Abstract
Understanding the current source-sink relationship between fibroblasts (FBs) and hiPSC-derived pacemaking cardiomyocytes (PCMs) is essential to create the appropriate niche for PCMs to properly function in cardiac tissue. It is unclear how fibroblasts support pacemaking functions or if they serve as a current sink, an insulator or a conductor. A tissue construct fabricated in vitro could be used as a model to study the current-source sink relationship between the two cell types. To engineer such tissue construct, our first goal was to test the proliferation of human induced pluripotent stem cell (hiPSC)-derived PCMs as an alternative to frequent differentiation by following the cardiomyocyte (CM) proliferation protocols designed for contractile CMs. This would improve the production of hiPSC-PCMs in cost and time without starting with hiPSCs each time. Resulting CMs were assessed for the cell type yield using flow cytometry using PCM marker of hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and CM marker of troponin T (TnT). I also immunostained to determine the phenotype using antibodies also targeting these markers in addition to a proliferative marker of Ki67. Our results have indicated that extracellular matrix (ECM), Matrigel, in conjunction with small molecule Wnt activator, CHIR 99021, best promotes cell proliferation in hiPSC-PCMs. The induced hiPSC-PCMs demonstrated a higher Ki67 expression compared to their controls, while inducing an immature phenotype in the cells that is similar to the reported response for contractile hiPSC-CMs.
Our second goal was to optimize bioprinting biopacemakers to further investigate the relationship between the two major cell types in the sinoatrial node (SAN). A tissue model with hiPSC-FBs, a current sink, and hiPSC-PCMs, a current source, was bioprinted to a desired dimension reflecting the porcine SAN. To demonstrate the distribution of PCM and FB clusters, the printed construct was immunostained using antibodies targeting HCN4, TnT, and vimentin (Vim) to assess cell placement. To ensure proper print, line widths of bioinks and cell densities were measured to determine the print resolution for our desired template. Our results demonstrate the settings that generated our desired constructs with some inconsistencies of line-to-line cell extrusion from the BIO X printer. Further, our study shed light on various complications associated with bioprinting that need further troubleshooting. This study is a first step in reproducing the desired organization of the cell types for studying cell-cell interactions in the maintenance of pacemaking function in engineered biopacemakers as well as the native pacemaking tissue in humans.