HIV-1 utilizes the CXCR4 chemokine receptor to infect multipotent hematopoietic stem and progenitor cells

HIV infection is characterized by gradual immune system collapse and hematopoietic dysfunction. We recently showed that HIV enters multipotent hematopoietic progenitor cells and establishes both active cytotoxic and latent infections that can be reactivated by myeloid differentiation. However, whether these multipotent progenitors include long-lived hematopoietic stem cells (HSCs) that could establish viral reservoirs for the life of the infected person remains unknown. Here we provide direct evidence that HIV targets long-lived HSCs and show that infected HSCs yield stable, multilineage engraftment in a xenograft model. Furthermore, we establish that the capacity to use the chemokine receptor CXCR4 for entry determines whether a virus will enter multipotent versus differentiated progenitor cells. Because HSCs live for the life span of the infected person and are crucial for hematopoietic health, these data may explain the poor prognosis associated with CXCR4-tropic HIV infection and suggest HSCs as long-lived cellular reservoirs of latent HIV.     

Lineage development of hematopoietic stem and progenitor cells

Hematopoietic stem cells have the potential to develop into multipotent and different lineage-restricted progenitor cells that subsequently generate all mature blood cell types. The classical model of hematopoietic lineage commitment proposes a first restriction point at which all multipotent hematopoietic progenitor cells become committed either to the lymphoid or to the myeloid development, respectively. Recently, this model has been challenged by the identification of murine as well as human hematopoietic progenitor cells with lymphoid differentiation capabilities that give rise to a restricted subset of the myeloid lineages. As the classical model does not include cells with such capacities, these findings suggest the existence of alternative developmental pathways that demand the existence of additional branches in the classical hematopoietic tree. Together with some phenotypic criteria that characterize different subsets of multipotent and lineage-restricted progenitor cells, we summarize these recent findings here.     

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.     

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.     

Reduced repression of cytokine signaling ameliorates age‐induced decline in hematopoietic stem cell function

Aging causes profound effects on the hematopoietic stem cell (HSC) pool, including an altered output of mature progeny and enhanced self‐propagation of repopulating‐defective HSCs. An important outstanding question is whether HSCs can be protected from aging. The signal adaptor protein LNK negatively regulates hematopoiesis at several cellular stages. It has remained unclear how the enhanced sensitivity to cytokine signaling caused by LNK deficiency affects hematopoiesis upon aging. Our findings demonstrate that aged LNK^?/? HSCs displayed a robust overall reconstitution potential and gave rise to a hematopoietic system with a balanced lineage distribution. Although aged LNK^?/? HSCs displayed a distinct molecular profile in which reduced proliferation was central, little or no difference in the proliferation of aged LNK^?/? HSCs was observed after transplantation when compared to aged WT HSCs. This coincided with equal telomere maintenance in WT and LNK^?/? HSCs. Collectively, our studies suggest that enhanced cytokine signaling can counteract functional age‐related HSC decline.     

Hepatic regeneration from hematopoietic stem cells

In recent years, numerous investigators have reported novel cellular fates of multipotent stem or progenitor cells. In this review, we discuss the unexpected observations that hematopoietic stem cells can contribute to the hepatocyte lineage in humans and in rodent models of liver disease and regeneration. A key unresolved issue regarding hepatic regeneration from hematopoietic stem cells is whether the mechanism occurs through transdetermination, cell fusion, or other processes. A better understanding of the various stem or progenitor cells of the hepatic lineage may facilitate cellular transplantation approaches for the correction of hepatic function in patients with end-stage liver disease.     

Deciphering the hierarchy of angiohematopoietic progenitors from human pluripotent stem cells

Identification of sequential progenitors leading to blood formation from pluripotent stem cells (PSCs) will be essential for understanding the molecular mechanisms of hematopoietic lineage specification and for development of technologies for in vitro production of hematopoietic stem cells (HSCs). It is well established that during development, blood and endothelial cells in the extraembryonic and embryonic compartments are formed in parallel from precursors with angiogenic and hematopoietic potentials. However, the identity and hierarchy of these precursors in human PSC (hPSC) cultures remain obscure. Using developmental stage-specific mesodermal and endothelial markers and functional assays, we recently identified discrete populations of angiohematopoietic progenitors from hPSCs, including mesodermal precursors and hemogenic endothelial cells with primitive and definitive hematopoietic potentials. In addition, we discovered a novel population of multipotent hematopoietic progenitors with an erythroid phenotype, which retain angiogenic potential. Here we introduce our recent findings and discuss their implication for defining putative HSC precursor and factors required for activation of self-renewal potential in hematopoietic cells emerging from endothelium.     

Lack of autophagy in the hematopoietic system leads to loss of hematopoietic stem cell function and dysregulated myeloid proliferation

The regulated lysosomal degradation pathway of autophagy prevents cellular damage and thus protects from malignant transformation. Autophagy is also required for the maturation of various hematopoietic lineages, namely the erythroid and lymphoid ones, yet its role in adult hematopoietic stem cells (HSCs) remained unexplored. While normal HSCs sustain life-long hematopoiesis, malignant transformation of HSCs or early progenitors leads to leukemia. Mechanisms protecting HSCs from cellular damage are therefore essential to prevent hematopoietic malignancies. By conditionally deleting the essential autophagy gene Atg7 in the hematopoietic system, we found that autophagy is required for the maintenance of true HSCs and therefore also of downstream hematopoietic progenitors. Loss of autophagy in HSCs leads to the expansion of a progenitor cell population in the bone marrow, giving rise to a severe, invasive myeloproliferation, which strongly resembles human acute myeloid leukemia (AML).     

The complex cartography of stem cell commitment

In this issue of Cell, a study by Adolfsson and coworkers (Adolfsson et al., 2005) provides insight into the early lineage commitment events of multipotent hematopoietic stem cells (HSCs). These studies demonstrate the importance of the Flt3 receptor tyrosine kinase as the earliest marker of hematopoietic cell fate commitment in that erythrocyte and megakaryocyte potentials are lost first as HSCs differentiate to lymphocyte progenitors.     

Gene profiling reveals association between altered Wnt signaling and loss of T‐cell potential with age in human hematopoietic stem cells

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T‐cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9‐DL1 in vitro co‐culture system to promote T‐cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T‐lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T‐lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T‐cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T‐progenitor cell subset derived during in vitro T‐lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age‐related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T‐cell generation and immune reconstitution following clinical transplantation.     

CD61 enriches long-term repopulating hematopoietic stem cells

Among the subsets that define hematopoietic stem cells (HSCs), CD34⁻ c-kit⁺ Sca-1⁺ lineage marker⁻ (CD34⁻KSL) cells are regarded as one of the populations that have the highest enrichment of HSCs in adult mouse bone marrow. Here, we demonstrate that long-term repopulating hematopoietic stem cells (LTR-HSCs) have high expression of CD61 (integrin β₃) within the CD34⁻KSL population. Approximately 60% of CD34⁻KSL cells showed high expression of CD61. CD61^HighCD34⁻KSL populations also exhibited significantly greater properties of HSC, such as expression of HSC markers, the side population (SP) phenotype, and ability for long-term repopulation. In both SP cells and non-SP (NSP) cells, CD61^HighCD34⁻KSL cells also contained significantly more LTR-HSCs than CD61^Low/−CD34⁻KSL cells. Our results indicate that CD61 is exploitable for HSC enrichment as a supportive positive cell surface marker.     

Nucleostemin is indispensable for the maintenance and genetic stability of hematopoietic stem cells

Nucleostemin is a nucleolar protein known to play a variety of roles in cell-cycle progression, apoptosis inhibition, and DNA damage protection in embryonic stem cells and tissue stem cells. However, the role of nucleostemin in hematopoietic stem cells (HSCs) is yet to be determined. Here, we identified an indispensable role of nucleostemin in mouse HSCs. Depletion of nucleostemin using short hairpin RNA strikingly impaired the self-renewal activity of HSCs both in vitro and in vivo. Consistently, nucleostemin depletion triggered apoptosis rather than cell-cycle arrest in HSCs. Furthermore, DNA damage accumulated during cultivation upon depletion of nucleostemin. The impaired self-renewal activity of HSCs induced by nucleostemin depletion was partially rescued by p53 deficiency but not by p16^Ink4a or p19^Arf deficiency. Taken together, our study demonstrates that nucleostemin protects HSCs from DNA damage accumulation and is required for the maintenance of HSCs.     

Adult mouse hematopoietic stem cells: purification and single-cell assays

Mouse hematopoietic stem cells (HSCs) are the best-studied stem cells because functional assays for mouse HSCs were established earliest and purification techniques for mouse HSCs have progressed furthest. Here we describe our current protocols for the purification of CD34-/lowc-Kit+Sca-1+lineage marker- (CD34-KSL) cells, the HSC population making up approximately 0.005% of bone marrow cells in adult C557BL/6 mice. Purified HSCs have been characterized at cellular and molecular levels. Since clonal analysis is essential for the study of self-renewal and lineage commitment in HSCs, here we present our single-cell colony assay and single-cell transplantation procedures. We also introduce our immunostaining procedures for small numbers of HSCs, which are useful for signal transduction analysis. The purification of CD34-KSL cells requires approximately 6 h. Initialization of single-cell culture requires approximately 1 h. Single-cell transplantation requires approximately 6 h. Single-cell immunostaining requires approximately 2 d.     

Influence of a dual-injection regimen,plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model

Background aimsInadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis.MethodsWe used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4⁺ fraction of CD34⁺ HSCs, we could improve engraftment. Human cord blood-derived CD34⁺ cells and human bone marrow-derived MSCs were used for these studies.ResultsWhen MSCs were transplanted 1 week before CD34⁺ cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34⁺ cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4⁺ cells in the CD34⁺ population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages.ConclusionsPrior MSC and HSC cotransplantation followed by manipulation of the CXCR4–SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.     

Evi-1 is a critical regulator for hematopoietic stem cells and transformed leukemic cells

Evi-1 has been recognized as one of the dominant oncogenes associated with murine and human myeloid leukemia. Here, we show that hematopoietic stem cells (HSCs) in Evi-1-deficient embryos are severely reduced in number with defective proliferative and repopulating capacity. Selective ablation of Evi-1 in Tie2(+) cells mimics Evi-1 deficiency, suggesting that Evi-1 function is required in Tie2(+) hematopoietic stem/progenitors. Conditional deletion of Evi-1 in the adult hematopoietic system revealed that Evi-1-deficient bone marrow HSCs cannot maintain hematopoiesis and lose their repopulating ability. In contrast, Evi-1 is dispensable for blood cell lineage commitment. Evi-1(+/-) mice exhibit the intermediate phenotype for HSC activity, suggesting a gene dosage requirement for Evi-1. We further demonstrate that disruption of Evi-1 in transformed leukemic cells leads to significant loss of their proliferative activity both in vitro and in vivo. Thus, Evi-1 is a common and critical regulator essential for proliferation of embryonic/adult HSCs and transformed leukemic cells.     

SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells

To improve our ability to identify hematopoietic stem cells (HSCs) and their localization in vivo, we compared the gene expression profiles of highly purified HSCs and non-self-renewing multipotent hematopoietic progenitors (MPPs). Cell surface receptors of the SLAM family, including CD150, CD244, and CD48, were differentially expressed among functionally distinct progenitors. HSCs were highly purified as CD150(+)CD244(-)CD48(-) cells while MPPs were CD244(+)CD150(-)CD48(-) and most restricted progenitors were CD48(+)CD244(+)CD150(-). The primitiveness of hematopoietic progenitors could thus be predicted based on the combination of SLAM family members they expressed. This is the first family of receptors whose combinatorial expression precisely distinguishes stem and progenitor cells. The ability to purify HSCs based on a simple combination of SLAM receptors allowed us to identify HSCs in tissue sections. Many HSCs were associated with sinusoidal endothelium in spleen and bone marrow, though some HSCs were associated with endosteum. HSCs thus occupy multiple niches, including sinusoidal endothelium in diverse tissues.     

Unique and independent roles for MLL in adult hematopoietic stem cells and progenitors

The Mixed Lineage Leukemia (MLL) gene is essential for embryonic hematopoietic stem cell (HSC) development, but its role during adult hematopoiesis is unknown. Using an inducible knockout model, we demonstrate that Mll is essential for the maintenance of adult HSCs and progenitors, with fatal bone marrow failure occurring within 3 weeks of Mll deletion. Mll-deficient cells are selectively lost from mixed bone marrow chimeras, demonstrating their failure to self-renew even in an intact bone marrow environment. Surprisingly, HSCs lacking Mll exhibit ectopic cell-cycle entry, resulting in the depletion of quiescent HSCs. In contrast, Mll deletion in myelo-erythroid progenitors results in reduced proliferation and reduced response to cytokine-induced cell-cycle entry. Committed lymphoid and myeloid cells no longer require Mll, defining the early multipotent stages of hematopoiesis as Mll dependent. These studies demonstrate that Mll plays selective and independent roles within the hematopoietic system, maintaining quiescence in HSCs and promoting proliferation in progenitors.     

Differentiation of hematopoietic stem cell and myeloid populations by ATP is modulated by cytokines

Extracellular nucleotides are emerging as important regulators of inflammation, cell proliferation and differentiation in a variety of tissues, including the hematopoietic system. In this study, the role of ATP was investigated during murine hematopoiesis. ATP was able to reduce the percentage of hematopoietic stem cells (HSCs), common myeloid progenitors and granulocyte–macrophage progenitors (GMPs), whereas differentiation into megakaryocyte–erythroid progenitors was not affected. In addition, in vivo administration of ATP to mice reduced the number of GMPs, but increased the number of Gr-1⁺Mac-1⁺ myeloid cells. ATP also induced an increased proliferation rate and reduced Notch expression in HSCs and impaired HSC-mediated bone marrow reconstitution in sublethally irradiated mice. Moreover, the effects elicited by ATP were inhibited by suramin, a P2 receptor antagonist, and BAPTA, an intracellular Ca²⁺ chelator. We further investigated whether the presence of cytokines might modulate the observed ATP-induced differentiation. Treatment of cells with cytokines (stem cell factor, interleukin-3 and granulocyte–monocyte colony stimulator factor) before ATP stimulation led to reduced ATP-dependent differentiation in long-term bone marrow cultures, thereby restoring the ability of HSCs to reconstitute hematopoiesis. Thus, our data suggest that ATP induces the differentiation of murine HSCs into the myeloid lineage and that this effect can be modulated by cytokines.