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

Authors:
Nikolay Turovets
Nikolay Turovets
University of California at Irvine
United States
Jeffrey Fair
Jeffrey Fair
University of Florida College of Medicine
Gainesville | United States
Richard West
Richard West
Van Andel Research Institute
United States
Alina Ostrowska
Alina Ostrowska
Wrocław Medical University
Poland
Ruslan Semechkin
Ruslan Semechkin
University of California
United States
Jeffrey Janus
Jeffrey Janus
Mayo Clinic
United States
Li Cui
Li Cui
School of Agriculture and Biology
China

Cell Transplant 2012 9;21(1):217-34. Epub 2011 Jun 9.

International Stem Cell Corporation, Oceanside, CA, USA.

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.

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http://dx.doi.org/10.3727/096368911X582723DOI Listing
August 2012
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