Generation of human induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer and scalable 3D suspension bioreactor cultures with reduced batch-to-batch variations

Abstract

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are promising candidates to treat myocardial infarction and other cardiac diseases. Such treatments require pure cardiomyocytes (CMs) in large quantities.Methods: In the present study we describe an improved protocol for production of hiPSC-CMs in which hiPSCs are first converted into mesodermal cells by stimulation of wingless (Wnt) signaling using CHIR99021, which are then further differentiated into CM progenitors by simultaneous inhibition of porcupine and tankyrase pathways using IWP2 and XAV939 under continuous supplementation of ascorbate during the entire differentiation procedure.Results: The protocol resulted in reproducible generation of >90% cardiac troponin T (TNNT2)-positive cells containing highly organized sarcomeres. In 2D monolayer cultures CM yields amounted to 0.5 million cells per cm2 growth area, and on average 72 million cells per 100 mL bioreactor suspension culture without continuous perfusion. The differentiation efficiency was hardly affected by the initial seeding density of undifferentiated hiPSCs. Furthermore, batch-to-batch variations were reduced by combinatorial use of ascorbate, IWP2, and XAV939.Conclusion: Combined inhibition of porcupine and tankyrase sub-pathways of Wnt signaling and continuous ascorbate supplementation, enable robust and efficient production of hiPSC-CMs.

Retinoic acid promotes metabolic maturation of human Embryonic Stem Cell-derived Cardiomyocytes

“…Studies on embryo biology are valuable resources, providing direct evidence for the mechanisms of stem cell differentiation 17 , 43 , 44 . As a ligand for the nuclear transcription factor RAR, RA exerts pleiotropic actions at different stages of heart development 14 .…”

Practical choice for robust and efficient differentiation of human pluripotent stem cells

“…The EB-based method is widely used to differentiate majority of cell lineages from the three germ layers (Table 1 ) and has an obvious advantage in improving the differentiation efficiency of some cells[ 8 - 10 ], such as hematopoietic progenitors[ 11 ] and melanocytes[ 12 ]. Combined with suspension bioreactor technology, this advantage can be further amplified for large-scale production[ 13 ]. Additionally, EB formation provides an excellent way to assess and manipulate developmental potential[ 7 ].…”

“…The omni-well-array culture platform can produce massive and miniaturized iPSC-derived liver buds on a clinically relevant large scale (> 10 8 )[ 50 ]. Hama et al[ 13 ] designed a protocol that generated > 90% hiPSC-derived CMs that yielded on average 72 million cells per 100 mL in a 3D bioreactor. These results show that the yield from the 3D suspension system is remarkable in contrast to the 2D system.…”

“…These results show that the yield from the 3D suspension system is remarkable in contrast to the 2D system. To test the reproducibility of the CM 3D differentiation protocol, a previous study compared biologically independent experiments with various passage numbers of iPSCs, and found minor inter-experimental variations[ 13 ]. Overall, the 3D differentiation culture appears to have advantages in differentiation efficiency and stability over the 2D system.…”

Automation, Monitoring, and Standardization of Cell Product Manufacturing

“…For example, scalable, reproducible, and biomimetic culture conditions are required to maintain cellular function during ex vivo culture ( Liu et al, 2017 ; Stephenson and Grayson, 2018 ; Ruiz et al, 2019 ; Serra et al, 2019 ). Further, large-capacity and automated bioreactor systems have the potential to reduce batch-to-batch variability and the use of expensive highly skilled labor ( Peroglio et al, 2018 ; Costariol et al, 2019 ; de Sousa Pinto et al, 2019 ; Hamad et al, 2019 ). In the case of allogeneic therapies, the aim is to scale up processes for numerous patients.…”