Quantitative proteomics reveal lineage-specific protein profiles in iPSC-derived Marfan syndrome smooth muscle cells

Cristiana Iosef,Samantha M P Churovich,Mamoru Arakawa,Tiffany K Koyano,Grayson Burdon,Michael P Fischbein,Alex R Dalal,Albert J Pedroza,Joshua O Orrick,Jason Z Cui,Yasushi Tashima,Mitchel Pariani

doi:10.1038/s41598-020-77274-w

Cristiana Iosef, Samantha M P Churovich + Show 10 more

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https://doi.org/10.1038/s41598-020-77274-w

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Journal: Scientific Reports	Publication Date: Nov 23, 2020
Citations: 9	License type: open-access

Affiliation: Stanford University, Pediatrics and Genetics

Abstract

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability. The lack of ample genotype–phenotype correlation hinders translational study development aimed at improving disease prognosis. In response to this need, an induced pluripotent stem cell (iPSC) disease model has been used to test patient-specific cells by a proteomic approach. This model has the potential to risk stratify patients to make clinical decisions, including timing for surgical treatment. The regional propensity for aneurysm formation in MFS may be related to distinct smooth muscle cell (SMC) embryologic lineages. Thus, peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSC) were differentiated into lateral mesoderm (LM, aortic root) and neural crest (NC, ascending aorta/transverse arch) SMC lineages to model MFS aortic pathology. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) proteomic analysis by tandem mass spectrometry was applied to profile LM and NC iPSC SMCs from four MFS patients and two healthy controls. Analysis revealed 45 proteins with lineage-dependent expression in MFS patients, many of which were specific to diseased samples. Single protein-level data from both iPSC SMCs and primary MFS aortic root aneurysm tissue confirmed elevated integrin αV and reduced MRC2 in clinical disease specimens, validating the iPSC iTRAQ findings. Functionally, iPSC SMCs exhibited defective adhesion to a variety of extracellular matrix proteins, especially laminin-1 and fibronectin, suggesting altered cytoskeleton dynamics. This study defines the aortic embryologic origin-specific proteome in a validated iPSC SMC model to identify novel protein markers associated with MFS aneurysm phenotype. Translating iPSC findings into clinical aortic aneurysm tissue samples highlights the potential for iPSC-based methods to model MFS disease for mechanistic studies and therapeutic discovery in vitro.

Highlights

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability
To model embryologic origin-specific susceptibility to causative FBN1 mutations, induced pluripotent stem cell (iPSC) from each MFS patient and healthy controls were differentiated through lateral mesoderm (LM) and neural crest (NC) progenitor stages followed by parallel smooth muscle cell (SMC) differentiation using a validated protocol (Fig. 1A)[23]
ICC detection of nuclear myocyte-specific enhancer factor 2C (MEF2C) and NK2 Homeobox 5 (NKX2.5) at this stage confirmed LM transition to secondary heart field identity[25]. Both NC- and LM-progenitor cells were further differentiated into iPSC derived SMCs using a chemically defined medium (CDM) formula with plateletderived growth factor (PDGF) and TGF-β1 for 12 days, when both LM and NC SMCs stained positively for SMC contractile proteins smooth muscle actin (SMA), transgelin (TAGLN) and calponin 1 (CNN1)

Summary

Introduction

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability. Each embryologic origin to study vascular diseases, including aneurysm formation[10,11,12,13] This unique model affords the opportunity to identify early pathologic mechanisms in aneurysm formation in vitro, in contrast to utilizing primary SMCs derived from aortic surgical specimens after aneurysms have already developed. We further validate the iPSC system as a faithful model for MFS pathology, underscoring its potential as an “aorta in a dish” system to predict clinical severity by molecular phenotyping rather than relying on serial radiographic imaging and generalized knowledge of FBN1 mutation subtypes This model system opens the door for future use of patient-derived iPSC in precision medicine strategies to tailor treatments for individual p atients[21,22]

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