Introduction. 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. However, the ephrinB2 signaling 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 Nipah virus (NiV) binds ephrinB2 with very high affinity and specificity, and can therefore compete or interfere with normal ephrinB2 interactions with its cognate Eph receptors. NiV envelope proteins pseudotyped onto lentiviral particles can also specifically transduced ephrinB2+ cells. Thus, using an arsenal of reagents based on engineered versions of this viral envelope protein, we had proposed to interrogate the role of the ephrinB2 signaling 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 ~20% of total SSEA4+ hESCs, even after repeated purification between passages. Thus, ephrinB2 does not mark for an independent, stable subpopulation of hESCs. Instead, ephrinB2 may be an intrinsic marker of stem cell heterogeneity; perhaps an emergent marker that arises from the statistical mechanics model of pluripotency as recently proposed by MacArthur and Lemischka (Cell, 2013).
EphrinB2 expression closely mirrored the upregulation of ectoderm markers, and to a lesser extent, mesoderm markers in a “spin embryoid body” (spin EB) assay, which we used as an in vitro surrogate assay for assessing pluripotency. Our results suggest that ephrinB2 signaling axis likely plays 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 signaling axis by generating stable hESCs (H9 and UCLA1) expressing the soluble NiV env glycoprotein (sNiV-G) or a short hairpin RNA against ephrinB2 (shB2). sNiV-G should bind avidly to ephrinB2 and antagonize the complex forward and reverse Eph receptor-Ephrin ligand signaling axis, while shB2 knocks down ephrinB2 mRNA expression. 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) and to a lesser extent, mesoderm differentiation (CD34) when assayed using the spin EB method under spontaneous differentiation or directed-mesoderm differentiation conditions, respectively.
Collectively, our results show that antagonizing the ephrinB2 signaling axis can affect the pluripotency of hESCs, specifically with regards to ectoderm and mesoderm differentiation. Interestingly, physically antagonizing the ephrinB2 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 signaling axis can play in ectoderm and mesendoderm differentiation.
In Year 3, we characterized the self-renewal, survival, and pluripotency of the sorted shB2 and shNT H9 hESCs (shNT is a non-targeted shRNA used as a comparison control for shB2). Parental H9, shNT and shB2 H9 cells showed no significant differences in self-renewal assays over 5 passages (data not shown). In vivo teratoma formation assays demonstrated no obvious qualitative differences in pluripotency as all three germ layers were observed in each H9 hESC line. However, in vivo teratoma formation assays are inherently variable and not amendable to easy quantification.
To address the impact of ephrinB2 antagonism on germ layer specification in a more quantitative and holistic fashion, we performed microarrays on H9, shNT, and shB2 hESCs, and on their derived EBs (days 6 and 13) under spontaneous differentiation conditions and compared their global gene expression profiles. Our analysis revealed a progressively larger number of genes were specifically up- or down-regulated in shEFNB2 cells compared to H9 and shNT cells on days 0, 6, and 13, respectively. Further analysis indicated that ephrinB2 knockdown in H9 hESCs may enhance the formation of mesoendoerm progenitors while inhibiting the differentiation of neuro-ectoderm lineages.
Finally, functional mesoderm-directed differentiation assays revealed that shEFNB2 hESCs have an increased propensity to differentiate into one specific sub-type of mesenchymal cells. In sum, the findings of this dissertation suggest that the heterogeneity of ephrinB2 expression and perturbation of ephrinB2 signaling may both be manipulated to enhance directed differentiation of hESCs in vitro.