Heart valve disease is a prevalent cardiac defect with limited medical treatment options. Surgical valve replacement, using mechanical or bioprosthetic valves, is common, but these options have drawbacks including the need for anticoagulation, limited durability, and lack of self-renewal. Induced pluripotent stem cell (iPSC)-based valve organoids or tissue-engineered heart valves (TEVs) offer a potential solution, requiring large numbers of functional valvular cells. Valvular cells include valvular endothelial cells (VECs) and valvular interstitial cells (VICs). While hPSCs can differentiate into various cell types, derivation of VECs has been limited by incomplete understanding of VEC biology. The endocardial cells are believed to originate from Isl1+/Kdr+ multipotent progenitors. During development, endocardial cushion cells (ECCs) at the atrioventricular canal and outflow tract either undergo EndoMT to become valve mesenchymal cells or adopt a VEC fate. Signaling pathways (BMP, FGF, WNT, NOTCH, TGF-β) and transcriptional regulation (e.g., NFATc1) are involved in this process. This study aimed to develop an efficient, chemically defined, xeno-free method for generating VECs from hPSCs and characterize their properties, addressing the need for a readily available source of cells for TEVs.

Existing literature highlights the challenges in treating valve disease and the potential of iPSC-derived valvular cells for tissue engineering. Studies have shown the importance of various signaling pathways and transcription factors in valvulogenesis, with a focus on the roles of BMP, FGF, WNT, NOTCH, and TGF-β signaling pathways and transcription factors such as NFATc1. However, detailed mechanisms of ECC induction and VEC fate specification remained unclear, along with the need for a robust and efficient method to generate large quantities of functional VECs from hPSCs for use in TEVs. This work builds upon existing knowledge by providing a novel approach to VEC generation and comprehensive characterization of these cells.

This study employed a two-stage, chemically defined, xeno-free differentiation protocol. First, hPSCs were differentiated into ISL1+/KDR+ CPCs by treatment with BMP4 and WNT agonists (WNT3a or CHIR99021) for 3 days, followed by bFGF and BMP4 for another 6 days. The efficiency of this step was assessed by qRT-PCR, immunofluorescence (IF), western blot (WB), and flow cytometry. Second, CPCs were differentiated into VELs using a combination of VEGFA, BMP4, and TGFβ1 for 6-9 days. The expression of ECC and VEC markers was monitored by qRT-PCR, IF, WB, and flow cytometry at different time points. Single-cell RNA sequencing (scRNA-seq) data from human embryos were re-analyzed to compare the differentiation trajectory with embryonic VEC development. Bulk RNA sequencing compared hPSC-derived VELs with primary VECs, HAECs, and HUVECs. Functional assays included tube formation, Ac-LDL uptake, shear stress response, and conversion to VIC-like cells. The interaction of hPSC-derived VELs with decellularized porcine aortic valve (DCV) scaffolds was evaluated by EdU incorporation, TUNEL assay, and long-term co-culture. Statistical analysis included paired and unpaired t-tests.

The study successfully generated ISL1+/KDR+ CPCs from hPSCs with high efficiency (>90% ISL1 positive and ~80% KDR positive). A one-step differentiation of CPCs into VELs was achieved using VEGFA/BMP4/TGFβ1. The combined treatment with BMP4 and TGFβ1 proved crucial for inducing ECC and subsequently VEC fates, likely by activating NOTCH signaling and upregulating NFATc1 and ATF3. scRNA-seq analysis revealed that the hPSC differentiation mirrored embryonic VEC development, with ECC-enriched genes (G1) upregulated early and VEC-enriched genes (G2) upregulated later. Transcriptome analysis showed high similarity between hPSC-derived VELs and primary VECs. Functional assays confirmed the endothelial nature of hPSC-derived VELs, including their ability to form tube-like structures, uptake Ac-LDL, respond to shear stress, and convert into VIC-like cells via EndoMT. hPSC-derived VELs showed superior proliferation and less apoptosis than primary VECs and HAECs when seeded onto DCV scaffolds. Long-term co-culture demonstrated successful endothelialization of DCVs by hPSC-derived VELs.

This study provides a significant advance in generating functional VECs from hPSCs. The findings demonstrate the crucial roles of TGFβ1 and BMP4 signaling in directing VEC fate, highlighting a previously unidentified role for ATF3 and KLF transcription factors. The close resemblance between the in vitro differentiation pathway and embryonic VEC development validates the model's physiological relevance. The superior proliferative capacity of hPSC-derived VELs compared to primary VECs makes them a promising cell source for TEVs. Although the hPSC-derived VELs showed high similarity to primary VECs, they may represent an immature form due to lack of hemodynamic stimulation in the in vitro model. Future research should focus on optimizing the differentiation protocol under conditions that mimic the physiological environment, including the incorporation of mechanical stimuli and potential contributions from neural crest cells.

This study presents an efficient and robust method for generating functional cardiac valve endothelial-like cells from human pluripotent stem cells. The generated VELs closely mimic the characteristics of primary VECs and demonstrate superior proliferative capacity, making them ideal candidates for applications in tissue engineering of heart valves. Future research could focus on optimizing the in vitro conditions to better mimic the in vivo environment and on investigating the potential contribution of neural crest cells to further enhance the maturity and function of the derived cells. This work provides a strong foundation for the development of autologous stem cell-based valve organoids for the treatment of heart valve disease.

The current study primarily focuses on the in vitro generation and characterization of VELs. The in vivo functionality and long-term effects of these cells require further investigation. The absence of hemodynamic stimuli in the in vitro system may lead to less mature VELs compared to native cells. While the study demonstrated the conversion of VELs to VIC-like cells, further studies are needed to fully characterize the properties of these VIC-like cells and to investigate their contributions to the functionality of a complete valve construct. The study also only examined the effects of mesenchymal lineages and further studies examining the effects of neural crest cells would be beneficial.