Hiroki Yoshihara

Thrombopoietin/MPL Signaling Regulates Hematopoietic Stem Cell Quiescence and Interaction with the Osteoblastic Niche

Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.

Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion.

Type I interferons (IFNs), a family of cytokines, orchestrate numerous biological and cellular processes. Although it is well known that type I IFNs are essential for establishing the host antiviral state, their role in hematopoietic homeostasis has not been studied. Here we show that type I IFNs induce proliferation and exhaustion in hematopoietic stem cells (HSCs) and that interferon regulatory factor-2 (IRF2), a transcriptional suppressor of type I IFN signaling, preserves the self-renewal and multilineage differentiation capacity of HSCs. HSCs were substantially less abundant in the bone marrow of Irf2−/− as compared to Irf2+/− mice. Irf2−/− HSCs showed enhanced cell cycling status and failed to produce hematopoietic cells in competitive repopulation assays, and the reconstituting capacity of Irf2−/− HSCs was restored by disabling type I IFN signaling in these cells. In wild-type mice, injection of poly(I:C), an inducer of type I IFN signaling, or IFN-α induced HSC proliferation, and chronic type I IFN signaling further reduced the number of quiescent HSCs. Notably, combined poly(I:C) and 5-fluorouracil (5-FU) treatment allowed exogenous HSC engraftment and hematopoietic reconstitution in WT mice. Our findings provide insight into the molecular basis for the maintenance of HSC quiescence and may lead to improvements in bone marrow transplantation and type I IFN–based therapies for viral infection and cancer.

p57(Kip2) and p27(Kip1) cooperate to maintain hematopoietic stem cell quiescence through interactions with Hsc70.

Cell cycle regulators play critical roles in the balance between hematopoietic stem cell (HSC) dormancy and proliferation. In this study, we report that cell cycle entry proceeded normally in HSCs null for cyclin-dependent kinase (CDK) inhibitor p57 due to compensatory upregulation of p27. HSCs null for both p57 and p27, however, were more proliferative and had reduced capacity to engraft in transplantation. We found that heat shock cognate protein 70 (Hsc70) interacts with both p57 and p27 and that the subcellular localization of Hsc70 was critical to maintain HSC cell cycle kinetics. Combined deficiency of p57 and p27 in HSCs resulted in nuclear import of an Hsc70/cyclin D1 complex, concomitant with Rb phosphorylation, and elicited severe defects in maintaining HSC quiescence. Taken together, these data suggest that regulation of cytoplasmic localization of Hsc70/cyclin D1 complex by p57 and p27 is a key intracellular mechanism in controlling HSC dormancy.

Isolation and characterization of endosteal niche cell populations that regulate hematopoietic stem cells

The endosteal niche is critical for the maintenance of hematopoietic stem cells (HSCs). However, it consists of a heterogeneous population in terms of differentiation stage and function. In this study, we characterized endosteal cell populations and examined their ability to maintain HSCs. Bone marrow endosteal cells were subdivided into immature mesenchymal cell-enriched ALCAM(-)Sca-1(+) cells, osteoblast-enriched ALCAM(+)Sca-1(-), and ALCAM(-)Sca-1(-) cells. We found that all 3 fractions maintained long-term reconstitution (LTR) activity of HSCs in an in vitro culture. In particular, ALCAM(+)Sca-1(-) cells significantly enhanced the LTR activity of HSCs by the up-regulation of homing- and cell adhesion-related genes in HSCs. Microarray analysis showed that ALCAM(-)Sca-1(+) fraction highly expressed cytokine-related genes, whereas the ALCAM(+)Sca-1(-) fraction expressed multiple cell adhesion molecules, such as cadherins, at a greater level than the other fractions, indicating that the interaction between HSCs and osteoblasts via cell adhesion molecules enhanced the LTR activity of HSCs. Furthermore, we found an osteoblastic marker(low/-) subpopulation in ALCAM(+)Sca-1(-) fraction that expressed cytokines, such as Angpt1 and Thpo, and stem cell marker genes. Altogether, these data suggest that multiple subsets of osteoblasts and mesenchymal progenitor cells constitute the endosteal niche and regulate HSCs in adult bone marrow.

Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells

During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized microenvironments termed stem cell niches. Cell-cell and cell-extracellular matrix interactions between HSCs and their niches are critical for the maintenance of HSC properties. Here, we analyzed the function of N-cadherin in the regulation of the proliferation and long-term repopulation activity of hematopoietic stem/progenitor cells (HSPCs) by the transduction of N-cadherin shRNA. Inhibition of N-cadherin expression accelerated cell division in vitro and reduced the lodgment of donor HSPCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. Cotransduction of N-cadherin shRNA and a mutant N-cadherin that introduced the silent mutations to shRNA target sequences rescued the accelerated cell division and reconstitution phenotypes. In addition, the requirement of N-cadherin for HSPC engraftment appears to be niche specific, as shN-cad-transduced lineage(-)Sca-1(+)c-Kit(+) cells successfully engrafted in spleen, which lacks an osteoblastic niche. These findings suggest that N-cad-mediated cell adhesion is functionally required for the establishment of hematopoiesis in the BM niche after BM transplantation.

Cadherin-Based Adhesion Is a Potential Target for Niche Manipulation to Protect Hematopoietic Stem Cells in Adult Bone Marrow

During postnatal life, hematopoietic stem cells (HSCs) are maintained in specialized bone marrow (BM) niches (Morrison and Spradling, 2008; Wilson and Trumpp, 2006; Yin and Li, 2006). Cadherins are major cell adhesion molecules responsible for Ca2+-dependent cell-cell interaction (Gumbiner, 1996), but the role of cadherin function, and more specifically N-cadherin, in HSC-niche interactions has been controversial (Li and Zon, 2010). In this study, we analyzed the expression of various cadherin (cad) genes in long-term HSCs (LT-HSCs) and subpopulations of the cells isolated from the endosteum.

Effects of methylenetetrahydrofolate reductase and reduced folate carrier 1 polymorphisms on high-dose methotrexate-induced toxicities in children with acute lymphoblastic leukemia or lymphoma

The authors investigated whether high-dose methotrexate-induced toxicity differed according to the presence of methylenetetrahydrofolate reductase (MTHFR) or reduced folate carrier 1 (RFC1) genetic polymorphism. The authors studied 15 children with acute lymphoblastic leukemia or lymphoblastic lymphoma who were treated using protocols that included high-dose methotrexate (3.0 g/m2), for an overall total of 43 courses. Methotrexate-induced toxicities and the plasma methotrexate concentrations were evaluated retrospectively. Hematologic toxicity was the most frequently observed toxicity, appearing in 87% of the patients. In a subset of patients (47%), elevation of liver transaminase levels showed a repeated tendency to develop. High plasma methotrexate concentrations at 48 hours after the methotrexate infusion were not significantly related to methotrexate-induced toxicities except for mucositis. A generalized estimating equation analysis revealed that vomiting during the high-dose methotrexate treatment was more pronounced in patients who had a larger number of G alleles at the RFC1 80G>A polymorphism. No significant differences in the development of other toxicities or in the plasma methotrexate concentrations were observed for the different MTHFR 677C>T or RFC1 80G>A polymorphisms. This study suggests but does not prove that the RFC1 80G>A polymorphism may contribute to interindividual variability in responses to high-dose methotrexate.

Niche regulation of hematopoietic stem cells in the endosteum.

During postnatal life, the bone marrow (BM) supports both the self‐renewal and differentiation of hematopoietic stem cells (HSCs) in specialized niches. The interaction of HSCs with their niches also regulates the quiescence of HSCs. HSC quiescence is critical to ensure lifelong hematopoiesis and to protect the HSC pool from myelotoxic insult and premature exhaustion under conditions of hematopoietic stress. Here we identified long‐term (LT)‐HSCs expressing the thrombopoietin (THPO) receptor, Mpl, as a quiescent population in adult BM. THPO was produced by bone‐lining cells in the endosteum. Inhibition and stimulation of the THPO/Mpl pathway produced opposite effects on the quiescence of LT‐HSC. Exogenous THPO transiently increased the quiescent LT‐HSC population, such as side‐population and pyronin Y–negative cells. In contrast, administration of an anti‐Mpl neutralizing antibody, AMM2, suppressed the quiescence of LT‐HSCs and enabled HSC engraftment without irradiation, indicating that inhibition of THPO/Mpl signaling reduces HSC–niche interactions. Moreover, it suggests that inhibiting the HSC–niche interaction could represent a novel technique for bone marrow transplantation without irradiation. Altogether, these data suggest that the THPO/Mpl signaling pathway is a novel niche component in the endosteum, and in the steady‐state condition, this signaling pathway plays a critical role in the regulation of LT‐HSCs in the osteoblastic niche.

Reconstitution activity of hypoxic cultured human cord blood CD34-positive cells in NOG mice

Hematopoietic stem cells (HSCs) reside in hypoxic areas of the bone marrow. However, the role of hypoxia in the maintenance of HSCs has not been fully characterized. We performed xenotransplantation of human cord blood cells cultured in hypoxic or normoxic conditions into adult NOD/SCID/IL-2Rgamma(null) (NOG) mice. Hypoxic culture (1% O(2)) for 6 days efficiently supported the maintenance of HSCs, although cell proliferation was suppressed compared to the normoxic culture. In contrast, hypoxia did not affect in vitro colony-forming ability. Upregulation of a cell cycle inhibitor, p21, was observed in hypoxic culture. Immunohistochemical analysis of recipient bone marrow revealed that engrafted CD34(+)CD38(-) cord blood HSCs were hypoxic. Taken together, these results demonstrate the significance of hypoxia in the maintenance of quiescent human cord blood HSCs.

Functional differences between two Tie2 ligands, angiopoietin-1 and -2, in regulation of adult bone marrow hematopoietic stem cells

OBJECTIVE Angiopoietin-1 (Ang-1) plays a critical role in the maintenance of hematopoietic stem cells (HSCs) in the bone marrow (BM) through its binding to the Tie2 receptor. Ang-2, another Tie2 ligand, is known to be an antagonist of Tie2/Ang-1 signaling in angiogenesis; however, its function in regulation of HSCs remains unclear. Here, we investigated the functional differences between Ang-1 and Ang-2 in the maintenance of HSCs. MATERIALS AND METHODS We treated mouse BM lineage(-)Sca-1(+)c-Kit(+) side population(+) cells with Ang-1 and/or Ang-2, and evaluated angiopoietin function by gene expression analysis, immunocytochemical staining of phosphorylated Akt, a colony-formation assay, and a long-term BM reconstitution assay. RESULTS Gene expression analysis and BM transplantation assay revealed that Ang-1 upregulated expression of p57, p18, Itgb1, Alcam, Tie2, Hoxb4, and Bmi1 genes in HSCs, while Ang-2 antagonized the effects of Ang-1. Ang-1 enhanced the phosphorylation of Akt, while Ang-2 again reduced the effect of Ang-1. The colony assay demonstrated that neither Ang-1, nor Ang-2 influenced the colony formation of HSCs. BM transplantation assay, following in vitro cultivation of HSCs with angiopoietins, showed that Ang-1 maintained long-term repopulating activity of HSCs, while the addition of Ang-2 interfered drastically with the effects of Ang-1. CONCLUSION Gene expression analysis and BM transplantation assay demonstrated that Ang-1 maintained HSC activity in an in vitro culture. In contrast, Ang-2 reversed the effects of Ang-1/Tie2 signaling in the regulation of long-term HSCs. Our data suggest that Ang-1 is a dominant ligand for the Tie2 receptor in long HSCs in BM.