title: Derivation of high-purity definitive endoderm from human parthenogenetic stem cells using an in vitro analog of the primitive streak.
abstract: 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.
authors.affiliation: International Stem Cell Corporation, Oceanside, CA, USA.
authors.forename: Nikolay
authors.initials: N
authors.lastname: Turovets
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Fair
authors.forename: Richard
authors.initials: R
authors.lastname: West
authors.forename: Alina
authors.initials: A
authors.lastname: Ostrowska
authors.forename: Ruslan
authors.initials: R
authors.lastname: Semechkin
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Janus
authors.forename: Li
authors.initials: L
authors.lastname: Cui
authors.forename: Vladimir
authors.initials: V
authors.lastname: Agapov
authors.forename: Irina
authors.initials: I
authors.lastname: Turovets
authors.forename: Andrey
authors.initials: A
authors.lastname: Semechkin
authors.forename: Marie
authors.initials: M
authors.lastname: Csete
authors.forename: Larissa
authors.initials: L
authors.lastname: Agapova
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D004707:Endoderm / Q000166:cytology*; D058750:Epithelial-Mesenchymal Transition; D005109:Extracellular Matrix; D018507:Gene Expression Regulation, Developmental; D022781:Hepatocytes; D006801:Humans; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis; D010312:Parthenogenesis; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D054240:Primitive Streak / Q000166:cytology
chemical_list: D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human