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Fluorescence-based tracing of transplanted intestinal epithelial cells using confocal laser endomicroscopy.
Bergenheim, Fredrik; Seidelin, Jakob B; Pedersen, Marianne Terndrup; Mead, Benjamin E; Jensen, Kim B; Karp, Jeffrey M; Nielsen, Ole Haagen.
Afiliação
  • Bergenheim F; Department of Gastroenterology, Herlev Hospital, University of Copenhagen, 2730, Herlev, Denmark. Fredrik.Bergenheim@regionh.dk.
  • Seidelin JB; Department of Gastroenterology, Herlev Hospital, University of Copenhagen, 2730, Herlev, Denmark.
  • Pedersen MT; Biotech Research and Innovation Center (BRIC), University of Copenhagen, DK-2200, Copenhagen, Denmark.
  • Mead BE; Broad Institute of Massachusetts, Institute of Technology and Harvard University, Cambridge, MA, 02139, USA.
  • Jensen KB; Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology and Harvard University, Cambridge, MA, 02139, USA.
  • Karp JM; Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
  • Nielsen OH; Institute for Medical Engineering and Science (IMES), Massachusetts Institute of Technology, Cambridge, MA, USA.
Stem Cell Res Ther ; 10(1): 148, 2019 05 27.
Article em En | MEDLINE | ID: mdl-31133056
BACKGROUND: Intestinal stem cell transplantation has been shown to promote mucosal healing and to engender fully functional epithelium in experimental colitis. Hence, stem cell therapies may provide an innovative approach to accomplish mucosal healing in patients with debilitating conditions such as inflammatory bowel disease. However, an approach to label and trace transplanted cells, in order to assess engraftment efficiency and to monitor wound healing, is a key hurdle to overcome prior to initiating human studies. Genetic engineering is commonly employed in animal studies, but may be problematic in humans due to potential off-target and long-term adverse effects. METHODS: We investigated the applicability of a panel of fluorescent dyes and nanoparticles to label intestinal organoids for visualization using the clinically approved imaging modality, confocal laser endomicroscopy (CLE). Staining homogeneity, durability, cell viability, differentiation capacity, and organoid forming efficiency were evaluated, together with visualization of labeled organoids in vitro and ex vivo using CLE. RESULTS: 5-Chloromethylfluorescein diacetate (CMFDA) proved to be suitable as it efficiently stained all organoids without transfer to unstained organoids in co-cultures. No noticeable adverse effects on viability, organoid growth, or stem cell differentiation capacity were observed, although single-cell reseeding revealed a dose-dependent reduction in organoid forming efficiency. Labeled organoids were easily identified in vitro using CLE for a duration of at least 3 days and could additionally be detected ex vivo following transplantation into murine experimental colitis. CONCLUSIONS: It is highly feasible to use fluorescent dye-based labeling in combination with CLE to trace intestinal organoids following transplantation to confirm implantation at the intestinal target site.
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Texto completo: 1 Base de dados: MEDLINE Assunto principal: Microscopia Confocal / Células Epiteliais / Fluorescência / Mucosa Intestinal Limite: Animals / Humans / Male Idioma: En Ano de publicação: 2019 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Microscopia Confocal / Células Epiteliais / Fluorescência / Mucosa Intestinal Limite: Animals / Humans / Male Idioma: En Ano de publicação: 2019 Tipo de documento: Article