Department of Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.
Stem Cells Transl Med. 2013 Jul;2(7):488-98. doi: 10.5966/sctm.2012-0130. Epub 2013 Jun 7.
Stem cells are generally collected using flow cytometry, but this method is not applicable when the cell surface marker is not well determined. Satellite cells, which are skeletal muscle stem cells, have the ability to regenerate damaged muscles and are expected to be applicable for treatment of muscle degeneration. Although the transcription factor Pax7 is a known specific marker of satellite cells, it is not located on the cell surface and therefore flow cytometry is not directly applicable. In the present study, we turned our attention to the stress tolerance of adult stem cells, and we propose long-term trypsin incubation (LTT) as a novel approach to collecting satellite cells from mouse and human skeletal muscles. LTT led to a remarkable increase in the ratio of Pax7(+) cells that retain normal myogenic stem cell function. In particular, human Pax7(+) cells made up approximately 30% of primary cultured cells, whereas after LTT, the ratio of Pax7(+) cells increased up to ∼80%, and the ratio of Pax7(+) and/or MyoD(+) myogenic cells increased to ∼95%. Once transplanted, LTT-treated cells contributed to subsequent muscle regeneration following repetitive muscle damage without additional cell transplantation. The stress tolerance of Pax7(+) cells is related to heat shock protein 27 and αB-crystallin, members of the small heat shock protein family. This approach, based on the stress resistance of adult stem cells, is a safe and inexpensive method of efficiently collecting human satellite cells and may also be used for collecting other tissue stem cells whose surface marker is unknown.
干细胞通常使用流式细胞术进行收集，但当细胞表面标志物未得到很好确定时，这种方法并不适用。卫星细胞是骨骼肌干细胞，具有再生受损肌肉的能力，有望适用于肌肉退化的治疗。虽然转录因子 Pax7 是卫星细胞的已知特异性标志物，但它并不位于细胞表面，因此流式细胞术不能直接应用。在本研究中，我们将注意力转向成年干细胞的应激耐受能力，我们提出长期胰蛋白酶孵育（LTT）作为一种从鼠和人骨骼肌中收集卫星细胞的新方法。LTT 导致 Pax7(+)细胞的比例显著增加，而这些细胞保留了正常的成肌干细胞功能。特别是，人 Pax7(+)细胞约占原代培养细胞的 30%，而经过 LTT 后，Pax7(+)细胞的比例增加到约 80%，并且 Pax7(+)和/或 MyoD(+)成肌细胞的比例增加到约 95%。一旦移植，LTT 处理过的细胞在重复肌肉损伤后有助于随后的肌肉再生，而无需额外的细胞移植。Pax7(+)细胞的应激耐受能力与热休克蛋白 27 和αB-晶体蛋白有关，它们是小热休克蛋白家族的成员。这种基于成年干细胞应激耐受能力的方法是一种安全且廉价的高效收集人卫星细胞的方法，也可用于收集其他表面标志物未知的组织干细胞。
