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Muse-ings on Reprogramming: Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts

By Stuart P. Atkinson

Many questions surrounding induced pluripotent stem cells (iPSCs) still remain, including a definitive answer to the question of the origin of the cells which ultimately undergo reprogramming. Two models exist; the stochastic model which suggests that random cells from an initial culture will eventually become iPSCs, and the elite model, which posits that only a few cells from within a culture have the ability to become reprogrammed, hence the low relative efficiency of the reprogramming process. In the elite model, it is supposed that such cells may be tissue-specific stem/progenitor cells which might already express certain factors required for the reprogramming process. A recent study in Stem Cells from the lab of Rudolf Jaenisch (Kim et al), also reported on the Stem Cells Portal (Reprogramming of Postnatal Neurons into Induced Pluripotent Stem Cells by Defined Factors) suggested that a pure terminally differentiated population of cells could be reprogrammed with the expression of Oct4, Sox2, Klf4 and Myc (OSKM) but only with expression of Rest and under conditions of p53 inhibition, giving some credence towards the stochastic model. However, a study in PNAS from the lab of Mari Dezawa at the Tohoku University Graduate School of Medicine, Sendai, Japan, suggest that only a small sub-population of cells from a naïve dermal fibroblast culture which express stem cell-like properties can be reprogrammed (Wakao et al), thus instead supporting the elite model.

Previous studies by the research group had identified an adult stem cell-like cell in dermal fibroblasts and bone marrow stromal cells which showed multilineage differentiation potential (Kuroda et al). These cells were stress tolerant, expressed pluripotency markers (NANOG, OCT4, and SOX2), were able to self-renew and importantly were not tumorigenic and for this reason were named “multilineage-differentiating stress-enduring cells “or Muse cells for short. These cells could be selected for from heterogeneous culture using SSEA3, which had been used in a similar study previously which showed that only SSEA3 cells could generate iPSCs (Byrne et al).

In the current study, Muse cells were selected from naïve human fibroblasts using SSEA3 and CD105, which comprised up to 2% of cells from an initial culture growing at 70-80% confluency. Growth of Muse cells as single-cell suspensions led to the formation of cell clusters similar to hESC-derived embryoid bodies (EBs) after 10 days, whereas non-Muse cells did not. Muse cell clusters were also positive for alkaline phosphatase (AP) staining and OCT4, SOX2 and NANOG expression; although the mRNA level of these pluripotency markers was markedly lower than observed in hESC and iPSCs derived from fibroblasts. However, these cells demonstrated self-renewal and could be differentiated towards ectodermal, mesodermal and endodermal lineages from a single cell, with no teratoma formation observed. Muse and non-Muse cells were then checked for their ability to undergo reprogramming to iPSCs using the retroviral or polycistronic transduction of OSKM. At 30 days post-transduction, Muse cells gave 8-times more colonies, but more importantly only the Muse cells gave colonies which resembled human embryonic stem cells (hESCs) morphologically, expressed TRA-1-81 and demonstrated endogenous SOX2 and NANOG mRNA expression. Further, after subsequent passaging of both types of iPSCs, only Muse cells gave iPSCs that expressed endogenous OCT4, SOX2 and NANOG mRNA and protein after subsequent passaging. Muse cells also undergo reprogramming roughly 30 times more efficiently than non muse cells (from ~0.001% to 0.03%).

This report suggests that Muse cells may be the primary cell source of reprogrammable cells, and can be easily selected for using SSEA3 and CD105. Does this settle the stochastic vs. elite model argument? Perhaps the reprogramming process lies somewhere between the two, as while only the Muse-cells could be reprogrammed, the efficiency was still relatively low (0.03%) suggesting that perhaps a certain degree of stochasticity is needed for the emergence of iPSCs from an elite starting cell population. However, even if the argument still remains open, which appears likely, this knowledge could perhaps allow for an increase in the efficiency of iPSC production through the better selection of target cells.



Reprogramming of postnatal neurons into induced pluripotent stem cells by defined factors.
Kim J, Lengner CJ, Kirak O, Hanna J, Cassady JP, Lodato MA, Wu S, Faddah DA, Steine EJ, Gao Q, Fu D, Dawlaty M, Jaenisch R.
Stem Cells. 2011 Jun;29(6):992-1000. doi: 10.1002/stem.641.

Multilineage-differentiating stress-enduring (Muse) cells are a primary source of induced pluripotent stem cells in human fibroblasts.
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.
Proc Natl Acad Sci U S A. 2011 Jun 14;108(24):9875-80.

Unique multipotent cells in adult human mesenchymal cell populations.
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.
Proc Natl Acad Sci U S A. 2010 May 11;107(19):8639-43.

Enhanced generation of induced pluripotent stem cells from a subpopulation of human fibroblasts.
Byrne JA, Nguyen HN, Reijo Pera RA.
PLoS One. 2009 Sep 23;4(9):e7118.