CRISPR-Corrected iPSCs Differentiated into Stretched SMCs

Dermal fibroblasts from a patient with MFS (Coriell Institute, GM21943) were reprogrammed into human induced pluripotent stem cells (iPSCs), and the mutation was corrected by CRISPR-Cas9 to generate an isogenic control.^{26 (link)} Control and Marfan iPSCs were grown on Vitronectin XF (STEMCELL Technologies) and maintained in mTeSR E8 media (STEMCELL Technologies). They were then differentiated into neural crest SMCs using the methods described previously.^{27 (link),28 (link)} After differentiation, the resulting SMCs were maintained in DMEM supplemented with 10% fetal bovine serum for at least 2 weeks before seeding onto UniFlex Culture Plates (FlexCell International Corporation). After 4 days, the SMCs were stretched for 48 hours using a Cyclic Stress Unit (FX5000 Tension System, FlexCell International Corporation), using a cyclic sine wave (60 cycles/min) and 10% elongation.

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Yin (殷晓科) X., Wanga S., Fellows A.L., Barallobre-Barreiro J., Lu R., Davaapil H., Franken R., Fava M., Baig F., Skroblin P., Xing Q., Koolbergen D.R., Groenink M., Zwinderman A.H., Balm R., de Vries C.J., Mulder B.J., Viner R., Jahangiri M., Reinhardt D.P., Sinha S., de Waard V, & Mayr M. (2019). Glycoproteomic Analysis of the Aortic Extracellular Matrix in Marfan Patients. Arteriosclerosis, Thrombosis, and Vascular Biology, 39(9), 1859-1873.

Publication 2019

Vitronectin XF mTeSR E8 media UniFlex Culture Plates FX5000 Tension System

Crispr Fetal bovine serum Fibroblasts Human induced pluripotent stem cells Induced pluripotent stem cells Mutation Neural crest Patient Sine Stemcell Vitronectin

Corresponding Organization : King's College London

Other organizations : Amsterdam University Medical Centers, University of Amsterdam, University of Cambridge, Wellcome/MRC Cambridge Stem Cell Institute, Netherlands Heart Institute, Thermo Fisher Scientific (United States), St George's, University of London, McGill University

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Variable analysis

independent variables

Stretching of SMCs using a Cyclic Stress Unit (FX5000 Tension System, FlexCell International Corporation), using a cyclic sine wave (60 cycles/min) and 10% elongation

dependent variables

Measured outcomes after 48 hours of stretching

control variables

Fibroblasts from a patient with MFS (Coriell Institute, GM21943) were reprogrammed into human induced pluripotent stem cells (iPSCs)
The mutation was corrected by CRISPR-Cas9 to generate an isogenic control
Control and Marfan iPSCs were grown on Vitronectin XF (STEMCELL Technologies) and maintained in mTeSR E8 media (STEMCELL Technologies)
The resulting SMCs were maintained in DMEM supplemented with 10% fetal bovine serum for at least 2 weeks before seeding onto UniFlex Culture Plates (FlexCell International Corporation)

controls

Isogenic control generated by CRISPR-Cas9 correction of the mutation in Marfan iPSCs

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