Abstract
In the stem cell field there is a lack of non invasive and fast methods to identify stem cell’s metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that the metabolic signature of NPSCs correlates with their differentiation potential, showing that neuronal progenitors and glial progenitors have a different free/bound NADH ratio. Reducing conditions in NPSCs correlates with their neurogenic potential, while oxidative conditions correlate with glial potential. For the first time we show that FLIM NADH metabolic fingerprint provides a novel, and quantitative measure of stem cell potential and a label-free and non-invasive means to identify neuron- or glial- biased progenitors.
Highlights
Current label-based methods used to describe cell phenotype have to date proven inadequate for accurately predicting the differentiation potential of many stem cell populations
In this work we discriminate different differentiation stages of neuronal progenitor stem cells and we identify their differentiation potential by measuring the level of metabolic activity of neural progenitor and stem cells (NPSCs) by Phasor analysis and Fluorescence lifetime microscopy of NADH
Oxidative conditions, such as oxidative stress induced by hydrogen peroxide (Figure 4b,d,f) and the inhibition of glycolysis (Figure 5b,d,f) shift the cellular phasor fluorescence lifetime microscopy (FLIM) signature toward the location of bound NADH
Summary
Current label-based methods used to describe cell phenotype have to date proven inadequate for accurately predicting the differentiation potential of many stem cell populations. The ability to direct differentiation of NPSCs towards neurons and to identify neuron-restricted progenitor cells may provide new therapeutic avenues for stroke, spinal cord injury and age-related cognitive conditions, such as Alzheimer’s and Parkinson’s diseases, which cause loss of neurons. The mammalian brain contains a population of neural stem cells (NSCs), which can self renew and differentiate to give raise to neurons, astrocytes and oligodendrocytes. They are relatively quiescent in adults [1], entering the cell cycle to produce more rapidly dividing progenitors that undergo limited rounds of proliferation and are more committed to specific neural lineages [2]. Astrocytes perform many different functions, including providing structural and nutrient support for neurons, secreting signaling molecules, and uptake and metabolism of neurotransmitters
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