Role of Nitric Oxide and Cyclic GMP in Stem Cells

描述（由申请人提供）：在本修订申请中，我们将主要关注研究NO和环GMP信号在人和小鼠胚胎干细胞增殖和分化为心肌细胞中的作用。我们的初步研究强烈表明一氧化氮和环GMP在小鼠和人类胚胎干细胞增殖和分化中的作用。在干细胞增殖和分化过程中，将检测小鼠和人干细胞（WA 09）中一氧化氮合酶（NOS 1、2、3）和可溶性鸟苷酸环化酶（NO受体）的mRNA（RT-PCR和Northerns）和蛋白质（Western和活性）。将通过免疫组织化学方法确认这些蛋白质和细胞标志物以及细胞分选的细胞定位。还将检查NOS、可溶性鸟苷酸环化酶、蛋白激酶G和环核苷酸磷酸二酯酶的激活剂和抑制剂的作用，以确认NO和cGMP在干细胞增殖和分化成心肌细胞中的作用。随着对干细胞和分化细胞中参与一氧化氮和环GMP合成和功能的蛋白质的存在或不存在的了解，我们期望能够设计各种药理学试剂的实验方案，如NO供体药物，环核苷酸类似物，磷酸二酯酶抑制剂，和鸟苷酸环化酶激活剂，其应当修饰干细胞增殖和分化成各种细胞谱系。这些干细胞和分化细胞的功能也将在几种心血管模型中进行检查。这对其他人和我们将来的临床研究来说应该是非常有价值的信息，目的是组织替代，也许是药物和基因递送系统。几位具有干细胞生物学和/或体内动物模型专业知识的同事将担任顾问，顾问和合作者。我们的初步实验和其他人的工作表明，我们提出的研究是可行的，很可能是成功的，以确定一氧化氮和环GMP在干细胞增殖，分化和功能的作用。

DESCRIPTION (provided by applicant): In this revised application, we will primarily focus on studying the role of NO and cyclic GMP signaling in proliferation and differentiation of human and mouse embryonic stem cells into cardiomyocytes. Our preliminary studies strongly suggest a role for nitric oxide and cyclic GMP in mouse and human embryonic stem cell proliferation and differentiation. The mRNA (RT-PCR and Northerns) and protein (Western and activity) in mouse and human stem cells (WA09) will be examined during stem cell proliferation and differentiation for nitric oxide synthases (NOS 1,2,3) and soluble guanylyl cyclase, the receptor for NO. The cellular localization will be confirmed with immunohistochemistry for these proteins and cellular markers and cell sorting. The effects of activators and inhibitors of NOS, soluble guanylyl cyclase, protein kinase G, and cyclic nucleotide phosphodieserases will also be examined to confirm the role of NO and cGMP in stem cell proliferation and differentiation into myocardial cells. With an understanding of the presence or absence of the proteins participating in nitric oxide and cyclic GMP synthesis and function in stem cells and differentiated cells with their in vitro culture, we expect to be able to design experimental protocols with various pharmacologic agents such as NO donor drugs, cyclic nucleotide analogues, phosphodieterase inhibitors, and guanylyl cyclase activators that should modify stem cell proliferation and differentiation into various cellular lineages. The function of these stem cells and differentiated cells will also be examined in several cardiovascular models. This should be extremely valuable information for future clinical studies by others and us for the purpose of tissue replacement and perhaps drug and gene delivery systems. Several colleagues with expertise in stem cell biology and/or in vivo animal models will serve as advisors, consultants, and collaborators. Our preliminary experiments and the work of others indicate that our studies proposed are feasible and are likely to be successful to determine the roles of nitric oxide and cyclic GMP in stem cell proliferation, differentiation, and function.