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

Nikolay Turovets, Jeffrey Fair, Richard West, Alina Ostrowska, Ruslan Semechkin, Jeffrey Janus, Li Cui, Vladimir Agapov, Irina Turovets, Andrey Semechkin, Marie Csete, Larissa Agapova

Research output: Contribution to journalArticle

12 Scopus citations

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-tomesenchymal 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.

Original languageEnglish (US)
Pages (from-to)217-234
Number of pages18
JournalCell Transplantation
Volume21
Issue number1
DOIs
StatePublished - Mar 16 2012
Externally publishedYes

Keywords

  • Definitive endoderm (DE)
  • Differentiation
  • Extracellular matrix (ECM)
  • Hepatocytes
  • Human embryonic stem cells (hESCs)
  • Human parthenogenetic stem cells (hpSCs)

ASJC Scopus subject areas

  • Biomedical Engineering
  • Cell Biology
  • Transplantation

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    Turovets, N., Fair, J., West, R., Ostrowska, A., Semechkin, R., Janus, J., Cui, L., Agapov, V., Turovets, I., Semechkin, A., Csete, M., & Agapova, L. (2012). Derivation of high-purity definitive endoderm from human parthenogenetic stem cells using an in vitro analog of the primitive streak. Cell Transplantation, 21(1), 217-234. https://doi.org/10.3727/096368911X582723