Origin and Fate of Myogenic Stem Cells

描述（由申请人提供）：未分化的干细胞存在于成人机体中。在大多数情况下，尚不清楚这些细胞是能够沿着多种途径分化还是沿着单一途径发育。此外，成体干细胞的起源是未知的。下面的研究重点是肌源性干细胞。在这些实验中需要验证的一般假设是，骨骼肌谱系的干细胞：(1)存在于早期鸡胚胎的外胚层中，(2)在原肠胚形成过程中被纳入缺乏骨骼肌的器官，(3)能够诱导多能干细胞形成肌肉。这些假设是基于先前的研究得出的，在这些研究中，来自外胚层和各种缺乏骨骼肌的胎儿器官的少量细胞在体内表达肌源性转录因子MyoD并在体外分化。对骨骼肌谱系的承诺将首先通过分析调节其他组织发育的转录因子是否在外胚层细胞中与MyoD共表达来测试。其次，利用G8抗体从鹌鹑胚胎中分离MyoD阳性外胚层细胞，植入发育中的心区和肢芽软骨区，分析心脏和软骨特异性分子的表达。为了检验胎儿器官中存在的肌源性干细胞是否来源于外胚层中表达MyoD的细胞，将从鹌鹑外胚层中分离出MyoD/G8阳性细胞，植入鸡外胚层，胚胎生长到胎儿阶段，并检查器官中是否存在鹌鹑细胞。利用RT-PCR和原位杂交技术对成年鸡和小鼠器官MyoD和Myf5的表达进行分析。最后，将MyoD/G8阳性细胞与鸡外胚层细胞和小鼠胚胎干细胞共培养，检测肌源性干细胞诱导多能干细胞形成肌肉的能力。这些实验将开始解决稳定编程细胞与多能干细胞结合时用于增强体内组织再生的可能性。

DESCRIPTION (provided by applicant): Undifferentiated stem cells are present in the adult organism. In most cases it is unclear whether these cells are capable of differentiating along multiple pathways or programmed to develop along a single pathway. Furthermore, the origin of adult stem cells is unknown. The following studies focus on myogenic stem cells. The general hypotheses to be tested in these experiments are that stem cells committed to the skeletal muscle lineage: (1) are present in the epiblast of the early chick embryo, (2) become incorporated into organs lacking skeletal muscle during gastrulation, and (3) are able to induce multipotent stem cells to form muscle. These hypotheses were formulated as a result of previous studies in which small numbers of cells from the epiblast and a variety of fetal organs lacking skeletal muscle express the myogenic transcription factor MyoD in vivo and differentiate in vitro. Commitment to the skeletal muscle lineage will be tested by first analyzing whether transcription factors that regulate the development of other tissues are co-expressed with MyoD in epiblast cells. Second, MyoD positive epiblast cells will be isolated from the quail embryo using the G8 antibody, implanted into the developing heart fields and chondrogenic region of the limb bud, then analyzed for the expression of cardiac and cartilage specific molecules. To test whether the myogenic stem cells present in fetal organs originate from cells that express MyoD in the epiblast, the MyoD/G8 positive cells will be isolated from the quail epiblast, implanted into the chick epiblast, the embryos grown to fetal stages, and organs examined for the presence of quail cells. Organs from adult chickens and mice will be analyzed by RT-PCR and in situ hybridization for the expression of MyoD and Myf5. Finally, the ability of myogenic stem cells to induce multipotent stem cells to form muscle will be tested by co-culturing MyoD/G8 positive cells with chick epiblast cells and mouse embryonic stem cells. These experiments will begin to address the possibility that stably programmed cells could be used to enhance tissue regeneration in vivo when combined with multipotent stem cells.