title: Long-term detection of fluorescently labeled human mesenchymal stem cell in vitro and in vivo by semi-automated microscopy.
abstract: The use of seeded scaffolds in regenerative medicine is limited by the low survival of transplanted mesenchymal stem cells (MSC). Current approaches aim at improving cell viability but require an adequate long-term detection of the transplanted cells. Unfortunately, commonly performed labeling techniques have not been validated for this purpose, and studies often reveal inconclusive results. Consequently, we intended to identify the most suitable method for long-term detection of human MSC (hMSC) in vitro and in vivo. hMSC were labeled using the vital stainings PKH26 and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) as well as enhanced green fluorescent protein (eGFP) transduction. Metabolic activity and relative fluorescence intensity (RFI) were quantified in vitro over 21 days at 8 time points using standardized semi-automated microscopy and flow cytometry. In vivo, cell seeded scaffolds were subcutaneously implanted in nude mice, and RFI was analyzed over 42 days at 5 time points. In vitro, PKH26 and CFDA-SE significantly reduced metabolic activity. RFI of both stainings significantly decreased after 1 day and further faded to <1% after 7 days. In contrast, labeling with eGFP showed no metabolic effect on hMSC, and no significant reduction of RFI over the total period of 21 days. In vivo, RFI of eGFP labeled cells reached a plateau phase after 21 days and displayed a 3.8-fold higher RFI compared with PKH26 and CFDA-SE on day 42 evaluated in 280 field of views per scaffold using three scaffolds for each labeling technique and time point. We conclude that PKH26 and CFDA-SE are unsuitable for long-term detection of hMSC. eGFP transduction, in turn, allows long-term detection of hMSC in vitro and in vivo. Our results suggest that eGFP is currently the best option among the fluorescent labeling techniques to follow the fate of transplanted hMSC.
authors.affiliation: Experimental Surgery and Regenerative Medicine, Department of Surgery, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Hans
authors.initials: H
authors.lastname: Polzer
authors.forename: Elias
authors.initials: E
authors.lastname: Volkmer
authors.forename: Maximilian M
authors.initials: MM
authors.lastname: Saller
authors.forename: Wolf C
authors.initials: WC
authors.lastname: Prall
authors.forename: Florian
authors.initials: F
authors.lastname: Haasters
authors.forename: Inga
authors.initials: I
authors.lastname: Drosse
authors.forename: David
authors.initials: D
authors.lastname: Anz
authors.forename: Wolf
authors.initials: W
authors.lastname: Mutschler
authors.forename: Matthias
authors.initials: M
authors.lastname: Schieker
mesh_terms: D000465:Algorithms; D000818:Animals; D001331:Automation; D001857:Bone Matrix / Q000378:metabolism; D002113:Calcification, Physiologic; D002460:Cell Line; D002470:Cell Survival; D058948:Cell Tracking; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D008819:Mice, Nude; D008856:Microscopy, Fluorescence / Q000379:methods*; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013778:Tetrazolium Salts; D013997:Time Factors; D054457:Tissue Scaffolds / Q000737:chemistry
chemical_list: C110656:2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; D005456:Fluorescent Dyes; D013778:Tetrazolium Salts; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins