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Generation of Novel Human Induced Pluripotent Stem Cell lines to study Proteinopathy in Cardiomyocytes

Semester: Summer 2023

Presentation description

Cardiac proteinopathy is a conformational disorder which results in accumulation of misfolded mutant proteins in the heart. Crystallin (CryAB) is a small heat shock protein which acts as a chaperone and participates in intermediate filament formation and maintenance. Missense mutation in CryAB, R120G, causes the formation of dense protein aggregates and is linked to familial desminopathy. Accumulation of R120G causes progressive muscle weakness, cataracts, and cardiac disorders leading to disability and death. Human Induced Pluripotent Stem cells (hiPSC) sourced from human fibroblasts, can reprogram into new cell types under appropriate growth conditions. This ethically sourced approach was chosen over other models as hiPSC derived cardiomyocytes (hiPSC-CM) can recapitulate human disease phenotypes. CryAB wild-type (WT) and R120G mutant genes were knocked-in to the genome of hiPSCs then cultured and differentiated into cardiomyocytes using stem cell differentiation media over a 24-day period. Specifically, cells were grown in vitronectin-coated plates and maintained in Essential 8 and Stem Flex media. HiPSC-CMs were treated with doxycycline to induce gene expression. Quantitative pcr (qPCR) was used to validate differentiation into cardiomyocytes by determining the mRNA expression of several cardiomyocyte-related genes (i.e., Tbx5, Actn1,Cryab, Hcn4, etc.) Next, immunocytochemistry was employed to determine the levels of the induced proteins: crystallin and mCherry. QPCR results show increased expression of cardiomyocyte markers in the differentiated cells vs. the non-differentiated cells. Confocal imaging revealed increased expression of both mCherry and CryAB aggregates in mutant R120G line but not in WT cells. While WT cell lines indicated increased expression of both mCherry and CryAB, the extent of protein expression was lower in the mutant line. Ongoing experiments involve culturing mutant lines in Matrigel-coated plates to achieve higher expression of proteins. HiPSC's will serve as an important tool in the study of clearing mutant induced aggregates.

Presenter Name: Taylor Marie James

Presentation Type: Poster
Presentation Format: In Person
Presentation #15
College: Health
School / Department: Nutrition and Integrative Physiology
Research Mentor: Rajeshwary Ghosh
Date | Time: Thursday, Aug 3rd | 10:30 AM