Efficient Generation and Cryopreservation of Cardiomyocytes Derived from Human Embryonic Stem Cells

Abstract

Aim Human embryonic stem cells (hESCs) represent a novel cell source to treat diseases such as heart failure and for use in drug screening. In this study, we aim to promote efficient generation of cardiomyocytes from hESCs by combining the current optimal techniques of controlled growth of undifferentiated cells and specific induction for cardiac differentiation. We also aim to examine whether these methods are scalable and whether the differentiated cells can be cryopreserved. Methods & results hESCs were maintained without conditioned medium or feeders and were sequentially treated with activin A and bone morphogenetic protein-4 in a serum-free medium. This led to differentiation into cell populations containing high percentages of cardiomyocytes. The differentiated cells expressed appropriate cardiomyocyte markers and maintained contractility in culture, and the majority of the cells displayed working chamber (atrial and ventricular) type electrophysiological properties. In addition, the cell growth and differentiation process was adaptable to large culture formats. Moreover, the cardiomyocytes survived following cryopreservation, and viable cardiac grafts were detected after transplantation of cryopreserved cells into rat hearts following myocardial infarctions. Conclusion These results demonstrate that cardiomyocytes of high quality can be efficiently generated and cryopreserved using hESCs maintained in serum-free medium, a step forward towards the application of these cells to human clinical use or drug discovery.

Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

“…microfabrication | heart regeneration | tissue engineering | cardiac toxicity | arrhythmia disease model D irected differentiation strategies for generating and preserving human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) are well-developed (1)(2)(3)(4)(5)(6)(7), and several CM cell-surface markers that can be used to enrich target subpopulations have been discovered (8,9). Although differentiation of hPSCs into contracting CMs is well-established, their maturation into adult-equivalent cells, and their formulation into functional adult-like tissue, remains an unmet challenge (5).…”

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

“…The hES cells were differentiated as previously described [25]. Briefly, WA07 hES cells were rinsed with 2 mL DPBS and incubated with 2 mL Versene (EDTA; Life Technologies) for 10 min at 37°C.…”

Cardiac Tissue Engineering

“…Finally, as summarized in section 1.3.2, the directed differentiation approach has led to high cardiomyocyte culture purities, and can be combined with all of the aforementioned purification strategies. Loss of teratoma forming ability hESC Percoll Gradient + directed differentiation 83% Loss of teratoma forming ability, contributed to repair in rat infarct model Genetic-based selection Muller et al, 2000) hESC MLC-2V FACS 93% N/A (Huber et al, 2007) hESC Activin A / BMP4 31% CMs Grafts were viable 4 weeks after (Xu et al, 2011) 18 transplantation onto rat hearts.…”