Investigation of developmental cardiac extracellular matrix on cardiac fibroblasts (CF) compaction and induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) differentiation and maturation
Chen, Gina.
2020
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Thesis (M.S.)--Tufts
University, 2020.
Submitted to the Dept. of Biomedical Engineering.
Advisor: Lauren Black.
Committee: David Kaplan, and James Van Deventer.
Keywords: Biomedical engineering, and Cellular biology.
Cardiac fibroblasts (CFs) and cardiomyocytes (CMs) are the two major cardiac cell types within the heart and both are greatly affected ... read moreduring and after myocardial infarction (MI). CFs take on an active and dynamic role in changes within the microenvironment. When CFs are activated to migrate to the injury site, they proliferate, breakdown the existing extracellular matrix (ECM) and deposit new ECM in the affected area. Generally, this leads to a highly collagenous scar tissue, which increases stiffness within the heart, preventing proper mechanical and electrical function. Studies have found that the activation of CFs to myofibroblasts may feature an intermediate step. These findings also suggest that the surrounding microenvironment may provide insight into CF activation and, thus, strategies for the prevention of cardiac fibrosis. CMs on the other hand are responsible for generating involuntary contractions and beating within the heart. Throughout the course of cardiac development, they lose their ability to proliferate. Thus, in the case of adults (but not necessarily in younger developmental ages), cardiac injury causes the death of these cells, which in turn negatively impacts the hearts ability to contract. To restore contractile function to the heart, induced pluripotent stem cells (iPSC) have proven to be an attractive autologous cell source and have been the focus of significant recent research. Differentiation of iPSC to iPSC-CMs has been successful; however, these cells tend to exhibit an immature, fetal-like phenotype. Previous research has shown that the compositional difference in developmental aged cardiac ECM (cECM) can impact not only the differentiation of iPSCs but also aid in maturation. We hypothesize that the developmental age of cECM can influence CF activation, as well as the differentiation and maturation of iPSC-CMs. To investigate CF activation and iPSC differentiation and maturation, we utilized a 3D fibrin hydrogel system combined with native cECM from fetal and adult hearts that provides a physiologically relevant in vitro model. For the CF studies, our results demonstrated a significant difference in compaction of the hydrogels with the developmental age of ECM incorporated. Analysis of Ki67 and alpha smooth muscle actin (αSMA) expression between developmental aged cECM groups showed some significance that may help to explain the mechanism for the differences in compaction; however, future studies should aim to look at markers other than αSMA in understanding CF activation. As for the impact of developmental aged cECM influence on differentiation and maturation of iPSC-CMs, conclusions cannot be drawn due to low biological replicates related to COVID-19 lab shutdown. However, we did find that in the samples analyzed, pluripotency of undifferentiated iPCS can be measured using our described methods. All in all, these studies indicate that developmental age of cardiac ECM has effects on both CFs and stem-cell derived CMs, which has implications for future ECM-based methods of cardiac repair.read less - ID:
- wp9890103
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