Cav-1 deletion impaired hematopoietic stem cell function

A tightly controlled balance between hematopoietic stem and progenitor cell compartments is required to maintain normal blood cell homeostasis throughout life, and this balance is regulated by intrinsic and extrinsic cellular factors. Cav-1 is a 22-kDa protein that is located in plasma membrane invaginations and is implicated in regulating neural stem cell and embryonic stem cell proliferation. However, the role of Cav-1 in hematopoietic stem cell (HSC) function is largely unknown. In this study, we used Cav-1^−/− mice to investigate the role of Cav-1 in HSCs function during aging. The results showed that Cav-1^−/− mice displayed a decreased percentage of B cells and an increased percentage of M cells in the bone marrow and peripheral blood, and these changes were due to an increased number of HSCs. FACS analysis showed that the numbers of Lin⁻Sca1⁺c-kit⁺ cells (LSKs), long-term HSCs (LT-HSCs), short-term HSCs and multipotent progenitors were increased in Cav-1^−/− mice compared with Cav-1^+/+ mice, and this increase became more pronounced with aging. An in vitro clonogenic assay showed that LT-HSCs from Cav-1^−/− mice had reduced ability to self-renew. Consistently, an in vivo competitive transplantation assay showed that Cav-1^−/− mice failed to reconstitute hematopoiesis. Moreover, a Cav-1 deletion disrupted the quiescence of LSKs and promoted cell cycle progression through G2/M phase. In addition, we found that Cav-1 deletion impaired the ability of HSCs to differentiate into mature blood cells. Taken together, these data suggest that Cav-1-deficient cells impaired HSCs quiescence and induced environmental alterations, which limited HSCs self-renewal and function.     

Angiopoietin-Like Protein 3 Promotes Preservation of Stemness during Ex Vivo Expansion of Murine Hematopoietic Stem Cells

Allogeneic hematopoietic stem cell (HSC) transplantations from umbilical cord blood or autologous HSCs for gene therapy purposes are hampered by limited number of stem cells. To test the ability to expand HSCs in vitro prior to transplantation, two growth factor cocktails containing stem cell factor, thrombopoietin, fms-related tyrosine kinase-3 ligand (STF) or stem cell factor, thrombopoietin, insulin-like growth factor-2, fibroblast growth factor-1 (STIF) either with or without the addition of angiopoietin-like protein-3 (Angptl3) were used. Culturing HSCs in STF and STIF media for 7 days expanded long-term repopulating stem cells content in vivo by ∼6-fold and ∼10-fold compared to freshly isolated stem cells. Addition of Angptl3 resulted in increased expansion of these populations by ∼17-fold and ∼32-fold, respectively, and was further supported by enforced expression of Angptl3 in HSCs through lentiviral transduction that also promoted HSC expansion. As expansion of highly purified lineage-negative, Sca-1⁺, c-Kit⁺ HSCs was less efficient than less pure lineage-negative HSCs, Angptl3 may have a direct effect on HCS but also an indirect effect on accessory cells that support HSC expansion. No evidence for leukemia or toxicity was found during long-term follow up of mice transplanted with ex vivo expanded HSCs or manipulated HSC populations that expressed Angptl3. We conclude that the cytokine combinations used in this study to expand HSCs ex vivo enhances the engraftment in vivo. This has important implications for allogeneic umbilical cord-blood derived HSC transplantations and autologous HSC applications including gene therapy.     

TAK1 (MAP3K7) Signaling Regulates Hematopoietic Stem Cells through TNF-Dependent and -Independent Mechanisms

A cytokine/stress signaling kinase Tak1 (Map3k7) deficiency is known to impair hematopoietic progenitor cells. However, the role of TAK1 signaling in the stem cell function of the hematopoietic system is not yet well defined. Here we characterized hematopoietic stem cells (HSCs) harboring deletion of Tak1 and its activators, Tak1 binding proteins 1 and 2 (Tab1 and Tab2) using a competitive transplantation assay in a mouse model. Tak1 single or Tab1/Tab2 double deletions completely eliminated the reconstitution activity of HSCs, whereas Tab1 or Tab2 single deletion did not cause any abnormality. Tak1 single or Tab1/Tab2 double deficient lineage-negative, Sca-1⁺, c-Kit⁺ (LSK) cells did not proliferate and underwent cell death. We found that Tnfr1 deficiency restored the reconstitution activity of Tak1 deficient bone marrow cells for 6–18 weeks. However, the reconstitution activity of Tak1- and Tnfr1-double deficient bone marrow cells declined over the long term, and the number of phenotypically identified long-term hematopoietic stem cells were diminished. Our results indicate that TAB1- or TAB2-dependent activation of TAK1 is required for maintenance of the hematopoietic system through two mechanisms: one is prevention of TNF-dependent cell death and the other is TNF-independent maintenance of long-term HSC.     

Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.     

Adiponectin Reduces Hepatic Stellate Cell Migration by Promoting Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) Secretion

Hepatic stellate cells (HSC) are central players in liver fibrosis that when activated, proliferate, migrate to sites of liver injury, and secrete extracellular matrix. Obesity, a known risk factor for liver fibrosis is associated with reduced levels of adiponectin, a protein that inhibits liver fibrosis in vivo and limits HSC proliferation and migration in vitro. Adiponectin-mediated activation of adenosine monophosphate-activated kinase (AMPK) inhibits HSC proliferation, but the mechanism by which it limits HSC migration to sites of injury is unknown. Here we sought to elucidate how adiponectin regulates HSC motility. Primary rat HSCs were isolated and treated with adiponectin in migration assays. The in vivo actions of adiponectin were examined by treating mice with carbon tetrachloride for 12 weeks and then injecting them with adiponectin. Cell and tissue samples were collected and analyzed for gene expression, signaling, and histology. Serum from patients with liver fibrosis was examined for adiponectin and tissue inhibitor of metalloproteinase-1 (TIMP-1) protein. Adiponectin administration into mice increased TIMP-1 gene and protein expression. In cultured HSCs, adiponectin promoted TIMP-1 expression and through binding of TIMP-1 to the CD63/β1-integrin complex reduced phosphorylation of focal adhesion kinase to limit HSC migration. In mice with liver fibrosis, adiponectin had similar effects and limited focal adhesion kinase phosphorylation. Finally, in patients with advanced fibrosis, there was a positive correlation between serum adiponectin and TIMP-1 levels. In sum, these data show that adiponectin stimulates TIMP-1 secretion by HSCs to retard their migration and contributes to the anti-fibrotic effects of adiponectin.     

Inhibition of c-Kit Is Not Required for Reversal of Hyperglycemia by Imatinib in NOD Mice

(1) Aim/Hypothesis

Recent studies indicate that tyrosine kinase inhibitors, including imatinib, can reverse hyperglycemia in non-obese diabetic (NOD) mice, a model of type 1 diabetes (T1D). Imatinib inhibits c-Abl, c-Kit, and PDGFRs. Next-generation tyrosine kinase inhibitors for T1D treatment should maintain activities required for efficacy while sparing inhibition of targets that might otherwise lead to adverse events. In this study, we investigated the contribution of c-Kit inhibition by imatinib in reversal of hyperglycemia in NOD mice.

(2) Methods

The T670I mutation in c-Kit, which confers imatinib resistance, was engineered into the mouse genome and bred onto the NOD background. Hematopoietic stem cells (HSCs) from NOD.c-Kit^T670I mice and NOD.c-Kit^wt littermates were expanded in the presence or absence of imatinib to verify imatinib resistance of the c-Kit^T670I allele. Diabetic mice were treated with imatinib at the onset of hyperglycemia for three weeks, and blood glucose was monitored.

(3 )Results

In vitro expansion of HSCs from NOD.c-Kit^wt mice was sensitive to imatinib, while expansion of HSCs from NOD.c-Kit^T670I mice was insensitive to imatinib. However, in vivo treatment with imatinib lowered blood glucose levels in both strains of mice.

(4) Conclusions/Interpretation

The HSC experiment confirmed that, in NOD.c-Kit^T670I mice, c-Kit is resistant to imatinib. As both NOD.c-Kit^T670I and NOD.c-Kit^wt mice responded comparably to imatinib, c-Kit inhibition does not substantially contribute to the efficacy of imatinib in T1D. Thus, we conclude that inhibition of c-Kit is not required in next-generation tyrosine kinase inhibitors for T1D treatment, and may be selected against to improve the safety profile.     

A non-redundant function of cyclin E1 in hematopoietic stem cells

A precise balance between quiescence and proliferation is crucial for the lifelong function of hematopoietic stem cells (HSCs). Cyclins E1 and E2 regulate exit from quiescence in fibroblasts, but their role in HSCs remains unknown. Here, we report a non-redundant role for cyclin E1 in mouse HSCs. A long-term culture-initiating cell (LTC-IC) assay indicated that the loss of cyclin E1, but not E2, compromised the colony-forming activity of primitive hematopoietic progenitors. Ccne1^−/− mice showed normal hematopoiesis in vivo under homeostatic conditions but a severe impairment following myeloablative stress induced by 5-fluorouracil (5-FU). Under these conditions, Ccne1^−/− HSCs were less efficient in entering the cell cycle, resulting in decreased hematopoiesis and reduced survival of mutant mice upon weekly 5-FU treatment. The role of cyclin E1 in homeostatic conditions became apparent in aged mice, where HSC quiescence was increased in Ccne1^−/− animals. On the other hand, loss of cyclin E1 provided HSCs with a competitive advantage in bone marrow serial transplantation assays, suggesting that a partial impairment of cell cycle entry may exert a protective role by preventing premature depletion of the HSC compartment. Our data support a role for cyclin E1 in controlling the exit from quiescence in HSCs. This activity, depending on the physiological context, can either jeopardize or protect the maintenance of hematopoiesis.     

Bone marrow derived cells in the tumour microenvironment contain cells with primitive haematopoietic phenotype

Infiltration of bone marrow derived cells is part of the angiogenic switch required for uncontrolled tumour growth. However, the nature of the tumour-infiltrating cells from bone marrow has not been fully elucidated. To investigate the phenotype of bone marrow derived cells within a tumour, we employed the Lewis lung carcinoma (LLC) murine tumour model. We followed bone marrow derivation of tumour-infiltrating cells through transplantation of CD45.2 bone marrow cells into pre-irradiated CD45.1 mice. We found robust CD45.2 donor type chimerism in bone marrow and blood of CD45.1 recipient tumour-bearing mice. Flow cytometric analysis of LLC tumours showed, in addition to previously described pro-angiogenic CD45⁺VEGFR2⁺‘endothelial progenitor cells’ (EPC), or CD45⁺Tie2⁺‘Tie2-expressing monocytes’ (TEM), incorporation of donor type lineage marker negative (Lin⁻) and Lin⁻Sca1⁺ undifferentiated haematopoietic cell types. Immunohistochemical analysis confirmed the extravasal location of the primitive haematopoietic cells. Flow-cytometric sorting of bone marrow cells and subsequent analysis in haematopoietic colony-forming assays revealed that cells with a Lin⁻Sca1⁺ phenotype, which were initially negative for VEGFR2 and Tie2, gave rise to VEGFR2⁺ and/or Tie2⁺ cells. Moreover, Lin⁻ bone marrow cells pre-labelled with the membrane dye PKH26 (a red fluorochrome) and transplanted i.v. into tumour-bearing mice were found to extravasate and incorporate into LLC tumours within 24 hrs. Thus, primitive haematopoietic precursors which are thought to be precursors of EPC and TEMs, constitute a part of the tumour microenvironment. This makes them an attractive target cell population for tumour-directed cellular therapies.     

Essential roles of mgcRacGAP in multilineage differentiation and survival of murine hematopoietic cells

MgcRacGAP, a negative regulator for Rho family GTPases, has been shown to play important roles in cytokinesis using several cell lines. However, the physiological role of mgcRacGAP in multilineage hematopoietic development remains unclear. Here, we conditionally ablated mgcRacGAP in vivo to clarify this issue. As the result, we found that normal hematopoietic development including proliferation and survival requires mgcRacGAP. We also found that depletion of mgcRacGAP in hematopoietic cells results in a marked decrease in c-Kit⁺Sca-1⁺Lin⁻ cells, suggesting that mgcRacGAP is required for the maintenance of the hematopoietic stem cells. In addition, B cells in which mgcRacGAP had been selectively ablated showed proliferation failure and fell into apoptosis. Taken together, mgcRacGAP is now shown to play a indispensable role in the development of hematopoietic cells in vivo.     

T lymphocyte development from fetal hematopoietic stem cells

Hematopoietic stem cells (HSCs) are isolated from bone marrow and fetal liver as Thy-1^lo Lin^- Sca-1⁺ cells. Both adult and fetal HSCs have similar stem cell activities. However, fetal HSCs differentiate more efficiently than adult HSCs into Vγ3 and Vγ4 cells without N nucleotide insertion in the fetal thymic microenvironment. Thus HSC themselves may lose some of their developmental potential during ontogeny. It is possible that only fetal, but not adult, HSCs can differentiate into the fetal types of hematopoietic cells, including Vγ3, Vγ4 T cells, CD5 B cells, and fetal type erythrocytes.     

An intensified systemic trafficking of bone marrow‐derived stem/progenitor cells in patients with pancreatic cancer

Various experimental studies indicate potential involvement of bone marrow (BM)-derived stem cells (SCs) in malignancy development and progression. In this study, we comprehensively analysed systemic trafficking of various populations of BM-derived SCs (BMSCs), i.e., mesenchymal, haematopoietic, endothelial stem/progenitor cells (MSCs, HSCs, EPCs respectively), and of recently discovered population of very small embryonic/epiblast-like SCs (VSELs) in pancreatic cancer patients. Circulating CD133⁺/Lin⁻/CD45⁻/CD34⁺ cells enriched for HSCs, CD105⁺/STRO-1⁺/CD45⁻ cells enriched for MSCs, CD34⁺/KDR⁺/CD31⁺/CD45⁻ cells enriched for EPCs and small CXCR4⁺CD34⁺CD133⁺ subsets of Lin⁻CD45⁻ cells that correspond to VSELs were enumerated and sorted from blood samples derived from 29 patients with pancreatic cancer, and 19 healthy controls. In addition, plasma levels of stromal-derived factor-1 (SDF-1), growth/inhibitory factors and sphingosine-1-phosphate (S1P; chemoattractants for SCs), as well as, of complement cascade (CC) molecules (C3a, C5a and C5b-9/membrane attack complex – MAC) were measured. Higher numbers of circulating VSELs and MSCs were detected in pancreatic cancer patients (P < 0.05 and 0.01 respectively). This trafficking of BMSCs was associated with significantly elevated C5a (P < 0.05) and C5b-9/MAC (P < 0.005) levels together with S1P concentrations detected in plasma of cancer patients, and seemed to be executed in a SDF-1 independent manner. In conclusion, we demonstrated that in patients with pancreatic cancer, intensified peripheral trafficking of selected populations of BMSCs occurs. This phenomenon seems to correlate with systemic activation of the CC, hepatocyte growth factor and S1P levels. In contrast to previous studies, we demonstrate herein that systemic SDF-1 levels do not seem to be linked with increased mobilization of stem cells in patients with pancreatic cancer.