Human Induced Pluripotent Stem Cell-Derived Non-Cardiomyocytes Modulate Cardiac Electrophysiological Maturation Through Connexin 43-Mediated Cell-Cell Interactions
| Autor: | Frank J. Secreto, Daniel J. Clemens, Timothy J. Nelson, Sherri M. Biendarra-Tiegs |
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| Rok vydání: | 2020 |
| Předmět: |
0301 basic medicine
Cell type Patch-Clamp Techniques induced pluripotent stem cells cardiac Cell Population Action Potentials Fluorescent Antibody Technique Gene Expression Connexin Cell Communication Biology Small hairpin RNA 03 medical and health sciences Paracrine signalling 0302 clinical medicine Original Research Reports health services administration medicine Humans Myocytes Cardiac Induced pluripotent stem cell education Cells Cultured health care economics and organizations education.field_of_study cell interactions Cell Differentiation differentiation Cell Biology Hematology Phenotype Electrophysiological Phenomena Cell biology 030104 developmental biology medicine.anatomical_structure Connexin 43 Culture Media Conditioned Female 030217 neurology & neurosurgery Developmental Biology |
| Zdroj: | Stem Cells and Development |
| ISSN: | 1557-8534 1547-3287 |
| DOI: | 10.1089/scd.2019.0098 |
| Popis: | The functional maturation status of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has a notable impact upon their use in pharmacological studies, disease modeling, and therapeutic applications. Non-cardiomyocytes (non-CMs) produced in the differentiation process have previously been identified as having an extrinsic influence upon hiPSC-CM development, yet the underlying mechanisms are not fully understood. Herein, we aimed to modulate electrophysiological properties of hiPSC-CMs within co-cultures containing varied proportions of non-CMs and investigate the nature of interactions between these different cell types. Therefore, we sorted cardiac differentiations on day 10 and subsequently replated the cells at ratios of 7:3, 1:1, 3:7, and 1:9 non-CMs to CMs. After a month of co-culture, we evaluated electrophysiological properties through the genetically encoded voltage indicator ArcLight. We ultimately identified that co-cultures with approximately 70%–90% CM purity demonstrated the highest action potential (AP) amplitude and maximum upstroke velocity by day 40 of differentiation, indicative of enhanced electrophysiological maturation, as well as more ventricular-like AP morphologies. Notably, these findings were distinct from those observed for co-cultures of hiPSC-CMs and dermal fibroblasts. We determined that the co-culture phenotypes could not be attributed to paracrine effects of non-CMs due to the inability of conditioned media to recapitulate the observed effects. This led to the further observation of a distinctive expression pattern of connexin 43 (Cx43) at cell-cell interfaces between both CMs and non-CMs. Depletion of Cx43 by short hairpin RNA (shRNA) specifically in the non-CM population within a co-culture environment was able to recapitulate electrophysiological phenotypes of a purer hiPSC-CM population. Collectively, our data demonstrate that abundant non-CM content exerts a significant negative influence upon the electrophysiological maturation of hiPSC-CMs through Cx43-mediated cell-cell-contacts, and thus should be considered regarding the future production of purpose-built hiPSC-CM systems. |
| Databáze: | OpenAIRE |
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