Background We aim to generate a line of "universal donor" human induced pluripotent stem cells (hi PSC s) that are nonimmunogenic and, therefore, can be used to derive cell products suitable for allogeneic transplantation. Methods and Results hi PSC s carrying knockout mutations for 2 key components (β2 microglobulin and class II major histocompatibility class transactivator) of major histocompatibility complexes I and II (ie, human leukocyte antigen [HLA] I/ II knockout hi PSC s) were generated using the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene-editing system and differentiated into cardiomyocytes. Pluripotency-gene expression and telomerase activity in wild-type ( WT ) and HLAI / II knockout hi PSC s, cardiomyocyte marker expression in WT and HLAI / II knockout hi PSC -derived cardiomyocytes, and assessments of electrophysiological properties (eg, conduction velocity, action-potential and calcium transient half-decay times, and calcium transient increase times) in spheroid-fusions composed of WT and HLAI / II knockout cardiomyocytes, were similar. However, the rates of T-cell activation before (≈21%) and after (≈24%) exposure to HLAI / II knockout hi PSC -derived cardiomyocytes were nearly indistinguishable and dramatically lower than after exposure to WT hi PSC -derived cardiomyocytes (≈75%), and when WT and HLAI / II knockout hi PSC -derived cardiomyocyte spheroids were cultured with human peripheral blood mononuclear cells, the WT hi PSC -derived cardiomyocyte spheroids were smaller and displayed contractile irregularities. Finally, expression of HLA -E and HLA -F was inhibited in HLAI / II knockout cardiomyocyte spheroids after coculture with human peripheral blood mononuclear cells, although HLA -G was not inhibited; these results are consistent with the essential role of class II major histocompatibility class transactivator in transcriptional activation of the HLA -E and HLA-F genes, but not the HLA -G gene. Expression of HLA -G is known to inhibit natural killer cell recognition and killing of cells that lack other HLAs. Conclusions HLAI / II knockout hi PSC s can be differentiated into cardiomyocytes that induce little or no activity in human immune cells and, consequently, are suitable for allogeneic transplantation.

We have shown that human cardiac muscle patches (hCMPs) containing three different types of cardiac cells-cardiomyocytes (CMs), smooth muscle cells (SMCs), and endothelial cells (ECs), all of which were differentiated from human pluripotent stem cells (hPSCs)-significantly improved cardiac function, infarct size, and hypertrophy in a pig model of myocardial infarction (MI). However, hPSC-derived CMs (hPSC-CMs) are phenotypically immature, which may lead to arrhythmogenic concerns; thus, since hPSC-derived cardiac fibroblasts (hPSC-CFs) appear to enhance the maturity of hPSC-CMs, we compared hCMPs containing hPSC-CMs, -SMCs, -ECs, and -CFs (4TCC-hCMPs) with a second hCMP construct that lacked hPSC-CFs but was otherwise identical [hCMP containing hPSC-CMs, -AECs, and -SMCs (3TCC-hCMPs)].

Channelrhodopsin-2 (ChR2)-based optogenetic technique has been increasingly applied to cardiovascular research. However, the potential effects of ChR2 protein overexpression on cardiomyocytes are not completely understood. The present work aimed to examine how the doxycycline-inducible lentiviral-mediated ChR2 expression may affect cell viability and electrophysiological property of neonatal rat ventricular myocyte (NRVM) cultures. Primary NVRMs were infected with lentivirus containing ChR2 or YFP gene and subjected to cytotoxicity analysis. ChR2-expressing cultures were then paced electrically or optically with a blue light-emitting diode, with activation spread recorded simultaneously using optical mapping. Results showed that ChR2 could be readily transduced to NRVMs by the doxycycline-inducible lentiviral system; however, high-level ChR2 (but not YFP) expression was associated with substantial cytotoxicity, which hindered optical pacing. Application of bromodeoxyuridine significantly reduced cell damage, allowing stimulation with light. Simultaneous optical Vm mapping showed that conduction velocity, action potential duration, and dVm/dtmax were similar in ChR2-expressing and control cultures. Finally, the ChR2-expressing cultures could be optically paced at multiple sites, with significantly reduced overall activation time. In summary, we demonstrated that inducible lentiviral-mediated ChR2 overexpression might cause cytotoxicity in NRVM cultures, which could be alleviated without impairing electrophysiological function, allowing simultaneous optical pacing and Vm mapping.

Engineered cardiac tissues fabricated from human induced pluripotent stem cells (hiPSCs) show promise for ameliorating damage from myocardial infarction, while also restoring function to the damaged left ventricular (LV) myocardium. For these constructs to reach their clinical potential, they need to be of a clinically relevant volume and thickness, and capable of generating synchronous and forceful contraction to assist the pumping action of the recipient heart. Design prerequisites include a structure thickness sufficient to produce a beneficial contractile force, prevascularization to overcome diffusion limitations and sufficient structural development to allow for maximal cell communication. Previous attempts to meet these prerequisites have been hindered by lack of oxygen and nutrient transport due to diffusion limits (100-200 μm) resulting in necrosis. This study employs a layer-by-layer (LbL) fabrication method to produce cardiac tissue constructs that meet these design prerequisites and mimic normal myocardium in form and function. Thick (>2 mm) cardiac tissues created from hiPSC-derived cardiomyocytes, -endothelial cells (ECs) and -fibroblasts (FBs) were assessed, in vitro, over a 4-week period for viability (<6% necrotic cells), cell morphology and functionality. Functional performance assessment showed enhanced t-tubule network development, gap junction communication as well as previously unseen, physiologically relevant conduction velocities (CVs) (>30 cm/s). These results demonstrate that LbL fabrication can be utilized successfully to create prevascularized, functional cardiac tissue constructs from hiPSCs for potential therapeutic applications.

Human induced-pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (hiPSC-CMs) via the GiWi method, which uses small-molecule inhibitors of glycogen synthase kinase (GSK) and tankyrase to first activate and then suppress Wnt signaling. However, this method is typically conducted in 6-well culture plates with two-dimensional (2D) cell sheets, and consequently, cannot be easily scaled to produce the large numbers of hiPSC-CMs needed for clinical applications. Cell suspensions are more suitable than 2D systems for commercial biomanufacturing, and suspended hiPSCs form free-floating aggregates (i.e., spheroids) that can also be differentiated into hiPSC-CMs. Here, we introduce a protocol for differentiating suspensions of hiPSC spheroids into cardiomyocytes that is based on the GiWi method. After optimization based on cardiac troponin T staining, the purity of hiPSC-CMs differentiated via our novel protocol exceeded 98% with yields of about 1.5 million hiPSC-CMs/mL and less between-batch purity variability than hiPSC-CMs produced in 2D cultures; furthermore, the culture volume could be increased ∼10-fold to 30 mL with no need for re-optimization, which suggests that this method can serve as a framework for large-scale hiPSC-CM production.

Direct cardiac reprogramming of fibroblasts into cardiomyocytes has emerged as a promising strategy to remuscularize injured myocardium. However, it is insufficient to generate functional induced cardiomyocytes from human fibroblasts using conventional reprogramming cocktails, and the underlying molecular mechanisms are not well studied.

Functional myocardium derived from human induced pluripotent stem cells (hiPSCs) can be impactful for cardiac disease modeling, drug testing, and the repair of injured myocardium. However, when hiPSCs are differentiated into cardiomyocytes, they do not possess characteristics of mature myocytes which limits their application in these endeavors. We hypothesized that mechanical and electrical stimuli would enhance the maturation of hiPSC-derived cardiomyocyte (hiPSC-CM) spheroids on both a structural and functional level, potentially leading to a better model for drug testing as well as cell therapy. Spheroids were generated with hiPSC-CM. For inducing mechanical stimulation, they were placed in a custom-made device with PDMS channels and exposed to cyclic, uniaxial stretch. Spheroids were electrically stimulated in the C-Pace EP from IONOptix for 7 days. Following the stimulations, the spheroids were then analyzed for cardiomyocyte maturation. Both stimulated groups of spheroids possessed enhanced transcript and protein expressions for key maturation markers, such as cTnI, MLC2v, and MLC2a, along with improved ultrastructure of the hiPSC-CMs in both groups with enhanced Z-band/Z-body formation, fibril alignment, and fiber number. Optical mapping showed that spheroids exposed to electrical stimulation were able to capture signals at increasing rates of pacing up to 4 Hz, which failed in unstimulated spheroids. Our results clearly indicate that a significantly improved myocyte maturation can be achieved by culturing iPSC-CMs as spheroids and exposing them to cyclic, uniaxial stretch and electrical stimulation.