Introduction:
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a promising technology for regenerative medicine and pharmacology. However, existing protocols are limited due to the high costs when generating cells at scale, to low yield, and to a lack of protocol replicability. Additionally, the use of animal-derived or recombinant proteins in cell differentiation creates concerns in human compatibility and presents technical challenges due to batch-to-batch variability.
Aim:
Develop a protocol for functional hiPSC-CM differentiation with high yield without the use of proteins.
Methods:
We expanded the CDM3 protocol by testing approximately 200 combinations of cell culture media during hiPSC differentiation. The differentiation was induced with a 24h incubation in 6 μM CHIR (d0), followed by 24 h in basal media (d1) and 48h in media supplemented with AA2P and Wnt-C59 (d2-d4). From day 4 onwards, cells were kept in media supplemented with insulin and AA2P, and in some conditions with polyvinyl alcohol. On day 8, hiPSC-CMs were antibiotically selected using geneticin. Tissues were imaged on day 13 to assess myocyte morphology and contraction. Videos were scored from 0 (dead/delaminated cells) to 9 (confluent tissues beating at least 4 times in a 5 sec interval).
Results:
We identified 6 basal media combinations that yield confluent monolayers on day 13. Out of these, 3 conditions consistently resulted in confluent contractile tissues.
Conclusions:
Our data demonstrate that a protein-free high yield hiPSC-CM differentiation is possible with the appropriate selection of basal media based on cellular nutritional requirements.
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