Muse cell

A muse cell (multi-lineage differentiating stress enduring cell) is a recently discovered non-tumorigenic pluripotent stem cell. They reside in mesenchymal tissues such as bone marrow, dermis and adipose tissue as well as in commercially obtainable mesenchymal cells (human fibroblasts and bone marrow).^[1] Muse cells are able to generate cells representative of all three germ layers from a single cell both spontaneously and under cytokine induction. 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.

Characteristics

• 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.
• Exhibit tissue repair effect when supplied to the blood stream.
• Can be collected from bone marrow, dermis, adipose tissue and commercially available fibroblasts.
• Comprise ~0.03% of bone marrow transplantation and several % of mesenchymal stem cell transplantation.
• Can be isolated as cells positive for SSEA-3, a well known human embryonic stem cell marker.
• Pluripotent stem cells can be directly obtained from normal human mesenchymal tissues without using artificial manipulations such as gene introduction.

Markers

Muse cells are identified as cells which are positive for SSEA-3+, a well known marker for undifferentiated human ES cells.^[2] They are also positive for general mesenchymal stem cell markers such as CD105, CD90 and CD29,^[1] so that 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. Muse cells do not express CD34 (hematopoietic and adipose stem cell markers) 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.^[3]

Differentiation capacity

In vitro

Muse cells can differentiate into:

1. Ectodermal- (cells positive for nestin, NeuroD, Musashi, neurofilament, MAP-2,^[4] melanocyte markers (tyrosinase, MITF, gf100, TRP-1, DCT) ^[5]),
2. Mesodermal- (brachyury, Nkx2-5, smooth muscle actin,^[1] osteocalcin, oil red-(+) lipid droplets,^[4] desmin ^[1])
3. Endodermal- (GATA-6, α-fetoprotein, cytokeratin-7,^[1] albumin ^[4]) lineages both spontaneously and under cytokine induction.^[1]

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.^[5] 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-specific cells according to the microenvironment to contribute to tissue regeneration when infused into the blood stream.^[1] This was shown in human Muse cells infused into immunodeficient SCID mice with fulminant hepatitis,^[1] muscle degeneration,^[1] skin injury,^[1] and spinal cord injury.^[6]

Non-tumorigenicity

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.^[4]

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.^[4] 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.^[7]

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.^[1] 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.^[8] Thus, pluripotent stem cells do not always show teratoma formation when transplanted in vivo.

Tissue repair

Muse cells behave as tissue repairing cells in vivo. When infused into the peripheral bloodstream of acute injury models, naive Muse cells migrate to damaged site, home into the site and then spontaneously differentiate into tissue-specific cells to replenish lost cells. This phenomenon was observed by the infusion of green fluorescent protein-labeled naive human Muse cells into an immunodeficient mouse model with fulminant hepatitis,^[1] skeletal muscle degeneration,^[1] skin injury ^[1] and spinal cord injury.^[6] Infused Muse cells integrated into each tissue and differentiated into human albumin- and human anti-trypsin-expressing hepatocytes in the liver,^[1] human dystrophin-expressing cells in the muscle,^[1] neurofilament-expressing cells in the spinal cord ^[6] and cytokerain14-expressing epidermal cells in the skin,^[1] respectively.

Muse cells have great advantages for regenerative medicine in because of their simplicity and effectiveness. Without need of induction or artificial manipulation, Muse cells are capable of repairing tissues when directly infused into the blood stream. Hence, the clinical applications of Muse cells appear promising.^[9] Precise conditions such as the number and source of Muse cells for each organ regeneration requires further investigation.

Basic characteristics

Location in vivo

Muse cells are not generated by cytokine induction or exogenous gene transfection. They are preexisting pluripotent stem cells that normally reside in mesenchymal tissues such as the bone marrow,^[1] dermis ^[4] and adipose tissue.^[10] 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.^[4]

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.^[9]

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).^[6]

Cluster forming of pluripotent Muse/Stem cell

Self-renewal

Muse cells are able to self-renew, maintaining their proliferative activity, pluripotency marker expression and a normal karyotype.^[6]

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 or from adipose tissue obtained by liposuction; a safe and non-invasive procedure often used for cosmetic surgery interventions. This 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
• 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.^[1] 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.^[6]
• 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.^[10]
• 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.^[11]

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:

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.

• 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) 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.^[1]
• 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.^[10]

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.^[4]
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 primary source of iPS cells

In 2009, a study showed that only SSEA-3+ cells generate induced pluripotent stem (iPS) cells in human fibroblasts.^[12] 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.^[3][4]

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.^[5]

Feasibility in regenerative medicine

• Bone marrow transplantation: Muse cells are a subpopulation of bone marrow cells. They represent a small population of mono-nucleated bone marrow cells (~0.03%).^[6] This means that they have already been supplied to patients many times all over the world in bone marrow transplantations; a well-known procedure that has been performed in clinics since 1958.^[13]
• Mesenchymal stem cell transplantation: Muse cells exist within cultured MSCs such as bone marrow mesenchymal stem cells and adipose-derived stem cells. MSC transplantation has been employed for repairing liver, heart, neural tissue, airway, skin, skeletal muscle, and intestine.^[14] Therefore, if Muse cells were purified or enriched, the effectiveness of currently performed MSC transplantation is expected to see vast improvements.
• Because Muse cells do not form teratomas in vivo, they could provide an ideal source of pluripotent stem cells for regenerative medicine and cell-based therapy.

See also

• Stimulus-triggered acquisition of pluripotency cell
• Induced stem cells

Further reading

• Wakao, S., Akashi, H., Kushida, Y. and Dezawa, M (2014). "Muse cells, newly found non-tumorigenic pluripotent stem cells, reside in human mesenchymal tissues". Pathology International. 64: 1–9. doi:10.1111/pin.12129.
• Uchida, H., Morita, T., Niizuma, K., Kushida, Y., Kuroda, Y., Wakao, S., Sakata, H., Matsuzaka, Y., Mushiake, H., Tominaga, T., Borlongan, C. V. and Dezawa, M. (2015). "Transplantation of Unique Subpopulation of Fibroblasts, Muse Cells, Ameliorates Experimental Stroke Possibly Via Robust Neuronal Differentiation". STEM CELLS. doi:10.1002/stem.2206.

References

1. Kuroda Y, Kitada M, Wakao S, Nishikawa K, Tanimura Y, Makinoshima H, Goda M, Akashi H, Inutsuka A, Niwa A, Shigemoto T, Nabeshima Y, Nakahata T, Nabeshima Y, Fujiyoshi Y, Dezawa M (2010). "Unique multipotent cells in adult human mesenchymal cell populations". Proc Natl Acad Sci U S A. 107 (19): 8639–43. Bibcode:2010PNAS..107.8639K. doi:10.1073/pnas.0911647107. PMC 2889306. PMID 20421459.
2. Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998). "Embryonic stem cell lines derived from human blastocysts". Science. 282 (5391): 1145–7. Bibcode:1998Sci...282.1145T. doi:10.1126/science.282.5391.1145. PMID 9804556.
3. Wakao S, Kitada M, Dezawa M (2013). "The elite and stochastic model for iPS cell generation: multilineage-differentiating stress enduring (Muse) cells are readily reprogrammable into iPS cells". Cytometry Part A. 83 (1): 18–26. doi:10.1002/cyto.a.22069. PMID 22693162.
4. Wakao S, Kitada M, Kuroda Y, Shigemoto T, Matsuse D, Akashi H, Tanimura Y, Tsuchiyama K, Kikuchi T, Goda M, Nakahata T, Fujiyoshi Y, Dezawa M (2011). "Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts". Proc Natl Acad Sci U S A. 108 (24): 9875–80. Bibcode:2011PNAS..108.9875W. doi:10.1073/pnas.1100816108. PMC 3116385. PMID 21628574.
5. Tsuchiyama K, et al. (2013). "Functional melanocytes are readily reprogrammable from multilineage-differentiating stress-enduring (muse) cells, distinct stem cells in human fibroblasts". J Invest Dermatol. 133 (10): 2425–35. doi:10.1038/jid.2013.172. PMID 23563197.
6. Wakao S, Kuroda Y, Ogura F, Shigemoto T, Dezawa M (2012). "Regenerative effects of mesenchymal stem cells: contribution of Muse cells, a novel pluripotent stem cell type that resides in mesenchymal cells". Cells. 1 (4): 1045–1060. doi:10.3390/cells1041045.
7. Kitada M, Wakao S, Dezawa M (2012). "Muse cells and induced pluripotent stem cell: implication of the elite model". Cell Mol Life Sci. 69 (22): 3739–50. doi:10.1007/s00018-012-0994-5. PMC 3478511. PMID 22527723.
8. Chou YF, Chen HH, Eijpe M, Yabuuchi A, Chenoweth JG, Tesar P, Lu J, McKay RD, Geijsen N (2008). "The growth factor environment defines distinct pluripotent ground states in novel blastocyst-derived stem cells". Cell. 135 (3): 449–61. doi:10.1016/j.cell.2008.08.035. PMC 2767270. PMID 18984157.
9. Kuroda Y, Wakao S, Kitada M, Murakami T, Nojima M, Dezawa M (2013). "Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells". Nat Protoc. 8 (7): 1391–415. doi:10.1038/nprot.2013.076. PMID 23787896.
10. Heneidi S, Simerman AA, Keller E, Singh P, Li X, Dumesic DA, Chazenbalk G (2013). "Awakened by cellular stress: isolation and characterization of a novel population of pluripotent stem cells derived from human adipose tissue". PLoS ONE. 8 (6): e64752. doi:10.1371/journal.pone.0064752. PMC 3673968. PMID 23755141.
11. Yang Z, Liu J, Liu H, Qiu M, Liu Q, Zheng L, Pang M, Quan F, Zhang Y (2013). "Isolation and characterization of SSEA3(+) stem cells derived from goat skin fibroblasts". Cell Reprogram. 15 (3): 195–205. doi:10.1089/cell.2012.0080. PMID 23668861.
12. Byrne, J.A., H.N. Nguyen, and R.A. Reijo Pera, Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts. PLoS One, 2009. 4(9): p. e7118.
13. Cosset JM (2002). "ESTRO Breur Gold Medal Award Lecture 2001: irradiation accidents-- lessons for oncology?". Radiother Oncol. 63 (1): 1–10. doi:10.1016/s0167-8140(02)00059-2. PMID 12065098.
14. Kuroda Y, Kitada M, Wakao S, Dezawa M (2011). "Bone marrow mesenchymal cells: how do they contribute to tissue repair and are they really stem cells?". Arch Immunol Ther Exp (Warsz). 59 (5): 369–78. doi:10.1007/s00005-011-0139-9. PMID 21789625.