We wish to identify determinants of endothelial lineage. Murine embryonic stem cells (mESC) were fused with human endothelial cells in stable, non-dividing, heterokaryons. Using RNA-seq it is possible to discriminate between human and mouse transcripts in these chimeric heterokaryons. We observed a temporal pattern of gene expression in the ESCs of the heterokaryons that recapitulated ontogeny, with early mesodermal factors being expressed before mature endothelial genes. A set of transcriptional factors not known to be involved in endothelial development was upregulated, one of which was POU class 3 homeobox 2 (Pou3f2). We confirmed its importance in differentiation to endothelial lineage via loss- and gain-of-function (LOF and GOF). Its role in vascular development was validated in zebrafish embryos using morpholino oligonucleotides. These studies provide a systematic and mechanistic approach for identifying key regulators in directed differentiation of pluripotent stem cells to somatic cell lineages.
Discovery of Novel Determinants of Endothelial Lineage: Insights from Chimeric HeterokaryonsPublicly available at the NCBI Gene Expression Omnibus (accession no. GSE84558).
- John P Cooke
- Wing Tak Wong
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: Zebrafish are kept according to the laboratory protocols described in Zebrafish: A Practical Approach (Oxford University Press, 2002). These protocols comply with the Guide for the Care and Use of Laboratory Animals, the American Association for the Accreditation of Laboratory Animal Care (AAALAC) standards, and the regulations set forth in the Animals Welfare Act (P.L. 89-544, as amended by P.L. 91-579 and P.L . 94-279). Veterinary care is provided on a 24 hours basis, including weekends and holidays, by a staff of veterinarians with specialties in laboratory animal medicine and anesthesiology, and licensed animal health technicians. Training classes are offered. All veterinary care is provided by Houston Methodist Research Institute, which is fully accredited by AAALAC (ID A4555-01) and holds an approved NIH Assurance and USDA License (start date 03/08/2013). Support includes quarantine rooms, sterile operating rooms, post-surgical recovery rooms, radiology and diagnostic laboratory services. All surgery procedures were performed under anesthesia with Tricaine 0.02 mg/ml.
- Gordana Vunjak-Novakovic, Columbia University, United States
© 2017, Wong et al.
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