Question

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Discussion

There are several approaches available for obtaining enriched cardiomyocyte populations from human PSCs. Ma et al. (2011) performed genetic-modification-based purification of cardiomyocytes (achieving >98% cardiomyocyte purity) from hiPSC derivatives, using the intrinsic MYH6 gene to express a blasticidin S resistance gene. Dubois et al. (2011) used a surface protein, signal-regulatory protein alpha (SIRPA), as a cardiac-specific marker in hiPSC derivatives prepared through a highly cardiogenic differentiation procedure. They purified cardiomyocytes (up to 98% purity) via FACS from sources comprising 40%–50% cardiomyocytes. The method we report here is a simple medium-exchanging procedure that enabled cardiomyocyte purification of up to 99% from a cell source comprising only 10% cardiomyocytes, with an estimated recovery of cardiomyocytes of 74.4 ± 12.1%, based on direct cell count before and after purification. Previously we reported a mitochondrial method for purifying cardiomyocytes to >99% purity via FACS. Our direct comparison of these two methods revealed a higher cardiomyocyte-yield-based efficiency for the lactate method than for the mitochondrial method. The lactate method has quantitative and economic advantages relative to other existing cardiomyocyte-purification methods by virtue of its simplicity and ease of application.

One question that arose from our studies is why ESCs die within a few hours under the lactate method but cardiomyocytes survive for much longer, even though both cell types use lactate for biomass synthesis. Through our investigations, we eliminated the possibility that lactate supplementation caused toxic extracellular or intracellular acidification. We propose that these differing properties may be a result of (1) the retrospective glycolytic pathway consuming two ATP molecules during conversion of a lactate molecule to G6P, and (2) ESCs not being able to effectively obtain ATP from glycolysis nor from OXPHOS under glucose-depleted conditions. Therefore, activation of the retrospective glycolytic pathway may accelerate a catastrophic balance of ATP supply and demand in ESCs, whereas cardiomyocytes can maintain cellular ATP homeostasis by producing more ATP via a highly active OXPHOS mechanism (Hattori et al., 2010).

A patient would theoretically require about 10⁹ cardiomyocytes in therapeutic applications of purified cardiomyocytes. In this study, we obtained approximately 5 × 10⁵ purified human cardiomyocytes per 177 cm² dish. Taking into account their postpurification proliferative capacities, rough estimates therefore suggest that 1 × 10⁹ cardiomyocytes could be obtained from 800 dishes (14.13 m²). This scale is close to the capacity of commercially available automatic large-scale culture systems and suggests that combining more sophisticated differentiation methods with our lactate method could facilitate realistic application of PSC-derived cardiomyocytes to human therapy.

Answer 1

Summary

1. The method we report here is a simple medium-exchanging procedure that enabled cardiomyocyte purification of up to 99% from a cell source comprising only 10% cardiomyocytes, with an estimated recovery of cardiomyocytes of 74.4 ± 12.1%, based on direct cell count before and after purification.

2. One question that arose from our studies is why ESCs die within a few hours under the lactate method but cardiomyocytes survive for much longer, even though both cell types use lactate for biomass synthesis.

3. (2011) performed genetic-modification-based purification of cardiomyocytes (achieving >98% cardiomyocyte purity) from hiPSC derivatives, using the intrinsic MYH6 gene to express a blasticidin S resistance gene.

4. Therefore, activation of the retrospective glycolytic pathway may accelerate a catastrophic balance of ATP supply and demand in ESCs, whereas cardiomyocytes can maintain cellular ATP homeostasis by producing more ATP via a highly active OXPHOS mechanism (Hattori et al., 2010).

5. We propose that these differing properties may be a result of (1) the retrospective glycolytic pathway consuming two ATP molecules during conversion of a lactate molecule to G6P, and (2) ESCs not being able to effectively obtain ATP from glycolysis nor from OXPHOS under glucose-depleted conditions.