Derivation of high-purity definitive endoderm from human parthenogenetic stem cells using an in vitro analog of the primitive streak.

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.

Human parthenogenetic stem cells produce enriched populations of definitive endoderm cells after trichostatin A pretreatment.

Human parthenogenetic stem cells (hpSC) hold great promise as a source of pluripotent stem cells for cell-based transplantation therapy due to their ethical method of derivation as well as the enhanced capacity for immunomatching with significant segments of the human population. We report here the directed differentiation of hpSC to produce enriched populations of definitive endoderm. Moreover, we find that treatment of undifferentiated hpSC by trichostatin A (TSA) before applying the directed differentiation protocol significantly increases the proportion of definitive endoderm cells in the final population. TSA-pretreated as well as non-TSA-treated hpSC undergoing differentiation toward definitive endoderm demonstrate a similar temporal sequence of gene expression to that which occurs in the course of definitive endoderm differentiation during vertebrate gastrulation and for differentiation of hESCs to definitive endoderm. Creation of the definitive endoderm lineages from hpSC represents the critical first step toward the development of hpSC-based cellular therapies for diseases of the liver or pancreas.