Date of Award

January 2017

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Natalia B. Ivanova

Second Advisor

Scharfe Curt

Abstract

The embryonic portion of the placenta originates from the trophectoderm (TE), one of the three lineages in the preimplantation blastocyst. Understanding the fundamental biology of the TE lineage is crucial for our ability to understand early pregnancy complications, infertility, and to make advances in personalized medicine for reproductive biology. The derivation of stem cells for the TE lineage in humans has proven surprisingly difficult. Here, we seek to fill this fundamental gap in knowledge by establishing stem cells for the TE through the process of lineage conversion. To better understand how TE stem cells (TSCs) form in the human embryo, we have performed transcriptome profiling of single cells isolated directly from human blastocysts. From this data, we identified fifteen transcription factors that are highly expressed specifically in TE cells. We therefore hypothesized that TSCs can be induced from human ES cells or fibroblasts by overexpressing a combination of TE-specific factors. To facilitate monitoring of the TE conversion, we used CRISPR/Cas9 mediated homologous recombination in human ES cells to fuse an mCherry reporter to the end of the endogenous GATA2/3 genes. We then overexpressed the 15 conversion factors using doxycycline (dox) inducible lentiviral constructs. Multiple mCherry+ clones with morphologies similar to those of mouse TSCs were isolated. RT-qPCR analyses of endogenous gene expression revealed that the three genes (EOMES, CDX2 and ELF5) that constitute the network for TSC self-renewal are highly induced in mCherry+ clones. Analysis of integrated transgenes revealed that EOMES and CEBPA were detected in all clones analyzed, suggesting that these two factors are required for the successful TE conversion in humans. Although our TSC-like cells can be passaged indefinitely in the presence of dox, they differentiate upon dox withdrawal, indicating that FGF4 alone is insufficient for TSC maintenance in humans. We conclude that human TSC-like cells can be obtained from hESCs via ectopic expression of key TE –specific factors. This work provides a foundation for future experiments which will include derivation of hTSCs from fibroblasts and ultimately from human embryos, determining the culture conditions that support in vitro propagation of hTSCs and their molecular and functional analyses.

Comments

This thesis is restricted to Yale network users only. This thesis is permanently embargoed from public release.

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