Identification, localization and characterization of hematopoietic stem cells and their niche.

摘要

To improve our ability to identify HSCs and their localization in vivo we compared the gene expression profiles of highly purified hematopoietic stem cells (HSCs) and non-self-renewing multipotent progenitors (MPPs). Cell surface receptors of the SLAM family, including CD150 and CD48, were differentially expressed among functionally distinct progenitors such that HSCs were highly purified as CD150+CD48cells whereas MPPs were CD150-CD48-. The ability to purify HSCs based on a simple combination of SLAM receptors allowed us to identify HSCs in tissue sections. Most HSCs were associated with sinusoidal endothelium in the spleen and bone marrow. This raised the possibility that HSCs reside in perivascular niches.;Our data suggesting that HSCs reside perivascularly was in contrast to studies which proposed that HSCs identified by the thymidine analogue bromo-deoxyuridine (BrdU)-label retention are associated with osteoblasts via N-cadherin-mediated homophilic adhesion. We therefore tested the hypotheses that N-cadherin was important for HSC maintenance and that HSCs could be identified by BrdU-label retention. We did not detect N-cadherin expression in HSCs by polymerase chain reaction, using anti-N-cadherin antibodies, or by beta-galactosidase staining of N-cadherin gene-trap mice. Moreover, N-cadherin deficiency did not affect bone marrow cellularity, the numbers of colony-forming progenitors, the frequency of HSCs, the ability of HSCs to sustain hematopoiesis over time, or their ability to reconstitute irradiated mice. These results indicate that HSCs do not depend on N-cadherin-mediated adhesion to osteoblasts for their maintenance. We tested whether HSCs might retain BrdU, either because they segregate chromosomes asymmetrically or because they divide slowly, by administering BrdU to newborn, cyclophosphamide/G-CSF mobilized, and normal adult mice for 4 to 10 days, followed by 70-days without BrdU. In each case, less than 6% of HSCs retained BrdU and less than 0.5% of BrdU-retaining hematopoietic cells were HSCs, revealing poor specificity and poor sensitivity as an FISC marker. Sequential administration of chloro-deoxyuridine (CIdU) and iodo-deoxyuridine (IdU) suggested that all HSCs segregate their chromosomes randomly. Division of individual HSCs in culture revealed no asymmetric segregation of label. HSCs therefore cannot be identified based on BrdU label-retention and BrdU-label retaining cells are highly unlikely to be HSCs.