muse cell muse cell a muse cell (multi-lineage differentiating stress enduring cell) is an endogenous non-cancerous pluripotent stem cell. they reside in the connective tissue of nearly every organ including the umbilical cord, bone marrow and peripheral blood. they are collectable from commercially obtainable mesenchymal cells such as human fibroblasts, bone marrow-mesenchymal stem cells and adipose-derived stem cells. muse cells are able to generate cells representative of all three germ layers from a single cell both spontaneously and under cytokine induction. expression of pluripotency genes and triploblastic differentiation are self-renewable over generations. muse cells do not undergo teratoma formation when transplanted into a host environment in vivo. this can be explained in part by their intrinsically low telomerase activity, eradicating the risk of tumorigenesis through unbridled cell proliferation. they were discovered in 2010 by mari dezawa and her research group. clinical trials for acute myocardial infarction, stroke, epidermolysis bullosa, spinal cord injury, amyotrophic lateral sclerosis, acute respiratory distress syndrome (ards) related to novel coronavirus (sars-cov-2) infection, are conducted by life science institute, inc., a group company of mitsubishi chemical holdings company. physician-led clinical trial for neonatal hypoxic-ischemic encephalopathy was also started. the summary results of a randomized double-blind placebo-controlled clinical trial in patients with stroke was announced. characteristics stress-tolerant. do not show tumorigenicity. resistant to genotoxic stresses due to efficient sensing of dna damage and activation of dna repair systems. can be isolated as cells positive for ssea-3, a well known human embryonic stem cell marker. needless to inform that the positive ssea-3 cells or muse cells are fresh for maximum one day after sorting and if you sort the same cells after 5 days for instance, you only collect about the same percentage of muse or positive signal you got before first sorting . pluripotent stem cells, which can generate various kinds of the cells representative of all three germ layers have the ability to self-renew. non-tumorigenic. low telomerase activity. selectively accumulate into damaged tissue by intravenous or local injections by sphingosine-1-phosphate (s1p)-s1p receptor 2 axis. replenish new functional cells through spontaneous differentiation into tissue-compatible cells after homing to damaged tissue. repair tissue by systemic administration. comprise ~0.03% of bone marrow transplantation and several % of mesenchymal stem cell transplantation. have immunosuppressive and immunomodulatory effect. pluripotent stem cells can be directly obtained from normal human tissues without using artificial manipulations such as gene introduction. donor-muse cells can be directly used for treatment without hla-matching test or immunosuppressant treatment due to the specific immune privilege. markers muse cells are identified as cells positive for ssea-3+, a well-known marker for undifferentiated human es cells. they are also positive for general mesenchymal stem cell markers such as cd105, cd90 and cd29. therefore, muse cells are double positive for pluripotent and mesenchymal stem cell markers. cell isolation by ssea-3 cell sorting can be done using ssea-3 antibody. their size is 13~15 μm in diameter. muse cells do not express cd34 (markers for hematopoietic stem cells, adipose stem cells, vsels) and cd117 (hematopoietic stem cells markers), snai1 and slug (skin-derived precursors markers), cd271 and sox10 (neural crest-derived stem cells markers), ng2 and cd146 (perivascular cells) or cd31 and von willebrand factor (endothelial progenitor markers). this indicates that muse cells do not belong to previously investigated stem cell types. differentiation capacity in vitro muse cells can differentiate into: ectodermal- (cells positive for nestin, neurod, musashi, neurofilament, map-2, melanocyte markers (tyrosinase, mitf, gf100, trp-1, dct)), mesodermal- (brachyury, nkx2-5, smooth muscle actin, osteocalcin, oil red-(+) lipid droplets, desmin) endodermal- (gata-6, α-fetoprotein, cytokeratin-7, albumin) lineages both spontaneously and under cytokine induction. in vivo muse cells are shown to home into the damage site by s1p-s1p recptor 2 axis and spontaneously differentiate into tissue-compatible cells according to the microenvironment to contribute to tissue regeneration when infused into the blood stream. this was shown in human muse cells infused into animal models with fulminant hepatitis, partial hepatectomy, muscle degeneration, epidermolysis bullosa, skin injury, stroke and spinal cord injury. non-tumorigenicity low telomerase activity muse cells are characterized by low telomerase activity, not a strong indicator of tumorigenicity. hela cells and human fibroblast-derived ips cells showed high telomerase activity while muse was at nearly the same level as that in somatic cells such as fibroblasts (these data are shown without running control for the telomerase activity, the comparison is not scientific thought). this indicates the non-tumorigenic nature of muse cells. expression of genes related to pluripotency and cell cycle the expression 'pattern' of genes related to pluripotency in muse cells was almost the same as that in es and ips cells, while the expression 'level' was much higher in es and ips cells and that in muse cells. in contrast, genes related to cell cycle progression and tumorigenicity in muse cells were at the same level as those in somatic cells, while the same genes were very high in es and ips cells. these gene expression pattern and level may explain why muse cells are pluripotent but without tumorigenic activity. transplantation into mouse testes unlike es and ips cells, transplanted muse cells in testes of immunodeficient mice -a commonly used experiment to test the tumorigenicity of stem cells- have not been reported to form teratomas, even after six months. thus, muse cells are pluripotent but are non-tumorigenic. similarly, epiblast stem cells cultured under certain conditions also do not form teratomas in testes, even though they show pluripotency in vitro. thus, pluripotent stem cells do not always show teratoma formation when transplanted in vivo. tissue repair muse cells act as tissue repairing cells in vivo. when systemically administrated, naive muse cells (without cytokine treatment or gene introduction) migrate to damaged site, home into the site and spontaneously differentiate into tissue-compatible cells to replenish new functional cells. this phenomenon was observed by the infusion of green fluorescent protein-labeled naive human muse cells into animal models with fulminant hepatitis, partial hepatectomy, muscle degeneration, skin injury, stroke and spinal cord injury. infused muse cells integrated into each damaged tissue and differentiated into human albumin- and human anti-trypsin-expressing hepatocytes in the liver, human dystrophin-expressing cells in the muscle, neurofilament and map-2-expressing neuronal cells in the spinal cord and brain, desmoglein-3-, cytokerain14- and cytokeratin 15-expressing epidermal cells in the skin, glomerular cells in the kidney, corneal epithelial cells in the cornea and physiologically functional cardiac cells in the heart, respectively. muse cells have great advantages for regenerative medicine. without need of cytokine induction or artificial gene manipulation, muse cells are capable of repairing tissues when directly infused into the blood stream. hence, the clinical applications of muse cells appear promising. precise conditions such as the number and source of muse cells for each organ regeneration requires further investigation. currently, life science institute, inc. and its parent company, mitsubishi chemical holdings, have established a cell-processing procedure that is compliant with the japanese good manufacturing practice (gmp) and good gene, cellular, and tissue-based products manufacturing practice (gctp) regulation. the muse cell preparation is currently being tested in nonclinical toxicity studies. basic characteristics pluripotency, namely pluripotent marker expression, triploblastic differentiation and self-renewability, are recognized in muse cells directly collected from bm aspirates, indicating that their characteristics are not newly acquired by in vitro manipulation nor are they modified under culture conditions. location in vivo muse cells are not generated by stress, cytokine induction or exogenous gene transfection. they are preexisting pluripotent stem cells that normally reside in the bone marrow, peripheral blood and connective tissue of every organ including the umbilical cord. ] in the bone marrow, they represent one out of 3000 mono-nucleated cells. other than mesenchymal tissues, muse cells locate in connective tissue of every organ and in the peripheral blood. duality muse cells behave as mesenchymal cells in adherent environments such as in connective tissue and adherent culture, and switch to pluripotent behavior when they are transferred to a suspension environment such as in the blood stream and suspension culture formation of clusters similar to embryoid body of es cells in suspension in cell suspension, muse cells begin to proliferate and to form clusters that are very similar to embryoid bodies formed from es cells in suspension. muse cell clusters are positive for pluripotency indicators such as alkaline phosphatase reactivities, nanog, oct3/4, sox2 and par4. one of remarkable properties of muse cells is that they are capable of forming clusters from a single cell in suspension. a single muse cell-derived cluster is shown to spontaneously generate cells representative of all three germ layers on a gelatin-coated dish, proving the pluripotency of muse cells. it is important to mention that non-muse population also generates cluster in same condition, but in lower percentage (50 muse vs 10 non-muse) proliferation speed muse cells proliferate at a speed of ~1.3 day/cell division in adherent culture. this is slightly slower than that of human fibroblasts (~1 day/cell division). self-renewal muse cells are able to self-renew, maintaining their proliferative activity, pluripotency marker expression and a normal karyotype. sources muse cells can be collected from bone marrow aspirate, whose collection is a well known procedure done daily in clinics. they can also be isolated from skin fibroblasts obtained via skin biopsy, adipose tissue obtained by liposuction and from the umbilical cord; a safe and non-invasive procedure often used for cosmetic surgery interventions easy accessibility of muse cells allows them to be auto- or allo-transplanted in regenerative clinical applications. muse cells are also isolated from commercially available mesenchymal cell cultures, which ensure their availability and accessibility. general sources. muse cells can be obtained from: bone marrow aspirate adipose tissue and liposuction dermis umbilical cord commercially available culture cells such as: bone marrow-derived mesenchymal stem cells fibroblasts adipose-derived stem cells bone marrow: bone marrow mononucleated cells contain ~0.03% of ssea-3/cd105 double positive muse cells. this ratio corresponds to one out of 3000 mono-nucleated cells. dermis: muse cells, detected as ssea-3-positive cells, are located sparsely in the connective tissues of organs. in the human dermis, muse cells are located in the connective tissues distributed in the dermis and hypodermis. their location is not related to particular structures such as blood vessels or dermal papilla. adipose tissue: recently, muse cells have been successfully isolated from adipose tissue and liposuction material. the characteristics of adipose tissue-derived muse cells were consistent with those of muse cells isolated from bone marrow aspirate and commercially available fibroblasts. chazenbalk et al. showed that muse cells spontaneously differentiated into cells representative of all three germ layers. bone marrow mesenchymal stem cells contain about 1% ssea-3 positive muse cells. human dermal fibroblasts contain around 1 to 5% ssea-3 positive muse cells. adipose-derived stem cells (lonza co.) has around 1 to 7% ssea-3 positive muse cells. the overall percentage of muse cells depends on the source of mesenchymal tissue as well as manipulation and number of mesenchymal cells utilized for isolation by cell culture technique. muse cells in different species: most of muse cell research has been done in human samples. recently, they have been isolated from goat skin fibroblasts. goat ssea3+ m-clusters showed stem cell-like morphological characters and normal karyotypes. also, they were consistently positive for pluripotency markers and alkaline phosphatase staining. goat muse cells showed triploblastic differentiation capability both in vivo and in vitro and remained undifferentiated over eight passages in suspension culture. collection methods muse cells can be collected by several techniques: cell sorting: by using ssea-3 single- or ssea-3/cd105 double-positivity, muse cells can be isolated from tissues and commercially obtained cultured cells. when muse cells are to be collected directly from tissue, cells are labeled with both ssea-3 and cd105. however, in the case of cultured mesenchymal cells, almost all cells in mscs are positive for mesenchymal markers such as cd105 and cd90. single labeling with ssea-3 is sufficient to collect muse cells. the procedure includes the following steps: preparation of mesenchymal cells from either dermal fibroblasts or fresh bone marrow-derived mononuclear cells. isolation of muse cells by facs as cells positive for ssea-3. m-cluster formation in suspension culture using single-cell suspension culture. the surface of the bottom of each culture dish or well must be coated with poly-hema to avoid adhesion of the cells. long-term trypsin (ltt) treatment: for large-scale usage of muse cells -for transplantation experiments for example- they could be enriched in naive cells by severe cellular stress conditions. the resulting population is called a muse-enriched cell (mec)