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Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Infertility is a frequent side effect of chemotherapy and/or radiotherapy and for some patients, cryopreservation of oocytes or embryos is not an option. ...

Weill Cornell Medical College

During development, endothelial cells (EC) display tissue-specific attributes that are unique to each vascular bed, as well as generic signaling mechanisms that are broadly applied to create a patent circulatory system. We have previously utilized human embryonic stem cells (hESC) to generate tissue-specific EC sub-types (Rafii et al., 2013) and identify pathways that govern growth and trans-differentiation potential of hESC-derived ECs (James et al., 2010). Here, we elucidate a novel Notch-dependent mechanism that induces endothelial to mesenchymal transition (EndMT) in confluent monolayer cultures of hESC-derived ECs. We demonstrate density-dependent induction of EndMT that can be rescued by the Notch signaling inhibitor DAPT and identify a positive feedback signaling mechanism in hESC-ECs whereby trans-activation of Notch by DLL4 ligand induces elevated expression and surface presentation of DLL4. Increased Notch activation in confluent hESC-EC monolayer cultures induces areas of EndMT containing transitional cells that are marked by increased Jagged1 expression and reduced Notch signal integration. Jagged1 loss of function in monolayer hESC-ECs induces accelerated feedback stimulation of Notch signaling, increased expression of cell-autonomous, cis-inhibitory DLL4, and EndMT. These data elucidate a novel interplay of Notch ligands in modulating pathway activation during both expansion and EndMT of hESC-derived ECs.

Notch hyper-activation drives trans-differentiation of hESC-derived endothelium.

Despite major advances in tissue cryopreservation and auto-transplantation, reperfusion ischemia and hypoxia have been reported as major obstacles to successful recovery of the follicular pool within grafted ovarian tissue. We demonstrate a benefit to follicular survival and function in human ovarian tissue that is co-transplanted with exogenous endothelial cells (ExEC). ExECs were capable of forming functionally perfused vessels at the host/graft interface and increased both viability and follicular volume in ExEC-assisted grafts with resumption of antral follicle development in long-term grafts. ExECs that were engineered to constitutively express anti-mullerian hormone (AMH) induced a greater proportion of quiescent primordial follicles than control ExECs, indicating suppression of premature mobilization that has been noted in the context of ovarian tissue transplantation. These findings present a cell-based strategy that combines accelerated perfusion with direct paracrine delivery of a bioactive payload to transplanted ovarian tissue.

Engineered endothelium provides angiogenic and paracrine stimulus to grafted human ovarian tissue.

Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during  differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs.

Laura Park

Center for Reproductive Medicine and Infertility

Weill Cornell Medical College

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Laura Park

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Weill Cornell Medical College

Co-transplantation of Human Ovarian Tissue with Engineered Endothelial Cells: A Cell-based Strategy Combining Accelerated Perfusion with Direct Paracrine Delivery

Center for Reproductive Medicine and Infertility