Stem cell survival is severely compromised by the thymidineanalog EdU (5-ethynyl-2′-deoxyuridine), an alternative to BrdU for proliferation assays and stem cell tracing

Abstract

Stem cell therapy has opened up the possibility of treating numerous degenerating diseases. However, we are still merely at the stage of identifying appropriate sources of stem cells and exploring their full differentiation potential. Thus, tracking the stem cells upon in vivo engraftment and during in vitro co-culture is very important and is an area of research embracing many pitfalls. 5-Ethynyl-2'-deoxyuridine (EdU), a rather new thymidine analog incorporated into DNA, has recently been suggested to be a novel highly valid alternative to other dyes for labeling of stem cells and subsequent tracing of their proliferation and differentiation ability. However, our results herein do not at any stage support this recommendation, since EdU severely reduces the viability of stem cells. Accordingly, we found that transplanted EdU-labeled stem cells hardly survive upon in vivo transplantation into regenerating muscle, whereas stem cells labeled in parallel with another dye survived very well and also participated in myofiber formation. Similar data were obtained upon in vitro myogenic culture, and further analysis showed that EdU reduced cell numbers by up to 88 % and increased the cell volume of remaining cells by as much as 91 %. Even at low EdU concentrations, cell survival and phenotype were substantially compromised, and the myogenic differentiation potential was inhibited. Since we examined both primary derived cells and cell lines from several species with the same result, this appears to be a common trait of EdU. We therefore suggest that EdU labeling should be avoided (or used with precaution) for stem cell tracing purposes.

A Simple Method to AssessIn VivoProliferation in Lung Vasculature with EdU: The Case of MMC-Induced PVOD in Rat

“…In some cases, users have to pay attention to the potential toxicity of this method. Indeed, Andersen et al [ 31 ] demonstrated that stem cell survival was severely compromised after EdU incorporation even at low EdU concentrations.…”

Transplanted Donor- or Stem Cell-Derived Cone Photoreceptors Can Both Integrate and Undergo Material Transfer in an Environment-Dependent Manner

“…The reasons for the discrepancy are not clear. Some reports have described potentially toxic effects of EdU, and we ( Warre-Cornish et al., 2014 ) and others ( Andersen et al., 2013 , Ligasova et al., 2015 , Neef and Luedtke, 2011 ) have reported detrimental effects on cell migration and survival. It is possible that such effects may account for the absence of integrated cells in the study by Wallace and colleagues ( Ortin-Martinez et al., 2017 ).…”

Developmental Patterning and Neurogenetic Gradients of Nurr1 Positive Neurons in the Rat Claustrum and Lateral Cortex

“…We investigate the distribution of EdU labeling and Nurr1 expression in rat pups at birth P0 ( Figure 1A ). Since EdU toxicity may adversely affect cellular health ( Andersen et al, 2013 ), we sought to identify the lowest dose of EdU that allows reliable birth dating while maximizing pup survival. We find that injections of 10 mg/kg or 25 mg/kg EdU show little effects on litter size, while higher doses correlate with reduced litter size ( Figure 1B ).…”