PB1302 - Lineage specific vascular smooth muscle cell differentiation from pluripotent stem cells: A developmental bias towards aortic calcification

Background: Vascular calcification is an active process that contributes to cardiovascular disease wherein the vascular smooth muscle cell (SMC) is the protagonist to disease progression. The developmental heterogeneity of SMC may attribute to the site-specific occurrence of calcification.

Aims: We aimed to generate and characterise various lineage SMCs from human induced pluripotent stem cells (iSMCs). Next, we aimed to determine whether the various lineage iSMCs would be capable to calcify and might there be differences in calcification between the various iSMCs.

Methods: For this study we performed stepwise lineage specification to generate iSMCs. Lateral mesoderm (LM), paraxial mesoderm (PM) and neural crest (NC) are induced before iSMC specification. Then iSMCs were subjected to calcification assay on various concentrations of calcium and serum. Next, we compared the calcification potential of the various lineages under optimised conditions against each other. Thereafter, we performed bulk RNA-sequencing on various iSMCs in calcification vs control experiments.

Results: Differentiation to various lineages, characterisation of iSMC subpopulations demonstrates that cells ubiquitously express canonical SMC markers. Next, we checked functionality with regards to calcification deposition. Either lowering serum concentration or increasing total calcium levels resulted in increased calcification in all cell types, confirming both functionality and dose response. On comparing calcification of the three different sub-populations, we found that LM iSMCs calcify more and faster than the two other lineages. No difference was found between calcification of NC or PM iSMCs. Upon performing RNA-sequencing DEGs are present between lineages. Further within the PC2 there is a drift for NC and PM populations that is absent in the LM-iSMCs.

Conclusion(s): Lateral mesoderm iSMCs calcify more and faster than both paraxial mesoderm and neural crest iSMCs in our disease-in-a-dish calcification model. This confers to aortic calcification that canonically starts at the aortic root. We are currently investigating mechanism and translation of our findings.