Human platelet lysate drives clinically compliant generation of vascular mural cells from human pluripotent stem cells

Background Vascular mural cells (MC) are essential components of vasculature, playing critical roles in tissue regeneration and cell therapy. The use of animal derived ancillary materials, like fetal bovine serum (FBS), in the induction of MC from human pluripotent stem cells (hPSCs), represents one of the biggest limitations to guarantee preclinical safety standards required to use this products in clinical settings. This study aimed to validate human platelet lysate (hPL) as a serum-free alternative for MC differentiation from hPSCs.

Comparison of MC differentiation efficiency from hiPSC using FBS vs hPL supplemented cultures was performed, along with functionality and gene expression assessment through bulk RNA sequencing.

Optimization of hPL concentration identified hPL1% as the most effective condition, yielding PDGFR-β+/CNN1+ MC, with a comparable efficiency to FBS10% and similar interaction with endothelial cells in vascular formation assays. However, distinct transcriptional profiles revealed that FBS10% and hPL1% drive differentiation toward different MC subphenotypes; hPL1% promoted contractile gene expression, while FBS10% enriched extracellular matrix pathways. Higher hPL concentrations further shifted differentiation toward cardiomyocytes.

In monolayer in vitro differentiation of MC from hiPSC, the differentiation efficiency using hPL 1% supplementation is equivalent to FBS 10%, while supporting a more contractile phenotype. These findings establish hPL as a xeno-minimized, clinically compliant substitute for FBS for hPSC-derived MC differentiation, an important breakthrough for regenerative medicine. Competing Interest Statement The authors have declared no competing interest. Abbreviations - MC - Vascular mural cells - FBS - fetal bovine serum - hPSCs - human pluripotent stem cells - hPL - human platelet lysate - GMP - Good Manufacturing Practice - hiPSCs - human induced pluripotent stem cells - EDTA - ethylenediaminetetraacetic acid - PBS - phosphate-buffered saline - BMP4 - bone morphogenetic protein 4 - HUVEC - human venous umbilical endothelial cells - HAoSMC - human aortic smooth muscle cells - EGM-2 - endothelial Cell Growth Medium 2 - SMGM - smooth muscle cell growth medium 2 - VEGF - vascular endothelial growth factor - PE - phycoerythrin - cTnT - anti–cardiac troponin - APC - allophycocyanin - DPBS - Dulbecco’s phosphate-buffered saline - vSMC - vascular smooth muscle cell - ACTA2 - anti alpha smooth muscle actin - CNN1 - calponin 1 - GO - gene ontology - MYH11 - myosin heavy chain 11 - TAGLN - transgelin - ECM - extracellular matrix