title: In vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy.
abstract: We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and temporal resolution to study in vivo hematopoiesis using the murine bone marrow transplant experimental model. Genetic combinatorial marking using lentiviral vectors encoding fluorescent proteins (FPs) enabled cell fate mapping through advanced microscopy imaging. Vectors encoding five different FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were used to concurrently transduce HSPCs, creating a diverse palette of color marked cells. Imaging using confocal/two-photon hybrid microscopy enables simultaneous high resolution assessment of uniquely marked cells and their progeny in conjunction with structural components of the tissues. Volumetric analyses over large areas reveal that spectrally coded HSPC-derived cells can be detected non-invasively in various intact tissues, including the bone marrow (BM), for extensive periods of time following transplantation. Live studies combining video-rate multiphoton and confocal time-lapse imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner.
authors.affiliation: Light Microscopy Core Facility, NHLBI/NIH; dmalide@nih.gov.
authors.forename: Daniela
authors.initials: D
authors.lastname: Malide
authors.affiliation: Hematology Branch, NHLBI/NIH.
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Métais
authors.affiliation: Hematology Branch, NHLBI/NIH.
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D058948:Cell Tracking / Q000379:methods*; D002999:Clone Cells / Q000737:chemistry / Q000166:cytology; D005260:Female; D005455:Fluorescent Antibody Technique / Q000379:methods*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods; D006412:Hematopoietic Stem Cells / Q000737:chemistry* / Q000166:cytology*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*
chemical_list: D008164:Luminescent Proteins