Thrombopoietin-Mpl Signal Induces Hematopoietic Stem Cells into Quiescent State in the Bone Marrow Niche.

Hematopoietic stem cells (HSCs) have the ability to self-renew and to differentiate to produce multi-lineage blood cells throughout the lifetime of individuals. Maintenance of these stem cell activities depends on the balance of intrinsic and extrinsic factors. The factors which regulate HSCs are provided by a microenvironment called stem cell niche, and interactions between HSCs and stem cell niche is critical for the maintenance of stem cell activities.

Recently, it has been reported that HSCs exist frequently aside of the trabecular bone surface in bone marrow (BM). We have previously reported that side-population (SP) in HSC fraction is in the G0 phase and anti-apoptotic stem cells, and contacts osteoblasts. (Arai et al., Cell 2004). To further investigate the regulation of quiescence, cell adhesion, and survival of HSCs, we tried to clone the quiescent HSCs specific molecules by microarray analysis of c-Kit⁺Sca-1⁺Lin⁻ (KSL)-SP vs. non-SP cells. We identified that Mpl, thrombopoietin (Tpo) receptor, was highly expressed in SP cells compared to non-SP cells. Tpo/Mpl signal is known as a physiological regulator of megakaryopoiesis, but the role of Tpo/Mpl signal in the maintenance of HSCs remains elusive.

In this study, we investigated the role of Tpo/Mpl signal on the regulation of HSCs in the niche. The frequency of Mpl⁺ cells in KSL, KSL-SP, and KSL-non SP cells were 50.4 %, 88.9 %, and 44.7 %, respectively. In addition, Tpo was expressed in osteoblastic cells in BM. Immunohistochemical staining of BM showed that Mpl⁺ HSCs adhered to the bone surface and bone-lining osteoblastic cells produced Tpo. These data suggest that Tpo/Mpl signal contributes to the HSCs-niche interaction. BM transplantation (BMT) assay demonstrated that Mpl⁺KSL cells showed high long-term reconstitution (LTR)-activity, whereas Mpl⁻KSL cells did not, suggesting that LTR-HSCs were enriched in Mpl⁺ fraction.

To investigate the function of Tpo/Mpl signal in HSCs, we performed CAFC assay and LTC-IC assay in the presence of anti-Mpl neutralizing antibody (AMM2). Inhibition of Tpo/Mpl signal reduced cobblestone formation and reduced LTC-IC formation. These data suggest that Tpo/Mpl signal maintained immature phenotypes of HSCs in vitro.

It was reported that Mpl deficient mice showed the defect of stem cell function. For rapid and transient inhibition of Mpl signaling in vivo, we administrated AMM2 into the adult mice. AMM2 did not affect the frequency of non-SP fraction, but transiently decreased frequency of SP in KSL after 6 days of injection. Moreover, the combination of AMM2 and 5-FU capacitated BMT without irradiation. In contrast, injection of Tpo increased KSL-SP cells. To understand the mechanism of Tpo/Mpl signal, we cultured Mpl⁺KSL cells in the presence of SCF and/or Tpo, and analyzed the gene expression. We found that Tpo treatment up-regulate β1-integrin and p57, but not p21, p27 or p18. As the up-regulation of p57 is essential for TGF-β induced cell cycle arrest in hematopoiesis, Tpo/Mpl signal may also be related to cell cycle arrest.

Altogether, these data suggest that Tpo/Mpl signal regulates HSCs-niche interaction and enhanced the quiescence of HSCs.