Directed Differentiation of Human Induced Pluripotent Stem Cells to Heart Valve Cells

Abstract

BACKGROUND: A main obstacle in current valvular heart disease research is the lack of high-quality homogeneous functional heart valve cells. Human induced pluripotent stem cells (hiPSCs)-derived heart valve cells may help with this dilemma. However, there are no well-established protocols to induce hiPSCs to differentiate into functional heart valve cells, and the networks that mediate the differentiation have not been fully elucidated. METHODS: To generate heart valve cells from hiPSCs, we sequentially activated the Wnt, BMP4, VEGF (vascular endothelial growth factor), and NFATc1 signaling pathways using CHIR-99021, BMP4, VEGF-165, and forskolin, respectively. The transcriptional and functional similarity of hiPSC-derived heart valve cells compared with primary heart valve cells were characterized. Longitudinal single-cell RNA sequencing was used to uncover the trajectory, switch genes, pathways, and transcription factors of the differentiation. RESULTS: An efficient protocol was developed to induce hiPSCs to differentiate into functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells. After 6-day differentiation and CD144 magnetic bead sorting, ≈70% CD144 + cells and 30% CD144 – cells were obtained. On the basis of single-cell RNA sequencing data, the CD144 + cells and CD144 – cells were found to be highly similar to primary heart valve endothelial cells and primary heart valve interstitial cells in gene expression profile. Furthermore, CD144 + cells had the typical function of primary heart valve endothelial cells, including tube formation, uptake of low-density lipoprotein, generation of endothelial nitric oxide synthase, and response to shear stress. Meanwhile, CD144 – cells could secret collagen and matrix metalloproteinases, and differentiate into osteogenic or adipogenic lineages like primary heart valve interstitial cells. Therefore, we identified CD144 + cells and CD144 – cells as hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells, respectively. Using single-cell RNA sequencing analysis, we demonstrated that the trajectory of heart valve cell differentiation was consistent with embryonic valve development. We identified the main switch genes (NOTCH1, HEY1, and MEF2C), signaling pathways (TGF-β, Wnt, and NOTCH), and transcription factors (MSX1, SP5, and MECOM) that mediated the differentiation. Finally, we found that hiPSC-derived valve interstitial-like cells might derive from hiPSC-derived valve endothelial-like cells undergoing endocardial-mesenchymal transition. CONCLUSIONS: In summary, this is the first study to report an efficient strategy to generate functional hiPSC-derived valve endothelial-like cells and hiPSC-derived valve interstitial-like cells from hiPSCs, as well as to elucidate the differentiation trajectory and transcriptional dynamics of hiPSCs differentiated into heart valve cells.

The Role of Stem Cells in the Treatment of Cardiovascular Diseases

“…Recently, Yang and colleagues generated chambered and vascularized cardiac organoids, which is highly promising for the above-mentioned purposes [235]. Interestingly, Cai et al developed a protocol to differentiate iPSCs towards heart valve cells, creating further opportunities to study valvular heart diseases [236]. Furthermore, iPSC-derived cardiomyocytes from individuals carrying genetic variants associated with the occurrence of cardiomyopathy enable studying the importance of these mutations in disease pathogenesis [237].…”

Empowering Valvular Heart Disease Research With Stem Cell–Derived Valve Cells

“…In this issue of Circulation, Cai et al introduce an innovative approach for simultaneous generation of both human iPSC-derived valvular endothelial cells (iVECs) and valvular interstitial cells (iVICs), 2 major valve resident cells instrumental to the pathogenesis of VHD. 8 In brief, iPSCs were subjected to treatment with Wnt (winglessrelated integration site) and BMP (bone morphogenetic protein) agonists to produce NKX2.5 + (NK2 homeobox 2.5) cardiac lateral plate mesodermal cells. These cells were then exposed to VEGF (vascular endothelial growth factor) and forskolin, leading to the emergence of iVECs (CD144 + ) and iVIC progenitors (CD144 -).…”

“…In contrast, Cai et al used relatively later-stage cardiac lateral plate mesoderm cells, characterized by high expression of NKX2-5 and HAND1 (heart and neural crest derivative expressed 1), to efficiently generate valve endocardial cells through VEGF and forskolin. 8 Notably, during endocardial development, evidence suggests that NKX2-5 can directly activate ETV2 (ETS transcription factor 2), a transcription factor essential for both endothelial and endocardial lineages. 11 Activated ETV2 then upregulates NFATC1 (nuclear factor of activated T cells 1) expression in NKX2.5 + progenitors, promoting endocardial development at the expense of myocardial lineages.…”

“…NOTCH signaling is crucial in various stages of heart valve biology, initially in the formation of endocardial cushion and the control of the EndoMT process, to the maintenance of quiescent valve cells in a noncalcific state. 8,10 Moreover, BMP10 was reported to be a potent driver of endocardial cell fate decision. 15 Therefore, it would be worthwhile to explore in future studies whether the combination of VEGF, forskolin, BMP10, and NOTCH agonists can lead to a more precisely defined endocardial cell fate (Figure ).…”

“…It is notable that NOTCH signaling is also required for the EndoMT process by repressing EC gene expression in the endocardium both in vivo and in vitro. 1,8 Although iVICs derived using both BMP2 and FGF2/ TGF-β express EndoMT markers (SNAI1 [snail family transcription repressor 1], TWIST1 [twist family bHLH transcription factor 1], SOX9 [SRY-box transcription factor 9], and MSX [msh homeobox]) and fibroblastselective markers (POSTN [periostin], ACTA2 [actin alpha 2, smooth muscle], S100A4 [S100 calcium binding protein A4], and COL1A1 [collagen, type I, alpha I]), a comprehensive transcriptome profile comparison with primary valvular interstitial cells was not conducted in these studies. 9,10 In contrast, on further differentiation of CD144cells into iVICs, Cai et al confirmed that the transcriptome profiles of their iVICs closely resembled those of primary valvular interstitial cells, but not of primary coronary artery fibroblasts, coronary artery smooth muscle cells, aortic fibroblasts, or aortic smooth muscle cells.…”