Analyses of stem cells in the chick Embryo and Fetus

鸡胚胎和胎儿的干细胞分析

• 批准号: 6315756
• 负责人: MINDY GEORGE-WEINSTEIN
• 金额: $ 16.1万
• 依托单位: PHILADELPHIA COLLEGE OF OSTEOPATHIC MED
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6315756
• 关键词: biomarker; cartilage development; cell differentiation; cell population study; cell sorting; cell type; chick embryo; embryo /fetus cell /tissue; gene expression; in situ hybridization; messenger RNA; monoclonal antibody; myogenesis; neurogenesis; stem cells; striated muscles; tissue /cell culture; transcription factor

DESCRIPTION (provided by applicant) Undifferentiated cells are present in the adult organism. In most cases, it is not clear whether these "stem 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. We propose to use the chick embryo as a model system with which to study these basic questions in stem cell biology. Previous experiments revealed that the skeletal muscle specific transcription factor MyoD is expressed in a small subpopulation of cells in several organs of the fetus and throughout the epiblast layer of the embryo that gives rise to all tissues of the body. When epiblast cells are placed in culture, some differentiate into muscle, chondroblasts, and neurons. These studies led to the hypotheses that stem cells for different lineages are present in the epiblast and their progeny are incorporated into a variety of mature tissues of the fetus. We will examine the epiblast and fetal organs for expression of mRNAs for Pax-l and NeuroM, transcription factors that regulate chondrogenesis and neurogenesis, respectively. In situ hybridizations will be performed with the recently developed and highly sensitive probes, fluorescently labeled DNA dendrimers. We will determine whether those cells that express MyoD in the epiblast and fetal organs are myogenic by marking them with the G8 antibody, placing them in culture for several days, and staining with an antibody to sarcomeric myosin. The potential of MyoD positive epiblast cells to differentiate into non-muscle cell types will be tested after isolating MyoD/G8 expressing cells from the quail embryo by fluorescence activated cell sorting, and implanting them into the chick embryo in the developing heart field and chondrogenic region of the limb bud. The fate of MyoD positive epiblast cells will be determined by isolating them from the quail epiblast with the G8 antibody, implanting them into the chick epiblast, culturing the embryo, and examining organs for the presence of quail cells. These studies will provide us with fundamental information regarding the extent of heterogeneity within the early epiblast and fetal tissues, the timing of development of stem cells for different lineages, and the extent to which they are committed to differentiate along a particular pathway.

描述（由申请人提供） 成人生物体中存在未分化的细胞。在大多数情况下，是 尚不清楚这些“干细胞”是否能够沿着 多个途径或编程以沿单个途径开发。此外， 成年干细胞的起源尚不清楚。我们建议使用小鸡胚胎 作为研究干细胞中这些基本问题的模型系统 生物学。先前的实验表明骨骼肌特异性 转录因子myod在细胞中的小亚群中表达 胎儿的几个器官以及整个胚胎的整个层 这引起了身体的所有组织。当将其中音细胞放入 培养，有些分化为肌肉，软骨细胞和神经元。这些 研究导致了不同谱系干细胞的假设是 在层细胞中存在及其后代。 胎儿的成熟组织。我们将检查层细胞和胎儿器官 MRNA用于PAX-L和NEUROM，调节的转录因子 软骨发生和神经发生。原位杂交将是 用最近开发和高度敏感的探针进行 荧光标记为DNA树枝状聚合物。我们将确定这些细胞是否 通过标记，在层状器官和胎儿器官中表达肌无力 使用G8抗体，将它们放在培养物中几天，然后染色 具有抗肌膜肌球蛋白的抗体。 MYOD阳性epiblast的潜力 细胞分化为非肌肉细胞类型，将在 通过荧光从鹌鹑胚胎中隔离Myod/g8表达细胞 激活的细胞分类，并将其植入小鸡胚胎中 发展肢体芽的心脏场和软骨形状区域。命运 myod阳性一个阳性细胞将通过将它们隔离为 用G8抗体的鹌鹑覆盖细胞，将它们植入小鸡epiblast， 培养胚胎，并检查器官是否存在鹌鹑细胞。 这些研究将为我们提供有关程度的基本信息 早期层细胞和胎儿组织内的异质性 干细胞的开发用于不同的谱系，以及它们的多大程度 致力于沿特定途径进行区分。