Clonal Differentiation of Skeletal Muscle–Derived CD34⁻/45⁻ Stem Cells Into Cardiomyocytes In Vivo

Abstract

The differentiation and/or therapeutic potential of skeletal muscle-derived stem cells for cardiac infarction have been studied extensively for use in cellular cardiomyoplasty, as injured cardiomyocytes exhibit limited regenerative capacity. We previously reported cardio-myogenic differentiation of skeletal muscle-derived CD34+/45(-) (Sk-34) stem cells after therapeutic transplantation. However, the clonal differentiation potential of these cells remains unknown. Here, we show that skeletal muscle-derived CD34(-)/45(-) (Sk-DN) stem cells, which are situated upstream of Sk-34 cells in the same lineage, exhibit clonal differentiation into cardiomyocytes after single cell-derived single-sphere implantation into myocardium. Sk-DN cells were enzymatically isolated from green fluorescent protein (GFP) transgenic mice and purified by flow cytometry, and were then clonally cultured in collagen-based medium with bFGF and EGF after clonal cell sorting. Single cell-derived single-sphere colonies of Sk-DN cells were directly implanted into the wild-type mouse myocardium. At 4 weeks after implantation, donor cells exhibited typical cardiomyocyte structure with the formation of gap-junctions between donor and recipient cells. Expression of specific mRNAs for cardiomyocytes, such as cardiac actin and GATA-4, Nkx2-5, Isl-1, Mef2, and Hand2, were also seen in clonal cell cultures of Sk-DN cells. Cell fusion-independent differentiation was also confirmed by bulk cell transplantation using Cre- and loxP (enhanced GFP)-mice. We conclude that Sk-DN cells can give rise to cardiac muscle cells clonally, and that skeletal muscle includes a practical cell source for cellular cardiomyoplasty.

Muscle-derived stem cells: isolation, characterization, differentiation, and application in cell and gene therapy

“…Unfortunately, until now, most studies have not shown that unexpected lineage differentiation is derived from the same single cell that differentiates into the expected cell type, and even when this has been achieved, the apparent lineage switch seen to occur in general at a low frequency. Recently, some studies have shown clonal multipotency of stem cells, including MDSCs, thereby ruling out cell fusion (Tamaki et al 2007b(Tamaki et al , 2010. Thus, we believe that stem cell plasticity involves rare cells but is a real phenomenon, and not a rare event (Jang and Sharkis 2005).…”

“…In addition, Tamaki et al (2008) have reported that freshly isolated skeletal-muscle-derived CD34+/45-cells can give rise to cardiomyocytes having desmosomes and intercalated discs associated with gapjunctions after transplantation into the MI zone, significantly contributing to functional recovery of the left ventricle. Further, they have performed clonal differentiation of skeletal muscle-derived CD34-/45-stem cells into cardiomyocytes in vivo (Tamaki et al 2010). Similarly, in patients with previous myocardial infarction, treatment with MDSCs in conjunction with coronary artery bypass has been shown to be safe and feasible and is associated with increased global and regional left ventricular function, improvement in the viability of cardiac tissue in the infarct area, and no induction of arrhythmias (Herreros et al 2003).…”

Plasticity and Physiological Role of Stem Cells Derived from Skeletal Muscle Interstitium: Contribution to Muscle Fiber Hyperplasia and Therapeutic Use

“…In contrast, when Sk-34 and Sk-DN cells are transplanted into a damaged cardiac muscle environment, they can give rise to cardiomyocytes having desmosomes and intercalated discs associated with gap-junctions that significantly contribute to functional recovery of the left ventricle [47]. This cardiomyogenic differentiation capacity was also confirmed to be clonal by cell transplantation and RT-PCR analysis of single cells and single cell-derived clonal cells [63].…”

Origin and hierarchy of basal lamina-forming and -non-forming myogenic cells in mouse skeletal muscle in relation to adhesive capacity and Pax7 expression in vitro

“…Details of the single and/or bulk-cell RT-PCR methods have been described previously (Tamaki et al 2008(Tamaki et al , 2010b. Specific primers used for the single-and bulk-cell RT-PCR are summarized in Tables 1 and 2.…”