Protein Kinase C Signaling and Pluripotent Stem Cell

描述（申请人提供）：多能干细胞具有发展成不同细胞类型的巨大潜力。然而，赋予干细胞维持多能性或分化为其他细胞类型能力的分子机制尚不清楚。我们发现，通过单一的、选择性的PKC抑制剂抑制蛋白激酶C （PKC）异构体可以维持小鼠胚胎干（ES）细胞的未分化表型，而不影响其多分化能力。此外，抑制PKC异构体允许从小鼠囊胚中衍生出生殖系胜任的胚胎干细胞，也促进了小鼠胚胎成纤维细胞向诱导多能干细胞的重编程。本探索性和发展性研究计划的目的是进一步验证该方法的有效性，以PKC信号通路为靶点，从大鼠获得、繁殖和维持新的胚胎干细胞，维持人类胚胎干细胞的多能性，并了解相关的分子机制。我们将在这项建议中提出两个具体目标。在第一个特定目标中，我们将验证PKC信号传导是决定维持哺乳动物胚胎干细胞多能性和分化的重要途径的假设，因此，抑制PKC异构体功能将有助于获得、繁殖和维持多能性干细胞，而不影响其完整的发育潜力。我们将测试大鼠胚胎干细胞的体内发育潜力，通过PKC抑制剂衍生和繁殖，将其注射到囊胚中产生嵌合体，并评估这些嵌合体的种系传播能力。此外，我们将确定PKC抑制是否维持人类胚胎干细胞的未分化表型，并将通过在免疫受损小鼠中发展畸胎瘤来评估多能性。在第二个特定目标中，我们将确定与PKC抑制介导的干细胞多能性维持相关的分子机制。我们将测试特异性PKC异构体/s的功能对诱导小鼠胚胎干细胞分化是否重要。此外，我们还将确定抑制胚胎干细胞中的PKC亚型是否能维持多能干细胞特异性染色质结构。我们预计本研究的成功完成将为靶向PKC信号通路的干细胞生物学和再生医学研究开辟新的领域。

DESCRIPTION (provided by applicant): Pluripotent stem cells have tremendous potential to develop into different cell types. However, molecular mechanisms that endow stem cells with the capacity to maintain pluripotency or to differentiate into other cell types are not well understood. We found that inhibition of protein kinase C (PKC) isoforms by a single, selective PKC inhibitor maintains the undifferentiated phenotype of mouse embryonic stem (ES) cells without affecting their multidifferentiation potency. Furthermore, inhibition of PKC isoforms permits derivation of germline-competent ES cells from mouse blastocysts and also facilitates reprogramming of mouse embryonic fibroblasts towards induced pluripotent stem cells. The objective of this exploratory and developmental research proposal is to further validate the efficacy of the approach, in which PKC signaling pathway will be targeted to derive, propagate and maintain new ES cells from rat, to maintain pluripotency of human ES cells, and to understand associated molecular mechanisms. We will institute two specific aims in this proposal. In the first Specific Aim we will test the hypothesis that PKC signaling is an important pathway to dictate maintenance of pluripotency vs. differentiation of mammalian ES cells and, therefore, inhibition of PKC isoform function will help to derive, propagate and maintain pluripotent stem cells without affecting their complete developmental potential. We will test in vivo developmental potential of rat ES cells, derived and propagated with PKC inhibitors, by injecting them into the blastocysts to generate chimera and by assessing those chimeras for germline transmission. In addition, we will determine whether PKC inhibition maintains undifferentiated phenotypes of human ES cells and will assess pluripotency by developing teratomas in immune-compromised mice. In the second Specific Aim we will determine molecular mechanisms that are associated with PKC inhibition-mediated maintenance of stem cell pluripotency. We will test whether function of specific PKC isoform/s is important to induce mouse ES cell differentiation. In addition, we will also determine whether inhibition of PKC isoforms in ES cells maintains pluripotent stem cell-specific chromatin structure. We predict successful completion of this proposal will open up new areas of research to target PKC signaling pathway for stem cell biology and regenerative medicine purposes.