneously differentiate into cell types that comprise the damaged tissue and replace the damaged/lost cells. This figure is reproduced from and with permission from Springer, copyright 2018 (1).

Figure 4. Concept of Muse cell clinical trials. Muse cell therapy comprises three simple steps, namely isolation of Muse cells from healthy donors, expansion to clinical scale for producing Muse cell preparation, and administration to patients by intravenous drip. Currently, clinical trials are being conducted for acute myocardial infarction, stroke, and epidermolysis bullosa. This figure is reproduced from Dezawa’s “Clinical Trials of Muse Cells” in Advances in Experimental Medicine and Biology with permission of Springer, copyright 2018. MUSE CELLS AS ENDOGENOUS REPARATIVE STEM CELLS 147

Increase in Peripheral Blood-Endogenous Muse vivo dynamics of these cells (10). Muse cells preferen- Cells Correlates with Tissue Damage tially homed to the post-infarct heart, mainly to the Endogenous Muse cells are thought to mobilize ischemic and border areas at day three and did not from the bone marrow into the peripheral blood and home to other intact organs except the lung, the first circulate throughout the body to widely distribute trap of intravenously injected cells (Figure 5) (10). On to organ connective tissues, where they participate the other hand, non-Muse MSCs mostly located in in tissue homeostasis through reparative functions the lung and did not home to the post-infarct heart (16,18). at day three (Figure 5). Similar findings are reported As mentioned, circulating peripheral blood (PB)- in liver and kidney disease models (6,7). Muse cells sense the site of damage via the S1P-S1PR2 Locally injected Muse cells also migrate to and inte- system. This was demonstrated clinically; an increase grate into the site of damage in stroke and skin injury in the serum S1P level in patients with acute myocar- dial infarction precedes an increase in PB-Muse cells Figure 5. In vivo dynamics of intravenously injected Muse and and usually occurs soon after onset. Following the non-Muse MSCs in a rabbit acute myocardial infarction model at three days after administration. Nano-lantern-labeled Muse increase in serum-S1P, the number of PB-Muse cells and non-Muse MSCs were injected by intravenous injection increases; in patients with stroke and acute myocar- into a rabbit model of acute myocardial infarction. Muse cells dial infarction, the increase may be up to ~30 times homed to the post-infarct heart at day three, while the majority higher than the baseline level at 24 hours after onset of non-Muse M SCs homed mainly to the lungs. The figure was reproduced from and with permission of AHA Journals, and gradually returns to the baseline level within ~3 copyright 2018 (10). weeks. Because of individual differences, however, some patients do not exhibit an increase in PB-Muse cells in the acute phase for up to seven days (16,18). The relation between the increase in PB-Muse cells and reparative effects was demonstrated in patients with acute myocardial infarction at the chronic phase six months after the onset (16). An increase in PB-Muse cells from the baseline level in patients models (11,12,15,19). In mouse/rat stroke models, in the acute phase statistically correlated with car- Muse cells topically injected into the infarct area diac function recovery and avoidance of heart failure migrate to the ischemic region (11,12,19). On the at six months, while patients whose PB-Muse cells other hand, control cells, either non-Muse MSCs or did not increase in the acute phase had lower car- non-Muse fibroblasts, do not remain in the host brain. diac function and a trend toward heart failure. Thus, In a mouse skin ulcer model, Muse cells injected sub- the increase in PB-Muse cells may be viewed as a cutaneously around the ulcer migrated to the skin consequence of the body’s protective reaction, trig- defect site and incorporated into the regenerated der- gering self-repair. mis and epidermis, while non-Muse MSCs did not remain in the skin—nor did they contribute to skin Exogenously Administered Muse Cells tissue reconstruction (15). Preferentially Home to the Site of Damage via the Several factors and ligands participate in Muse cell S1P-S1PR2 System migration and homing, including stromal-derived Preferential homing of exogenous Muse cells to the factor 1 (SDF-1)- CXCR4 receptor (6). S1P-S1PR2, site of damage after intravenous injection is reported however, is the main system that regulates the migra- in several animal models (6,7,10,13,14). Intravenous tion and homing of Muse cells to the damaged site injection of Nano-lantern–labeled Muse cells and (10). non-Muse mesenchymal stem cells (MSCs; control S1P is a signaling sphingolipid and a bioactive cells, the remainder of MSCs after removing the Muse lipid mediator. S1P is less abundant in tissue flu- cells [non-Muse MSCs]) into a rabbit acute myocar- ids, creating an S1P gradient, and is produced from dial infarction model clearly revealed contrasting in 148 DEZ AWA sphingomyelin by an enzymatic reaction cascade cells in the glomerulus; and recover renal function converting sphingomyelin to ceramide, sphingosine, for up to ~7 weeks without immunosuppression (7). and, finally, S1P (20). While sphingomyelin locates Based on these findings, clinical trials to evaluate widely in the plasma membrane, nucleus, lysosomes, the treatment effects of intravenous administration and mitochondria, S1P is produced in the cytosol of allogeneic Muse cell preparations without HLA- and plasma membrane at baseline levels and pro- matching and immunosuppressant administration for duction is highly activated when cells are damaged. acute myocardial infarction, stroke, and epidermol- In the cell membrane, sphingomyelin locates mainly ysis bullosa are currently being conducted. Indeed, in the outer leaflet (21). When the cell is intact, con- like the immunomodulatory effects of MSCs, Muse verting enzymes in the cytoplasm are not accessible cells activate regulatory T cells, suppress dendritic to the outer leaflet of the cell membrane because the cell differentiation, and produce interferon gam- inner leaflet of the cell membrane lies between them. ma-induced indoleamine-2,3 dioxygenase (7,9,10). Once the cell membrane is damaged, however, the Notably, however, Muse cells express HLA-G, an active production of S1P begins. immunotolerance factor expressed in immune-privi- Active production of S1P in damaged tissue was leged organs such as the placenta, thymus, ovary, and demonstrated in a rabbit acute myocardial infarction testis (22). HLA-G is also associated with reduced model 24 hours after onset. Consistently, intrave- inflammation and immune responses as well as nously administered Muse cells mainly homed to with tolerogenic properties through interactions the border area rather than the infarct or intact area with inhibitory receptors on dendritic cells, natural in the post-infarct heart (10). killer cells, and T cells (23). The expression ratio in Among five S1PR1~S1PR5 subtypes, S1PR2 is Muse cells is ~90%, which is remarkably higher than the receptor most highly expressed in Muse cells. that in other stem cells (10). Human ES and iPS cells Consistently, in vitro in a Boydon chamber, Muse do not express HLA-G (24,25), and less than 20% of cells migrated toward an S1PR2-specific agonist, adult bone marrow-derived MSCs express HLA-G SID46371153, in a dose-dependent manner, and (26). HLA-G is suggested to promote graft tolerance JTE-013, an antagonist particularly selective for in heart transplantation (27). Thus, high expression S1PR2, inhibited the migration of Muse cells toward of HLA-G, together with the immunomodulatory a post-infarct heart slice culture in a dose-depen- effects of Muse cells, may contribute to their escape dent manner (10). In vivo, both co-injection of from immunologic attack during the early phase of JTE-013 and Muse cells and suppression of S1PR2 tissue integration. gene expression in Muse cells substantially attenu- ated the migration and homing of Muse cells into the Muse Cells Replace Damaged/Lost Cells by post-infarct tissue after intravenous injection in an Spontaneous Differentiation into Tissue- acute myocardial infarction model (10). These find- Constituent Cells after Homing ings together confirm the strong central role of the An outstanding feature of Muse cells relevant to S1P-S1PR2 axis in the specific homing of systemi- their reparative functions is their ability to spon- cally administered Muse cells to the site of damage. taneously and simultaneously differentiate into multiple cell types that comprise the tissue to which Immunomodulatory Effects of Muse Cells they home (Figure 3). In a rabbit acute myocardial Allogeneic Muse cells home into the post-infarct infarction model, homed Muse cells spontaneously heart after intravenous injection, spontaneously dif- differentiated into cardiomyocytes and vascular ferentiate into cardiomyocytes, and remain in the cells without fusing with host cells, thereby repair- tissue as functional cardiac cells for over six months, ing the post-infarct heart tissue (10). Functionality even without immunosuppression (10). Similarly, of the Muse cell-derived cardiomyocytes was demon- xenograft human Muse cells are engrafted into dam- strated by the expression of typical cardiac markers, aged glomeruli after intravenous injection; replace troponin-I and sarcomeric α-actinin, and connec- damaged podocytes, mesangial cells, and endothelial tions to neighboring cardiomyocytes by connexin MUSE CELLS AS ENDOGENOUS REPARATIVE STEM CELLS 149

43. Most importantly, GCaMP3-labeled Muse cells markers of liver progenitor cells, at day two and that engrafted into the ischemic region exhibited expressed mature hepatocyte markers HepPar1, increased GCaMP3 fluorescence during systole and albumin, and anti-trypsin within two weeks (13). decreased fluorescence during diastole, synchronous In the rabbit acute myocardial infarction model, elec- with cardiac electrical excitation, demonstrating elec- trophysiologic functionality as cardiomyocytes was trophysiologic functionality of the spontaneously observed at two weeks (10). This rapid progression differentiated Muse cells as cardiomyocytes. of events is in sharp contrast to the in vitro differ- In a mouse chronic kidney disease model, Muse entiations of other stem cells such as ES, iPS, and cells differentiated into podocytes (positive for MSCs, which require at least several weeks to several WT-1 and podocin), mesangial cells (megsin), and months of induction procedures to generate mature endothelial cells (CD31 and von Willebrand fac- differentiated cells. Therefore, the mechanism of in tor), components of the glomerulus, without fusion, vivo differentiation in Muse cells is presumed to dif- and improved creatinine clearance, urine protein, fer from that of ES and iPS cells, and MSCs. and plasma creatinine (7). In a mouse liver cirrho- sis model, Muse cells differentiated into albumin-, Pleiotropic Effects of Muse Cells HepPar-1-, and anti-trypsin-positive hepatocytes In addition to their differentiation and immuno- without fusing with the host hepatocytes and also modulatory capacities, Muse cells have paracrine, expressed cytochrome P450, family 1, subfamily A, anti-apoptotic, and fibrolysis/anti-fibrosis effects. polypeptide2, and glucose-6-phosphatase, enzymes They produce cytokines and trophic factors, such as related to drug metabolism and glycolysis. The ani- tissue growth factor-alpha, tissue growth factor-beta, mals injected with Muse cells had increased serum insulin-like growth factor-1, HGF, vascular endothe- albumin and decreased total bilirubin levels, sug- lial growth factor, epidermal growth factor, leukemia gesting functionality of Muse cells as hepatocytes inhibitory factor, and corticotropin-releasing hor- (6). In a mouse stroke model, Muse cells sponta- mone (6,7,10,15,28). These cytokines have multiple neously differentiated into neuronal cells (~65% of functions, including anti-apoptotic effects and acti- engrafted cells) and oligodendrocytes (~25%), and vation of endogenous tissue progenitors. In fact, incorporated into the pyramidal tract, including the less apoptosis and increased proliferative activity pyramidal decussation, and sensory tracts. This led of endogenous tissue progenitor cells is observed to motor function recovery and electrophysiologic in Muse cell-treated animal models of chronic kid- improvement in somatosensory evoked potentials ney disease and acute myocardial infarction (7,10). (12). In a muscle degeneration model, Muse cells dif- The production capacity for vascular endothelial ferentiated into dystrophin (+) skeletal muscle cells growth factor and HGF in Muse cells contributes (2); in an aortic aneurism model, Muse cells differ- to neovascularization through the incorporation of entiated into endothelial cells and smooth muscle Muse cells into vessels and their subsequent differen- cells (14); and in a damaged skin model, Muse cells tiation into vascular cells (7). In fact, animal models differentiated into keratinocytes (2). of acute myocardial infarction and chronic kidney Untreated naïve Muse cells were used in all of the disease receiving Muse cell injections exhibited sig- above-described models, and unlike general ES and nificantly higher neovascularization in repaired iPS cell transplantation, they had not been pre-treated tissues compared to controls (7,10). with cytokines or gene introduction for differenti- Muse cell-injected animals consistently exhibit sig- ation into purposive cells prior to administration. nificantly lower levels of fibrosis (6,7,10). Muse cells Another important feature is that the differentia- produce matrix metalloprotease-1, -2, and -9, which tion is initiated swiftly after homing. In a rat stroke are involved in fibrolysis and suppression of fibrosis. model, homed Muse cells expressed early neuronal markers already at day three and mature markers at Clinical Trials of Muse Cells day seven (11). In a liver damage model, Muse cells Because Muse cells are non-tumorigenic and expressed CK19, DLK, OV6, and alpha-fetoprotein, 150 DEZ AWA collectable from accessible sources, they are feasi- in general clinics. Muse cells provide the possibility ble for clinical trials from the viewpoint of safety. of supplying regenerative medicine to patients in In addition, Muse cells comprise several percent of outpatient clinics by intravenous administration and MSCs that are already widely applied to clinical tri- could therefore transform current medical practice. als, suggesting lower safety concerns. Therefore, Muse cells have groundbreaking poten- Compared with ES and iPS cells, Muse cells have tial for innovative medicine. unique advantages. First, Muse cells do not require genetic manipulation to newly acquire pluripotency, CONCLUSION unlike iPS cells, or to differentiate into purposive cells, Muse cells may safely provide clinically relevant unlike ES and iPS cells, because they are already plu- regenerative effects compatible with the ‘body’s ripotent and able to spontaneously differentiate into natural repair systems’ by a simple, cost-effective tissue-constituent cells after homing. Second, ES and strategy—collection of Muse cells, large-scale expan- iPS cells, irrespective of their differentiation state, sion, and intravenous administration. do not home to the target tissue when administered intravenously. Therefore, they need to be delivered ACKNOWLEDGMENTS directly to the target site either by surgical operation The studies reported in this work were supported or local injection. Muse cells are able to preferentially by Grants-in-Aid from the New Energy and Industrial home to the target tissue following intravenous injec- Technology Development Organization, Japan tion. Based on these unique characteristics, Muse Agency for Medical Research and Development, and cells are applicable for clinical treatment by simple collaborative research development with Life Science procedures: collection of Muse cells from the tissue Institute, Inc. source, expansion, and intravenous administration to patients. REFERENCES The usability of allogeneic cells is an important 1. Wakao S, Kushida Y, Dezawa M. Basic char- point for clinical applications. While autologous cells acteristics of Muse cells. Adv Exp Med Biol. have fewer problems regarding immunorejection, 2018;1103:13-41. they are not applicable to acute phase patients, and 2. Kuroda Y, Kitada M, Wakao S, Nishikawa cells from aged patients or patients with serious ill- K, Tanimura Y, Makinoshima H, Goda M, ness will not be available. Another advantage of Muse Akashi H, Inutsuka A, Niwa A, Shigemoto cells is that allogeneic Muse cells remain as functional T, Nabeshima Y, Nakahata T, Nabeshima cells in the host tissue for a longer period without Y-I, Fujiyoshi Y, Dezawa M. Unique mul- immunosuppression. tipotent cells in adult human mesenchymal Life Science Institute, Inc., a group company of cell populations. Proc Natl Acad Sci U S A. Mitsubishi Chemical Holdings Corporation, suc- 2010;107(19):8639-8643. ceeded in clinical grade Muse cell preparation and 3. Wakao S, Kitada M, Kuroda Y, Shigemoto T, started phase I and II clinical trials in 2018 for acute Matsuse D, Akashi H, Tanimura Y, Tsuchiyama myocardial infarction, stroke, and epidermolysis K, Kikuchi T, Goda M, Nakahat T, Fujiyoshi bullosa patients under the approval of a regulatory Y, Dezawa M. Multilineage-differentiating organization, Pharmaceuticals and Medical Devices stress-enduring (Muse) cells are a primary Agency, in Japan. All of these clinical trials are being source of induced pluripotent stem cells in conducted by intravenous administration of healthy human fibroblasts. Proc Natl Acad Sci U S A. donor-derived Muse cell preparations (Figure 4). 2011;108(24):9875-9880. Regenerative medicine is a promising new strategy 4. Amin M, Kushida Y, Wakao S, Kitada M, for curing intractable diseases. It is costly, however, Tatsumi K, Dezawa M. Cardiotrophic growth and requires multiple step manipulations with a long factor-driven induction of human Muse cells preparatory period. Furthermore, invasive routes of into cardiomyocyte-like phenotype. Cell administration, such as by surgery, are not available Transplant. 2018;27(2):285-298. MUSE CELLS AS ENDOGENOUS REPARATIVE STEM CELLS 151

5. Tsuchiyama K, Wakao S, Kuroda Y, Ogura F, subpopulation of fibroblasts, Muse cells, Nojima M, Sawaya N, Yamasaki K, Aiba S, ameliorates experimental stroke possibly via Dezawa M. Functional melanocytes are readily robust neuronal differentiation. Stem Cells. reprogrammable from multilineage-differenti- 2016;34(1):160-173. ating stress-enduring (muse) cells, distinct stem 12. Uchida H, Niizuma K, Kushida Y, Wakao cells in human fibroblasts. J Invest Dermatol. S, Tominaga T, Borlongan CV, Dezawa M. 2013;133(10):2425-2435. Human Muse cells reconstruct neuronal cir- 6. Iseki M, Kushida Y, Wakao S, Akimoto T, cuitry in subacute lacunar stroke model. Stroke. Mizuma M, Motoi F, Asada R, Shimizu S, 2017;48(2):428-435. Unno M, Chazenbalk G, Dezawa M. Muse 13. Katagiri H, Kushida Y, Nojima M, Kuroda Y, cells, nontumorigenic pluripotent-like stem Wakao S, Ishida K, Endo F, Kume K, Takahara cells, have liver regeneration capacity through T, Nitta H, Tsuda H, Dezawa M, Nishizuka specific homing and cell replacement in a SS. A distinct subpopulation of bone marrow mouse model of liver fibrosis. Cell Transplant. mesenchymal stem cells, Muse cells, directly 2017;26(5):821-840. commit to the replacement of liver compo- 7. Uchida N, Kushida Y, Kitada M, Wakao S, nents. Am J Transplant. 2016;16(2):468-483. Kumagai N, Kuroda Y, Kondo Y, Hirohara Y, 14. Hosoyama K, Wakao S, Kushida Y, Ogura F, Kure S, Chazenbalk G, Dezawa M. Beneficial Maeda K, Adachi O, Kawamoto S, Dezawa M, effects of systemically administered human Saiki Y. Intravenously injected human mul- Muse cells in Adriamycin nephropathy. J Am tilineage-differentiating stress-enduring cells Soc Nephrol. 2017;28(10):2946-2960. selectively engraft into mouse aortic aneurysms 8. Leng Z, Sun D, Huang Z, Tadmori I, Chiang and attenuate dilatation by differentiating into N, Kethidi N, Sabra A, Kushida Y, Fu YS, multiple cell types. J Thorac Cardiovasc Surg. Dezawa M, He X, Young W. Quantitative anal- 2018;155(6):2301-2313 e2304. ysis of SSEA3+ cells from human umbilical 15. Kinoshita K, Kuno S, Ishimine H, Aoi N, Mineda cord after magnetic sorting. Cell Transplant. K, Kato H, Doi K, Kanayama K, Feng J, Mashiko 2019:963689719844260. T, Kurisaki A, Yoshimura K. Therapeutic poten- 9. Gimeno ML, Fuertes F, Barcala Tabarrozzi AE, tial of adipose-derived SSEA-3-positive Muse Attorressi AI, Cucchiani R, Corrales L, Oliveira cells for treating diabetic skin ulcers. Stem Cells TC, Sogayar MC, Labriola L, Dewey RA, Perone Transl Med. 2015;4(2):146-155. MJ. Pluripotent nontumorigenic adipose 16. Tanaka T, Nishigaki K, Minatoguchi S, Nawa T, tissue-derived muse cells have immunomodula- Yamada Y, Kanamori H, Mikami A, Ushikoshi tory capacity mediated by transforming growth H, Kawasaki M, Dezawa M, Minatoguchi S. factor-beta1. Stem Cells Transl Med. 2016. Mobilized Muse cells after acute myocar- 10. Yamada Y, Wakao S, Kushida Y, Minatoguchi dial infarction predict cardiac function and S, Mikami A, Higashi K, Baba S, Shigemoto T, remodeling in the chronic phase. Circ J. Kuroda Y, Kanamori H, Amin M, Kawasaki 2018;82(2):561-571. M, Nishigaki, Taoka M, Isobe T, Muramatsu 17. Alessio N, Squillaro T, Ozcan S, Di Bernardo C, Dezawa M, Minatoguchi S. S1P-S1PR2 G, Venditti M, Melone M, Peluso G, Galderisi axis mediates homing of Muse cells into dam- U. Stress and stem cells: adult Muse cells tol- aged heart for long-lasting tissue repair and erate extensive genotoxic stimuli better than functional recovery after acute myocardial mesenchymal stromal cells. Oncotarget. infarction. Circ Res. 2018;122(8):1069-1083. 2018;9(27):19328-19341. 11. Uchida H, Morita T, Niizuma K, Kushida Y, 18. Hori E, Hayakawa Y, Hayashi T, Hori S, Okamoto Kuroda Y, Wakao S, Sakata H, Matsuzaka S, Shibata T, Kubo M, Horie Y, Sasahara M, Y, Mushiake H, Tominaga T, Borlongan Kuroda S. Mobilization of pluripotent mul- CV, Dezawa M. Transplantation of unique tilineage-differentiating stress-enduring cells 152 DEZ AWA

in ischemic stroke. J Stroke Cerebrovasc Dis. G, Dezawa M, Galderisi U. The secretome of 2016;25(6):1473-1481. MUSE cells contains factors that may play a role 19. Yamauchi T, Kuroda Y, Morita T, Shichinohe H, in regulation of stemness, apoptosis and immu- Houkin K, Dezawa M, Kuroda S. Therapeutic nomodulation. Cell Cycle. 2017;16(1):33-44. effects of human multilineage-differentiating stress enduring (MUSE) cell transplanta- tion into infarct brain of mice. PLoS One. 2015;10(3):e0116009. 20. Rivera J, Proia RL, Olivera A. The alli- ance of sphingosine-1-phosphate and its receptors in immunity. Nat Rev Immunol. 2008;8(10):753-763. 21. Ghasemi R, Dargahi L, Ahmadiani A. Integrated sphingosine-1 phosphate signaling in the central nervous system: from physio- logical equilibrium to pathological damage. Pharmacol Res. 2016;104:156-164. 22. Ferreira LMR, Meissner TB, Tilburgs T, Strominger JL. HLA-G: at the interface of maternal-fetal tolerance. Trends Immunol. 2017;38(4):272-286. 23. Rizzo R, Bortolotti D, Bolzani S, Fainardi E. HLA-G molecules in autoimmune diseases and infections. Front Immunol. 2014;5:592. 24. Drukker M, Katz G, Urbach A, Schuldiner M, Markel G, Itskovitz-Eldor J, Reubinoff B, Mandelboim O, Benvenisty N. Characterization of the expression of MHC proteins in human embryonic stem cells. Proc Natl Acad Sci U S A. 2002;99(15):9864-9869. 25. Kim EM, Manzar G, Zavazava N. Human iPS cell-derived hematopoietic progenitor cells induce T-cell anergy in in vitro-gen- erated alloreactive CD8(+) T cells. Blood. 2013;121(26):5167-5175. 26. Nasef A, Mathieu N, Chapel A, Frick J, Francois S, Mazurier C, Boutarfa A, Bouchet S, Gorin NC, Thierry D, Fouillardd L. Immunosuppressive effects of mesenchymal stem cells: involvement of HLA-G. Transplantation. 2007;84(2):231-237 27. Lila N, Amrein C, Guillemain R, Chevalier P, Latremouille C, Fabiani JN, Dausset J, Carosella ED, Carpentier A. Human leukocyte antigen-G expression after heart transplantation is asso- ciated with a reduced incidence of rejection. Circulation. 2002;105(16):1949-1954. 28. Alessio N, Ozcan S, Tatsumi K, Murat A, Peluso