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New Probe Aims to Aid Pluripotent Stem Cell Culture

Review of “A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency” from Stem Cells Translational Medicine by Stuart P. Atkinson

The culture of human pluripotent stem cells (hPSCs) can be a long and laborious process and, even with the highest level of care, unwanted cells in the process of losing their pluripotent characteristics always sneak through into the next culture dish. While this may sound like a small annoyance, such cells can affect the quality of the entire culture and may affect experiments or the production of a stem cell-based therapy.

So how can we improve the detection, selection, and avoidance of such pesky non-pluripotent cells? ? Hiroshi Takemori (National Institutes of Biomedical Innovation, Osaka, Japan) knew that cells change their mode of energy production during differentiation [1, 2] which alters their mitochondrial membrane potential. Therefore, a fluorescent dye which is sensitive to such changes can highlight unwanted cells in an hPSC culture. 

Now, in a new study published in Stem Cells Translational Medicine, the Takemori lab describe the application of the fluorescent JC-1 probe [3] to human embryonic stem cell (hESC) cultures in the hope that it will enhance our ability to culture high-quality PSCs and enhance any future therapeutic applicability [4].

Using feeder-free conditions, the study first demonstrated that well-maintained hESC cultures relied on glycolysis for energy production while poorly maintained cultures contained higher levels of cells which instead drifted towards the use of oxidative phosphorylation. This shift caused an increase in mitochondrial membrane potential which causes the JC-1 probe to form multimers and emit a red signal instead of a green signal. Therefore, the cultures of poorly maintained cells presented with higher levels of red fluorescence than well-maintained cultures and cell-tracking experiments suggested that those cells exhibiting red fluorescence lost their pluripotent gene expression (Oct3/4) and expressed differentiation-associated gene markers (Vimentin) (See Figure).

A splash of color in the tissue culture hood may be a good thing for PSC scientists as this new dye could aid in the maintenance of high-quality hESCs and the often tricky-to-work-with human induced pluripotent stem cells (hiPSCs). Indeed, the dye may also be of use in monitoring the emergence of truly pluripotent colonies during the reprogramming process. 


  • Folmes CD, Dzeja PP, Nelson TJ, et al. Metabolic plasticity in stem cell homeostasis and differentiation. Cell Stem Cell 2012;11:596-606.
  • Choi HW, Kim JH, Chung MK, et al. Mitochondrial and metabolic remodeling during reprogramming and differentiation of the reprogrammed cells. Stem Cells Dev 2015;24:1366-1373.
  • Smiley ST, Reers M, Mottola-Hartshorn C, et al. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc Natl Acad Sci U S A 1991;88:3671-3675.
  • Kumagai A, Suga M, Yanagihara K, et al. A Simple Method for Labeling Human Embryonic Stem Cells Destined to Lose Undifferentiated Potency. Stem Cells Transl Med 2016;5:275-281.