Public Summary of Scientific Progress
Introduction. EphrinB2 and ephB4 are cell surface molecules that mediate and transduce signaling cascades upon interaction with each other. Cell-cell contacts between ephrinB2 and ephB4 expressing cells provide guidance cues for cell migration and boundary formation in many developmental systems such as the formation of neurons and blood vessels. Importantly, ephrinB2 has been determined to be a molecular marker of “stemness” and is expressed in human embryonic stem cells, neural stem cells and hematopoietic stem cells. Despite much evidence from model systems that ephrinB2/ephB4 axis may be intimately involved in ESC fate (survival, self-renewal, and pluripotency), this particular axis has not been carefully studied in human ESC due to the lack of highly specific reagents to block cognate ephrinB2-ephB4 interactions.
Intriguingly, the envelope protein from an exotic and highly lethal virus called Nipah virus (NiV), binds ephrinB2 more “tightly” than the EphB4 receptor, and can therefore compete or interfere with normal ephrin-B2-EphB4 interactions. NiV envelope proteins pseudotyped onto lentiviral particles can also specifically transduced EphrinB2 expressing cells. Thus, using an arsenal of reagents based on engineered versions of this viral envelope protein, which retains the ephrinB2 binding properties without the virulence of the actual virus, we had proposed to interrogate the role of the ephrinB2-ephB4 axis in regulating hESC’s ability to proliferate, self-renew, and differentiate into any cell type that make up the human body.
In Year 1, using NiV envelope mediated lentiviral transduction to mark ephrinB2+ hESCs, we found that ephrinB2+ cells were homeostatically maintained at 5-20% of total SSEA4+ hESCs, even if ephrinB2+ cells were purified to near homogeneity (>85%) between passages. These results indicate that ephrinB2 does not mark for a stable distinct subpopulation of hESCs; instead, ephrinB2 expression might represent a marker for intrinsic stem cell heterogeneity that needs to be maintained at a certain percentage of hESCs in culture in order for the line to maintain all the cardinal properties of stem cells. However, the subpopulation of ephrinB2+ hESCs do appear to have decreased self-renewal capacity, although they maintained the ability to form teratomas, albeit smaller ones, when injected in SCID mice. Using a “spin embryoid body” (spin EB) assay as an in vitro surrogate assay for pluripotency and monitoring the time-course and expression levels of various germlayer differentiation markers after formation of spin EBs, we found that ephrinB2 expression closely mirrored the upregulation of ectoderm markers (the germ layer that forms cells like neurons), and to a lesser extent, mesoderm markers (the germ layer that forms cells like endothelial cells and hematopoietic stem cells). These results suggest that ephrinB2-EphB4 interactions likely play a role in regulating ectoderm and mesoderm formation, and that antagonizing this axis using our Nipah envelope based reagents will illuminate these early differentiation processes.
In Year 2, we examined the effects of antagonizing the ephrinB2-ephB4 axis by generating stable hESCs (H9 and UCLA1 cell lines) expressing the soluble NiV attachment glycoprotein (sNiV-G) or a short hairpin RNA against ephrinB2 (shB2). sNiV-G binds to ephrinB2 and should prevent bi-directional signaling via the ephrinB2-ephB4 axis, while shB2 knocks down ephrinB2 mRNA expression by 50-80%. sNiV-G expressing hESCs gradually lose their pluripotency markers (SSEA4 and Oct-4) while upregulating ectoderm markers like Pax6 by 100-fold. On the other hand, hESCs expressing the shB2 exhibited marked defects in ectoderm differentiation (pax6 and NeuroD) when assayed using the spin EB method under spontaneous differentiation protocols. When the spin EB method was performed under directed-mesoderm differentiation, shB2 hESCs showed a 10-fold decrease in CD34 levels compared to control hESCs, indicating a defect in endothelial cell and/or hematopoietic cell differentiation.
Collectively, our results show that antagonizing the ephrinB2-ephB4 axis can affect the pluripotency of hESCs, specifically with regards to ectoderm and mesoderm differentiation. Interestingly, physically antagonizing the ephrinB2-ephB4 signaling in trans (via secreted sNiV-G binding to ephrinB2) and knocking down ephrinB2 expression in cis (via shB2 mediated decrease in ephrinB2 mRNA) appears to reveal the different roles that ephrinB2-ephB4 axis can play in ectoderm and mesoderm differentiation. Future experiments will examine these putative differences in greater detail, and also confirm their phenotype on hESC pluripotency in vivo via the use of teratoma formation assays.