title: Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.
abstract: We demonstrate a methodology for tracing the clonal history of hematopoietic stem and progenitor cells (HSPCs) behavior in live tissues in 4 dimensions (4D). This integrates genetic combinatorial marking using lentiviral vectors encoding various fluorescent proteins (FPs) with advanced imaging methods. Five FPs: Cerulean, EGFP, Venus, tdTomato, and mCherry were concurrently used to create a diverse palette of color-marked cells. A key advantage of imaging using a confocal/2-photon hybrid microscopy approach is the simultaneous assessment of uniquely 5FP-marked cells in conjunction with structural components of the tissues at high resolution. Volumetric analyses revealed that spectrally coded HSPC-derived cells can be detected noninvasively in various intact tissues, including the bone marrow, for extensive periods of time after transplantation. Live studies combining video-rate multiphoton and confocal imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. This methodology has applications in the understanding of clonal architecture in normal and perturbed hematopoiesis.
authors.affiliation: Light Microscopy Core Facility. National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA. dmalide@nih.gov
authors.forename: Daniela
authors.initials: D
authors.lastname: Malide
authors.forename: Jean-Yves
authors.initials: JY
authors.lastname: Métais
authors.forename: Cynthia E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000818:Animals; D058948:Cell Tracking / Q000379:methods; D002478:Cells, Cultured; D060965:Clonal Evolution / Q000502:physiology*; D002999:Clone Cells / Q000166:cytology; D005260:Female; D005456:Fluorescent Dyes / Q000032:analysis* / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*; D021621:Imaging, Three-Dimensional; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods; D041681:NIH 3T3 Cells; D013194:Staining and Labeling / Q000379:methods; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism / Q000648:ultrastructure*
chemical_list: D005456:Fluorescent Dyes