Better tools for improved understanding of mitochondrial polarization in stem cells

Mitochondria play an important role in cells by providing energy, regulating the cell cycle and regulating programmed cell death. However, the capacity to study mitochondria in a quantitative manner and within the live cell is still rather limited. Fluorescence Lifetime Imaging Microscopy (FLIM) enables a deeper understanding of mitochondrial function.

The method is based on the environment-sensitive (polarity, temperature, and other conditions) emission of fluorescent dyes, proteins and nanoparticles. Modern FLIM microscopes allow for the measurement of fluorescence lifetime in the sub-nanosecond time range and can be used to reconstruct two- and three-dimensional images of live cells, tissues or organoids. This method is already actively used in studies of tissue oxygenation and hypoxia, pH and cellular redox status.

The authors of the paper, published in Cytometry Part A, found that several well-known dyes, such as tetramethylrhodamine methyl ester (TMRM) and SYTO family of dyes, significantly improve traditional microscopy methods by examining mitochondria in a FLIM mode.

“The work started by attempting to perform FLIM with green-fluorescent SYTO dyes to analyze the DNA and chromatin compaction in live cells. To our surprise, we found that most dyes did not show exclusive nuclear staining, the dye also resided in the mitochondria, in a membrane potential-dependent manner. Thus, the project was suddenly driven in a new and exciting direction”, recalls Ruslan Dmitriev, group leader at the Institute for Regenerative Medicine, Sechenov University.

The study reports that fluorescence lifetime-dependent detection of mitochondrial polarisation (changes in mitochondrial membrane potential) allows for distinguishing cell types more accurately, continuously monitoring ‘mitochondria at work’, and facilitates assessing cell oxygenation (hypoxia). Using FLIM, researchers confirmed the increase of mitochondrial polarisation at the border between G1 and S phases, – the critical moment for cell cycle progression. Alterations in the normal cell cycle progression are directly related to a reduced ability of the tissue to regenerate (i.e. aging) and proliferation of cancer cells.

FLIM becomes an even more powerful approach when it is applied to three-dimensional tissue models, such as stem cell-derived intestinal organoids. Such ‘mini-gut’ tissue is a multicellular structure with the diversity of intestinal epithelium cell types including stem cells, enterocytes, Paneth, enteroendocrine and goblet cells. Organoid cultures enable the study of the gastrointestinal tract, its interactions with the microbiota and pharmaceutical drugs, and are very useful for studying disease such as diabetes, colitis and cancer. The study demonstrated that FLIM is well-suited for analysis of live intestinal organoid culture and enables discrimination of cells within stem cell niche and monitoring of their proliferation. Multiplexed monitoring of mitochondrial polarization and cell cycle S phase helped to find different subpopulations of stem cells. At the same time, studies of live three-dimensional objects using FLIM require improved data analysis algorithms and the use of state-of-the-art microscopes.

The reported approach opens bright prospects for tissue engineering with stem cells including monitoring of their quality. It should promote the development of new tissue engineering methods and microscopy techniques and stimulate basic research in the stem cell field.

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