Next generation of ALDH substrates and their potential to study maturational lineage biology in stem and progenitor cells

Abstract

High aldehyde dehydrogenase (ALDH) activity is a feature of stem cells from normal and cancerous tissues and a reliable universal marker used to isolate them. There are numerous ALDH isoforms with preferred substrate specificity variably expressed depending on tissue, cell type, and organelle and cell status. On the other hand, a given substrate may be metabolized by several enzyme isoforms. Currently ALDH activity is evidenced by using Aldefluor, a fluorescent substrate likely to be metabolized by numerous ALDH isoforms. Therefore, isolation techniques based on ALDH activity detection select a heterogeneous population of stem or progenitor cells. Despite active research in the field, the precise role(s) of different ALDH isoforms in stem cells remains enigmatic. Understanding the metabolic role of different ALDH isoform in the control of stem cell phenotype and cell fate during development, tissue homeostasis, or repair, as well as carcinogenesis, should open perspectives to significant discoveries in tissue biology. In this perspective, novel ALDH substrates are being developed. Here we describe how new substrates could be instrumental for better isolation of cell population with stemness potential and for defining hierarchy of cell populations in tissue. Finally, we speculate on other potential applications.

Keywords: ALDH activity; AldeRed-588-A; Aldefluor; liver progenitor cell.

Fig. 1.

The AldeRed-588-A substrate provides a useful tool to select stem cells and opens up prospects for the next generation of aldehyde dehydrogenase (ALDH) substrates. A: Aldefluor and AldeRed-588-A have the same capacity to isolate an ALDH^bright cell population enriched in stem cells from a heterogeneous mixture of cells. B: AldeRed-588-A can be used for multicolor applications to fractionate ALDH^bright cells in the presence of green fluorophores, including the Aldefluor reagent and cells expressing enhanced green-fluorescent protein (eGFP) and can be visualized by fluorescent microscopy. C: emerging opportunities in generating preferred labeled substrates with different fluorescent probes. D: distinguishing distinct cell populations from a digested tissue on the basis of the specificity of the ALDH substrate might help in fractionating the different category of cells with ALDH activity referring to a certain physiological state. ALDH^bright, cells with high ALDH activity; ALDH^dim, low-ALDH-activity fraction; FSC, forward-scattered light; SSC, side-scattered light.

Fig. 2.

Opportunities in refining the metabolic hierarchy of stem cells and their progeny in the liver. A: general representation of the cell fate determination of the hepatic stem/progenitor cells (HSPCs) to the hepatocytic lineage and their organization is represented. B: to follow the fate of the stem cells and their progeny in the liver upon injury, OPN-iCreERT2;ROSA26RYFP mice could be used. All sorted cells are by definition green, and the green color is therefore not shown to avoid confusion. Reconstitution of the cell fate can be done by sorting yellow fluorescent protein (YFP)⁺ fractions at different time points after injury. C: hypothetically, combination of ALDH activity (here, red substrate) with genetic tracing allows an additional fractionation of the YFP⁺ population. A hypothetical scenario is given, which illustrates a potential bias in ALDH activity depending on cell fate. Red color has been added in cytoplasm for a simplistic view. D: as in C, but now using a mixture of 2 distinct fluorescent-labeled substrates (red and blue), which could even further refine the molecular features of the YFP⁺ sorted cells based on additional subfractionations. Red or blue colors in cytoplasm of cells represent the ALDH^bright populations. ALDH^int and ALDH^dim (for each substrate) are not represented to lighten the figure. With these additional parameters, it will definitively be possible to fractionate further the 3 ALDH^bright populations. ALDH^int, intermediate aldehyde dehydrogenase activity; Aldh^Red, red ALDH activity based on the use of Substrate A-Red; Aldh^Blue, blue ALDH activity based on the use of Substrate B-Blue; Red^Bright, high red ALDH activity; BlueBright: high blue ALDH activity; Red^BrightBlue^Bright, high red and high blue ALDH activities.