A Novel Transcription Factor-Driven Approach for Mural Progenitor Cells Generation

PROJECT SUMMARY/ABSTRACT Mural cells, including pericytes and smooth muscle cells, are critical for vascular development, function, and stability. Dysregulation of mural cells can lead to vascular abnormalities, emphasizing the need for generating functional mural cells to explore novel therapeutic approaches in vascular disorders, tissue repair, and regenerative medicine. Human induced pluripotent stem cells (iPSCs) offer a promising method for obtaining patient-specific mural cells; however, conventional chemical-based differentiation methods are limited in scope and precision. Inducible transcription factors (TFs) have gained traction as a differentiation strategy, offering precise temporal control and the potential for simultaneous differentiation of multiple cell types. However, identifying TFs for mural cell differentiation remains challenging. Our overarching goal is to develop TF-driven strategies for the effective differentiation of human iPSCs into competent vascular cells for regenerative medicine. Our group previously demonstrated the successful generation of vascular endothelial cells (iECs) from iPSCs using ETV2, a pioneer TF. More recently, we identified that another TF, NK3 Homeobox 1 (NKX3.1), facilitates the generation of mural progenitor cells (iMPCs) from iPSCs. These iMPCs display crucial mural cell characteristics and mature into fully differentiated mural cells upon interaction with ECs. Our group has also developed a novel vascular organoid (VO) model that allows concurrent co-differentiation of iPSCs into iECs and iMPCs and facilitates the maturation of iMPCs. Our central hypothesis is that NKX3.1 dictates mural cell lineage fate, and its activation can effectively generate iMPCs, introducing a novel method for creating an unlimited supply of functional mural cells for regenerative medicine. To test these hypotheses and elucidate the mechanisms underlying NKX3.1-driven mural cell differentiation and maturation, we propose three specific aims. In Aim-1, we seek to elucidate the transcriptional regulation of iMPC specification using NKX3.1. Aim-2 delves into the mechanisms of mural cell maturation in the VO model, examining how EC interactions affect iMPC maturation. In Aim-3, we seek to evaluate the therapeutic potential of iMPCs in ischemic tissues, determining whether iMPCs can act as a source of functional perivascular cells in vivo, supporting EC engraftment and contributing to the stability of newly generated blood vessels. We anticipate that these investigations will uncover the molecular underpinnings of mural cell lineage determination and functionality, ultimately advancing our understanding of mural cell development and therapeutic potential. This knowledge will pave the way for patient- specific strategies for treating vascular disorders and advancing vascular regenerative medicine.