Generation of Highly Purified Human Cardiomyocytes from Peripheral Blood Mononuclear Cell-Derived Induced Pluripotent Stem Cells

Abstract

Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine.

Quantitative Assessment of Sialo‐Glycoproteins and N‐Glycans during Cardiomyogenic Differentiation of Human Induced Pluripotent Stem Cells

“…For the application of human pluripotent stem‐cell‐derived CMs (hPSC CMs), it must be assured that the generated tissue grafts contain only pure CMs. Residual stem cells could lead to teratoma formation after transplantation, and contaminating cells types could affect the functionality of the produced graft . Pure populations of CMs are also essential for drug discovery and drug safety testing .…”

Cardiac differentiation of human pluripotent stem cells in scalable suspension culture

“…Efficient genetic enrichment of hPSC-derived CMs in suspension culture has been demonstrated requiring genetic engineering of hPSCs 28 . Alternatively, metabolic purification of CMs in glucose-free, lactate-supplemented medium in stirred suspension culture was published 29 , but the reduction of nonCMs is accompanied by the substantial loss of CMs as well, which limits the overall yield of the method 30 .…”

Selection of human induced pluripotent stem cells lines optimization of cardiomyocytes differentiation in an integrated suspension microcarrier bioreactor

“…Overall, our process yielded > 40 CMs per seeded hiPSC (Table 2D), which is at least eightfold more than other reports. They ranged from 1 to 5 CMs/hPSC [10,13,14,27,28,38,57,60], and 35% more than our previous report [58]. This difference in CM yields is mainly attributed to the high proliferative capacity in the initial expansion phase (~16fold) and differentiation phase (~3-fold), as well as > 96% of Troponin T obtained after the recovery phase.…”