Jiao Jiao1, Weihua Tian1, Ping Qiu1, Elizabeth L Norton1, Michael M Wang2, Y Eugene Chen1, Bo Yang3. 1. Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich. 2. Department of Neurology, Michigan Medicine, Ann Arbor, Mich; VA Ann Arbor Healthcare System, Ann Arbor, Mich. 3. Department of Cardiac Surgery, Michigan Medicine, Ann Arbor, Mich. Electronic address: boya@med.umich.edu.
Abstract
OBJECTIVE: The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. METHODS: The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1-/- (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1-/- and wild type cells. RESULTS: The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1-/-NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1-/- NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1-/-CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1-/-ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. CONCLUSIONS: NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve.
OBJECTIVE: The NOTCH1 gene mutation has been identified in bicuspid aortic valve patients. We developed an in vitro model with human induced pluripotent stem cells (iPSCs) to evaluate the role of NOTCH1 in smooth muscle and endothelial cell (EC) differentiation. METHODS: The iPSCs were derived from a patient with a normal tricuspid aortic valve and aorta. The NOTCH1 gene was targeted in iPSCs with the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 nuclease (Cas9) system. The NOTCH1-/- (NOTCH1 homozygous knockout) and isogenic control iPSCs (wild type) were differentiated into neural crest stem cells (NCSCs) and into cardiovascular progenitor cells (CVPCs). The NCSCs were differentiated into smooth muscle cells (SMCs). The CVPCs were differentiated into ECs. The differentiations of SMCs and ECs were compared between NOTCH1-/- and wild type cells. RESULTS: The expression of NCSC markers (SRY-related HMG-box 10 and transcription factor AP-2 alpha) was significantly lower in NOTCH1-/-NCSCs than in wild type NCSCs. The SMCs derived from NOTCH1-/- NCSCs showed immature morphology with smaller size and decreased expression of all SMC-specific contractile proteins. In NOTCH1-/-CVPCs, the expression of ISL1, NKX2.5, and MYOCD was significantly lower than that in isogenic control CVPCs, indicating impaired differentiation from iPSCs to CVPCs. The NOTCH1-/-ECs derived from CVPCs showed significantly lower expression of cluster of differentiation 105 and cluster of differentiation 31 mRNA and protein, indicating a defective differentiation process. CONCLUSIONS: NOTCH1 is critical in SMC and EC differentiation of iPSCs through NCSCs and CVPCs, respectively. NOTCH1 gene mutations might potentially contribute to the development of thoracic aortic aneurysms by affecting SMC differentiation in some patients with bicuspid aortic valve.
Authors: Maximilian W Schenk; Sam Humphrey; A S Md Mukarram Hossain; Mitchell Revill; Sarah Pearsall; Alice Lallo; Stewart Brown; Samuel Bratt; Melanie Galvin; Tine Descamps; Cong Zhou; Simon P Pearce; Lynsey Priest; Michelle Greenhalgh; Anshuman Chaturvedi; Alastair Kerr; Fiona Blackhall; Caroline Dive; Kristopher K Frese Journal: Nat Commun Date: 2021-11-17 Impact factor: 14.919