Muse cell


A Muse cell is an endogenous non-cancerous pluripotent stem cell. They reside in the connective tissue of nearly every organ, 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 and spinal cord injury are conducted by ., a group company of Mitsubishi Chemical Holdings company.

Characteristics

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 and CD117, Snai1 and Slug, CD271 and Sox10, NG2 and CD146 or CD31 and von Willebrand factor. This indicates that Muse cells do not belong to previously investigated stem cell types.

Differentiation capacity

In vitro

Muse cells can differentiate into:
  1. Ectodermal-,
  2. Mesodermal-
  3. Endodermal- lineages both spontaneously and under cytokine induction.
Recently, Tsuchiyama et al. showed that human dermal fibroblast-derived Muse cells were efficiently differentiated into melanin-producing functional melanocytes by a cocktail of cytokines. These cells maintained their melanin producing activity even after transplantation into the skin.

In vivo

Muse cells are shown to home into the damage site 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, 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. 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

. When systemically administrated, naive Muse cells 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 stroke, and cytokerain14-expressing epidermal cells in the skin, 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 and Good Gene, Cellular, and Tissue-based Products Manufacturing Practice 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 mesenchymal tissues such as the bone marrow, dermis and adipose tissue. 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

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.

Self-renewal

Muse cells are able to self-renew, maintaining their proliferative activity, pluripotency marker expression and a normal karyotype.

Collection methods

Muse cells can be collected by several techniques:
  1. Preparation of mesenchymal cells from either dermal fibroblasts or fresh bone marrow-derived mononuclear cells.
  2. Isolation of Muse cells by FACS as cells positive for SSEA-3.
  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.
There are major differences between Muse cells and non-Muse cells in present within mesenchymal cell population. When mesenchymal cells are separated into Muse and non-Muse cells by SSEA-3 cell sorting, the following differences are observed:
  1. Muse cells, SSEA-3 form clusters 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.
  2. Basic expression level of pluripotency genes in non-Muse cells is very low or undetectable level compared to Muse cells.
  3. 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 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.

Derived melanocytes

Human dermal fibroblast-derived Muse cells are shown to be a practical source for melanocyte induction. A cytokine induction system consisting of Wnt3a, SCF, ET-3, bFGF, linoleic acid, cholera toxin, L-ascorbic acid, 12-O-tetradecanoylphorbol 13-acetate, insulin, transferrin, selenium, and dexamethasone was applied to both human dermal fibroblast-derived Muse and non-Muse cells. Only Muse cells differentiated into L-DOPA reactive functional melanocytes. A three-dimensional culture model was used to assess Muse cell-derived melanocytes. In that model, the dermis was mimicked by collagen type 1 and normal human dermal fibroblasts, while epidermis was mimicked by keratinocytes and Muse cell-derived melanocytes. Furthermore, Muse cell-derived melanocytes showed melanin production. Moreover, when Muse cell-derived melanocytes was transplanted onto the back skin of severe combined immunodeficient mice, they integrated to the basal layer of the epidermis producing melanin in vivo.

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, MAP-2, GFAP, and O4, 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 -3a, bone morphogenetic proteins -2/4, and transforming growth factor 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 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 -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, 3 x 104 human dermal-Muse cells topically injected into three sites in the infarct area 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 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 and human Glc-6-Pase 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, cholangiocytes, sinusoidal endothelial cells, and Kupffer cells after migrating and homing into the injured liver. Non-Muse bone marrow MSCs are not detected in the liver from the early stage 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, mesangial cells, and endothelial cells 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.

Muse cells in clinical data

Muse cells are present in the human bone marrow of healthy donors. The number of peripheral blood-Muse cells is drastically elevated in stroke patients 24 h after onset. In acute myocardial infarction patients, peripheral blood-Muse cells significantly increase 24 h after onset, concomitant with an increase in serum sphingosine-1-phosphate, and return to baseline levels with 2~3 weeks. Importantly, patients with an increased peripheral blood-Muse cell number in the acute phase show cardiac function recovery and avoidance of heart failure at 6 months after onset, suggesting the reparative function of endogenous Muse cells.

Regenerative medicine