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dc.contributor Altman, Robin en_US
dc.contributor Bauer, Gerhard en_US
dc.contributor.advisor Mulligan, Kimberly en_US
dc.contributor.author Smithers, Regan
dc.date.accessioned 2021-05-17T17:25:20Z
dc.date.issued 2021-05-17
dc.date.submitted 2020-05-05
dc.identifier.uri http://hdl.handle.net/10211.3/218999
dc.description Project (M.S., Biological Science (Stem Cell))--California State University, Sacramento, 2020. en_US
dc.description.abstract Cardiovascular disease has remained the leading cause of death globally for decades. Despite great progress in basic science and clinical research, current therapies remain largely palliative and fail to address cardiomyocyte (CM) loss. The limited regenerative capacity of the human heart coupled with complex cardiovascular pathophysiology has ushered in a new era of research rooted in stem cell biology. Human induced pluripotent stem cells (hiPSCs) have emerged as a foundational avenue for regenerative cell therapy, drug discovery, and disease modeling. Their inherent ability to be expanded indefinitely and directly differentiated into CMs offers an unprecedented opportunity for treatment and research. However, current hiPSC-derived CM (hiPSC-CM) differentiation protocols yield heterogenous cultures lacking mature phenotypes. Mixed populations of atrial, ventricular, and pacemaking CMs are highly problematic for clinical studies, as they pose severe safety risks due to CM subtypes having distinct functions. Addressing these limitations is critical for successful development of safe and efficacious stem cell-derived therapeutics for cardiovascular medicine. Advances in our understanding of embryonic heart development have guided hiPSC-CM differentiation strategies. Researchers have developed small molecule- and chemical-based protocols that target key signaling pathways involved in cardiogenesis. In early differentiation, Wnt activation by small molecule, CHIR99021, or a combination of bone morphogenic protein (BMP) 4 and Activin A (Act A) have been used to promote specification of hiPSCs towards cardiac mesoderm, followed by treatment with IWR1 (Wnt inhibitor), insulin growth factor, fibroblast growth factor, retinoic acid (RA), and SB-431542 (SB; TGF-β inhibitor) to direct differentiation to the cardiac lineage. Combinations of these small molecule and biochemicals have successfully generated up to 98% CMs, but no protocol to date can derive homogenous CM subtype populations needed for clinical application. Given this encumbrance, researchers have begun to consider all aspects of cardiac development, beyond cell signaling. Recent publications have highlighted the importance of extracellular matrix (ECM) proteins and substrate mechanics in stem cell differentiation and phenotype development. Modern differentiation protocols are typically performed on rigid, polystyrene (PS) cell culture treated plates that are not representative of a physiological cellular microenvironment. Moreover, recently our lab has shown distinct differences in the ECM of the pacemaking sinoatrial node (~ 16kPa) versus the ECM of the contractile left ventricle (~ 5kPa), further suggesting possible implications of ECM properties in hiPSC-CM subtype specification. Cellular microenvironment plays an essential role in cardiac development, although its impact on hiPSC-CM fate remains largely unknown. Here, we have used a developmental-biology-guided approach to investigate the temporal effects of biochemical and biomechanical interactions on hiPSC-CM specification into pacemaking and contractile subtypes. Molecular analyses revealed that yes-associated protein (YAP), downstream effector of the Hippo pathway and important regulator of cellular mechanotransduction, is differentially expressed and localized in hiPSCs cultured on glass versus elastic substrate of physiological stiffness. Genetic characterizations indicate that a physiological microenvironment significantly enhances cardiac mesoderm specification, while altering the subtype-specific gene expression profiles of hiPSC-CMs. We further identify temporal manipulation of YAP localization through small molecule activator XMU as a potential target for exploiting substrate mechanics to drive differentiation into a desired CM subtype. Together, these findings provide unique insight into the specification of contractile and pacemaking CMs through modulation of biochemical and biomechanical cues. en_US
dc.description.sponsorship Biological Sciences en_US
dc.language.iso en_US en_US
dc.subject Stem cells en_US
dc.subject Microenvironment en_US
dc.subject Biochemical and biomechanical cues en_US
dc.subject Pacemaking and contractile CMs en_US
dc.title Small molecule-induced cardiomyocyte differentiation en_US
dc.type Project en_US
dc.description.embargoterms 1 year en_US
dc.date.embargountil 2022-05-17T17:25:20Z

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