Development of Pluripotent Amniotic Stem Cell Banks for Disease Modeling

描述（由申请人提供）： 用转录因子Oct 4（也称为Pou 5 f1）、Sox 2、Klf 4和Myc 1直接重编程体细胞产生与胚胎干（ES）细胞具有显著相似性的诱导多能干（iPS）细胞。当细胞的来源是受各种疾病影响的患者时，iPS细胞有望成为研究疾病渐进发病机制的极好模型。然而，最近有证据表明，来自成体组织的iPS细胞保留了其体细胞组织来源的残留DNA甲基化特征，这似乎限制了它们的分化潜力。人羊水细胞用于染色体和孟德尔遗传病的产前诊断已有数十年的历史。AF中有一个表达酪氨酸激酶受体c-Kit的细胞亚群似乎具有干细胞样特性。在发育和功能上，它们代表了一类具有介于胚胎干细胞和成体干细胞之间的中间特征的干细胞。我们的假设是，表达c-kit的AF细胞可以使用比成人细胞所需的更少的外源性蛋白质进行重编程，并且所得的iPS细胞将具有与胚胎干细胞比成人衍生的iPS细胞更相似的甲基化模式。这种多能干细胞系将为研究各种遗传疾病的潜在机制提供模型，从而导致新的诊断和治疗方式。人羊水可以提供能够产生更适用的疾病特异性iPS细胞的细胞来源。我们的具体目标是：1）在细胞遗传学诊断证实存在遗传异常后，表征从AF获得的细胞亚群; 2）确定对目标1中鉴定的AF细胞亚群进行重编程以产生iPS细胞的最低要求。我们的长期目标是开发代表不同遗传相关疾病的AF衍生iPS细胞系库。 公共卫生相关性： 我们建议从诊断后遗留的羊水细胞中创建干细胞系，用于遗传疾病的研究。这种干细胞系将提供极好的模型，用于研究各种遗传疾病的潜在机制，从而导致新的诊断和治疗方式。

DESCRIPTION (provided by applicant): Direct reprogramming of somatic cells with the transcription factors Oct4 (also called Pou5f1), Sox2, Klf4 and Myc1 yields induced Pluripotent Stem (iPS) cells with marked similarity to Embryonic Stem (ES) cells. When the source of the cells is patients affected by various disorders iPS cells promise to be excellent models to study the gradual pathogenesis of disease. However, there is recent evidence that iPS cells derived from adult tissues retain residual DNA methylation signatures characteristic of their somatic tissue of origin, which appears to restrict their differentiation potential. Human amniotic fluid (AF) cells have been used for prenatal diagnosis of chromosomal and Mendelian disorders for decades. There is a subpopulation of cells in AF that expresses the tyrosine kinase receptor c-Kit appear to have stem cell-like properties. Developmentally and functionally, they represent a class of stem cells with intermediate characteristics between embryonic and adult stem cells. Our hypotheses are that c-kit expressing AF cells can be reprogrammed using fewer exogenous proteins than necessary with adult cells and that the resulting iPS cells will have methylation patterns more similar to embryonic stem cells than to adult derived iPS cells. Such pluripotent stem cell lines would provide models with which to investigate the underlying mechanisms of a variety of genetic diseases, leading to new diagnostic and treatment modalities. Human amniotic fluid may provide a source of cells capable of generating more applicable disease-specific iPS cells. Our specific aims are: 1) characterize subpopulations of cells obtained from AF after cytogenetic diagnosis has confirmed the presence of genetic abnormalities; 2) determine minimal requirements for reprogramming AF cell subpopulations identified in Aim 1 to generate iPS cells. Our long-term goal is to develop banks of AF derived iPS cell lines representing different genetically related diseases. PUBLIC HEALTH RELEVANCE: We propose to create stem cell lines for the study of genetic diseases from amniotic fluid cells left over after diagnosis. Such stem cell lines would provide excellent models with which to investigate the underlying mechanisms of a variety of genetic diseases, leading to new diagnostic and treatment modalities.