Using induced pluripotent stem cells to investigate elastic fiber assembly

National Heart, Lung, And Blood Institute

Investigators

Abstract

We have developed induced pluripotent stem cell (iPS) lines from fibroblasts and peripheral blood cells obtained from elastin-deficiency patients and healthy individual. Currently, we are working to generate iPS-derived vascular smooth muscle cells (iVSMC) from these lines using chemically defined and serum free media. The differentiated cells remain relatively immature and express SMA, SM22a, SMEMb, and calponin, but do not express additional maturity markers like MYH11. As in human and mouse tissues, elastin is expressed by our cell lines for only a brief period, with initial elastin mRNA expression on day 2 and peaking on day 3 of differentiation and disappears by day 5. Elastin protein was detectable in cells by day 5 and in the matrix in robust quantities by day 9. Additionally, the protein appeared to remain within the cell for a prolonged time, not entering the matrix until other elastic fiber assembly genes such as FBLN4, FBLN5 and LOX are up-regulated. Our study first demonstrated that iPS-derived vascular smooth muscle cells expressed ELN in early phase of differentiation, while ELN deposition machinery was initiated by the presence of other elastic fiber assembly genes. Single cell RNA-seq study further confirms the transition of TBXT+ mesoderm cells to SM22a+ iVSMC from day 2 to day 3 of differentiation. More than 90% of elastin-expressing cells co-express SM22a at day 3 (ELN+/SM22a+) revealing they are of smooth muscle origin. Gene enrichment analysis demonstrates an increased level of genes involved in signal-recognition particle (SRP)-dependent protein-membrane targeting as well as extracellular matrix generation in ELN+/SM22a+ cells. Interestingly, principal component analysis on differentiated cells from Day 1, Day 2 and Day3 revealed unique populations of cells between each time points, i.e. there were minimal intermediate cells and within each time point, the cells were very homogenous. In order to identify the unique gene expression profile of ELN-expression cells in our platform, we are currently performing numerous bioinformatic analysis by comparing the expression profile of ELN-expressing cells and ELN negative cells in each time point we studied. Until today, we have identified 21 genes that are unique in ELN-expressing cells at Day 3 of differentiation. Concurrently, we are developing an organoid-based method to study the functional deficiency and pathological mechanism of ELN-deficient iPSC-derived SMCs. We have successfully optimized our unique protocol with the adoption of the same base serum-free media used in our 2d differentiation culture. These 20-day old organoids were transplanted into immunocompromised mice and survived through 10 weeks. The research is still ongoing. We are hoping to generate SMC-surrounded arterioles and arteries in these transplanted organoids. With the use of ELN-deficient iPSCs, any functional deficiency and potentially its mechanism can be elucidated using this organoid-based in vivo model.