Muscle Physiology and Cell Biology Unit, Department of Regenerative Medicine, Division of Basic Clinical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa, 259-1193, Japan.
Cell Tissue Res. 2011 Apr;344(1):147-68. doi: 10.1007/s00441-010-1127-9. Epub 2011 Jan 29.
As a novel approach to distinguish skeletal myogenic cell populations, basal lamina (BL) formation of myogenic cells was examined in the mouse compensatory enlarged plantaris muscles in vivo and in fiber-bundle cultures in vitro. MyoD(+) myogenic cells located inside the regenerative muscle fiber BL were laminin(-) but interstitial MyoD(+) cells were laminin(+). This was also confirmed by electron microscopy as structural BL formation. Similar trends were observed in the fiber-bundle cultures including satellite cells and interstitial myogenic cells and laminin(+) myogenic cells predominantly showed non-adhesive (non-Ad) behavior with Pax7(-), whereas laminin(-) cells were adhesive (Ad) with Pax7(+). Moreover, non-Ad/laminin(+) and Ad/laminin(-) myotubes were also observed and the former type showed spontaneous contractions, while the latter type did not. The origin and hierarchy of Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells were also examined using skeletal muscle interstitium-derived CD34(+)/45(-) (Sk-34) and CD34(-)/45(-) (Sk-DN) multipotent stem cells, which were composed of non-committed myogenic cells with a few (<1%) Pax7(+) cells in the Sk-DN cells at fresh isolation. Both cell types were separated by Ad/non-Ad capacity in repetitive culture. As expected, both Ad/Pax7(+)/laminin(-) and non-Ad/Pax7(-)/laminin(+) myogenic cells consistently appeared in the Ad and non-Ad cell culture. However, Ad/Pax7(+)/laminin(-) cells were repeatedly detected in the non-Ad cell culture, while the opposite phenomenon did not occur. This indicates that the source of non-Ad/ Pax7(-)/laminin(+) myogenic cells was present in the Sk-34 and Sk-DN stem cells and they were able to produce Ad/ Pax7(+)/ laminin(-) myogenic cells during myogenesis as primary myoblasts and situated hierarchically upstream of the latter cells.
作为一种区分骨骼肌细胞群体的新方法，本研究在体内再生的比目鱼肌和体外纤维束培养中检测了成肌细胞的基底膜（BL）形成。位于再生肌纤维 BL 内的 MyoD(+)成肌细胞为层粘连蛋白(-)，但间质 MyoD(+)细胞为层粘连蛋白(+)。电镜也证实了这种结构 BL 的形成。在纤维束培养中也观察到了类似的趋势，包括卫星细胞和间质成肌细胞，并且层粘连蛋白(+)成肌细胞主要表现为非黏附（非 Ad）行为，Pax7(-)，而层粘连蛋白(-)细胞为黏附（Ad），Pax7(+)。此外，还观察到非 Ad/层粘连蛋白(+)和 Ad/层粘连蛋白(-)肌管，前者类型表现出自发收缩，而后者类型则没有。使用源自骨骼肌间质的 CD34(+)/45(-)（Sk-34）和 CD34(-)/45(-)（Sk-DN）多能干细胞也检查了 Ad/Pax7(+)/层粘连蛋白(-)和非 Ad/Pax7(-)/层粘连蛋白(+)成肌细胞的起源和层次，这些细胞由非定型的成肌细胞组成，在 Sk-DN 细胞新鲜分离时只有少量（<1%）的 Pax7(+)细胞。这两种细胞类型在重复培养中通过 Ad/非 Ad 能力分离。正如预期的那样，在 Ad 和非 Ad 细胞培养中都出现了 Ad/Pax7(+)/层粘连蛋白(-)和非 Ad/Pax7(-)/层粘连蛋白(+)成肌细胞。然而，在非 Ad 细胞培养中反复检测到 Ad/Pax7(+)/层粘连蛋白(-)细胞，而相反的现象并未发生。这表明非 Ad/ Pax7(-)/层粘连蛋白(+)成肌细胞的来源存在于 Sk-34 和 Sk-DN 干细胞中，它们在成肌过程中作为原代成肌细胞能够产生 Ad/ Pax7(+)/层粘连蛋白(-)成肌细胞，并位于后者细胞的上游层次。
