It is generally accepted that muscle satellite cells are myogenic progenitors functioning as stem cells that can regenerate skeletal muscle while retaining capacity for self renewal. In the current studies both isolated human fetal myoblasts and adult satellite cells appeared committed to muscle lineage despite displaying multipotency. However, the developmental pathway by which satellite cells are derived has not been well defined and remains controversial [42
]. Recent evidence supports the idea that in the mouse, satellite cells arise from pre-existing Pax 7-positive cells during fetal development and do not appear to develop separately from a unique cell lineage [45
]. Recently, human satellite cells have also been shown to express Pax 7 [46
] but it is not clear if this effector plays a similar role in the human and may not be a useful marker for satellite cells in human muscle [46
Much progress in understanding muscle development and satellite cell function has been obtained mostly in the mouse. However, at present only limited studies have focused on human muscle development. One difficulty has been efficient isolation of human fetal and adult myogenic progenitors. Other studies have shown that cultures of human myoblasts from adult muscle can be contaminated with non-myogenic cells including fibroblastic [47
] and pericyte-like cells [48
]. Similarly, fetal human myoblast preparations also appear to contain non-muscle cells and frequently clonal populations are used to eliminate contamination issues [49
]. In the current study, we isolated human fetal and satellite muscle stem cells by taking advantage of the high expression of the α7 integrin as a marker for the myogenic lineage. The α7-positive fetal cells were capable of fusion and could differentiate into myotubes with high efficiency. We show that both fetal and adult human cells can be easily be purified and expanded in vitro to obtain large numbers of differentiation-competent myoblasts that might be suitable for engineering into other tissues.
The α7 integrin is an important adhesion receptor whose initial expression in primary myotubes and secondary myoblasts occurs early during development [24
], and remains strongly expressed in quiescent satellite cells in adult muscle [24
]. In fully developed muscle the integrin forms essential adhesions at costameric and myotendous junctions. Relatively low levels of α7 in myoblasts ensured motility during muscle development and regeneration whereas high levels are needed for efficient MTJ formation after muscle differentiation[53
]. Previous studies on this differentiation-specific adhesion receptor have been performed primarily in rodent muscle with limited studies in human muscle development. The present studies establish that both fetal and adult myogenic progenitor cells strongly express the α7 integrin. Detailed analysis of differentiation markers suggests that the cultures of α7 positive fetal cells represent myogenic cells at different developmental stages. For example, the populations were typically heterogeneous with a significant subpopulation of freshly isolated or passaged α7 positive cells positive for MyoD or desmin. The cells that were negative for nuclear MyoD staining may represent more primitive precursors. When cells were examined immediately after dissociation from tissue but prior to culture, a significant number showed positivity for desmin and this expression was maintained during the primary and subsequent passages. This is consistent with earlier studies of human fetal muscle, where immunochemical analysis at 20 and 29 week stages identified desmin expression in undifferentiated myoblasts [55
]. Desmin-positive cells may represent a more differentiated lineage of myoblast precursor cells known to be present in fetal muscle. In human adult satellite cells, desmin does not appear to be strongly expressed but is one of the first proteins expressed following activation [56
]. Cultures of α7 positive fetal cells were also uniformly positive for muscle markers that include c-Met and M-cadherin. CD-56/NCAM was found to be expressed in the population, but expression was heterogeneous, with a spread in expression levels. Importantly, expression was increased following continued passaging as initially isolated cells were mostly negative. Cells also expressed Notch and its inhibitor, Numb. Rando and collaborators [37
] have recently shown that active Notch is important in the expansion of satellite progenitors and is modulated by Numb, that permits progression to more differentiated myoblast stage. The expression of both Notch and Numb would be consistent with a mixed set of cells that are in different levels of myogenic differentiation. These data reinforce the findings that the cell population from the muscle stem pool is heterogeneous.
Our studies showed that α7-positive cells isolated from human fetal muscle and expanded in culture generally follow marker profiles seen in previous studies of adult satellite cells isolated from mouse or human muscle. Many of these markers are expressed in quiescent or activated/proliferating mouse satellite cells (Pax-7, c-Met, M-cadherin, NCAM) while others are specific for activated cells (MyoD, Myf-5, and desmin) [6
]. Yet other work has shown that a subpopulation of satellite cells lack Myf5, CD34 or M-cadherin expression and may be represent poorly differentiated stem cells [42
] and additional studies indicate that most cells express Myf5 and M-cadherin, but a small fraction of cells are negative for all [5
]. In previous studies of human adult and fetal myoblasts, heterogeneity was also reported for satellite markers including Pax-7, myogenin, MyoD, and Myf5 [46
]. Our results using individual cell clones replicated this heterogeneity suggesting that the diversity in cell phenotype is not simply due to the presence of multiple cell lineages in the population. Individual clones of α7 positive cells displayed a similar divergence in phenotype where the cells consisted of a mixture of more differentiated, desmin-positive cells with a variable fraction of the population being desmin-negative. This implies that subsets for the human fetal myogenic cells may be the precursor for satellite cells.
Recent studies have suggested that adult somatic stem cells may be capable of converting to other tissue lineages. This complex process involves the switching of one cell type to another and occurs with the loss of differentiated tissue markers and the acquisition of new tissue-specific characteristics. Our results indicate that isolated α7-positive cells are clonogenic and capable of expansion and have multipotent renewal potential. This is consistent with the presence of the multipotent stem cells in the α7-positive population. These cells have a full complement of muscle-specific markers such as M-cadherin and c-Met, yet they did not appear to be fully committed to the myogenic lineage and appear capable of differentiating to the osteogenic and adipocytic pathways. The results are similar to other recent studies that indicate that mouse satellite cells are multipotent and are capable of differentiating to other cell types after induction [22
]. More recently, human myoblasts were found to be capable of differentiation to osteoblasts and other cell fates [11
]. It is interesting that the populations display heterogeneity for mature myoblasts such as MyoD and desmin yet other cells do not. In the present studies, following BMP-2 treatment the expression of these markers is downregulated and a majority of the cells appear to progress along a osteoblastic lineage. It remains unclear if these myogenic cells after switching to osteoblastic cells still retain stem cell-like capacity.
In isolated human fetal myoblasts, the α7 integrin is highly expressed and was found to mediate adhesion to laminins, the major attachment proteins in the surrounding basement membrane. In contrast, we found that fetal cells did not express high levels of the α2 integrin that binds interstitial collagens and consequently these cells displayed poor adhesion to this substrate. Others have shown poor adhesion of myoblasts to collagen [61
]. Following BMP-2 treatment, the fetal cells were induced to differentiate along the osteogenic lineage. Expression of the α7 integrin was lost while the α2 integrin became strongly expressed and promoted increased binding to type I collagen. Adult satellite cells also express the α7 integrin and following BMP-2 induction also show a similar osteogenic differentiation and concurrent switch in integrin profiles (data not shown). In osteoblasts, the α2β1 integrin that binds type I collagen, the major bone matrix protein, facilitates osteoblastic differentiation and function [62
]. Thus, the coordinate regulation of adhesion receptors may be important for controlling adhesion and migration of precursor cells following injury and facilitate their correct positioning within the tissue microenvironment.
In conclusion, we have described a method to isolate multipotent human myoblasts based on their expression of α7 integrin as a surface marker. The α7-positive myoblasts expressed multiple established muscle specific markers, were capable of efficient myogenesis, but also had the potential to differentiate along multiple cell lineages. This indicates that α7 integrin expression is not only useful to identify differentiation-competent cells, but may also identify multipotent capability and suggests these progenitors have stem cell-like properties. Little is known about how extracellular signals are able to induce differentiation from one mesenchymal lineage to another. However, the modulation of expressed adhesion receptors may be an important mechanism by which stem and progenitor cells are recruited to target tissues. Importantly, specific ECM-integrin interactions by resident stem cells found in each tissue microenvironment may provide the proper cue for terminal differentiation and tissue-specific regeneration. Understanding how integrin expression is modulated as myogenic stem cells progress along specific differentiation pathways (e.g., muscle and bone) may provide insights into the hierarchy of the molecular mechanisms and signaling networks that regulate cell fate.