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Clonal analysis of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

Friday, November 08, 2013 — Poster Session III

10:00 a.m. – 12:00 p.m.

FAES Academic Center (Upper-Level Terrace)




  • D. Malide
  • J.-Y. Metais
  • C.E. Dunbar


We demonstrate a methodology for tracing the clonal history of hematopoietic stem and progenitor cells (HSPCs) behavior in live tissues in four-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/two-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 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 imaging in 4D demonstrate the possibility of dynamic cellular and clonal tracking in a quantitative manner. Our approach allows clear tracking of color-marked hematopoietic clones readily identifiable in tissues of recipient for extended periods of time, bringing a new tool for assessing the plasticity that HSCPs possess. This methodology has applications in the understanding of clonal architecture in normal and perturbed hematopoiesis and is of clinical relevance for chemotherapy, transplantation and gene therapy.

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