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An Induced Pluripotent Stem Cell Model of Cell-ECM Interactions During Heart Development in Down Syndrome Rachel S

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An Induced Pluripotent Stem Cell Model of Cell-ECM Interactions During Heart Development in Down Syndrome Rachel S An Induced Pluripotent Stem Cell Model of Cell-ECM Interactions During Heart Development in Down Syndrome Rachel S. Reeser1, Alyssa K. Salazar1, Jessica McPhee1, Kendra M. Prutton2, Jeffrey G. Jacot, PhD1 1Department of Bioengineering, 2Department of Pharmacology, University of Colorado | Anschutz Medical Campus, Aurora, Colorado, USA. Introduction Results Conclusions Nearly 50% of individuals born with Down Characterization of Down Syndrome and Control iPSC and iPSC-CM 1. iPSC characterization confirmed presence of extra st syndrome (DS), also known as trisomy 21, A Control iPSC-CM B Down Syndrome iPSC-CM 21 chromosome in DS iPSC and increased are born with a congenital heart defect A cTnT DAPI relative expression of COL6A1/2 in DS iPSC-CM (CHD)1. The most common type of CHD in DS is a septal defect, where a hole develops in the 2. Proliferation of Down syndrome iPSC-CM septum between the upper and/or lower decreases as COLVI concentration increases. chambers of the heart (Fig 1). Despite the high Since COL6A1/2 genes are upregulated in Down frequency of DS-CHD, the underlying Fig 1: Left to right syndrome iPSC-CM, increased COLVI expression in mechanisms dysregulating cardiac septation in shunting of blood cTnT the endocardial cushions could hinder cardiac cell through a ventricular DAPI Down syndrome remain unknown septal defect proliferation during heart development, predisposing *** An investigation into partial trisomy 21 cases with CHD **** the DS fetus to a septal defect. B C revealed a narrowed candidate region on chromosome 21 for DAPI OCT3/4 TRA-1-81 DAPI SSEA-4 DAPI 3. iPSC-CM from Down syndrome exhibit decreased increased DS-CHD risk, encoding for type VI collagen migratory ability on COLVI compared to control – (COLVI) and the Down Syndrome Cell Adhesion Molecule iPSC Control suggesting that altered migration of DS cardiac cells (DSCAM). When upregulated in Down syndrome, these genes have the potential to alter cardiac cell-ECM interactions by on COLVI could increase risk of a septal defect. influencing the microenvironment in which cells adhere, DS DS Clinical Relevance: Individuals with Down syndrome iPSC migrate and proliferate to form the septum. Indeed, studies are 2000x more likely to develop a septal heart defect. have found that cell seeding of DS fibroblasts on COLVI Identification of altered cell-ECM interactions that increases cell-ECM adhesion compared to other ECM Fig. 3: Characterization of Down syndrome increase risk of CHDs in Down syndrome could inform substrates2. and control iPSC lines. (A) Representative Fig. 4: Characterization of Down syndrome and Control image of G-banding karyotype analysis of DS future studies and therapies to treat and prevent CHD in Therefore, I hypothesize that upregulation of COLVI and iPSC-CM. Immunofluorescent staining of (A) control and (B) iPSC (B) Representative immunofluorescent this population. DSCAM alters cardiac cell adhesion, migration and Down syndrome iPSC-CM using cardiac troponin T, cTnT, staining of pluripotency markers OCT3/4 proliferation, ultimately predisposing the developing heart (orange) and nuclear stain, DAPI (blue). (C) Relative expression (green, nuclear marker), TRA-1-81 (green, to a septal defect. To test this hypothesis, cell-ECM of cardiac and COLVI genes of control (dots) and DS (grey) surface marker), SSEA-4 (orange) and nuclear interactions were investigated using an iPSC-based model iPSC-CM using RT-qPCR. Bars represented as mean with SEM. stain DAPI (blue) of control and DS iPSC. * p< 0.05, *** p<0.0001, **** p<0.00001 Future Work Cell-ECM Interactions: Proliferation and Migration on Type VI Collagen Mechanotransductive Response to Approach MGEL COL1 FN 10COLVI * Biomechanical Stimuli A B EdU EdU 1. Quantify DS and Control iPSC-CM cell-cell and cell-ECM cTnT cTnT DAPI CM Down syndrome iPSC Model of - DAPI adhesion on COLVI using Atomic Force Microscopy protocols previously established by our lab Control Cell-ECM Interactions iPSC 2. COLVI upregulation may stiffen the surrounding ECM of Renal-derived Methodsurine epithelial Reprogramming Induction of Differentiate cardiac tissue and alter the mechanotransductive response to cells Factors Pluripotency to CM CM mechanical stimuli. Therefore, future obJectives will utilize this - Klf4 DS DS Lin28 iPSC-CM model to identify potentially dysregulated Oct4 Cardiomyocytes iPSC Nanog Induced pluripotent Epithelial Cells c-Myc mechanotransductive pathways during cardiac septation in Sox2 stem cells Down syndrome: Individual with Fig. 5: Proliferation of Down syndrome iPSC-CM on ECM components. (A) Representative images of proliferation over Down syndrome 48h using immunofluorescent staining of EdU (green), cTnT (orange), and DAPI (blue) of DS iPSC-CM on ECM components. Images on 25COLVI, 50COLVI, 100COLVI not shown. (B) Quantitative analysis of DS and control iPSC-CM proliferation over 48h. MGEL: matrigel; COL1: type I collagen; FN: fibronectin; 10COLVI: 10ug/mL type VI collagen; 25COLVI: 25ug/mL; 50COLVI: 50ug/mL; 100COLVI: 100ug/mL. Bars represent mean with SEM; * indicates p < 0.05 A 0 hours 24 hours 48 hours 72 hours B C * CM - Control Fig. 2: iPSC-Derived Cardiomyocyte Model of Cell-ECM iPSC Fig. 7: Control and DS iPSC-CM Response to Biomechanical Interactions during Heart Development. Robust age- and Stimuli on COLVI. After exposure to increased substrate stiffness sex-matched lines from male and female individuals with and * CM * and mechanical stretch whilst seeded on COLVI, cardiac cells will be without DS were given by the Linda Crnic Institute for Down - DS DS used to measure changes in gene expression, migration, syndrome and Gates Center for Regenerative Medicine. iPSC iPSC proliferation, and hypertrophy. Innate differences in the response to were differentiated into beating cardiomyocytes (iPSC-CM) biomechanical stimuli could underlie the pathogenesis of DS-CHDs. using a small molecule, monolayer based protocol. DS and Fig. 6: Migration of Down syndrome and control iPSC-CM on ECM Components. (A) Representative brightfield FUNDING, ACKNOWLEDGEMENTS AND REFERENCES control iPSC-CM are used to detect differences in migration images of DS and control iPSC-CM migration over 72 hours following scratch. Data analyzed from live-imaging This research was supported by NIH T32 HL072738-17S1 funding. iPSC lines and proliferation on COLVI and other ECM components present techniques rendered (B) displacement (um) and (C) mean velocity (um/hour) of DS (black) and control (grey) iPSC-CM. were given by the Linda Crnic Institute and Gates Center for Regenerative during cardiac septation. MGEL: matrigel (a control); COL1: type I collagen; FN: fibronectin; COLVI: type VI collagen. Medicine References: 1. Freeman, S. B. et al. Am. J. Med. Genet. (1998); 2. Bars represent mean with SEM; * indicates p < 0.05; Jongewaard, I. N. et al. Am. J. Med. Genet. (2002).
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