Differentiation of Human Induced-Pluripotent Stem Cells into Smooth-Muscle Cells: Two Novel Protocols

• Published in: PloS one 2016-01, Vol.11 (1), p.e0147155-e0147155
• Main Authors: Yang, Libang, Geng, Zhaohui, Nickel, Thomas, Johnson, Caitlin, Gao, Lin, Dutton, James, Hou, Cody, Zhang, Jianyi
• Format: Article
• Language: English
• Subjects: Actin; Assessments; Biomedical engineering; Calcium-Binding Proteins - genetics; Calponin; Calponins; Carbachol; Cardiology; Cell Culture Techniques; Cell Differentiation - genetics; Cell Differentiation - physiology; Cell migration; Collagen; Collagen Type I - genetics; Connexin 43; Connexin 43 - genetics; Coronary vessels; Cytometry; Differentiation; Epigenetics; Fibroblasts; Flow cytometry; Genotype & phenotype; Humans; Induced Pluripotent Stem Cells - cytology; Induced Pluripotent Stem Cells - metabolism; Insulin; Intermediate filament proteins; Medical schools; Medicine; Metabolism; Microfilament Proteins - genetics; mRNA; Muscle contraction; Muscle proteins; Muscle Proteins - genetics; Muscles; Myocytes, Smooth Muscle - cytology; Myocytes, Smooth Muscle - metabolism; Myosin; Myosin Heavy Chains - genetics; Physiology; Pluripotency; Populations; RNA; Smooth muscle; Stem cells; Tissue engineering; Vascular endothelial growth factor; Vimentin; Vimentin - genetics
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0147155

Conventional protocols for differentiating human induced-pluripotent stem cells (hiPSCs) into smooth-muscle cells (SMCs) can be inefficient and generally fail to yield cells with a specific SMC phenotype (i.e., contractile or synthetic SMCs). Here, we present two novel hiPSC-SMC differentiation protocols that yield SMCs with predominantly contractile or synthetic phenotypes. Flow cytometry analyses of smooth-muscle actin (SMA) expression indicated that ~45% of the cells obtained with each protocol assumed an SMC phenotype, and that the populations could be purified to ~95% via metabolic selection. Assessments of cellular mRNA and/or protein levels indicated that SMA, myosin heavy chain II, collagen 1, calponin, transgelin, connexin 43, and vimentin expression in the SMCs obtained via the Contractile SMC protocol and in SMCs differentiated via a traditional protocol were similar, while SMCs produced via the Sythetic SMC protocol expressed less calponin, more collagen 1, and more connexin 43. Differences were also observed in functional assessments of the two SMC populations: the two-dimensional surface area of Contractile SMCs declined more extensively (to 12% versus 44% of original size) in response to carbachol treatment, while quantification of cell migration and proliferation were greater in Synthetic SMCs. Collectively, these data demonstrate that our novel differentiation protocols can efficiently generate SMCs from hiPSCs.