IFN-gamma negatively modulates self-renewal of repopulating human hemopoietic stem cells

Whereas multiple growth-promoting cytokines have been demonstrated to be involved in regulation of the hemopoietic stem cell (HSC) pool, the potential role of negative regulators is less clear. However, IFN-gamma, if overexpressed, can mediate bone marrow suppression and has been directly implicated in a number of bone marrow failure syndromes, including graft-vs-host disease. Whether IFN-gamma might directly affect the function of repopulating HSCs has, however, not been investigated. In the present study, we used in vitro conditions promoting self-renewing divisions of human HSCs to investigate the effect of IFN-gamma on HSC maintenance and function. Although purified cord blood CD34(+)CD38(-) cells underwent cell divisions in the presence of IFN-gamma, cycling HSCs exposed to IFN-gamma in vitro were severely compromised in their ability to reconstitute long-term cultures in vitro and multilineage engraft NOD-SCID mice in vivo (>90% reduced activity in both HSC assays). In vitro studies suggested that IFN-gamma accelerated differentiation of targeted human stem and progenitor cells. These results demonstrate that IFN-gamma can negatively affect human HSC self-renewal.     

Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells

Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood production for their lifetime. Appropriate control of HSC self-renewal is crucial for the maintenance of hematopoietic homeostasis. Here we show that activation of p38 MAPK in response to increasing levels of reactive oxygen species (ROS) limits the lifespan of HSCs in vivo. In Atm(-/-) mice, elevation of ROS levels induces HSC-specific phosphorylation of p38 MAPK accompanied by a defect in the maintenance of HSC quiescence. Inhibition of p38 MAPK rescued ROS-induced defects in HSC repopulating capacity and in the maintenance of HSC quiescence, indicating that the ROS-p38 MAPK pathway contributes to exhaustion of the stem cell population. Furthermore, prolonged treatment with an antioxidant or an inhibitor of p38 MAPK extended the lifespan of HSCs from wild-type mice in serial transplantation experiments. These data show that inactivation of p38 MAPK protects HSCs against loss of self-renewal capacity. Our characterization of molecular mechanisms that limit HSC lifespan may lead to beneficial therapies for human disease.     

Differentiating embryonal stem cells are a rich source of haemopoietic gene products and suggest erythroid preconditioning of primitive haemopoietic stem cells

The difficulties associated with studying molecular mechanisms important in hemopoietic stem cell (HSC) function such as the problems of purifying homogeneous stem cell populations, have prompted us to adapt the murine ES cell system as an in vitro model of HSC generation and function. We now report that careful analysis of the time course of HSC generation in differentiating ES cells allows them to be used as a source of known and novel hemopoietic gene products. We have generated a subtracted library using cDNA from ES cells collected just prior to and just following the emergence of HSCs. Analysis of this library shows it to be a rich source of known hemopoietic and hemopoietic related gene products with 44% of identifiable cDNAs falling into these camps. We have demonstrated the value of this system as a source of novel genes of relevance to HSC function by characterizing a novel membrane protein encoding cDNA that is preferentially expressed in primitive hemopoietic cells. Intriguingly, further analysis of the known components of the subtracted library is suggestive of erythroid preconditioning of the ES cell-derived HSC. We have used dot-blot and in situ analysis to indicate that this erythroid preconditioning is probably restricted to primitive but not definitive HSC.     

Wnt signaling in the niche enforces hematopoietic stem cell quiescence and is necessary to preserve self-renewal in vivo

Wingless (Wnt) is a potent morphogen demonstrated in multiple cell lineages to promote the expansion and maintenance of stem and progenitor cell populations. Wnt effects are highly context dependent, and varying effects of Wnt signaling on hematopoietic stem cells (HSCs) have been reported. We explored the impact of Wnt signaling in vivo, specifically in the context of the HSC niche by using an osteoblast-specific promoter driving expression of the paninhibitor of canonical Wnt signaling, Dickkopf1 (Dkk1). Here we report that Wnt signaling was markedly inhibited in HSCs and, unexpectedly given prior reports, reduction in HSC Wnt signaling resulted in reduced p21Cip1 expression, increased cell cycling, and a progressive decline in regenerative function after transplantation. This effect was microenvironment determined, but irreversible if the cells were transferred to a normal host. Wnt pathway activation in the niche is required to limit HSC proliferation and preserve the reconstituting function of endogenous hematopoietic stem cells.     

Adiponectin stimulates proliferation of adult hippocampal neural stem/progenitor cells through activation of p38 mitogen-activated protein kinase (p38MAPK)/glycogen synthase kinase 3β (GSK-3β)/β-catenin signaling cascade

Adiponectin is the most abundant adipokine secreted from adipocytes. Accumulating evidence suggests that the physiological roles of adiponectin go beyond its metabolic effects. In the present study, we demonstrate that adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) are expressed in adult hippocampal neural stem/progenitor cells (hNSCs). Adiponectin treatment increases proliferation of cultured adult hNSCs in a dose- and time-dependent manner, whereas apoptosis and differentiation of adult hNSCs into neuronal or glial lineage were not affected. Adiponectin activates AMP-activated protein kinase and p38 mitogen-activated protein kinase (p38MAPK) signaling pathways in adult hNSCs. Pretreatment with the p38MAPK inhibitor SB203580, but not the AMP-activated protein kinase inhibitor Compound C, attenuates adiponectin-induced cell proliferation. Moreover, adiponectin induces phosphorylation of Ser-389, a key inhibitory site of glycogen synthase kinase 3β (GSK-3β), and this effect can be blocked by inhibition of p38MAPK with SB203580. Levels of total and nuclear β-catenin, the primary substrate of GSK-3β, were increased by adiponectin treatment. These results indicate that adiponectin stimulates proliferation of adult hNSCs, via acting on GSK-3β to promote nuclear accumulation of β-catenin. Thus, our studies uncover a novel role for adiponectin signaling in regulating proliferation of adult neural stem cells.     

The role of AdipoR1 and AdipoR2 in liver fibrosis

Activation of the adiponectin (APN) signaling axis retards liver fibrosis. However, understanding of the role of AdipoR1 and AdipoR2 in mediating this response is still rudimentary. Here, we sought to elucidate the APN receptor responsible for limiting liver fibrosis by employing AdipoR1 and AdipoR2 knock-out mice in the carbon tetrachloride (CCl₄) model of liver fibrosis. In addition, we knocked down receptor function in primary hepatic stellate cells (HSCs) in vitro. Following the development of fibrosis, AdipoR1 and AdipoR2 KO mice had no quantitative difference in fibrosis by Sirius red staining. However, AdipoR2 KO mice had an enhanced fibrotic signature with increased Col1-α1, TGFß-1, TIMP-1, IL-10, MMP-2 and MMP-9. Knockdown of AdipoR1 or AdipoR2 in HSCs followed by APN treatment demonstrated that AdipoR1 and AdipoR2 did not affect proliferation or TIMP-1 gene expression, while AdipoR2 modulated Col1-α1 and α-SMA gene expression, HSC migration, and AMPK activity. These finding suggest that AdipoR2 is the major APN receptor on HSCs responsible for mediating its anti-fibrotic effects.     

Switch of cadherin expression from E- to N-type during the activation of rat hepatic stellate cells

The activation of hepatic stellate cell (HSC) is a common pathway leading to hepatic fibrosis. However, the molecular mechanisms underlying HSC activation remain obscure. To elucidate the nature of the HSC activation, we investigated the expression of E-cadherin and its switch to N-cadherin during rat HSC activation, in vivo and in vitro. Immunohistochemical and immunocytochemical staining were performed to identify the expressions of E-cadherin, N-cadherin, and β-catenin in rat HSCs, in vivo and in vitro. Serial changes in the expressions of these adhesion molecules during the spontaneous activation of cultured rat HSCs were also demonstrated by RT-PCR and by immunoblotting. E-cadherin and β-catenin were expressed on opposing cell membranes of GFAP-positive rat HSCs and adjacent hepatocytes in vivo, and between desmin-positive rat HSCs in vitro. With the progression of rat HSC activation in tissue and in culture, E-cadherin disappeared gradually, whereas N-cadherin appeared at the cell periphery. The results of RT-PCR and immunoblotting were concordant with immunocytochemistry findings. In conclusion, resting rat HSCs express E-cadherin and β-catenin both in vivo and in vitro, and E-cadherin switches to N-cadherin during HSC activation. These results suggest that HSC activation represents transdifferentiation from an epithelial to a mesenchymal phenotype.     

Hierarchical and ontogenic positions serve to define the molecular basis of human hematopoietic stem cell behavior

The molecular basis governing functional behavior of human hematopoietic stem cells (HSCs) is largely unknown. Here, using in vitro and in vivo assays, we isolate and define progenitors versus repopulating HSCs from multiple stages of human development for global gene expression profiling. Accounting for both the hierarchical relationship between repopulating cells and their progenitors, and the enhanced HSC function unique to early stages of ontogeny, the human homologs of Hairy Enhancer of Split-1 (HES-1) and Hepatocyte Leukemia Factor (HLF) were identified as candidate regulators of HSCs. Transgenic human hematopoietic cells expressing HES-1 or HLF demonstrated enhanced in vivo reconstitution ability that correlated to increased cycling frequency and inhibition of apoptosis, respectively. Our report identifies regulatory factors involved in HSC function that elicit their effect through independent systems, suggesting that a unique orchestration of pathways fundamental to all human cells is capable of controlling stem cell behavior.     

Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo

Hoxa5 is preferentially expressed in haematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs), and is more highly expressed in expanding HSCs. To date, little is known regarding the role of Hoxa5 in HSCs and downstream progenitor cells in vivo. In this study, we show that increased expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Hoxa5 differentially modifies the cell cycle of HSCs and lineage committed progenitor cells, depending on the cellular context. Hoxa5 drives HSCs, but not MPPs, through the cell cycle and arrests erythroid progenitor cells in G0 phase. Although the HSC pool shrinks after overexpression of Hoxa5, HSCs sustain the abilities of self-renewal and multipotency. In vivo, Hoxa5 has two effects on erythropoiesis: it causes a predominance of mature erythroid lineage cells and the partial apoptosis of erythroid progenitors. RNA-seq indicates that multiple biological processes, including erythrocyte homeostasis, cell metabolism, and apoptosis, are modified by Hoxa5. The results of this study indicate that Hoxa5 is a key regulator of the HSC cell cycle, and the inappropriate expression of Hoxa5 in lineage-committed progenitor cells leads to aberrant erythropoiesis.     

Enforced expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo

Hoxa5 is preferentially expressed in haematopoietic stem cells (HSCs) and multipotent progenitor cells (MPPs), and is more highly expressed in expanding HSCs. To date, little is known regarding the role of Hoxa5 in HSCs and downstream progenitor cells in vivo. In this study, we show that increased expression of Hoxa5 in haematopoietic stem cells leads to aberrant erythropoiesis in vivo. Hoxa5 differentially modifies the cell cycle of HSCs and lineage committed progenitor cells, depending on the cellular context. Hoxa5 drives HSCs, but not MPPs, through the cell cycle and arrests erythroid progenitor cells in G0 phase. Although the HSC pool shrinks after overexpression of Hoxa5, HSCs sustain the abilities of self-renewal and multipotency. In vivo, Hoxa5 has two effects on erythropoiesis: it causes a predominance of mature erythroid lineage cells and the partial apoptosis of erythroid progenitors. RNA-seq indicates that multiple biological processes, including erythrocyte homeostasis, cell metabolism, and apoptosis, are modified by Hoxa5. The results of this study indicate that Hoxa5 is a key regulator of the HSC cell cycle, and the inappropriate expression of Hoxa5 in lineage-committed progenitor cells leads to aberrant erythropoiesis.