Romero-Ramos Marina, Vourc'h Patrick, Young Henry E, Lucas Paul A, Wu Young, Chivatakarn Onanong, Zaman Rumina, Dunkelman Noushin, el-Kalay Mohammad A, Chesselet Marie-Françoise
Department of Neurology, UCLA School of Medicine, Los Angeles, California 90095, USA.
J Neurosci Res. 2002 Sep 15;69(6):894-907. doi: 10.1002/jnr.10374.
Lineage uncommitted pluripotent stem cells reside in the connective tissue of skeletal muscle. The present study was carried out with pluripotent stem cells (PPSCs) isolated from 6-month old rat muscle. Before differentiation, these cells were vimentin+, CD90+, CD45-, and varied in their expression of CD34. The PPSCs were expanded as non-adherent aggregates under similar conditions to those used to generate neurospheres from embryonic or neural stem cells. The PPSC-derived neurospheres were positive for nestin, an early marker present in neuronal precursors, and expressed the two alternative mRNA forms of the neuroectodermal marker Pax-6, as well as mRNA for Oct-4, a gene related to the pluripotentiality of stem cells. To confirm their neural potential, PPSC-derived neurospheres were plated on coated coverslips under varying conditions: Neurobasal medium with N2 or B27, and either NT3 or BDNF. After 4-6 days the cells expressed neuronal (Tuj1+, NF68), astrocytic (GFAP) and oligodendrocytic (MOSP+, MBP+) markers, both by immunocytochemistry and RT-PCR. In addition, PPSCs were cultured as monolayers under adherent conditions, exposed to growth factors and defined differentiating conditions for 5 hr, and subsequently kept for 2 days in a maturation medium. At this point they gave rise to a mixed population of early neural progenitors (Nestin+ or NG2+), immature and mature neurons (Tuj1+ and NF145+) and myelin producing oligodendrocytes (CNPase + and MOSP+). Our study shows that PPSCs present in adult muscle can overcome germ lineage restrictions and express the molecular characteristics of brain cells. Therefore, PPSCs isolated from adult muscle could provide a novel source for autologous cell replacement in neurodegenerative and demyelinating diseases.
未定向的多能干细胞存在于骨骼肌的结缔组织中。本研究使用从6个月大的大鼠肌肉中分离出的多能干细胞（PPSC）进行。在分化之前，这些细胞波形蛋白阳性、CD90阳性、CD45阴性，且CD34的表达各不相同。PPSC作为非贴壁聚集体进行扩增，培养条件与用于从胚胎干细胞或神经干细胞生成神经球的条件相似。PPSC衍生的神经球对巢蛋白呈阳性，巢蛋白是神经元前体细胞中的一种早期标志物，并且表达神经外胚层标志物Pax - 6的两种可变mRNA形式，以及与干细胞多能性相关的Oct - 4基因的mRNA。为了确认它们的神经潜能，将PPSC衍生的神经球在不同条件下接种到包被的盖玻片上：添加N2或B27的Neurobasal培养基，以及NT3或BDNF。4 - 6天后，通过免疫细胞化学和逆转录 - 聚合酶链反应，细胞表达神经元标志物（Tuj1阳性、NF68阳性）、星形胶质细胞标志物（GFAP）和少突胶质细胞标志物（MOSP阳性、MBP阳性）。此外，将PPSC在贴壁条件下作为单层培养，暴露于生长因子并在特定分化条件下培养5小时，随后在成熟培养基中培养2天。此时，它们产生了早期神经祖细胞（巢蛋白阳性或NG2阳性）、未成熟和成熟神经元（Tuj1阳性和NF145阳性）以及产生髓磷脂的少突胶质细胞（CNPase阳性和MOSP阳性）的混合群体。我们的研究表明，成年肌肉中的PPSC可以克服种系限制并表达脑细胞的分子特征。因此，从成年肌肉中分离出的PPSC可为神经退行性疾病和脱髓鞘疾病的自体细胞替代提供一种新的来源。
