Characterization of Endoderm Stem Cells Derived from Murine ESCs

鼠 ESC 来源的内胚层干细胞的表征

• 批准号: 7658494
• 负责人: Jon S Odorico
• 金额: $ 18.56万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-22 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7658494
• 关键词: Address; Alveolar Cell; Animals; Back; Beta Cell; Cell Adhesion Molecules; Cell Line; Cell Separation; Cells; Characteristics; Coculture Techniques; Commit; Cultured Cells; Data; Derivation procedure; Development; ES Cell Line; Embryo; Endoderm; Endoderm Cell; Environment; Epithelial Cells; Epithelium; Generations; Genes; Germ Layers; Glucose; Goals; Growth; Growth Factor; Hepatocyte; Human; Immunodeficient Mouse; In Vitro; Injection of therapeutic agent; Insulin; Islets of Langerhans; Knowledge; Length; Liver; Lung; Magnetism; Morphology; Mus; Nodule; Organ; Organ Donor; Pancreas; Pattern; Phenotype; Population; Population Heterogeneity; Primitive Streaks; Primitive foregut structure; Production; Protocols documentation; Publishing; Replacement Therapy; Signal Transduction; Sorting - Cell Movement; Source; Stem cell transplant; Stem cells; Structure of beta Cell of islet; Teratoma; Testing; Therapeutic; Time; Tissues; To specify; Transplantation; Undifferentiated; Visceral; base; blastomere structure; cell type; embryonic stem cell; improved; in vivo; pancreas development; public health relevance; segregation; stem cell population; tumorigenic

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.

描述（由申请人提供）：胚胎干细胞（ESCs）被设想为可移植胰岛的来源，以缓解供体器官的短缺。尽管最近取得了许多进展，但将ESCs分化为均匀的葡萄糖反应胰岛素分泌β细胞的方案尚未开发。人们普遍认为，这些种群的生产首先需要产生最终的内胚层。因此，内胚层限制干细胞系的成功衍生和稳定繁殖将是胰腺、肝脏和肺细胞发展的重要一步，用于细胞替代疗法，并提高我们对这些组织如何从胚层正常形成的理解。我们开发了一个简单的方案，使用磁激活细胞分选（MACS），以上皮细胞粘附分子（EpCAM）表达为基础，从分化小鼠ESCs的异质培养物中分离内胚层细胞。该方案包括阴性选择去除未分化的ESCs和内脏内胚层细胞，阳性选择保留表达EpCAM的细胞。当分选的细胞皮下植入免疫缺陷小鼠时，EpCAM+PDX1+小结节的形成与胚胎胰腺上皮高度相似。来自这些结节的细胞可以在体外广泛繁殖，并具有内胚层干细胞（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限制基因，而FGSCs为OCT4-，高表达Sox17、Foxa2和PDX1。移植回动物体内的EndSCs和FGSCs再现了原来的结节表型，证明了它们在体外的稳定性。我们假设只有从MACS分离中恢复的OCT4+EpCAM+SSEA1-细胞才能产生体内源性内胚层细胞系。在这里，我们将通过facs分选OCT4+EpCAM+SSEA1-细胞，使用OCT4GFP ESC系直接验证这一假设。来自facs分类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的细胞类型（包括β细胞）的能力。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中，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ESCs向最终内胚层和胰腺谱系的分化效率，而且有助于更好地理解胚层分离和早期胚胎细胞命运的决定。公共卫生相关性：我们的初步研究已经导致分化小鼠胚胎干细胞（ESCs）细胞系的发展，这些细胞系似乎与前肠内胚层有关。这些细胞系是无致瘤性的，在培养中生长良好，在这里，我们建议阐明这些内胚层细胞系的细胞来源，并探索它们在体外分化为胰腺、肝脏和肺细胞的能力。这些知识将有助于建立分离和分化人类内胚层细胞的方案，以生产用于移植治疗的功能细胞。