International Stem Cell Corporation, Oceanside, CA, USA.
Cell Transplant. 2012;21(1):217-34. doi: 10.3727/096368911X582723. Epub 2011 Jun 9.
Human parthenogenetic stem cells (hpSCs) are pluripotent stem cells with enormous potential as cell sources for cell-based therapies: hpSCs may have histocompatibilty advantages over human embryonic stem cells (hESCs) and derivation of hpSCs does not require viable blastocyst destruction. For translation of all pluripotent stem cell-based therapies, derivation of differentiated cell products that are not contaminated with undifferentiated cells is a major technical roadblock. We report here a novel method to derive high-purity definitive endoderm (DE) from hpSCs, based on reproducing features of the normal human embryonic microenvironment. The method mimics the developmental process of transition through a primitive streak, using a differentiation device that incorporates a three-dimensional extracellular matrix (ECM) combined with a porous membrane. Treatment of undifferentiated hpSCs above the membrane results an epithelial-to-mesenchymal transition (EMT); thus, responsive cells acquire the ability to migrate through the membrane into the ECM, where they differentiate into DE. Importantly, the resultant DE is highly purified, and is not contaminated by undifferentiated cells, as assessed by OCT4 expression using immunocytochemistry and flow cytometry. The functional properties of the DE are also preserved by the process: DE differentiated in the device can generate a highly enriched population of hepatocyte-like cells (HLCs) characterized by expression of hepatic lineage markers, indocyanine green clearance, glycogen storage, cytochrome P450 activity, and engraftment in the liver after transplantation into immunodeficient mice. The method is broadly applicable and we obtained purified DE using hESCs, as well as several hpSC lines. The novel method described here represents a significant step toward the efficient generation of high-purity cells derived from DE, including hepatocytes and pancreatic endocrine cells, for use in regenerative medicine and drug discovery, as well as a platform for studying cell fate specification and behavior during development.
人孤雌生殖干细胞（hpSCs）是多能干细胞，具有作为基于细胞的治疗的细胞来源的巨大潜力：hpSCs 可能比人胚胎干细胞（hESCs）具有组织相容性优势，并且 hpSCs 的衍生不需要有活力的囊胚破坏。对于所有基于多能干细胞的治疗的翻译，衍生出未分化细胞污染的分化细胞产品是一个主要的技术障碍。我们在此报告一种从 hpSCs 中衍生高纯度的确定内胚层（DE）的新方法，该方法基于复制正常人类胚胎微环境的特征。该方法通过使用一种包含三维细胞外基质（ECM）和多孔膜的分化装置来模拟通过原始条纹的发育过程来模拟过渡的发育过程。在膜上方处理未分化的 hpSCs 会导致上皮-间充质转化（EMT）；因此，响应性细胞获得通过膜迁移到 ECM 中的能力，在那里它们分化为 DE。重要的是，通过免疫细胞化学和流式细胞术评估 OCT4 表达，所得的 DE 高度纯化，并且没有未分化细胞的污染。该过程还保留了 DE 的功能特性：在装置中分化的 DE 可以产生富含肝样细胞（HLC）的高度富集群体，其特征在于表达肝谱系标记物、吲哚菁绿清除、糖原储存、细胞色素 P450 活性以及在移植到免疫缺陷小鼠后的肝脏内植入。该方法具有广泛的适用性，我们使用 hESCs 以及几种 hpSC 系获得了纯化的 DE。这里描述的新方法代表了朝着有效产生高纯度 DE 衍生细胞的重要一步，包括肝细胞和胰腺内分泌细胞，用于再生医学和药物发现，以及用于研究细胞命运特化和发育过程中的行为的平台。
