[show abstract][hide abstract] ABSTRACT: The regenerative capacity of most tissues declines dramatically after embryonic development and during post-natal life. The underlying mechanisms of this phenomenon are incompletely understood. In a recent issue of Cell, Shyh-Chang and colleagues provide experimental evidence that Lin28 prolongs youthful regenerative capacity by increasing oxidative glucose metabolism (Shyh-Chang et al, ).
[show abstract][hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) are a more heterogeneous population than previously thought. Extensive analysis of reconstitution kinetics after transplantation now permits new classifications of HSCs based on lineage balance. Previously unrecognized classes of HSCs, such as myeloid- and lymphoid-biased HSCs, have emerged. However, varying nomenclature has been used to describe these cells, promoting confusion in the field. To establish a common nomenclature, we propose a re-classification of short-, intermediate-, and long-term (ST, IT, and LT) HSCs defined as: ST <6 months, IT >6 months, and LT >12. We observe that myeloid-biased HSCs or α cells overlap with LT-HSCs, whereas lymphoid-biased HSCs or γ/δ cells overlap with ST-HSCs, suggesting that HSC lifespan is linked to cell differentiation. We also suggest that HSC heterogeneity prompts reconsideration of long-term (>4 months) multilineage reconstitution as the gold standard for HSC detection. In this review, we discuss relationships among ST-, IT-, and LT-HSCs relevant to stem cell heterogeneity, hierarchical organization, and differentiation pathways.
[show abstract][hide abstract] ABSTRACT: Consensus holds that hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs) of reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells in a stepwise manner. Using a single-cell transplantation system and marker mice, we unexpectedly found myeloid-restricted progenitors with long-term repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or common myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired daughter cell assays combined with transplantation revealed that HSCs can give rise to HSCs via symmetric division or directly differentiate into MyRPs via asymmetric division (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be essential for fast responses to ablation stress. Our results show that loss of self-renewal and stepwise progression through specific differentiation stages are not essential for lineage commitment of HSCs and suggest a revised model of hematopoietic differentiation.
[show abstract][hide abstract] ABSTRACT: Fluorescent-protein transgenic mice are useful for obtaining marked somatic cells to study kinetics of development or differentiation. Fluorescence-marked hematopoietic stem cells in particular are commonly used for studying hematopoiesis. However, as far as we know, no transgenic mouse line is described in which a fluorescent protein is stably and constitutively expressed in all hematopoietic cells, including erythrocytes and platelets. Using the random segregation of provirus (RSP) method, we generated from retrovirally transduced mouse embryonic stem cells a transgenic mouse line expressing a red/orange fluorescent protein, Kusabira Orange (KuO). KuO transgenic mouse line cells carry only one proviral integration site and stably express KuO in all hematopoietic-lineage elements, including erythrocytes and platelets. Moreover, bone-marrow transplantation in KuO transgenic mice demonstrated normal hematopoieisis. KuO transgenic mice likely will prove useful for study of hematopoiesis that includes erythropoiesis and megakaryopoiesis.
Biochemical and Biophysical Research Communications 05/2013; · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: HSC fate decisions are regulated by cell-intrinsic and cell-extrinsic cues. The latter cues are derived from the BM niche. Membrane-type 1 matrix metalloproteinase (MT1-MMP), which is best known for its proteolytic role in pericellular matrix remodeling, is highly expressed in HSCs and stromal/niche cells. We found that, in MT1-MMP(-/-) mice, in addition to a stem cell defect, the transcription and release of kit ligand (KitL), stromal cell-derived factor-1 (SDF-1/CXCL12), erythropoietin (Epo), and IL-7 was impaired, resulting in a trilineage hematopoietic differentiation block, while addition of exogenous KitL and SDF-1 restored hematopoiesis. Further mechanistic studies revealed that MT1-MMP activates the hypoxia-inducible factor-1 (HIF-1) pathway via factor inhibiting HIF-1 (FIH-1) within niche cells, thereby inducing the transcription of HIF-responsive genes, which induce terminal hematopoietic differentiation. Thus, MT1-MMP in niche cells regulates postnatal hematopoiesis, by modulating hematopoietic HIF-dependent niche factors that are critical for terminal differentiation and migration.
[show abstract][hide abstract] ABSTRACT: Checkpoints that limit stem cell self-renewal in response to DNA damage can contribute to cancer protection but may also promote tissue aging. Molecular components that control stem cell responses to DNA damage remain to be delineated. Using in vivo RNAi screens, we identified basic leucine zipper transcription factor, ATF-like (BATF) as a major component limiting self-renewal of hematopoietic stem cells (HSCs) in response to telomere dysfunction and γ-irradiation. DNA damage induces BATF in a G-CSF/STAT3-dependent manner resulting in lymphoid differentiation of HSCs. BATF deletion improves HSC self-renewal and function in response to γ-irradiation or telomere shortening but results in accumulation of DNA damage in HSCs. Analysis of bone marrow from patients with myelodysplastic syndrome supports the conclusion that DNA damage-dependent induction of BATF is conserved in human HSCs. Together, these results provide experimental evidence that a BATF-dependent differentiation checkpoint limits self-renewal of HSCs in response to DNA damage.
[show abstract][hide abstract] ABSTRACT: The tumour suppressor p53 activates Puma-dependent apoptosis and p21-dependent cell-cycle arrest in response to DNA damage. Deletion of p21 improved stem-cell function and organ maintenance in progeroid mice with dysfunctional telomeres, but the function of Puma has not been investigated in this context. Here we show that deletion of Puma improves stem- and progenitor-cell function, organ maintenance and lifespan of telomere-dysfunctional mice. Puma deletion impairs the clearance of stem and progenitor cells that have accumulated DNA damage as a consequence of critically short telomeres. However, further accumulation of DNA damage in these rescued progenitor cells leads to increasing activation of p21. RNA interference experiments show that upregulation of p21 limits proliferation and evolution of chromosomal imbalances of Puma-deficient stem and progenitor cells with dysfunctional telomeres. These results provide experimental evidence that p53-dependent apoptosis and cell-cycle arrest act in cooperating checkpoints limiting tissue maintenance and evolution of chromosomal instability at stem- and progenitor-cell levels in response to telomere dysfunction. Selective inhibition of Puma-dependent apoptosis can result in temporary improvements in maintenance of telomere-dysfunctional organs.
[show abstract][hide abstract] ABSTRACT: Throughout life, one's blood supply depends on sustained division of hematopoietic stem cells (HSCs) for self-renewal and differentiation. Within the bone marrow microenvironment, an adhesion-dependent or -independent niche system regulates HSC function. Here we show that a novel adhesion-dependent mechanism via integrin-β3 signaling contributes to HSC maintenance. Specific ligation of β3-integrin on HSCs using an antibody or extracellular matrix protein prevented loss of long-term repopulating (LTR) activity during ex vivo culture. The actions required activation of αvβ3-integrin "inside-out" signaling, which is dependent on thrombopoietin (TPO), an essential cytokine for activation of dormant HSCs. Subsequent "outside-in" signaling via phosphorylation of Tyr747 in the β3-subunit cytoplasmic domain was indispensable for TPO-dependent, but not stem cell factor-dependent, LTR activity in HSCs in vivo. This was accompanied with enhanced expression of Vps72, Mll1, and Runx1, 3 factors known to be critical for maintaining HSC activity. Thus, our findings demonstrate a mechanistic link between β3-integrin and TPO in HSCs, which may contribute to maintenance of LTR activity in vivo as well as during ex vivo culture.
[show abstract][hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) reside in both bone marrow (BM) and spleen in adult mice. However, whether BM and spleen HSCs are functionally similar is not known. Spleen HSCs were compared with BM HSCs by various assays.
Whole BM and spleen cells were quantitatively analyzed by competitive repopulation. Single-cell transplantation was performed with HSCs purified from BM and spleen. A parabiosis model was used to distinguish organ-specific HSCs from circulating HSCs. The cell cycle was analyzed with pyronin Y staining and bromodeoxyuridine uptake.
Repopulating and self-renewal potentials were similar on a clonal basis between BM and spleen HSCs, whereas the HSC frequency in the spleen was significantly lower than that in the BM. Analysis of parabiotic mice suggested that most HSCs are long-term residents in each organ. Cell-cycle analysis revealed that spleen HSCs cycle twice as frequently as do BM HSCs, suggesting that G(0) phase length is longer in BM HSCs than in spleen HSCs. The cycling difference between BM and spleen HSCs was also observed in mice that had been reconstituted with BM or spleen cells, suggesting that HSC quiescence is regulated in an organ-specific manner.
Spleen HSCs and BM HSCs are functionally similar, but their cycling behaviors differ.
[show abstract][hide abstract] ABSTRACT: Histiocytic sarcoma (HS), a rare hematological malignancy, is an aggressive neoplasm that responds poorly to therapy. The molecular etiology and pathology of this disease remain unclear, hampering the development of an effective therapy, and there remains a need for more, and more realistic, animal models. HS cells typically show a histiocytic (ie, tissue macrophage-like) morphology and express histiocyte/macrophage markers in the absence of lymphocyte markers. In this study, we report that Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice develop HS, but do not exhibit elevated incidence of other types of tumors. These mutant mice showed earlier mortality than wild-type (WT) or the other mutant mice, and this mortality was associated with HS. In total, 17 of 21 tumor-bearing Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice necropsied at 25-66 weeks of age showed multiple organ spread, with osteolytic lesions and orthotopic invasion from the bone marrow to skeletal muscle. Tumors from the mice were transplantable. In addition, all Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice, but only a small proportion of Dok-3(-/-) mice and no Dok-1(-/-)Dok-2(-/-) mice, exhibited abnormal accumulation of macrophages in the lung on necropsy at 8-12 weeks of age. Macrophages derived from Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice displayed an exaggerated proliferative response to macrophage colony-stimulating factor (M-CSF) or granulocyte- macrophage colony-stimulating factor (GM-CSF) compared with WT and mutant controls. Together, these findings indicate that Dok-1, Dok-2, and Dok-3 cooperatively suppress aggressive HS, and commend Dok-1(-/-)Dok-2(-/-)Dok-3(-/-) mice as a useful model for the study of this neoplasia.
[show abstract][hide abstract] ABSTRACT: Fus is the gene for a member of the FET family of RNA-binding proteins often involved in chromosomal translocations to generate oncogenic fusion genes in human cancers. Fus participates in multiple cellular functions, including RNA processing and transport, transcriptional regulation, and genome integrity. However, its role in hematopoiesis remains obscure. In this study, we examined its role in the self-renewal of hematopoietic stem cells (HSCs).
HSCs in Fus(-/-) fetal livers were analyzed for proliferative capacity in vitro and long-term repopulating capacity in recipient mice. Radiation sensitivity of Fus(-/-) HSCs was evaluated in recipient mice repopulated by Fus(-/-) fetal liver cells.
Fus(-/-) fetal livers developed normally, except for a mild reduction in numbers of hematopoietic stem and progenitor cells compared to wild-type. The proliferation and differentiation of Fus(-/-) hematopoietic progenitors were normal in vitro. However, the number of colony-forming cells present in long-term cocultures of Fus(-/-) hematopoietic progenitors and stromal cells was significantly reduced. Fus(-/-) HSCs had an impaired long-term repopulating capacity and failed to repopulate in tertiary recipient mice. Fus(-/-) HSCs were highly susceptible to radiation both in vitro and in vivo and showed retardation of radiation-induced DNA damage repair.
Our findings define Fus as a novel regulator of self-renewal and radioprotection of HSCs and also implicate it in stress-resistance and maintenance of the genomic integrity of HSCs.
[show abstract][hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) have been extensively characterized based on functional definitions determined by experimental transplantation into lethally irradiated mice. In mice, HSCs are heterogeneous with regard to self-renewal potential, in vitro colony-forming activity, and in vivo behavior. We attempted prospective isolation of HSC subsets with distinct properties among CD34(-/low) c-Kit+Sca-1+Lin- (CD34-KSL) cells. CD34-KSL cells were divided, based on CD150 expression, into three fractions: CD150high, CD150med, and CD150neg cells. Compared with the other two fractions, CD150high cells were significantly enriched in HSCs, with great self-renewal potential. In vitro colony assays revealed that decreased expression of CD150 was associated with reduced erythroblast/megakaryocyte differentiation potential. All three fractions were regenerated only from CD150high cells in recipient mice. Using single-cell transplantation studies, we found that a fraction of CD150high cells displayed latent and barely detectable myeloid engraftment in primary-recipient mice but progressive and multilineage reconstitution in secondary-recipient mice. These findings highlight the complexity and hierarchy of reconstitution capability, even among HSCs in the most primitive compartment.
Journal of Experimental Medicine 06/2010; 207(6):1173-82. · 13.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The nature of the in vivo cellular events underlying thrombus formation mediated by platelet activation remains unclear because of the absence of a modality for analysis. Lymphocyte adaptor protein (Lnk; also known as Sh2b3) is an adaptor protein that inhibits thrombopoietin-mediated signaling, and as a result, megakaryocyte and platelet counts are elevated in Lnk-/- mice. Here we describe an unanticipated role for Lnk in stabilizing thrombus formation and clarify the activities of Lnk in platelets transduced through integrin alphaIIbbeta3-mediated outside-in signaling. We equalized platelet counts in wild-type and Lnk-/- mice by using genetic depletion of Lnk and BM transplantation. Using FeCl3- or laser-induced injury and in vivo imaging that enabled observation of single platelet behavior and the multiple steps in thrombus formation, we determined that Lnk is an essential contributor to the stabilization of developing thrombi within vessels. Lnk-/- platelets exhibited a reduced ability to fully spread on fibrinogen and mediate clot retraction, reduced tyrosine phosphorylation of the beta3 integrin subunit, and reduced binding of Fyn to integrin alphaIIbbeta3. These results provide new insight into the mechanism of alphaIIbbeta3-based outside-in signaling, which appears to be coordinated in platelets by Lnk, Fyn, and integrins. Outside-in signaling modulators could represent new therapeutic targets for the prevention of cardiovascular events.
The Journal of clinical investigation 01/2010; 120(1):179-90. · 15.39 Impact Factor
[show abstract][hide abstract] ABSTRACT: Generation of induced pluripotent stem cells (iPSCs) generally uses fibroblastic cells, but other cell sources may prove useful in both research and clinical settings. Although proof of cellular origin requires genetic-marker identification in both target cells and established iPSCs, somatic cells other than mature lymphocytes mostly lack such markers. Here we show definitive proof of direct reprogramming of murine hematopoietic cells with no rearranged genes. Using iPSC factor transduction, we successfully derived iPSCs from bone marrow progenitor cells obtained from a mouse whose hematopoiesis was reconstituted from a single congenic hematopoietic stem cell. Established clones were demonstrated to be genetically identical to the transplanted single hematopoietic stem cell, thus proving their cellular origin. These hematopoietic cell-derived iPSCs showed typical characteristics of iPSCs, including the ability to contribute to chimerism in mice. These results will prompt further use of hematopoietic cells for iPSC generation while enabling definitive studies to test how cellular sources influence characteristics of descendant iPSCs.
[show abstract][hide abstract] ABSTRACT: Side population (SP) cell analysis and sorting have been successfully applied to hepatocellular carcinoma (HCC) cell lines to identify a minor cell population with cancer stem cell properties. However, the molecular mechanisms operating in SP cells remain unclear. The polycomb gene product BMI1 plays a central role in the self-renewal of somatic stem cells in a variety of tissues and organs and seems to be implicated in tumor development. In this study, we determined the critical role of BMI1 in the maintenance of cancer stem cells with the SP phenotype in HCC cell lines. BMI1 was preferentially expressed in SP cells in Huh7 and PLC/PRF/5 HCC cells compared with the corresponding non-SP cells. Lentiviral knockdown of BMI1 considerably decreased the number of SP cells in both Huh7 and PLC/PRF/5 cells. Long-term culture of purified SP cells resulted in a drastic reduction in the SP subpopulation upon the BMI1 knockdown, indicating that BMI1 is required for the self-renewal of SP cells in culture. More importantly, the BMI1 knockdown abolished the tumor-initiating ability of SP cells in nonobese diabetic/severe combined immunodeficiency mice. Derepression of the INK4A and ARF genes that are major targets for BMI1 was not necessarily associated with impaired self-renewal of SP cells caused by BMI1 knockdown. In conclusion, our findings define an important role for BMI1 in the maintenance of tumor-initiating SP cells in HCC. BMI1 might be a novel therapeutic target for the eradication of cancer stem cells in HCC.
Cancer Research 11/2008; 68(19):7742-9. · 8.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: Angiogenesis and lymphangiogenesis are complex phenomena that involve the interplay of several growth factors and receptors. Recently, we have demonstrated that in Keratin-14 (K14) promoter-driven Vegf-A transgenic (Tg) mice, not only angiogenesis but also lymphangiogenesis is stimulated. However, the mechanism by which VEGFR1 is involved in lymphangiogenesis remains unclear.
To examine how important the tyrosine kinase (TK) of VEGFR1 is in lymphangiogenesis in K14 Vegf-A Tg mice, we crossed the K14 Vegf-A Tg mice with VEGFR1-TK-deficient mice to generate double mutant K14 Vegf-A Tg Vegfr1 tk(-/-) mice. K14 Vegf-A Tg Vegfr1 tk(-/-) mice exhibit a remarkable decrease in lymphangiogensis as well as angiogenesis in subcutaneous tissues. To address the mechanism underlying the decrease in lymphangiogensis, we investigated the recruitment of monocyte-macrophage-lineage cells into the skin. The recruitment of VEGFR1-expressing macrophages driven by VEGF-A was reduced in K14 Vegf-A Tg Vegfr1 tk(-/-) mice. Vegf-A Tg mice that received VEGFR1-TK-deficient bone marrow showed a reduction of macrophage recruitment, lymphangiogenesis and angiogenesis compared with those in K14 Vegf-A Tg mice.
VEGFR1 signaling promotes lymphangiogenesis as well as angiogenesis mainly by increasing bone marrow-derived macrophage recruitment.