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a Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, Pittsburgh, Pennsylvania, USA;
b Institute for Cellular Therapeutics, University of Louisville, Louisville, Kentucky, USA;
c Division of Immunogenetics, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA;
d Department of Bioengineering, University of Pittsburgh, Pittsburgh, Pennsylvania, USA;
e Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
Key Words. Hematopoietic stem cells • Bone marrow transplantation • In vivo optical imaging • Fluorescence microscopy
Nadir Askenasy, M.D., Frankel Laboratory of Bone Marrow Transplantation, Center of Pediatric Hematology Oncology, Schneider Children's Medical Center of Israel, 14 Kaplan Street, Petach Tikva 49202, Israel. Telephone: 972-3-641-1475; Fax: 972-3-641-1475; e-mail: anadir{at}012.net.il or askenasy{at}andrew.cmu.edu
The process of hematopoietic stem and progenitor cell (HSPC) seeding in recipient bone marrow (BM) early after transplantation is not fully characterized. In vivo tracking of HSPCs, labeled with PKH dyes, through an optical window surgically implanted on the mouse femur revealed that transplanted cells cluster in the recipient BM. Within the first day after intravenous injection, 86 ± 6% of the cells seeded in clusters (p < 0.001 versus scattered cells) in the endosteal surfaces of the epiphyses. The primary clusters were formed by concomitant seeding of 6-10 cells over an area of ~70 µm, and secondarily injected cells did not join the already existing clusters but formed new clusters. Major antigen-disparate HSPCs participated in formation of the primary clusters, and T lymphocytes were also incorporated. After 4 to 5 days, some cellular clusters were observed in the more central regions of the BM, where the brightness of PKH fluorescence decreased, indicating cellular division. These later clusters were classified as secondary, assuming that the mechanisms of migration in the BM might be different from those of primary seeding. Some clusters remained in the periphery of the BM and retained bright fluorescence, indicating cellular quiescence. The number of brightly fluorescent cells in the clusters decreased exponentially to two to three cells after 24 days (p < 0.001). The data suggest that the hematopoietic niche is a functional unit of the BM stromal microenvironment that hosts seeding of a number of transplanted cells, which form a cluster. This may be the site where auxiliary non-HSPC cells, such as T lymphocytes, act in support of HSPC engraftment.
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