Robust differentiation of human pluripotent stem cells into mural progenitor cells via transient activation of NKX3.1

Mural cells are central to vascular integrity and function. In this study, we demonstrate the innovative use of the transcription factor NKX3.1 to guide the differentiation of human induced pluripotent stem cells into mural progenitor cells (iMPCs). By transiently activating NKX3.1 in mesodermal intermediates, we developed a method that diverges from traditional growth factor-based differentiation techniques. This approach efficiently generates a robust iMPC population capable of maturing into diverse functional mural cell subtypes, including smooth muscle cells and pericytes. These iMPCs exhibit key mural cell functionalities such as contractility, deposition of extracellular matrix, and the ability to support endothelial cell-mediated vascular network formation in vivo. Using bulk RNA sequencing, we compared gene expression profiles of induced mural progenitor cells (iMPC_NKX3.1, #13-15) derived from hiPSCs (#7-9) with primary vascular smooth muscle cells (hpVSMC, #10-12) and mesenchymal cells (hbMSC, #4-6, control group). Additionally, we contrasted iMPCs with induced smooth muscle cells (iSMCs, #16-18) generated via a different growth factor-driven protocol to study their differentiation into mature smooth muscle cells or pericytes. To assess differentiation, we conducted a 7-day co-culture of iMPCs with endothelial cells (coECiMPC, #1-3). Employing scRNA sequencing, we investigated iPSC differentiation into iMPCs and explored mural cell heterogeneity due to EC interaction. Our study sampled crucial stages: day 0 (iPSCs, #22), day 2 (MePCs after mesodermal differentiation, #20), day 4 (iMPCs post-NKX3.1 activation, #21), and day 11 post 7-day iMPC-EC co-culture (#19).