Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification

Abstract

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

Keywords: calcification; cardiovascular diseases; induced pluripotent stem cells; proteomics and bioinformatics; smooth muscle cells (SMCs); stem cell differentiation and reprogramming.

FIGURE 1

Differentiation timeline of iPSCs into iSMCs. (A) Schematic of the differentiation timeline and protocol. (B) Brightfield microscopy images of the differentiation time course from iPSCs to mesoderm and then to iSMCs (magnification = 20×). Representative images of n = 3 differentiation replicates.

FIGURE 2

Characterization of iPSCs at day 0 and iSMCs at day 10. (A) Brightfield microscopy image (20× magnification) of iPSC colony morphology, and immunofluorescent staining (20× magnification) for DAPI (blue), pluripotency marker NANOG (green), and merged. (B) Flow cytometry analysis on iPSC pluripotency marker TRA-1-60; left side (blue dots) representative flow cytometry analysis, right side (pink dots) flow cytometry detected percentage of stem cell marker positive cells. (C) Brightfield microscopy of iSMCs at day 10. Immunofluorescent staining for DAPI (blue), αSMA (red), MYH11 (white) and merged. (D) Flow cytometry analysis on iPSC marker TRA-1-60, EC marker CD31, and iSMC marker CD140b; left side representative flow cytometry analysis, right side flow cytometry detected percentage of stem cell marker, EC marker, and SMC marker. n = 3 differentiation replicates; mean ± SEM; scale bars = 100 μm (20× magnification).

FIGURE 3

Immunocytochemistry on iPSC markers and SMC markers during differentiation. (A) iPSC markers NANOG (red) and OCT4 (green) with DAPI (blue) shows that OCT4 expression is maintained throughout the differentiation time course. (B) SMC markers αSMA (red) and MYH11 (green) with DAPI (blue) shows that by day 7 the cells express high levels of SMC marker proteins that persists through day 10. A total of 10× magnification, scale bars = 100 μm; representative images.

FIGURE 4

High-dimensional cluster analysis of the proteome describing the iPSC to iSMC transition. (A) K-means clustering analysis with 20 clusters describing the combined time-resolved protein abundances of n = 3 differentiation replicates. (B) Condition composition of the clusters shows equal distribution of each differentiation replicate in each cluster: yellow = replicate 1, pink = replicate 2, purple = replicate 3. (C) Expression of top 10 shared proteins between experimental replicates at each time point (day 0 = cluster 14; day 3 = cluster 3; day 5 = cluster 20; day 7 = cluster 5; day 10 = cluster 10) normalized by time point. String analysis on the shared proteins of each cluster was used to analyze GO terms.

FIGURE 5

Functional enrichment of day-specific proteins accurately maps differentiation trajectory. Proteins were identified as day-specific with increase z-score abundance and function enrichment analysis was performed showing biological processes active on each day: Day 0, Day 3, Day 5, Day 7, and Day 10. (A) Day 0 major nodes include ribose phosphate and ribonucleotide metabolic processes. (B) Day 3 major nodes include RNA processing and Wnt signaling. (C) Day 5 nodes include chromatin assembly, disassembly, and remodeling. (D) Day 7 nodes include cornification and mitochondrion localization. (E) Day 10 nodes include extracellular matrix organization, regulation of cell morphogenesis, cell junction assembly, and cell-substrate adhesion.

FIGURE 6

Day-to-day differential protein abundance analysis. Volcano plots of increased of previous day (shown on right with positive log fold change) vs. adjacent day (shown on left with -negative log fold change). (A) Day 0 vs. Day 3, (B) Day 3 vs. Day 5, (C) Day 5 vs. Day 7, (D) Day 7 vs. Day 10, (E) Day 0 vs. Day 10. (F) Bar chart showing the functional enriched processes on Day 0 and Day 10.

FIGURE 7

Induced-smooth muscle cells (iSMCs) calcify under osteogenic media (OM) conditions. (A) Study design and Alizarin Red S staining on iSMCs cultured in normal media (NM) and osteogenic media (OM) during three independent differentiations on Days 10 and 17. Bright red staining under OM conditions represents calcification in OM while no staining is observed in NM indicative of no calcification. (B) Study design and representative images of Alizarin Red S staining on primary SMCs cultured in normal media (NM) and osteogenic media (OM). (C) Pearson’s correlation between mean protein abundance between iSMCs and primary SMCs cultured with and without the inclusion of the protein vimentin in the analyses under NM. (D) Similar plot under OM. (E) Pearson correlations of iSMCs with primary SMCs, without the inclusion of the protein vimentin at days 10 and 17 in NM and OM.