Newly formed skeletal muscle fibers are prone to false positive immunostaining by rabbit antibodies

Andersen, Ditte Caroline; Kliem, Anette; Jensen, Charlotte Harken

doi:10.1016/j.acthis.2009.08.001

Cited by 1 publication

(2 citation statements)

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“…It is well known that engraftment of stem cells is greatly enhanced by tissue damage. As previously described [2,3,11], we therefore performed a stab lesion in the m. gastrocnemius and immediately thereafter transplanted 10 5 DiI/EdU-labeled EPCs into five injection sites-four outside the lesion and one centered in the middle. At day 7 post transplantation, we dissected the engrafted muscles (n =6) and performed immunohistochemistry and EdU detection in order to trace donor EPCs.…”

Section: Resultsmentioning

confidence: 99%

“…In order to track the stem cells in the differentiating environments, fluorescent dyes of various types have been developed for stem cell labeling [1]. However, stem cell tagging is often questioned due to issues such as diffusion of small tracers like green flourescent protein (GFP) into the surroundings or fading of dye fluorescents as well as analysis settings, which all may result in erroneous conclusions on stem cell potential [2][3][4][5]. Most often, labeling dyes reside within the cytoplasm of the labeled cell, and some even associate to cytoplasmic structures, whereas only a few dyes localize to the nucleus.…”

Section: Introductionmentioning

confidence: 99%

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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

et al. 2013

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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.

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Section: Resultsmentioning

confidence: 99%