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.     

Functional ramifications for the loss of P-selectin expression on hematopoietic and leukemic stem cells

Hematopoiesis is a tightly regulated biological process that relies upon complicated interactions between blood cells and their microenvironment to preserve the homeostatic balance of long-term hematopoietic stem cells (LT-HSCs), short-term HSCs (ST-HSCs), multipotent progenitors (MPPs), and differentiated cells. Adhesion molecules like P-selectin (encoded by the Selp gene) are essential to hematopoiesis, and their dysregulation has been linked to leukemogenesis. Like HSCs, leukemic stem cells (LSCs) depend upon their microenvironments for survival and propagation. P-selectin plays a crucial role in Philadelphia chromosome-positive (Ph(+)) chronic myeloid leukemia (CML). In this paper, we show that cells deficient in P-selectin expression can repopulate the marrow more efficiently than wild type controls. This results from an increase in HSC self-renewal rather than alternative possibilities like increased homing velocity or cell cycle defects. We also show that P-selectin expression on LT-HSCs, but not ST-HSCs and MPPs, increases with aging. In the absence of P-selectin expression, mice at 6 months of age possess increased levels of short-term HSCs and multipotent progenitors. By 11 months of age, there is a shift towards increased levels of long-term HSCs. Recipients of BCR-ABL-transduced bone marrow cells from P-selectin-deficient donors develop a more aggressive CML, with increased percentages of LSCs and progenitors. Taken together, our data reveal that P-selectin expression on HSCs and LSCs has important functional ramifications for both hematopoiesis and leukemogenesis, which is most likely attributable to an intrinsic effect on stem cell self-renewal.     

Quantification of self-renewal capacity in single hematopoietic stem cells from normal and Lnk-deficient mice

Despite being a hallmark of hematopoietic stem cells (HSCs), HSC self-renewal has never been quantitatively assessed. Establishment of a clonal and quantitative assay for HSC function permitted demonstration that adult mouse HSCs are significantly heterogeneous in degree of multilineage repopulation and that higher repopulating potential reflects higher self-renewal activity. An HSC with high repopulating potential could regenerate approximately 1000 HSCs, whereas the repopulating activity of regenerated HSCs on average was significantly reduced, indicating extensive but limited self-renewal capacity in HSCs. Comparisons of wild-type mice with mutant mice deficient in the signal adaptor molecule Lnk showed that not only HSC numbers but also the self-renewal capacity of some HSCs are markedly increased when Lnk function is lost. Lnk appears to control HSC numbers by negatively regulating HSC self-renewal signaling.     

Luteinizing hormone signaling restricts hematopoietic stem cell expansion during puberty

The number and self‐renewal capacity of hematopoietic stem cells (HSCs) are tightly regulated at different developmental stages. Many pathways have been implicated in regulating HSC development in cell autonomous manners; however, it remains unclear how HSCs sense and integrate developmental cues. In this study, we identified an extrinsic mechanism by which HSC number and functions are regulated during mouse puberty. We found that the HSC number in postnatal bone marrow reached homeostasis at 4 weeks after birth. Luteinizing hormone, but not downstream sex hormones, was involved in regulating HSC homeostasis during this period. Expression of luteinizing hormone receptor (Lhcgr) is highly restricted in HSCs and multipotent progenitor cells in the hematopoietic hierarchy. When Lhcgr was deleted, HSCs continued to expand even after 4 weeks after birth, leading to abnormally elevated hematopoiesis and leukocytosis. In a murine acute myeloid leukemia model, leukemia development was significantly accelerated upon Lhcgr deletion. Together, our work reveals an extrinsic counting mechanism that restricts HSC expansion during development and is physiologically important for maintaining normal hematopoiesis and inhibiting leukemogenesis.     

Nbs1‐mediated DNA damage repair pathway regulates haematopoietic stem cell development and embryonic haematopoiesis

ObjectivesDNA damages pose threats to haematopoietic stem cells (HSC) maintenance and haematopoietic system homeostasis. Quiescent HSCs in adult mouse bone marrow are resistant to DNA damage, while human umbilical cord blood‐derived proliferative HSCs are prone to cell death upon ionizing radiation. Murine embryonic HSCs proliferate in foetal livers and divide symmetrically to generate HSC pool. How murine embryonic HSCs respond to DNA damages is not well‐defined.Materials and methodsMice models with DNA repair molecule Nbs1 or Nbs1/p53 specifically deleted in embryonic HSCs were generated. FACS analysis, in vitro and in vivo HSC differentiation assays, qPCR, immunofluorescence and Western blotting were used to delineate roles of Nbs1‐p53 signaling in HSCs and haematopoietic progenitors.ResultsNbs1 deficiency results in persistent DNA breaks in embryonic HSCs, compromises embryonic HSC development and finally results in mouse perinatal lethality. The persistent DNA breaks in Nbs1 deficient embryonic HSCs render cell cycle arrest, while driving a higher rate of cell death in haematopoietic progenitors. Although Nbs1 deficiency promotes Atm‐Chk2‐p53 axis activation in HSCs and their progenies, ablation of p53 in Nbs1 deficient HSCs accelerates embryonic lethality.ConclusionsOur study discloses that DNA double‐strand repair molecule Nbs1 is essential in embryonic HSC development and haematopoiesis. Persistent DNA damages result in distinct cell fate in HSCs and haematopoietic progenitors. Nbs1 null HSCs tend to be maintained through cell cycle arrest, while Nbs1 null haematopoietic progenitors commit cell death. The discrepancies are mediated possibly by different magnitude of p53 signaling.     

Niche-less maintenance of HSCs by 2i

Maintenance of hematopoietic stem cells (HSCs) in vitro has been believed to be difficult due to a lack of complete understanding of HSC quiescence maintained by the niche. Recent evidence suggests that in vitro maintenance of human and mouse long-term HSCs (LT-HSCs) is possible through dual inhibition (2i) of both GSK-3 and mTOR in the absence of cytokines, serum, or feeder cells.Hematopoietic stem cells (HSCs) are generally quiescent, and have the ability to self-renew or to differentiate into mature blood cells. Despite recent advances, it has not been possible to maintain functional long-term HSCs (LT-HSCs) outside the hematopoietic niche, because mechanisms by which HSC quiescence is maintained by the niche¹ have not been fully understood. There have been many attempts to expand HSCs and hematopoietic progenitor cells in vitro using hematopoietic cytokines combined with factors, including Wnt activators^2,3,4, glycogen synthase kinase 3 (GSK-3) inhibitors⁵, Notch ligand, HoxB4, prostaglandin E2, aryl hydrocarbon receptor antagonists, angiopoietin-like proteins, or pleiotrophin^6,7. However, all studies have required hematopoietic cytokines, which may promote lineage commitment at the expense of LT-HSC maintenance.Huang et al.⁸ previously reported that disruption of GSK-3 in hematopoietic cells in mice leads to an increase in the number of HSCs through Wnt activation, and that the subsequent depletion of LT-HSCs occurs because inhibition of GSK-3 also activates mammalian target of rapamycin (mTOR) (Figure 1A). The mTOR pathway is recognized as an established nutrient sensor, and nutrient-sensing systems are associated with HSC homeostasis. Indeed, HSCs reside in a low-perfusion environment in the bone marrow with low oxygen and low nutrition. Activation of mTOR has been shown to increase the proliferation of committed progenitors at the cost of HSC maintenance (Figure 1A), indicating that low nutrient availability is an essential characteristic of the niche. Thus, Huang et al. hypothesized that low nutrient availability might contribute to HSC maintenance.Open in a separate windowFigure 1Schematic diagrams of maintenance of LT-HSCs. (A) Disruption of GSK-3 results in HSC self-renewal through Wnt activation in a β-catenin-dependent manner. However, in assays of LT-HSC function, disruption of GSK-3 leads to depletion of HSCs through activation of mTOR. GSK-3 regulates both Wnt and mTOR signalings in HSCs⁸. (B) GSK-3 inhibition-induced mTOR activation is attributable to HSC depletion, which can be prevented by mTOR inhibition⁹. (C) In vitro maintenance of LT-HSCs is possible by dual inhibition (2i) of GSK-3 and mTOR under cytokine-free, serum-free, feeder-free conditions⁹. CHIR99021 and lithium are GSK-3 inhibitors, and rapamycin is mTOR inhibitor.Recently, Huang et al.⁹ clearly demonstrate that human and mouse LT-HSCs can be maintained in vitro by inhibiting both GSK-3 and mTOR, in the absence of cytokines, serum, or feeder cells (Figure 1B). Moreover, the combination of two clinically approved inhibitors, lithium (GSK-3) and rapamycin (mTOR) (Figure 1C), increases the number of functional LT-HSCs in mice. First, Huang et al.⁹ determined whether dual inhibition (2i) of GSK-3 and mTOR would be sufficient for maintaining HSCs in vitro. They cultured mouse c-Kit⁺ or Lin⁻Sca1⁺c-Kit⁺ (LSK) cells in X-VIVO 15 (Lonza) (which is chemically defined, serum-free, hematopoietic cell medium) supplemented with inhibitors of GSK-3 (CHIR99021 or lithium) and mTOR (rapamycin) for 7 days in the absence of cytokines, serum, or feeder cells. They subsequently assessed the hematopoietic potential of the cultured HSCs by competitive repopulation assay. It was confirmed that HSCs cultured with 2i maintained long-term reconstitution potential, and that the frequency of HSCs was similar to that in uncultured c-Kit⁺ cells. Similarly, they confirmed that the effects of 2i on LT-HSCs were recapitulated in human HSCs that are present in umbilical cord blood CD34⁺ cells. To explore the mechanism by which 2i preserves HSCs, they also investigated cell cycle status in mouse LSK cells. They found an increased percentage of quiescent cells by 2i, suggesting that the maintenance of LSK cells by 2i is the result of increased dormancy in vitro. Finally, they demonstrated that GSK-3 and mTOR inhibition increases mouse LT-HSCs in vivo. They treated mice with lithium and rapamycin for 2 weeks, and found that both the overall bone marrow cellularity and the absolute number of LT-HSCs increased in the treatment group. In a competitive repopulation assay, the absolute number of competitive rescue units was increased by 2-fold in bone marrow of treated mice.The above findings by Huang et al.⁹ are outstanding, but many questions need to be answered in future studies. i) The authors examined 2i cultures for 7 days in vitro (and 2 weeks in vivo), and it would be interesting to examine how long it is possible to maintain LT-HSCs in vitro under 2i condition. However, as the authors mentioned⁹, prolonged activation of Wnt signaling might be associated with transformation in vitro, and might have the risk of inducing colorectal cancers and leukemias when GSK-3 inhibitors are administered in vivo¹⁰. Nevertheless, lithium (GSK-3 inhibitor) has been used to treat bipolar disorder for over 50 years and is not associated with an increased risk of malignancies¹¹, as the authors pointed out⁹. ii) In cytokine-free medium, is there cytokine production by HSCs or progenitor cells? It may be possible that cytokine production would contribute to the maintenance of LT-HSCs in an autocrine or paracrine manner. iii) Although feeder cells and/or serum are not defined factors for culture, it would be of interest to investigate whether 2i culture in the presence of supporting cells would further improve the maintenance of LT-HSCs. Some extrinsic regulators for HSC quiescence¹², such as N-cadherins, could contribute to LT-HSC maintenance in cooperation with 2i. iv) In addition to iii), hypoxic environment is known to be an extrinsic regulator for HSC quiescence¹², as the bone marrow niche is a low-perfusion environment. Hypoxic culture might synergize with 2i. v) When human ESCs/iPSCs are induced to differentiate into HSCs, it is difficult to capture true human LT-HSCs in vitro. If this is due to inability to maintain human LT-HSCs in vitro, it would be interesting to examine whether 2i culture would enable in vitro induction and maintenance of transplantable LT-HSCs derived from human ESCs/iPSCs.In summary, dual inhibition (2i) of GSK-3 and mTOR allows for the maintenance of human and mouse LT-HSCs in vitro (Figure 1C), and this may resolve the difficulty in culturing HSCs, which in turn, may improve basic research of HSCs (e.g., gene editing in vitro) and human HSC transplantation outcomes. Furthermore, although the effect of 2i on expansion of HSCs is relatively small, a combination of 2i drugs may increase human clinical trials^1,6,7 that use 2i in vivo for the aim of increasing the number of LT-HSCs, since 2i drugs are known as clinically tolerated medications. Insights gained from the discovery of 2i for HSC maintenance may lead to great benefits for patients with hematologic disorders, hopefully in the near future.     

Kit regulates maintenance of quiescent hematopoietic stem cells

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (LSK)CD34(-)Flt3(-) long-term HSCs by 12 wk of age, whereas LSKCD34(+)Flt3(-) short-term HSCs and LSKCD34(+)Flt3(+) multipotent progenitors were less affected. Whereas homing and initial reconstitution of Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/W41) HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation Kit(W41/W41) HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.     

Disease mutations in RUNX1 and RUNX2 create nonfunctional, dominant-negative, or hypomorphic alleles

Monoallelic RUNX1 mutations cause familial platelet disorder with predisposition for acute myelogenous leukemia (FPD/AML). Sporadic mono- and biallelic mutations are found at high frequencies in AML M0, in radiation-associated and therapy-related myelodysplastic syndrome and AML, and in isolated cases of AML M2, M5a, M3 relapse, and chronic myelogenous leukemia in blast phase. Mutations in RUNX2 cause the inherited skeletal disorder cleidocranial dysplasia (CCD). Most hematopoietic missense mutations in Runx1 involve DNA-contacting residues in the Runt domain, whereas the majority of CCD mutations in Runx2 are predicted to impair CBFbeta binding or the Runt domain structure. We introduced different classes of missense mutations into Runx1 and characterized their effects on DNA and CBFbeta binding by the Runt domain, and on Runx1 function in vivo. Mutations involving DNA-contacting residues severely inactivate Runx1 function, whereas mutations that affect CBFbeta binding but not DNA binding result in hypomorphic alleles. We conclude that hypomorphic RUNX2 alleles can cause CCD, whereas hematopoietic disease requires more severely inactivating RUNX1 mutations.     

Regulation of hematopoiesis and the hematopoietic stem cell niche by Wnt signaling pathways

Hematopoietic stem cells （HSCs） are a rare population of cells that are responsible for life-long generation of blood cells of all lineages. In order to maintain their numbers, HSCs must establish a balance between the opposing cell fates of self-renewal （in which the ability to function as HSCs is retained） and initiation of hematopoietic differentiation. Multiple signaling pathways have been implicated in the regulation of HSC cell fate. One such set of pathways are those activated by the Wnt family of ligands. Wnt signaling pathways play a crucial role during embryogenesis and deregulation of these pathways has been implicated in the formation of solid tumors. Wnt signaling also plays a role in the regulation of stem cells from multiple tissues, such as embryonic, epidermal, and intestinal stem cells. However, the function of Wnt signaling in HSC biology is still controversial. In this review, we will discuss the basic characteristics of the adult HSC and its regulatory microenvironment, the ＂niche＂, focusing on the regulation of the HSC and its niche by the Wnt signaling pathways.     

Functional compensation between hematopoietic stem cell clones in vivo

In most organ systems, regeneration is a coordinated effort that involves many stem cells, but little is known about whether and how individual stem cells compensate for the differentiation deficiencies of other stem cells. Functional compensation is critically important during disease progression and treatment. Here, we show how individual hematopoietic stem cell (HSC) clones heterogeneously compensate for the lymphopoietic deficiencies of other HSCs in a mouse. This compensation rescues the overall blood supply and influences blood cell types outside of the deficient lineages in distinct patterns. We find that highly differentiating HSC clones expand their cell numbers at specific differentiation stages to compensate for the deficiencies of other HSCs. Some of these clones continue to expand after transplantation into secondary recipients. In addition, lymphopoietic compensation involves gene expression changes in HSCs that are characterized by increased lymphoid priming, decreased myeloid priming, and HSC self‐renewal. Our data illustrate how HSC clones coordinate to maintain the overall blood supply. Exploiting the innate compensation capacity of stem cell networks may improve the prognosis and treatment of many diseases.