Abstract 12671: Distinct Metabolic Flow Enables Large-Scale Purification of Pluripotent Stem Cell-Derived Cardiomyocytes
Background Mass production of highly purified cardiomyocytes is a critical bottleneck in realizing heart regenerative therapy. Our recently established non-genetic purification method of cardiomyocytes using mitochondrial dye is efficient but not suitable to produce clinical-scale cardiomyocytes due to the usage of FACS. This study was designed to establish a large-scale purification method for pluripotent stem cell (PSC)-derived cardiomyocytes.
Methods and results (1) We first approached this issue by focusing on the possible metabolic differences between cardiomyocytes and embryonic stem cells (ESCs) by transcriptome and metabolome analyses. Transcriptome analysis delineated marked differences in isozyme expression and mRNA levels between metabolic pathways including glycolysis and the TCA cycle. Fluxome analysis using [13C]-labeled glucose revealed that ESCs used glucose mainly for amino acid and nucleotide synthesis, while cardiomyocytes used glucose primarily for ATP production via TCA cycle. (2) Lactate was discarded by ESC, but it was incorporated into cardiomyocytes. (3) Taken together, we developed a novel method for purifying the bulk of ESC-derived cardiomyocytes using original culture media (glucose depleted and with lactate). This culture media enabled us to obtain >99% pure cardiomyocytes which did not form teratoma when transplanted into immunodeficient mice testes. (4) Electrophysiological analysis of the purified cardiomyocytes using microelectrode recordings and multi-electrode array system showed normal properties. (5) We investigated the molecular mechanism to determine why cardiomyocytes can survive for the long period in the lactate-supplemented and glucose-starved medium by fluxome analysis using [13C]-labeled lactate. Surprisingly, they can produce some biomass including nucleic acids and amino acids from lactate via gluconeogenic pathway, but ESCs could not.
Conclusions In this study, we report a novel method to purify a bulk of cardiomyocytes from PSC-derivatives based on the findings from transcriptome and fluxome analyses. We believe that our novel method termed “Lactate Method” will resolve the bottleneck and directly facilitate human heart-regenerative therapies.
- © 2011 by American Heart Association, Inc.