Muse cell

Characteristics

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.

In vitro

Muse cells can differentiate into:

1. Ectodermal- (cells positive for nestin, NeuroD, Musashi, neurofilament, MAP-2, melanocyte markers (tyrosinase, MITF, gf100, TRP-1, DCT) ),
2. Mesodermal- (brachyury, Nkx2-5, smooth muscle actin, osteocalcin, oil red-(+) lipid droplets, desmin )
3. Endodermal- (GATA-6, α-fetoprotein, cytokeratin-7, albumin ) 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.

Low telomerase activity

Muse cells are characterized by low telomerase activity, a strong indicator of tumorigenicity. Hela cells and human fibroblast-derived iPS cells showed high telomerase activity while Muse were 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

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 wit 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 Corporation Group, have established a cell-processing procedure that is compliant with the Japansese 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 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.

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.

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.

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:

1. 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.
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 (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.

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.

Regenerative medicine