population. the best conditions for muse enrichment have been described as long trypsin incubation for 16 hours in skin fibroblasts and long trypsin incubation for 8 hours in bone marrow mesenchymal stem cells. however, the practical procedure for transplantation or differentiation purposes is the isolation of muse cells from a bulk culture of skin fibroblasts or bone marrow mscs as cells positive for ssea-3. severe cellular stress treatment (scst): muse cells can be isolated from lipoaspired fat by subjection to severe stress conditions that eliminate all other cell types except for muse cells, which survive as a feature of their capacity for stress endurance. the resulting cell population contains a high number of muse cells and therefore there is no need for cell sorting. the stress conditions included; long incubation with collagenase, low temperature, serum deprivation, and severe hypoxia for 16 hours. finally, the digested material is centrifuged and the pellet is re-suspended in pbs and incubated with a red blood cell lysis buffer. muse cells isolated by this method have been found to be distinct population from adipose stem cells. basic difference from other mesenchymal stem cells there are major differences between muse cells and non-muse cells in present within mesenchymal cell population. when mesenchymal cells (sometimes called mesenchymal stem cells) are separated into muse and non-muse cells by ssea-3 cell sorting, the following differences are observed: muse cells, ssea-3(+) form clusters (which are similar to embryoid bodies of es cells) from a single cell in suspension, while non-muse cells, ssea-3(-) do not proliferate successfully in suspension and thus do not form these distinctive clusters. basic expression level of pluripotency genes in non-muse cells is very low or undetectable level compared to muse cells. non-muse cells do not exhibit tissue reparation when infused into the blood stream. while they do not integrate into the damaged tissue, they may indirectly contribute to tissue regeneration by their production of cytokines, trophic factors and anti-inflammatory factors. muse cells as a primary source of ips cells in 2009, a study showed that only ssea-3+ cells generate induced pluripotent stem (ips) cells in human fibroblasts. in 2011, it was suggested that ips cells are generated only from muse cells. when the technique for generation of ips cells was applied to both muse and non-muse cells, ips cells were successfully generated only from muse cells. in contrast, non-muse cells did not show elevation in sox2 and nanog, master genes of pluripotent stem cells, even after receiving the four yamanaka factors. these results support the elite model of ips cell generation rather than the stochastic model. divergent from their muse cell origin, ips cells showed tumorigenecity. since muse cells are originally pluripotent without tumorigenic activity, what the yamanaka factors newly conferred to muse cells was not 'pluripotency' but tumorigenic activity. these results collectively suggest that only preexisting cells with promising pluripotency can be programmed into ips cells. differentiation ability of muse cells in vitro muse cells from different sources are capable of in vitro differentiation into various cell types. melanocytes: human dermal fibroblast-derived muse cells are a practical source for melanocyte induction. application of a cytokine induction system comprising wnt3a, scf, et-3, basic fibroblast growth factor, linoleic acid, cholera toxin, l-ascorbic acid, 12-o-tetradecanoylphorbol 13-acetate, insulin, transferrin, selenium, and dexamethasone to both human dermal fibroblast-derived muse and non-muse cells induces only the muse cells into l-dopa–reactive functional melanocytes capable of melanin production in a 3d cultured skin model. the application of a set of cytokines also differentiates dermal-muse cells into melanocytes. keratinocytes human adipose tissue-derived muse cells differentiate into keratinocytes by spontaneous differentiation on a gelatin culture dish or by cytokine induction containing bone morphogenetic protein-4 and all trans retinoic acid. neuronal cells: human bone marrow- and fibroblast-derived muse cells spontaneously differentiate into neural lineage cells with a lower proportion on a gelatin culture. cells expanded from single muse cell-derived clusters on gelatin-coated culture dishes express the neural markers nestin (1.9%), map-2 (3.8%), gfap (3.4%), and o4 (2.9%), suggesting the ability of muse cells to differentiate into neural-lineage cells. the cells positive for map-2 or gfap were increased following induction with basic fibroblast growth factor, forskolin, and ciliary neurotrophic factor. liver cells: muse cells can spontaneously differentiate in vitro into hepatocyte lineage cells positive for dlk, alpha-fetoprotein, cytokeratin 19, and cytokeratin 18 on gelatin-coated culture dishes. in the presence of insulin-transferrin-selenium, dexamethasone, hepatocyte growth factor, and fibroblast growth factor-4, muse cells differentiate into alpha-fetoprotein(+), albumin(+) cells. glomerular cells: muse cells differentiate in vitro into renal lineage-cells with increased expression of developmental renal markers wt1 and eya1 compared with non-muse cells after 3 weeks following the application of a cytokine induction cocktail containing all trans retinoic acid, activin a, and bone morphologic protein-7. cardiac cells: treatment of muse cells with 5’ -azacytidine in suspension culture; then transferring the cells onto adherent culture and treatment with early cardiac differentiation factors wingless-int (wnt)-3a, bone morphogenetic proteins (bmp)-2/4, and transforming growth factor (tgf) b 1; further treatment with late cardiac differentiation cytokines including cardiotrophin-1 converted muse cells into cardiomyocyte-like cells that expressed a -actinin and troponin-i with a striation-like pattern. adipocytes & osteocytes: expanded cells from muse clusters differentiate into adipocytes by the application of 1-methyl-3-isobutylxanthine, dexamethasone, insulin, and indomethacin. these induced adipocytes contain lipid droplets and stain positive for oil red o. in addition, muse cluster expanded cells differentiate into osteoblasts positive for osteocalcin using dexamethasone, ascorbic acid, and β-glycerophosphate. in vivo reparative effect of muse cells muse cells from different sources demonstrate reparative effects in animal disease models. acute myocardial infarction model rabbit autograft, allograft, and xenograft (human) bone marrow-muse cells were intravenously administrated in a rabbit acute myocardial infarction model. in vivo dynamics of muse cells showed preferential homing of the cells to the postinfarct heart at 3 days and 2 weeks, with ≈14.5% of injected muse cells estimated to be engrafted into the heart at 3 days. the migration and homing of the muse cells were shown to be mediated through the s1p (sphingosine monophosphate)-s1pr2 axis. after homing, muse cells spontaneously differentiated into cells positive for cardiac markers, such as cardiac troponin-i, sarcomeric α-actinin, and connexin-43, and vascular markers, and gcamp3-labeled muse cells that engrafted into the ischemic region exhibited increased gcamp3 fluorescence during systole and decreased fluorescence during diastole, suggesting their functionality as working cardiomyocytes. infarct size was reduced by ≈52%, and the ejection fraction was increased by ≈38% compared with vehicle injection at 2 months, ≈2.5 and ≈2.1 times higher, respectively, than that induced by mesenchymal stem cells. muse cell allografts and xenografts efficiently engrafted and recovered functions, and allografts remained in the tissue and sustained functional recovery for up to 6 months without immunosuppression. stroke and intracerebral hemorrhage models: the neural regeneration capability of muse cells has been demonstrated in several models. in a rat stroke model induced by ischemic-reperfusion of middle cerebral artery occlusion (mcao), 3 x 104 human dermal-muse cells topically injected into three sites in the infarct area (each site received 1 x 104 muse cells) delivered statistically significant functional recovery compared to vehicle and non-muse fibroblast cell-injected groups after ~2.5 months. the functional recovery was supported by the incorporation of human muse cells into rat pyramidal and sensory tracts with normalized hind limb somatosensory evoked potentials. similarly, topically injected human bone marrow-muse cells integrate into infarct region and replenish new neuronal cells and oligodendrocytes in mouse permanent mcao and mouse lacunar stroke models. in the mouse lacunar stroke model, human muse cells-derived neuronal cells integrated into the pyramidal tract, leading to statistically meaningful functional recovery. in a mouse intracerebral hemorrhage model, topically injected human bone marrow-muse cells spontaneously differentiate into neuronal cells. mice recovered motor function and spatial learning and memory ability. liver cirrhosis and partial hepatectomy models: intravenously injected human bone marrow-derived muse cells are able to repair an immunodeficient mouse (scid) model of ccl4-induced liver cirrhosis. human muse cells spontaneously differentiate in vivo into hepatocytes without fusing with host hepatocytes, and express mature functional markers such as human cyp1a2 (detoxification enzyme) and human glc-6-pase (enzyme for glucose metabolism) at 8 weeks after homing. human bone marrow-derived muse cells injected intravenously into a partial hepatectomy model in scid mice differentiate spontaneously into major liver components, namely hepatocytes (74.3% of green fluorescent protein-positive integrated muse cells), cholangiocytes (17.7%), sinusoidal endothelial cells (2.0%), and kupffer cells (6.0%) after migrating and homing into the injured liver. non-muse bone marrow mscs are not detected in the liver from the early stage (~ 1 week) to the end-point in either model. chronic kidney disease model: human bone marrow-derived muse cells injected intravenously repair scid and balb/c mouse models of focal segmental glomerulosclerosis without added immunosuppression. injected human muse cells preferentially integrate into the damaged glomeruli and spontaneously differentiate into cells expressing markers of podocytes (podocin; ~31%), mesangial cells (megsin; ~13%), and endothelial cells (cd31; ~41%) without fusing with host glomerular cells; attenuate glomerular sclerosis and interstitial fibrosis; and induce the recovery of renal function, including creatinine clearance. skin ulcers in diabetes mellitus: human adipose tissue-derived muse-rich cells significantly accelerate wound healing in skin ulcers of a mouse type 1 diabetes model. subcutaneously injected human muse cells integrate into the epidermis and dermis and differentiate into keratinocytes, vascular endothelial cells, and other cell types in the dermis. ulcers treated with human muse cells heal faster with a thick epidermal layer than those treated with non-muse cells, with a wound closure duration even shorter than that in wild-type mice. aortic aneurism model: therapeutic efficacy of intravenous injection of human bone marrow-muse cells into a scid mouse aortic aneurysm model was evaluated. at 8 weeks, infusion of human muse cells attenuated aneurysm dilation, and the aneurysmal size in the muse group corresponded to approximately 45.6% in the vehicle group. infused muse cells were shown to migrate into aneurysmal tissue from the adventitial side and penetrated toward the luminal side. histologic analysis demonstrated robust preservation of elastic fibers and spontaneous differentiation of muse cells into endothelial cells and vascular smooth muscle cells. epidermolysis bullosa model type xvii collagen (col17)-knockout (ko) mice that simulate junctional eb and recurrent skin injuries received 5.0 × 10^4 human muse cells or human non-muse-mesenchymal stem cells (mscs) by intravenous injection into the tail vein. ex vivo imaging of dissected injured skin confirmed the homing of injected muse cells but not of non-muse-mscs. human muse cells homed to the mouse epidermis expressed keratin 14 and human desmoglein-3. notably, all the mice in the muse group showed the linear deposition of human type vii col (hcol7) at the injury site of the mouse skin wheremuse-derived cells were intensively integrated. similarly, four of the five mice in the muse group showed the deposition of human col17 in association with muse cell-derived basal cells. neonatal hypoxic-ischemic encephalopathy model seven-day-old rats underwent ligation of the left carotid artery then were exposed to 8% oxygen for 60 min, and 72 hours later intravenously transplanted with 1 × 10^4 of human-muse and -non-muse cells, collected from bone marrow-mesenchymal stem cells as stage-specific embryonic antigen-3 (ssea-3)+ and -, respectively, or saline (vehicle) without immunosuppression. muse cells distributed mainly to the injured brain at 2 and 4 weeks, and expressed neuronal and glial markers until 6 months. in contrast, non-muse cells lodged in the lung at 2 weeks, but undetectable by 4 weeks. magnetic resonance spectroscopy and positron emission tomography demonstrated that muse cells dampened excitotoxic brain glutamatergic metabolites and suppressed microglial activation. muse cell-treated group exhibited significant improvements in motor and cognitive functions at 4 weeks and 5 months. intravenously transplanted muse cells afforded functional benefits in experimental hie possibly via regulation of glutamate metabolism and reduction of microglial activation. amyotrophic lateral sclerosis model in g93a-transgenic als mice, intravenous injection of 5.0 × 10^4 muse cells revealed successful homing to the lumbar spinal cords, mainly at the pia-mater and underneath white matter, and exhibited glia-like morphology and gfap expression. in contrast, such homing or differentiation were not recognized in human mesenchymal stem cells but were instead distributed mainly in the lung. relative to the vehicle groups, the muse group significantly improved scores in the rotarod, hanging-wire and muscle strength of lower limbs, recovered the number of motor neurons, and alleviated denervation and myofiber atrophy in lower limb muscles. these results suggest that muse cells homed in a lesion site-dependent