Generating iPSC-Derived Smooth Muscle Cells

Human undifferentiated iPSCs reprogrammed from skin fibroblasts of a WBS patient (WS1-iPSC line C; refs. 65 (link), 66 (link)), an ELN-mutant nonsyndromic SVAS patient (ELN1; ref. 66 (link)), and a control human (CT2; ref. 66 (link)) were used to generate iPSC-SMC progenitor cell lines via an embryoid body stage (65 (link), 66 (link)). The age and sex of iPSC donors are provided in Supplemental Table 2. As described previously (67 (link)), the SMC progenitor cells were expanded in Matrigel-coated 6-well plates in smooth muscle growth medium (Medium 231, growth supplement) (Life Technologies). Cells were passaged when they reached 80% to 90% confluence. To induce differentiation, the iPSC-SMC progenitors were trypsinized and plated in smooth muscle differentiation medium (Medium 231, differentiation supplement) (Life Technologies) on 0.1% gelatin–coated 6-well plates for 6 days. RNA was isolated before and after SMC differentiation and utilized for qRT-PCR analysis of SMC markers. Protein lysates from the differentiated iPSC-SMCs were collected and utilized for Western blotting.

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Dave J.M., Chakraborty R., Ntokou A., Saito J., Saddouk F.Z., Feng Z., Misra A., Tellides G., Riemer R.K., Urban Z., Kinnear C., Ellis J., Mital S., Mecham R., Martin K.A, & Greif D.M. (2022). JAGGED1/NOTCH3 activation promotes aortic hypermuscularization and stenosis in elastin deficiency. The Journal of Clinical Investigation, 132(5), e142338.

Publication 2022

Medium 231

Cell body Cells Donors Fibroblasts Gelatin Growth plates Human Ipsc Matrigel Patient Progenitor cells Protein Skin Smooth muscle Supplement Svas

Corresponding Organization : Cardiovascular Research Center

Other organizations : Yale University, Stanford University, University of Pittsburgh, Institut thématique Génétique, génomique et bioinformatique, Hospital for Sick Children, Washington University in St. Louis

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Variable analysis

independent variables

Reprogrammed iPSC lines from skin fibroblasts of a WBS patient (WS1-iPSC line C), an ELN-mutant nonsyndromic SVAS patient (ELN1), and a control human (CT2)

dependent variables

Differentiation of iPSC-SMC progenitor cell lines
Expression of SMC markers (measured by qRT-PCR)
Protein expression (measured by Western blotting)

control variables

Culture conditions (Matrigel-coated 6-well plates, smooth muscle growth medium, and smooth muscle differentiation medium)
Passage of cells (80-90% confluence)
Duration of differentiation (6 days)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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