Osteoblast Lineage Progression from Embryonic Stem Cells

描述（由申请人提供）：本提案旨在通过提高我们对局部微环境在人胚胎干（ES）细胞分化中的作用的理解，专门响应再生牙科医学中的RFA。我们将用RFA中概述的另一个关键概念来补充体外方法，即在体内设计的生物材料支架上研究hES细胞分化。我们研究的长期目标是开始理解指示hES细胞成骨分化的信号。基于我们团队的研究兴趣和干细胞生物学知识的主要空白，我们将评估人类ESCs向成骨细胞的谱系进展可以由局部微环境信号控制的中心假设。我们将首先确定饲养层和/或促成骨生长因子在体外引导hES细胞向成骨样细胞谱系进展中的影响。在第二个目标中，我们计划使用谱系特异性转基因驱动超生命标记基因来分离、操纵和跟踪细胞的命运，以便它们可以作为一个同质群体被分离和研究。一旦确定了促进hES细胞向骨细胞分化的特定组织培养条件，我们将使用更生理学相关的模型系统，通过控制设计的生物材料上的微环境来研究人类ES细胞在体内的成骨细胞分化。在我们的第三个目标中，我们将通过将未分化的hES细胞和成骨细胞谱系选择的细胞移植到体内确定的位置，确定局部微环境线索如何调节体内胚胎干细胞的命运。

DESCRIPTION (provided by applicant): This proposal is designed to specifically respond to the RFA in Regenerative Dental Medicine by improving our understanding of the role of the local microenvironment on human embryonic stem (ES) cell differentiation. We will compliment in vitro approaches with another key concept outlined in the RFA, that is, the study of hES cell differentiation on designed biomaterial scaffolds in vivo. The long term goal of our research is to begin to understand the signals that specify osteoblast differentiation of hES cells. Based on the research interests of our team and the major gaps in knowledge of stem cell biology, we will evaluate the central hypothesis that the lineage progression of human ESCs to osteoblasts can be controlled by signaling from the local microenvironment. We will first determine the influence of the feeder layer and/or pro-osteogenie growth factors in directing lineage progression of hES cells to osteoblast-like cells in vitro. In the second aim, we plan to use lineage specific transgenes driving supravital marker genes to isolate, manipulate and follow the fate of cells so that they can be isolated and studied as a homogeneous population. Once specific tissue culture conditions are identified that promote bone cell differentiation from the hES cells, we will use a more physiologically relevant model system to study osteoblast differentiation of human ES cells in vivo by controlling the microenvironment on designed biomaterials. In our third aim we will determine how local microenvironmental cues regulate the fate of ES cells in vivo by transplanting both undifferentiated hES cells and osteoblast lineage-selected cells to defined in vivo locations. These studies will further our understanding of the biology of lineage segregation during normal development and provide essential information for the development of future tissue regeneration strategies.