Student Theses and Dissertations

Date of Award

2006

Document Type

Thesis

RU Laboratory

Brivanlou Laboratory

Keywords

human embryonic stem cells, embryonic chimeras, TGF beta signaling, mouse blastocysts

Abstract

Human embryonic stem cells (hESCs) are a unique population of cells derived from a 6 day old human embryo that can be maintained indefinitely in vitro and have the ability to differentiate to all adult cell types. In addition to their potential for cell based therapies in the treatment of disease and injury, the broad developmental capacity of hESCs offers potential for studying the origins of all human cell types. Embryonic stem cells were first derived from mouse embryos (mESCs), and years of work have demonstrated their utility to developmental research, but relatively little is known about human ESCs. The experiments described below address two fundamental questions in hESC biology: First, what are the molecular signaling pathways that are relevant to hESC "sternness"? And second, can hESCs contribute to specialized human cell types in the context of mouse embryogenesis? Because it plays a prominent role in the early cell fate decisions of embryonic development, we examined the role of TGFP superfamily signaling in hESCs. W e found that, in undifferentiated cells, the TGFp/activin/nodal branch is activated (through the signal transducer Smad2/3) while the BMP/GDF branch (Smadl/5) is only active in isolated mitotic cells. Upon early differentiation, Smad2/3 signaling is decreased while Smadl/5 signaling is activated. We next tested the functional role of TGFp/activin/nodal signaling in hESCs and found that it is required for the maintenance of markers of the undifferentiated state. We extended these findings to show that Smad2/3 activation is required downstream of Wnt signaling, which we have previously shown to be sufficient to maintain the undifferentiated state of hESCs. Strikingly, we show that in ex vivo mouse blastocyst cultures, Smad2/3 signaling is also required to maintain the inner cell mass (from which stem cells are derived). These data reveal a critical role for TGFP signaling in the earliest stages of cell fate determination and demonstrate an interconnection between TGFP and Wnt signaling in these contexts. To date, the emergence of specialized cells from hESCs has commonly been studied in tissue culture or upon injection into adult mice, yet these methods have stopped short of demonstrating the potential exhibited by mESCs, which can give rise to every cell type when combined with embryos at the blastocyst stage. Due to obvious barriers precluding the use of human embryos in similar cell mixing experiments with hESCs, human/non-human chimeras may need to be generated for this purpose. In order to define the developmental potential of hESCs in the context of embryogenesis, we explored the ability of hESCs to engraft into mouse blastocysts. In advance of these cell mixing experiments, we derived a new hESC line, RUES1, and characterized its marker expression, functional characteristics and gene expression profiles. Using this new line, we showed that hESCs engrafted into mouse blastocysts, where they, proliferated and differentiated in vitro and persisted in mouse/human embryonic chimeras that implanted and developed in the uterus of pseudopregnant foster mice. Embryonic chimeras generated in this way offer the opportunity to study the behavior of specialized human cell types in a non-human animal model. Our data demonstrate the feasibility of this approach, using mouse embryos as a surrogate for hESC differentiation.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.

Permanent URL

http://hdl.handle.net/10209/229

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