Spp1 (Osteopntin) Is Essential for the Early Niche Formation in the Bone Marrow.

Hematopoietic stem cells (HSCs) have a potential to differentiate into variety types of blood cells, and have an ability of self-renewing in the bone marrow (BM). All these fates are regulated by intrinsic genetic pathways in the micro-enviroment (niche) where HSCs are located. Osteoblasts are thought to be the hematopoietic niche which regulates HSC function in the BM. To clarify the molecular basis of hematopoietic niche system, we compared the two somatic stem cells (Germ line stem cell (GS) and HSC) using cDNA suppression subtractive hybridization, and identified 12 stem cell-specific genes which are expressed both in the HSCs and GS. Among these genes, we analyzed Spp1, since it is a major glycoprotein produced by osteoblast and it has been reported to be a negative regulator of hematopoietic niche size in the BM.

In situ Hybridization (ISH) with Spp1-specific probes demonstrated that Spp1-expressing osteobalsts were located at the endosteal region of trabecular bone (TB). In the TB of adult mouce, Spp1-expressing hematopoietic cells attached with Spp1-expressing osteobalsts. Double ISH analysis revealed that Spp1 and Tie2 were partially co-expressed in the TB. These data suggests that Spp1 is an important molecule of hematopoietic niche system which were produced and interacted both HSCs and niche cell.

When we analyzed spp1 knock out mouse (Spp1−/−), adult Spp1−/− mice (8 weeks) appeared to be normal and fertile. However, the abnormal TB formation was observed in the younger Spp1−/− mice (2 weeks). A number of osteobalsts were increased and formed a multi-layer of osteoblasts at the endosteal region of TB. Along with the morphological change of the TB, distribution pattern of CD34, Tie2 and Bmi-1 expressing HSC was unusual. Tie2 expressing cells of Spp1−/− mouse were mainly located at vascular-enriched zone in the bone marrow, but few Tie2 expressing cells at osteoblastic zone. These data suggest that HSC and osteobalst do not form the correct niche in the Spp1−/− TB, and Spp1 is thought to be essential for the initial niche formation. To confirm the function of Spp1 at the initiation phase of niche formation, we performed the bone marrow transplantation. Mononuclear cells (1×10⁵ cells) from Ly5.1 mice were transplanted into lethally irradiated Ly5.2 mice. At 12 days after transplantation, donor hematopoietic cells were observed in the recipient TB. At this day 12, the expression of Spp1 was up-regulated in the endosteal region of TB, however, Spp1 expression was decreased in the course of reconstitution. Thus, the findings regarding the hematopoietic niche formation in Spp1−/− mice and in the irradiated mice allow to understand the Spp1 function in the initial phase of niche formation in the bone marrow.