(M-511) Vascular Engineering Through Hydrogels and Organoids to Model Sexual Dimorphisms During Development

Introduction:: Understanding of sexual dimorphisms in the microvasculature is lacking, though it has been demonstrated that sex differences are present in vascular anatomy within the cerebrovasculature. Moreover, females and males experience different underlying causes for certain CVDs, such as coronary microvascular disease, with decreased blood flow to the heart more prevalent in young women. The sexual dimorphic mechanisms underlying CVD pathophysiology are poorly understood, so there is a need for sex-specific research into the biological mechanisms of CVD. Towards understanding sexual dimorphisms in disease mechanisms, characterization of naturally occurring sex-specific differences within the cardiovascular system could inform which discovered differences are a result of the diseased state versus those which are inherently present in healthy female and male vasculature. Human-induced pluripotent stem cells (hiPSCs) allow for in vitro modeling of the vasculature to identify the genes and regulatory pathways responsible for sexual dimorphisms. In mature female cells, X chromosome inactivation (XCI) occurs on all but one X chromosome to prevent toxic doses of X-linked genes. We expect female hiPSCs to not express XIST, the XCI marker, if they have been completely reprogrammed. However, previous works showed that female hiPSCs often express XIST, suggesting they are partially differentiated. The incomplete X chromosome reactivation in female hiPSCs during reprogramming may impede their ability to differentiate into vascular cells when modeling healthy sexual dimorphisms when compared to male hiPSCs. We test the overarching hypothesis that sex-specific mechanisms underlie endothelial cell differentiation, maturation, and vascular morphogenesis.

Materials and Methods:: We engineer 3D microvascular tissue in hydrogel systems and create vascular organoids to test our hypothesis. For the hydrogel systems, female and male endothelial cells are derived from hiPSCs (iECs) through an established small-molecule differentiation protocol. Efficacy and phenotype of iEC derivation are assessed through flow cytometry and immunofluorescent (IF) staining for multiple vascular markers. We measure the expression of XIST in female cells before and after differentiation through RT-qPCR to confirm female hiPSCs express XIST. iECs are encapsulated in 3D hydrogels to form networks and are characterized by IF and confocal imaging to characterize tube formation. RNA is extracted from 2D iECs cells and engineered networks in hydrogels for bulk RNA-seq and qRT-PCR to identify upregulated and downregulated pathways within the vascular tissues. For the vascular organoid generation, female and male hiPSCs are made to form hiPSC aggregates and then subjected to a fifteen-day, small-molecule differentiation protocol to create blood vessel organoids. Network formation of endothelial cells and mural cells within organoids is assessed through IF through multiple vascular markers. RNA is extracted from vascular organoids for to further identify and corroborate upregulated and downregulated signaling pathways using qRT-PCR. Three biological samples (N=3) in technical triplicates are analyzed from two female and two male hiPSC lines C3-1, 480, C1-2, and 479 in our assays.

Results, Conclusions, and Discussions:: Using hydrogel systems, collected data indicate female and male hiPSCs differentiate into ECs with similar efficacy in 2D. Specifically, there does not appear to be a statistically significant difference between the percentage of female and male iECs expressing CD31 and Vascular Endothelial Cadherin (VECad) when comparing female and male hiPSCs reprogrammed by the same methodology. The iECs also express EC markers, including CD31, von Willebrand Factor, and VECad, indicative of their lineage specification (Fig 1A). This data suggests that sex does not modulate EC fate from hiPSCs and confirms reliable sources of iECs for the engineering of in vitro vascular networks to explore sexual dimorphisms. Ongoing experiments examine the dynamics of 3D vascular morphogenesis of female and male iECs in hydrogel systems. Specifically, we are embedding female and male iECs in collagen I hydrogels to observe differences in sprouting and network formation. Preliminary data displays male iECs sprout quicker than female iECs, yet female iECs networks are sustained for longer time periods, suggesting differences in the behavior of female and male iECs in 3D systems. During blood vessel organoid generation, when hiPSC lines are seeded at the same cell density for aggregate formation, male hiPSC aggregates are often larger than female hiPSC aggregates. Following differentiation, female and male vascular organoids express mural cell and EC markers and demonstrate network formation, providing an additional model for investigating sexual dimorphisms which occur during vascular development (Fig 1B). Future directions include the use of different polymeric hydrogels used for the engineering of the vascular tissue to permit modeling different vascular tissue behavior, such as the use of polymers with dynamic crosslinks that more readily facilitate vascular remodeling by iECs to more closely recapitulate regeneration in vitro. Additionally, engineering blood vessels for disease modeling in vitro or therapeutic use in female and male patients in the future may require different considerations based on identified sex-linked differences. The engineered female and male vascular tissues lay a foundation for testing of the sex-specific effects of exogenous hormones and various drugs on the vasculature.

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