Development of a human VSMC in vitro platforms for SVEP1 vasoconstrictive investigations
A limiting factor in smooth muscle contraction experiments is the loss of membrane channels and GPCRs within days of culturing following tissue extraction25-27. To overcome this issue, we developed a human iPSC-derived vascular smooth muscle cells (iVSMC) model with iPSCs differentiated into a mesodermal phenotype as a monolayer prior to differentiation into specialised VSMC phenotype28. iPSC pluripotency gene expression is stopped by day 4 (SF. 5B, 1&5). Cells differentiate into primitive streak cells (days 2-4, SF. 5B, 2&6) and mesodermal progenitors (days 3-6, SF. 5B, 3), with 94% of cells CD140+ at day 8 (SF. 5B, 7). After a further 12 days culture in TGFβ and PDGF supplemented media, the iVSMCs express a panel of smooth muscle contractile markers (SF. 5C), reliably physically contract a collagen gel (SF. 5D, 1), and display an increase in [Ca2+]i in response to a panel of GPCR vasoconstrictors (SF.5D, 2) compared to the limited contractile responses seen in cultured primary human VSMCs (SF. 5D, 3).
To interrogate the role of SVEP1 integrin α4β1 and α9β1 in VSMC contraction we used 2 complimentary methods. Gene expression ofSVEP1 , ITGA9 and ITGA4 were knocked down using siRNA in differentiated iVSMCs. We achieved a knockdown efficiency between 60-90% at the RNA level, with protein knockdown confirmed for integrin α4 and α9 by western blotting and immunofluorescence, and SVEP1 by immunofluorescence alone (SF. 6). We were unable to detect a reliable band of the correct molecular weight to reliably quantify SVEP1 protein expression. In addition to siRNA depletion of SVEP1 , we generatedSVEP1-/- knockout iPSCs using CRISPR-Cas9 which contain a 1 base pair deletion at position 130 in the coding sequence within exon 1 of SVEP1 (SF.7). This isogenic pair of iPSCs were then differentiated into iVSMCs and used in calcium release experiments.