RHESUS ES CELLS: A MODEL TO STUDY PANCREAS DEVELOPMENT

全面了解控制人类胰腺发育的遗传程序和胚胎诱导事件将有助于我们加深对先天性胰腺畸形的理解，并有助于制定合理的糖尿病治疗策略。最近在小鼠和鸡身上进行的实验已经对胰腺器官发生的机制有了重要的了解。然而，这些物种和人类之间早期发育途径的关键差异使这些数据对人类发育的直接适用性受到质疑。此外，人类发育中的功能研究受到直接操纵人类胚胎的伦理影响的限制。因此，一个以非人类灵长类动物为基础的更“人类”的模型将在研究人类发育方面有广泛的应用。最近，从非人灵长类动物(恒河猴和普通绒猴)中分离出了胚胎干细胞系，并研究了它们在体内分化为三层胚胎生殖层(外胚层、中胚层和内胚层)的潜力。然而，到目前为止，它们在体外进行谱系限制分化的能力还没有得到彻底的测试。相比之下，小鼠胚胎干细胞在体外的神经、造血和肌肉分化已经得到了广泛的研究。最近，小鼠ES细胞在培养中经历内胚层和胰腺分化的能力也被证明。因此，我们将验证胰腺内分泌和/或外分泌谱系规范可以在诱导分化的恒河猴ES细胞中重现的假设。本研究的具体目的是：L)确定内胚层相关基因和胰腺外分泌/内分泌特异性基因在恒河猴ES细胞体外分化中的表达模式，以及2)确定恒河猴ES细胞在免疫低下小鼠ES细胞来源的肿瘤中分化为胰腺系的潜能。将结合RT-PCR分析、Northern杂交、核糖核酸酶保护分析、原位杂交和免疫荧光研究来表征胰腺基因的表达模式。这些研究将为完善人类胰腺发育的体外灵长类动物模型提供基础。这样的模型系统将对研究胰腺分化的控制机制和开发新的基于细胞的治疗糖尿病的策略具有重要意义。

A comprehensive knowledge of the genetic programs and embryonic inductive events regulating human pancreas development would advance our understanding of congenital pancreatic malformations and aid in developing rational treatment strategies for diabetes mellitus. Significant insights to the mechanisms of pancreatic organogenesis have been gained through recent experiments in mice and chickens. However, critical differences in early developmental pathways between these species and humans places the direct applicability of these data to human development in question. Furthermore, functional studies in human development are restricted by the ethical implications of directly manipulating human embryos. Consequently, a more "human" model based on nonhuman primates would have broad application for studying human development. Recently, embryonic stem (ES) cell lines from nonhuman primates (rhesus macaque and common marmoset) have been derived and their potential for differentiation into derivatives of all three embryonic germ layers (ectoderm, mesoderm, and endoderm) in vivo has been characterized. Their capacity for lineage-restricted differentiation in vitro, however, has not been thoroughly tested to date. in contrast, neural, hematopoietic and muscle lineage differentiation of mouse ES cells in vitro has been extensively studied. Recently, the ability of mouse ES cells to undergo elements of endoderm and pancreatic differentiation in culture has also been demonstrated. Therefore, we will test the hypothesis that pancreatic endocrine and/or exocrine lineage specification can be recapitulated in rhesus ES cells induced to differentiate. The specific aims of this proposal are: l) To determine the expression pattern of endoderm related genes as well as pancreatic exocrine/endocrine-specific genes in rhesus ES cells differentiating in vitro, and 2) To determine the potential of rhesus ES cells for pancreatic lineage differentiation in ES cell derived tumors in immunocompromised mice. A combination of RT-PCR analysis, Northern hybridization, ribonuclease protection assay, in situ hybridization, and immunofluorescence studies will be used to characterize the pattern of pancreatic gene expression. These studies will provide a basis for the refinement of an in vitro primate model of human pancreatic development. Such a model system would have important implications for investigating the mechanisms controlling pancreatic differentiation and for developing new cell-based strategies for treating diabetes.