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.