Abstract 1070: Mitochondrial Membrane Potential Measurement Dye is Applicable for Purification of Mouse and Marmoset Embryonic Stem Cell-Derived Cardiomyocytes
Genetic engineering of human embryonic stem (ES) cells highlights difficulties such as the validation of safety and extrinsic gene silencing. Thus, we developed a novel purification method for ES-derived cardiomyocytes (CM) without the necessity for genetic modification. From our experience with primary cultured rat CM, we noticed that the dyes for monitoring mitochondrial membrane potential could stain CM much stronger than the other cells. This study was designed to investigate whether or not the application of such dyes could facilitate the separation of CM from the other cells.
[Methods and Results]
Preliminarily, we demonstrated population studies of embryonic and postnatal rat heart cells by comparing several dyes. We separated the populations and performed immunohistochemical analysis (sarcomeric actinin and Nkx2.5). From these results, we selected tetramethylrhodamine methyl (TMRM), which separated the CM from the other cells with a 34.2-fold higher intensity.
Whole rat embryo (E11.5) culture was examined with 50 nM TMRM-containing medium and revealed that the beating heart was selectively stained in vivo.
We observed that the TMRM fluorescent intensity of mouse ES-derived CM reached a plateau 15 days after EB formation.
From the cell population study of day 15 EB cells, the population with the highest fluorescent intensity was 6.0-fold higher than the other cells. Immunohistochemical analysis revealed that this population was consisted of only CM.
Furthermore, we performed a scaled-up version, and obtained a large number of CM enough for producing 1cm2 of confluent CM cell sheet (99.2% purity).
Finally, we also studied the effectiveness of this method for primate ES cells. Recently, we established new marmoset ES cell lines. They can differentiate CM after day 12–14 EB. TMRM is selectively accumulated in CM of day 20–30 EB.
The population study of day 23 EB cells revealed that the CM population had a 7.6-fold higher fluorescent intensity than the other cells. In contrast to mice CM, marmoset CM continued to proliferate for more than 10 days after purification.
[Conclusion] Our newly developed purification method is relatively safe for cardiomyocytes, highly efficient and it holds advantages for future therapeutic use.