Origin and Fate of Myogenic Stem Cells

描述（由申请人提供）：未分化干细胞存在于成体生物体中。在大多数情况下，尚不清楚这些细胞是否能够沿着沿着多个途径分化或编程为沿着沿着单个途径发育。此外，成体干细胞的起源尚不清楚。以下研究集中在肌源性干细胞。在这些实验中要测试的一般假设是，干细胞定向于骨骼肌谱系：（1）存在于早期鸡胚的外胚层中，（2）在原肠胚形成期间并入缺乏骨骼肌的器官中，以及（3）能够诱导多能干细胞形成肌肉。这些假设是根据以前的研究制定的，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。将通过首先分析调节其他组织发育的转录因子是否与上胚层细胞中的MyoD共表达来测试对骨骼肌谱系的承诺。第二，使用G8抗体从鹌鹑胚胎中分离MyoD阳性上胚层细胞，植入发育中的心脏区域和肢芽的软骨形成区域，然后分析心脏和软骨特异性分子的表达。为了测试存在于胎儿器官中的肌源性干细胞是否来源于在上胚层中表达MyoD的细胞，将从鹌鹑上胚层中分离MyoD/G8阳性细胞，植入鸡上胚层中，胚胎生长至胎儿阶段，并检查器官是否存在鹌鹑细胞。将通过RT-PCR和原位杂交分析来自成年鸡和小鼠的器官的MyoD和Myf 5的表达。最后，通过将MyoD/G8阳性细胞与鸡外胚层细胞和小鼠胚胎干细胞共培养来测试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，以解决稳定编程的细胞可用于增强组织再生在体内时，与多能干细胞的可能性。

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.