Fractionated Osteoblastic Niche Cells Enhances the Homing Activity of Hematopoietic Stem Cells.

Abstract 560

Hematopoietic stem cells (HSCs) retain quiescent state in the adult bone marrow (BM), interacting with specialized niches along the endosteum (osteoblastic niche) and in perivascular sites adjacent to endothelial and reticular cells (perivascular niche). We have previously reported that the regulations of HSCs in the osteoblastic niche through the receptor-cytokine, cell-to-cell, and cell-to-extracellular matrix (ECM) interactions are critical for the maintenance of cell cycle quiescence. However, osteoblast population is heterogeneous in terms of the degree of differentiation and function of each cell. In this study, we characterized the cellular components of osteoblastic niche and examined the ability for the maintenance of HSCs. We found that CD45–CD31–Ter119– endosteal population can be subdivided into three fractions, ALCAM+Sca-1–, ALCAM–Sca-1+, and ALCAM–Sca-1– cells. Expression of osteoblast markers and differentiation potential of these three fractions revealed that osteoblasts were enriched in the Sca-1– populations. On the other hand, ALCAM–Sca-1+ cells had characteristics of mesenchymal progenitor cells (MPCs). Microarray analysis showed that ALCAM+Sca-1– fraction tend to highly express the genes related to cell-to-cell or cell-to-ECM adhesions, such as N-cadherin, Osteopontin, and Alcam, compared to other fractions, indicating that this population might regulate HSCs through the physiological interactions. On the other hand, ALCAM–Sca-1+ fraction highly expressed the genes related to growth factor and cytokine that are involved in the regulation of both quiescence and proliferation of long-term-HSCs, such as Angiopoietin-1, Flt3l, Cxcl12, Thrombopoietin, and Kitl. These data rise the possibility that multiple cell populations in endosteum collaborate as a complex to regulate the balance between HSC proliferation and quiescence in the endosteum in adult BM. Next we examined whether these fractionated cells could maintain the long-term repopulation (LTR) activity of HSCs in vitro. BM Lin–Sca-1+Kit+ (LSK) cells were cocultured with each fractionated endosteal population without growth factors. After 2 days of coculture, Ly5.1+ cells were sorted and transplanted into lethally irradiated mice. We found that all three fractions maintained LTR-activity of LSK cells. In particular, LSK cells cocultured with ALCAM+Sca-1– cells showed significantly higher LTR-activity compared to that cocultured with other fractions. Then we analyzed the gene expression implicated in the homing and lodgment of HSCs. Q-PCR array analysis revealed that upregulation of Cxcr4, integrins, Cd44, N-cadherin and Alcam was induced in LSK cells cocultured with Sca-1– populations. In particular, ALCAM+Sca-1– cells significantly upregulated N-cadherin expression in HSCs. These data suggest that osteoblasts increased homing activity of HSCs in culture or enriched cell population that had high homing activity. Furthermore, the cell-to-cell adhesion between HSCs and ALCAM+Sca-1– cells enhanced the interaction of HSCs with niche complex and maintained self-renewal activity of HSCs.