From Stem Cell to Cardiomyocyte: Differentiation and Evaluation of Human Induced Pluripotent Stem Cell Cardiomyocytes
Human induced pluripotent stem cells (hiPSC) are a revolutionary technology that, for the first time, offer a renewable source of human cells in a dish that retain the genetic background from the patient from which they were derived. hiPSCs can be differentiated to cardiomyocytes with high fidelity, which serves as a way to study human cardiac disease as animal models do not fully recapitulate human disease and human cardiac tissue is available only in limited quantities. While hiPSC-CMs are relatively immature, they function as an important way to model human cardiac disease.
In this study, my objective was to utilize hiPSC to generate hiPSC-cardiomyocytes (hiPSC-CMs) using a 2D small molecule differentiation protocol, that mimics the cues in cardiac development, to generate hiPSC-CMs and evaluate relevant cardiac proteins that include those in the sarcomere, calcium handling apparatus, cytoskeleton, and mitochondria.
I successfully used control hiPSCs to generate hiPSC-CMs utilizing a 2D small molecule differentiation protocol that modulates the WNT signaling pathway. The hiPSC-CMs started beating in the dish at day 18, while this protocol typically results in beating hiPSC-CMs at day 8-12 of differentiation. At day 30 of differentiation I utilized flow cytometry to assess the percentage of cardiac troponin T, a sarcomeric marker that defines CMs. In the two wells that I assessed, I identified that there were 35.4% and 58.5% cardiac troponin T positive cells, which reflects the percentage of hiPSC-CMs generated. Subsequently, I performed immunofluorescence for cTnT (sarcomere), MLC2v (sarcomere), SERCA (calcium handling), PLB (calcium handling), RYR2 (calcium handling), CasQ (calcium handling), SLN (calcium handling), Mfn2 (mitochondria), MCU (mitochondria), Dys (cytoskeletal) I identified that these important cardiac proteins are expressed in the hiPSC-CMs at day 110 of differentiation.
In summary, I was able to utilize a 2D small molecule differentiation protocol to generate hiPSC-CMs. I demonstrated that CMs were generated through flow cytometry and immunofluorescence. These results are important for future studies which will include assessing the maturity of hiPSC-CMs compared to adult human tissue and for cardiac disease modeling.