Identifying Stem-like Cells Using Mitochondrial Membrane Potential

Electrical qualities of stem cells. Measurement of the voltage difference across the inner mitochondrial membrane (ΔΨm) predicts ‘stemness.’ ΔΨm serves as the electromotive force for ATP synthesis in all eukaryotic cells, but high voltage predicts cell differentiation, function. and death.

Therapies that are based on living cells promise to improve treatments for metastatic cancer and for many degenerative diseases. Lasting treatment of these maladies may require the durable persistence of cells. Long-term engraftment of cells – for months or years – and the generation of large numbers of progeny are characteristics of stem cells. Most approaches to isolate viable hematopoetic stem cells and therapeutically active T cells are based on immunophenotyping using highly multicolored flow cytometry. However, these methods do not directly measure the metabolic features of cells, which are known to be important in predicting cell fate.

Recent work by Nicholas Restifo, M.D., of CCR’s Surgery Branch, and his colleagues provides a novel method of identifying cells with more stem-like characteristics, which are more likely to be effective in cell-based treatments. The researchers showed that a cell’s mitochondrial membrane potential is a measure of its stem-like character. They employed a fluorescent dye that accumulates in the mitochondrial matrix in proportion to the membrane potential of the mitochondria to divide cells by fluorescence-activated cell sorting into populations with either low or high mitochondrial membrane potential (low-ΔΨm or high-ΔΨm&m, respectively). Using a pmel-1 T cell receptor transgenic mouse line that expresses CD8+ T cells that recognize the gp100 melanocyte antigen, the investigators isolated CD8+ T cells based on their mitochondrial membrane potential following inoculation of the mice with a vaccinia virus containing gp100. They then introduced an equal number of low- or high-ΔΨm T cells into wild type mice with the vaccinia virus and characterized the CD8+ T cells produced. Mice injected with low-ΔΨm T cells showed a higher percentage of stem-cell memory T cells and central memory T cells, while the high-ΔΨm T cell recipients showed a higher percentage of effector memory T cells as determined by cell surface markers.

The scientists further investigated the properties and functionality of the low- and high-ΔΨm T cell populations. Using RNA-sequencing, quantitative PCR, and metabolic profiling, they showed that low-ΔΨm cells contain expression profiles and metabolite levels consistent with previously characterized memory CD8+ T cells, including a preference for fatty acid oxidation over glycolysis. High-ΔΨm cells showed a greater reliance on glycolytic flux and higher oxygen consumption rates, consistent with an effector T cell phenotype. The metabolomic distinctions between these two subpopulations support the use of T cell isolation based on differences in mitochondrial membrane potential to specifically identify memory T cells (low-ΔΨm) or effector T cells (high-ΔΨm). Additionally, the researchers isolated central memory T cells via cell surface markers and then further separated them based on mitochondrial membrane potential. Mice treated with low-ΔΨm cells showed more engraftment than mice treated with high-ΔΨm T cells. Here, mitochondrial membrane potential does not simply confirm cell identity as predicted by surface markers but can identify a distinct fraction of more stem-like T cells from a population of cells that otherwise appear homogenous.

The investigators then explored the relationship between mitochondrial membrane potential and stem-like characteristics in other cell types, including effector T cell populations and hematopoietic stem cells (HSC). In both cases, they observed enhanced survival and proliferation in cells with low-ΔΨm. The scientists next generated effector T cell subsets to investigate their cytokine production. Within each subset of effector T cells examined, cytokine production increased in high-ΔΨm cells. These results are consistent with distinct roles for T cells with differing ΔΨm and indicate that increased ΔΨm may be necessary to achieve full effector T cell function, which requires inflammatory cytokines and mTOR signaling. They then examined the link between mTOR activation and increased ΔΨm levels in cells and found that low-ΔΨm T cells showed decreased basal mTORC1 signaling. Interleukin-2 exposure during T cell priming produced an increase in ΔΨm, which was inhibited by the addition of rapamycin, an mTOR inhibitor.

Next, the researchers examined the relationship between oxidative stress and ΔΨm in CD8+ T cells, effector T cells, and HSC. Low-ΔΨm cells consistently showed lower levels of oxidative stress compared to high-ΔΨm cells. Low-ΔΨm CD8+ T cells had a large reserve of oxidized glutathione and increased transcript levels for reactive oxygen species-related enzymes, such as catalase and superoxide dismutase 1 and 2. These cells also show less DNA damage as measured by serine 139 phosphorylation on histone H2A.X. In contrast, high-ΔΨm T cells showed upregulation in genes for DNA replication, DNA repair, and cell cycle arrest. They confirmed these observations in other T cell subsets and HSC and found that T cell exhaustion markers were upregulated in high-ΔΨm cells. These cells also showed increased markers for cell death, including Bax expression and Annexin V staining.

Finally, the low-ΔΨm CD8+ T cells demonstrated superior long-term persistence and antitumor activity., Madhu Sukumar, Ph.D., first author of the study and a postdoctoral fellow, CCR’s Surgery Branch, found that cells with low-ΔΨm CD8+ cells could still be found to be persisting in mice for 300 after transfer. Perhaps most importantly, low-ΔΨm pmel-1 CD8+ T cells showed better antitumor activity and an increased ability to reduce tumor size when transferred to wild-type mice with subcutaneous B16 melanomas. While these initial findings were observed in mouse model systems, the investigators confirmed a relationship between low-ΔΨm and less differentiated T cells by studying human T cells as well. This work marks a new method for the selection of T cells that maintain a stem-like state. The ability to isolate these cells could improve the treatment of cancers using cell-based treatments.

Summary Posted: Tue, 12/01/2015


Sukumar M, Liu J, Mehta GU, Patel SJ, Roychoudhuri R, Crompton JG, Klebanoff CA, Ji Y, Li P, Yu Z, Whitehill GD, Clever D, Eil RL, Palmer D, Mitra S, Rao M, Keyvanfar K, Schrump D, Wang E, Marincola F, Gattinoni L, Leonard WJ, Muranski P, Finkel T, and Restifo NP. Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy. Cell Metabolism. December 7, 2015. PubMed Link