Characterization of Endoderm Stem Cells Derived from Murine ESCs

描述(申请人提供)：胚胎干细胞(ESCs)被设想为一种可移植胰岛的来源，以缓解供体器官短缺的问题。尽管最近取得了许多进展，但将ESCs与均一的葡萄糖反应性胰岛素分泌β细胞池区分开来的方案尚未开发出来。人们普遍认为，这些种群的产生首先需要产生最终的内胚层。因此，内胚层限制性干细胞系的成功衍生和稳定繁殖将是发展胰腺、肝脏和肺细胞用于细胞替代治疗的重要一步，并有助于提高我们对这些组织通常是如何从胚层形成的理解。我们开发了一种简单的方法，使用磁激活细胞分选(MACS)，根据上皮细胞黏附分子(EpCAM)的表达，从分化的小鼠ESCs的异种培养中分离内胚层承诺细胞。该方案包括去除未分化的ESCs和内脏内胚层细胞的阴性选择，以及保留表达EpCAM的细胞的阳性选择。当分选的细胞被放置到免疫缺陷小鼠的皮下时，小的EpCAM+PDX1+结节产生了非常像胚胎胰腺上皮的结节。这些结节中的细胞可以在体外广泛繁殖，并具有内皮干细胞(EndSC)或前肠限制性干细胞(FGSC)群体的特征，这取决于体内生长的持续时间。EndSCs为OCT4+，表达一些早期内胚层受限基因，而FGSCs为OCT4-，高表达Sox17、Foxa2和PDX1。移植回动物体内的EndSCs和FGSCs重现了原始的结节表型，证明了它们在体外的稳定性。我们推测，只有从MACS分离中回收的OCT4+EpCAM+SSEA1-细胞才能产生体内来源的内皮细胞系。在这里，我们将通过FACS-使用OCT4GFP ESC系对OCT4+EpCAM+SSEA1-细胞进行分选来直接验证这一假设。来自FACS分选群体的细胞系将与现有的MACs来源的EndSCs和FGSCs进行比较。我们还将研究EndSCs和FGSCs在体外分化为特定内胚层来源细胞类型的能力，包括β细胞。为了促进分化，我们将在胚胎胰腺环境中培养细胞，将细胞暴露于相关的生长因子信号，并根据最近发表的方案培养细胞。这些研究不仅有助于提高胚胎干细胞向明确的内胚层和胰腺系的分化效率，而且有助于更好地理解生殖层分离和早期胚胎细胞的命运决定。公共卫生相关性：我们的初步研究已经导致从分化的小鼠胚胎干细胞(ESCs)发展出似乎是前肠内胚层承诺的细胞系。这些细胞系不会致瘤，在培养中生长良好，在此我们建议阐明这些内皮细胞系的细胞来源，并探索它们在体外分化为胰腺、肝脏和肺细胞的能力。这一知识将有助于建立分离和分化人内胚层承诺细胞的方案，以生产用于移植治疗的功能细胞。

DESCRIPTION (provided by applicant): Embryonic stem cells (ESCs) are envisioned as a source of transplantable pancreatic islets to alleviate the donor organ shortage. Despite many recent advances, protocols to differentiate ESCs to homogeneous pools of glucose-responsive insulin-secreting beta cells have yet to be developed. It is widely accepted that the production of these populations will first require generation of definitive endoderm. Thus, the successful derivation and stable propagation of endoderm-restricted stem cell lines would be a significant step forward in the development of pancreas, liver, and lung cells for cell replacement therapies and for improving our understanding of how these tissues are normally formed from this germ layer. We have developed a simple protocol, using magnetic-activated cell sorting (MACS), to isolate endoderm-committed cells from heterogeneous cultures of differentiated murine ESCs on the basis of epithelial cell adhesion molecule (EpCAM) expression. The protocol includes negative selection to remove undifferentiated ESCs and visceral endoderm cells, and positive selection to retain cells that express EpCAM. When sorted cells are placed sub-cutaneously into immunodeficient mice, small EpCAM+PDX1+ nodules highly reminiscent of embryonic pancreatic epithelium develop. Cells from these nodules can be extensively propagated in vitro and have the characteristics expected of an endodermal stem cell (EndSC) or foregut- restricted stem cell (FGSC) population, depending on the duration of growth in vivo. The EndSCs are OCT4+ and express some early endoderm-restricted genes, whereas FGSCs are OCT4- and have high expression of Sox17, Foxa2, and PDX1. EndSCs and FGSCs transplanted back into animals recapitulate the original nodule phenotype, demonstrating their stability in vitro. We hypothesize that only the OCT4+EpCAM+SSEA1- cells recovered from the MACS separation give rise to the in vivo-derived endodermal cell lines. Here, we will directly test this hypothesis by FACS-sorting OCT4+EpCAM+SSEA1- cells using an OCT4GFP ESC line. Cell lines derived from the FACS-sorted population will be compared to the existing MACS-derived EndSCs and FGSCs. We will also study the ability of EndSCs and FGSCs to differentiate in vitro to specific endoderm-derived cell types, including beta cells. To promote differentiation, we will culture cells in an embryonic pancreas environment, expose cells to relevant growth factor signals, and culture cells according to recently published protocols. These studies will contribute not only to improved differentiation efficiency of ESCs to definitive endoderm and pancreatic lineages, but also to a better understanding of germ layer segregation and early embryonic cell fate decisions. PUBLIC HEALTH RELEVANCE: Our preliminary studies have led to the development of cell lines from differentiated mouse embryonic stem cells (ESCs) that appear to be foregut endoderm-committed. These cell lines are non-tumorigenic and grow well in culture, and here we propose to elucidate the cell of origin of these endodermal cell lines and explore their ability to differentiate into pancreatic, liver, and lung cells in vitro. This knowledge will help in establishing protocols for the isolation and differentiation of human endoderm-committed cells for the production of functional cells to be used in transplantation therapies.