Ear mesenchymal stem cells (EMSC) can differentiate into spontaneously contracting muscle cells

Abstract

We have previously shown that cells isolated from the outer ears of adult mice are a source of mesenchymal stem cells that can be induced to differentiate into adipo-, osteo-, and chondrocytes. In this study, we demonstrate that ear mesenchymal stem cells (EMSC) express stromal cell-associated markers (CD44, CD73) and stem cell marker Sca-1 and can be differentiated into spontaneously contracting muscle cells. Treatment of cells with epidermal growth factor (EGF) change their morphology from fibroblast shapes into stick-like structures that show repeated spontaneous contractions. Under conditions that promote myogenic differentiation, EMSC expressed mRNA for myoD and ventricular specific myosin light chain (MLC-2v) and protein for connexin 43, sarcomeric alpha-actinin, myocyte enhancer factor 2c (MEF2c), myosin heavy chain (MyHC), myogenin, and sarco-endoplasmic reticulum Ca(2+)ATPase (SERCA) 1. However, the cells were negative for Nkx2.5, GATA4, and ANP. Intracellular Ca(2+) transients in spontaneously beating EMSC, visualized by Fluo-3AM, showed a frequency of Ca(2+) oscillations ranging over 28-59/min (mean 41.17 +/- SEM 1.54). We also demonstrated that small pieces of ear tissues (ear punches) collected from live mice provide sufficient numbers of EMSC to isolate, culture and differentiate them into myocytes. Due to the ease of acquiring an expanding repertoire of differentiated EMSC cell types by a noninvasive surgical procedure, we conclude that the ear may prove to be a potential source of autologous cells for regenerative medicine, as supported by the fact that ears are one of the best sources of cells for somatic cell nuclear transfer (SCNT).

Cell Growth Characteristics, Differentiation Frequency, and Immunophenotype of Adult Ear Mesenchymal Stem Cells

“…mice showed similar in vitro differentiation potentials, suggesting that EMSCs are necessary but not suffi cient for the regeneration in ear punched model [12]. Immunophenotype analysis of the cells [13] revealed that EMSCs are positive for stem cell (stem cell antigen-1; Sca-1) and stromal markers (CD90, CD73, CD44), but negative for hematopoietic markers (CD45, CD4); however, the study was limited only to cells cultured at passage 0. The present study was undertaken to extend the characteristics of EMSCs.…”

“…Previous studies in our laboratory showed that both the external murine ears [12] and ear punches obtained during standard procedure used for marking live animals [13] are a source of MSCs. Termed ear mesenchymal stem cells (EMSCs), these cells have the characteristics of stem cells including the ability to self-renew and to commit into adipocyte, osteocyte, chondrocyte, and myocyte lineages at the clonal level [12][13][14].…”

“…Termed ear mesenchymal stem cells (EMSCs), these cells have the characteristics of stem cells including the ability to self-renew and to commit into adipocyte, osteocyte, chondrocyte, and myocyte lineages at the clonal level [12][13][14]. Moreover, cells isolated from the ears of regenerative (FOXN1-defi cient-nude) and non-regenerative (wild-type)…”

Diet-Induced Obesity in Stem Cell Antigen-1 KO Mice

“…Red blood cell lysis buffer (Sigma Co., St. Louis, MO) was used to remove erythrocyte contamination before analyzing by flow cytometry. Cells isolated from the fat depots were incubated with phycoerythrin-conjugated antiSca-1, antiCD44, antiCD45, antiCD73, antiCD90.1, antiCD-90.2, and antiCD117 antibodies (BD Pharmingen, San Diego, CA), as previously described [17]. The flow cytometry assay was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA) and data were analyzed with a Macintosh G5 workstation (Apple Computer, Cupertino, CA), which contains Cellquest graphics software (Becton Dickinson) for data acquisition and analysis.…”

Reprogramming mouse ear mesenchymal stem cells (EMSC) expressing the Dlk1-Dio3 imprinted gene cluster

“…Ear mesenchymal stem cells (EMSC) were first isolated as a primary culture from the external ear tissue of adult mice and shown to possess the capacity for selfrenewal and differentiation into chondrocytes, osteocytes, adipocytes [18] and myocytes [19]. EMSC also serve as a simple model for adipogenesis [20] with potentially broader applications for regenerative medicine [21].…”