N-Cadherin Induces Hematopoietic Stem Cells in a Quiescent State in the Bone Marrow Niche.

Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout the lifetime of individuals. Interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining the stem cell properties, including self-renewal capacity and the ability of differentiation into single or multiple lineages. The niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules, and regulate stem cell fates. Recently, it was clarified that long-term bone marrow (BM) repopulating (LTR) HSCs exist frequently in BM trabecular bone surface, and that N-cadherin ⁺ spindle-shaped osteoblasts (OBs) are identified as a major niche component. We found that side-population (SP) in c-Kit ⁺Sca-1 ⁺Lin ⁻(KSL) fraction, which is the quiescent HSC in the OB niche, expressed N-cadherin. Expression of N-cadherin in both of the quiescent HSCs and OBs thought to be essential for an adherens junction between HSCs and OBs in the niche. However, the role of N-cadherin in hematopoiesis is still unclear.

In this study, we focused on the function of N-cadherin in the maintenance of the stem cell specific property, such as cell adhesion, quiescence, and LTR-activity. To clarify the function of N-cadherin in hematopoiesis, we prepared the retroviruses expressing wild-type N-cadherin, transfected retroviruses into OP9 stromal cell line and KSL cells, and performed the coculture. After coculture of KSL cells with OP9 cells, long-term culture-initiating cells (LTC-ICs) were maintained on OP9 cells overexpressing WT-N-cadherin (OP9/WT-NCAD). In addition, overexpression of WT-N-cadherin in both of the KSL cells and stromal cells enhanced cobblestone formation. N-cadherin overexpressing KSL cell showed slow-cell division from the single cell, when they cultured on OP9/WT-N-cedherin or N-cadherin-Fc protein coated plates, suggesting that N-cadherin-mediated cell-cell adhesion between HSCs and stromal cells enhances the quiescence of HSCs and keeps HSCs in immature state in in vitro. To clarify the role of N-cadherin in the BM reconstitution ability of HSC, we transfected control-IRES-GFP, WT-N-cadherin-IRES-GFP and N-cedherin/390Δ-IRES-GFP retrovirus into the Ly5.1 BM mononuclear cells and transplanted into lethally irradiated Ly5.2 mice. N-cedherin/390Δ, which is a mutant N-cadherin with a deletion at the extracellular domain, exhibits a dominant negative effect on the activity of endogenous cadherins. Control and WT-N-cadherin expressing cell reconstitute the recipient mice BM, while N-cadherin/390Δ expressing cells did not. It suggests that the adhesion between HSCs and BM niche cell is indispensable for the LTR-activity. In addition, we found that WT-N-cadherin overexpressing HSCs were enriched in the SP fraction after 4 months of BM transplantation, indicating that N-cadherin-mediated cell adhesion induced HSCs in the quiescent and kept quiescent HSCs in the niche.

Altogether, these observations suggest that N-cadherin is a critical niche factor for the maintenance of the quiescence and self-renewal activity of HSCs. N-cadherin promotes tight adhesion of HSCs to the niche and keeps HSCs in the quiescent state