Human embryonic stem cells (hESC) have an inexhaustible ability to divide and renew, and under the appropriate conditions, differentiate and change into any cell type in the body. This balance between pluripotency and self-renewal is a complex and carefully choreographed response of the hESC to local microenvironmental cues. Understanding the molecular regulators of this balance, and the various signals that are integrated by hESC to maintain their pluripotency and self-renewal characteristics are critical for the expansion and differentiation of hESC to specific cell types.
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, binds ephrinB2 more “tightly” than the EphB4 receptor, and can therefore compete or interfere with normal ephrin-B2-EphB4 interactions. 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.
hESCs prefer to grow in clusters and propagate as a complex and dynamic ecosystem of cells where any given cell may have different capacity for pluripotency or self-renewal. We first asked if ephrinB2 was homogenously expressed on hESCs, and if not, does ephrinB2 mark for a subpopulation within hESC cultures with distinct properties? To do this, we infected hESCs with GFP-reporter lentiviruses bearing Nipah envelope proteins (NiVpp for NiV pseudotyped particles), which can only infect ephrinB2+ cells. We found that NiVpp consistently infected only 5-20% of hESCs through primary and secondary rounds of infection even though we can purify the initially infected subpopulation to near homogeneity (>85%) between rounds of infection. This suggests that ephrinB2 is not a stable cell surface marker for a distinct subpopulation of cells. However, ephrinB2+ hESCs do appear to represent a subpopulation of hESCs with decreased self-renewal capacity when subjected to the appropriate tests. Interestingly, these NiVpp infected hESCs still maintained the ability to form teratomas, albeit smaller ones, when injected in SCID mice. In toto, our results show that pluripotency and self-renewal are distinct and dissociable properties of hESCs and they do not necessarily reside within one particular cell in the hESC culture.
Next we sought to determine whether the ephrin2-EphB4 axis plays a role in regulating the ability of hESCs to differentiate into the three major germ layers that make up the cells of the various organs and tissues in the human body. Differentiation is a carefully choreographed molecular and cellular response to local environmental determinants. In vitro formation of embryoid bodies, where expression of genetic markers for all three germ layers can be detected, is surrogate in vitro assay for pluripotency. Under standard conditions, embryoid bodies form extremely heterogenous spherical clusters that make it difficult to reproducibly quantify any differences in germ layer commitment that might result as a consequence of antagonizing EphrinB2-EphB4 interactions. Thus, we optimized a “spin embryoid body” assay where the number of hESC per embroid body formed could be carefully controlled. Under these conditions, ephrin-B2 expression increased dramatically between days 10-15, closely mirroring 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). Enoderm markers (the germ layer that forms cells like those that line the gut) are dramatically downregulated during the first 10 days, and do not peak until days 15-20. These exciting results from our first year suggest 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.