Skeletal Muscle–Derived CD34⁺/45^-and CD34^-/45^-Stem Cells Are Situated Hierarchically Upstream of Pax7⁺Cells

Abstract

The hierarchical relationship of skeletal muscle-derived multipotent stem cells sorted as CD34(+)/CD45(-) (Sk-34) and CD34(-)/CD45(-) (Sk-DN) cells, which have synchronized reconstitution capacities for blood vessels, peripheral nerves, and muscle fibers, was examined. Expression of Sca-1 and CD34 (typical state of freshly isolated Sk-34 cells) in Sk-DN cells was examined using in vitro culture and in vivo cell implantation. Sk-DN cells sequentially expressed Sca-1 and CD34 during cell culture showing self-maintenance and/or self-renewal-like behavior, and are thus considered hierarchically upstream of Sk-34 cells in the same lineage. Sk-34 and Sk-DN cells were further divided into small and large cell fractions by cell sorting. Immunocytochemistry using anti-Pax7 was performed at the time of isolation (before culture) and revealed that only 1% of cells in the large Sk-DN cell fraction were positive for Pax7, while Sk-34 cells and 99% of Sk-DN cells were negative for Pax7. Therefore, putative satellite cells were possibly present in the large Sk-DN cell fraction. However, serial analysis of Pax7 expression by RT-PCR and immunocytochemistry for single and 2 to >40 clonally proliferated Sk-34 and Sk-DN cells revealed that both cell types expressed Pax7 after several asymmetric cellular divisions during clonal-cell culture. In addition, production of satellite cells was seen after muscle fiber formation following Sk-34 or Sk-DN cell transplantation into damaged muscle, and even in the nonmuscle tissue environment (beneath the renal capsule). Thus, Sk-DN cells are situated upstream of Sk-34 cells and both cells can produce Pax7+ cells (putative satellite cells) after cellular division.

Anabolic-androgenic steroid does not enhance compensatory muscle hypertrophy but significantly diminish muscle damages in the rat surgical ablation model

“…Myogenic cells located inside the basal lamina are undoubtedly satellite cells. We also reported the myogenic cells originated in the interstitium of skeletal muscle (Tamaki et al 2002a(Tamaki et al , b, 2003b(Tamaki et al , 2005(Tamaki et al , 2007a and their multimyogenic potential for skeletal-cardiac-smooth muscle cells (Tamaki et al 2008a) and relationship to satellite cells (Tamaki et al 2008b). In either case, it is likely that AAS loading does not directly accelerate myogenic cell activations itself.…”

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

“…We also identified the two stem cell populations from the interstitial spaces of skeletal muscle using the fluorescence activating cell sorting (FACS) method as CD34 + /45 − (Sk-34) and CD34 − /45 − (Sk-DN) cells and demonstrated typical colony formation with their myogenic, endothelial and adipogenic differentiation potential on culture in vitro (Tamaki et al 2002a;. In the freshly isolated state, there were no Pax7 + cells in the Sk-34 cells and less than 1% in the large cell fraction of Sk-DN cells (Tamaki et al 2008). Similarly, vessel wall-derived myogenic cells were identified as multi-potent mesenchymal stem cells termed "mesoangioblasts" Cossu and Bianco 2003;Minasi et al 2002).…”

“…Therefore, we also aimed to determine the origin and hierarchy of BL + and BL − myogenic cells. In this context, we used mouse skeletal muscle interstitiumderived multipotent Sk-34 and Sk-DN stem cells, as these are composed of Pax7 − and Pax7 + myogenic cells in a freshly isolated state (Tamaki et al 2008). Formation of BL and contractile components and expression of myogenic determination factors were examined by plain and immuneelectron microscopy and by immunohistochemistry during repeated conditioning cell cultures.…”

Antioxidant Levels Represent a Major Determinant in the Regenerative Capacity of Muscle Stem Cells

“…More recently, a skeletal muscle precursor population was isolated via FACS sorting CD45 Ϫ , Sca1, Mac1, CXCR4 ϩ , and ␤1 integrin ϩ subpopulations of satellite cells (Cerletti et al, 2008), and a myogenic endothelial cell population in skeletal muscle was initially reported based on CD34 ϩ and CD45 Ϫ expression, which was shown to improve cardiac function after myocardial infarction (Tamaki et al, 2002(Tamaki et al, , 2008a. Tamaki et al (2007Tamaki et al ( , 2008b have also clonally isolated so-called skeletal double-negative cells (Sk-DN), which are CD34 Ϫ /CD45 Ϫ , and have demonstrated their multipotency in vitro and in vivo. Our group has reported a similar population of so-called myoendothelial cells isolated from human skeletal muscle based on their coexpression of CD56, CD34, and CD144 (Zheng et al, 2007).…”