Infection Of Hematopoietic Stem Cells Research Articles

Human T-cell leukemia virus type 1 infects multiple lineage hematopoietic cells in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) infects mainly CD4+CCR4+ effector/memory T cells in vivo. However, it remains unknown whether HTLV-1 preferentially infects these T cells or this virus converts infected precursor cells to specialized T cells. Expression of viral genes in vivo is critical to study viral replication and proliferation of infected cells. Therefore, we first analyzed viral gene expression in non-human primates naturally infected with simian T-cell leukemia virus type 1 (STLV-1), whose virological attributes closely resemble those of HTLV-1. Although the tax transcript was detected only in certain tissues, Tax expression was much higher in the bone marrow, indicating the possibility of de novo infection. Furthermore, Tax expression of non-T cells was suspected in bone marrow. These data suggest that HTLV-1 infects hematopoietic cells in the bone marrow. To explore the possibility that HTLV-1 infects hematopoietic stem cells (HSCs), we analyzed integration sites of HTLV-1 provirus in various lineages of hematopoietic cells in patients with HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) and a HTLV-1 carrier using the high-throughput sequencing method. Identical integration sites were detected in neutrophils, monocytes, B cells, CD8+ T cells and CD4+ T cells, indicating that HTLV-1 infects HSCs in vivo. We also detected Tax protein in myeloperoxidase positive neutrophils. Furthermore, dendritic cells differentiated from HTLV-1 infected monocytes caused de novo infection to T cells, indicating that infected monocytes are implicated in viral spreading in vivo. Certain integration sites were re-detected in neutrophils from HAM/TSP patients at different time points, indicating that infected HSCs persist and differentiate in vivo. This study demonstrates that HTLV-1 infects HSCs, and infected stem cells differentiate into diverse cell lineages. These data indicate that infection of HSCs can contribute to the persistence and spread of HTLV-1 in vivo.

Infection of hematopoietic stem cells by Leishmania infantum increases erythropoiesis and alters the phenotypic and functional profiles of progeny

Immunosuppression is a well-established risk factor for Visceral Leishmaniasis. Post-immunosuppression leishmaniasis is characterized by an increase of parasite burden, hematopoietic disorders and unusual clinical manifestations. Although there are many reports on bone marrow findings in VL, less is known about the relationship between parasite dynamics in this organ and the function of either hematopoietic stem cells and progenitor cells themselves. In the present study, we tackle these issues using a new approach of infecting human stem cells derived from bone marrow with L. infantum. Using this strategy, we show that human hematopoietic stem cells (hHSC) are able to phagocytize L. infantum promastigotes and release modulatory and pro-inflammatory cytokines, mainly TNF-α. Our results demonstrated that L. infantum infection in vitro enhances hematopoiesis, favoring the development of erythrocitic lineage through a mechanism yet unknown. Moreover, we found that L. infantum infection alters the phenotypic profile of the hematopoietic progeny; modifying the surface markers expression of differentiated cells. Thus, our study represents a rare opportunity to monitor the in vitro differentiation of human stem cells experimentally infected by L. infantum to better understand the consequences of the infection on phenotypic and functional profile of the cell progeny.

Reconstitution of Mice with Modified Hematopoietic Stem Cells.

Stem cell transplantation is well established in humans for the treatment of hematopoietic disease, including hematopoietic malignancies. Similar direct transplant procedures can readily be performed in mice; these procedures can be paired with retroviral infection to introduce exogenous genes or to silence endogenous genes in a subset of cells in the murine hematopoietic system. The resulting mice are chimeric for cells bearing a specific alteration. This approach has the advantage of examining tumorigenesis on a largely wild-type background (if only a subset of cells are infected), a situation that more accurately parallels the human situation. Additionally, tumor development occurs within the appropriate native microenvironment. Here, we describe the isolation and retroviral infection of hematopoietic stem cells (HSCs), as well as the reconstitution and monitoring of tumor formation in lethally irradiated recipient mice. This protocol requires a source of long-term HSCs; these can include either stimulated adult bone marrow or fetal liver—the site of primitive hematopoiesis.

Migration of Cells From the Yolk Sac to Hematopoietic Tissues After Transplantation of Early and Mid Gestation Canine Fetuses

Migration of Cells From the Yolk Sac to Hematopoietic Tissues After Transplantation of Early and Mid Gestation Canine Fetuses

Hematopoietic stem/precursor cells as HIV reservoirs

Although latent HIV-1 infection in CD4+ T cells contributes to HIV persistence, there is mounting evidence that other viral reservoirs exist. Here, we review recent data suggesting that the infection of hematopoietic progenitor cells creates additional reservoirs for HIV in vivo. New studies suggest that some types of hematopoietic progenitor cells have the potential to generate reservoirs for HIV. This review focuses on two types that can be infected by HIV in vitro and in vivo: multipotent hematopoietic progenitor cells in the bone marrow and circulating mast cell progenitors. Of these two types, only CD34+ bone marrow cells have been shown to harbor latent provirus in HIV-positive individuals with undetectable viral loads on highly active antiretroviral therapy (HAART). Latent infection of these long-lived cell types may create a significant barrier to HIV eradication; the infection of hematopoietic stem cells in particular could lead to an HIV reservoir that does not appreciably decay over the lifespan of the host. To eradicate HIV infection, it will be necessary to purge all viral reservoirs in the host. The findings highlighted here suggest that multipotent hematopoietic progenitor cells and possibly tissue mast cells may constitute significant reservoirs for HIV that must be addressed in order to eliminate HIV infection. Future studies are needed to determine which types of CD34+ cells are infected in vivo and whether infected CD34+ cells contribute to residual viremia in people with undetectable viral loads on HAART.

Abstract 405: HTLV-1 infection of humanized SCID mice recapitulates adult T-cell leukemia/lymphoma (ATLL) development

Abstract Human T-lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4+/CD25+ T cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATLL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+ HP/HSCs) and that infection of CD34+ HPCs has dramatically different biological effects in comparison to infection of mature T lymphocytes. To determine if HTLV-1 infection of CD34+ HP/HSCs recapitulates leukemogenesis in vivo, human hematopoiesis was reconstituted in NOD/SCID mice by injection of human CD34+ HPCs infected ex vivo with HTLV-1. Humanized NOD/SCID (HU-SCID) mice infected with HTLV-1 (HTLV-1-HU-SCID) consistently developed CD4+CD25+ T cell lymphomas with clinical characteristics associated with ATLL at ∼10 weeks post-reconstitution and show significantly elevated levels of HTLV-1 infected human CD4+ T cells in the thymus, mesenteric lymph node, spleen and peripheral blood. Lymphoma cells successfully engrafted in naïve NOD/SCID mice when injected into the peritoneal cavity and maintain the expression of viral proteins, gp46env and p19gag. Moreover these infected mice showed hyperproliferation of infected human stem cells (CD34+CD38−) in the bone marrow suggesting that HP/HSCs represents viral reservoir target cells which maintain HTLV-1 infection for extended periods of time in vivo. We speculate that HTLV-1 infection of hematopoietic stem cells establishes a virally-infected “cancer stem cell” which subsequently gives rise ATL in patients. Notably, CD34+ HPCs isolated from HTLV-1 infected patient PBL demonstrate proviral integrations, suggesting that these cells harbor infection in humans. CD34+ HPCs transduced with a lentivirus vector expressing the HTLV-1 Tax gene (Tax1) also results in CD4+/CD25+ T cell leukemia/lymphoma in HU-SCID mice, suggesting that Tax1 expression is sufficient for lymphomagenesis. HTLV-1 infection of humanized NOD/SCID mice represents a novel in vivo model which recapitulates viral lymphomagenesis and provides a compelling system to investigate and characterize molecular events in human stem cells in the initiation and progression to ATLL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 405.

HTLV-1 infection of Humanized SCID mice recapitulates Adult T-cell Leukemia/Lymphoma (ATLL) development

Human T-lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T-cell leukemia/lymphoma (ATLL), an aggressive CD4+/CD25+T cell malignancy. The early molecular events induced by HTLV-1 infection as well as the role of various viral genes in the induction of leukemia remain unclear, predominantly due to the lack of an animal model that recapitulates ATLL development. We have previously demonstrated that HTLV-1 is capable of infecting human hematopoietic progenitor and stem cells (CD34+HP/HSCs) and that infection of CD34+HPCs has dramatically different biological effects in comparison to infection of mature T lymphocytes. To determine if HTLV-1 infection of CD34+HP/HSCs recapitulates leukemogenesis in vivo, human hematopoiesis was reconstituted in NOD/SCID mice by injection of human CD34+HPCs infected ex vivo with HTLV-1. Humanized NOD/SCID (HU-SCID) mice infected with HTLV-1 (HTLV-1-HU-SCID) consistently developed CD4+CD25+T cell lymphomas with clinical characteristics associated with ATLL at ~10 weeks post-reconstitution and show significantly elevated levels of HTLV-1 infected human CD4+T cells in the thymus, mesenteric lymph node, spleen and peripheral blood. Lymphoma cells successfully engrafted in naive NOD/SCID mice when injected into the peritoneal cavity and maintain the expression of viral proteins, gp46env and p19gag. Moreover these infected mice showed hyperproliferation of infected human stem cells (CD34+CD38-) in the bone marrow suggesting that HP/HSCs represents viral reservoir target cells which maintain HTLV-1 infection for extended periods of time in vivo. We speculate that HTLV-1 infection of hematopoietic stem cells establishes a virally-infected ''cancer stem cell'' which subsequently gives rise ATL in patients. Notably, CD34+HPCs isolated from HTLV-1 infected patient PBL demonstrate proviral integrations, suggesting that these cells harbor infection in humans. CD34+HPCs transduced with a lentivirus vector expressing the HTLV-1 Tax gene (Tax1) also results in CD4+/CD25+T cell leukemia/lymphoma in HU-SCID mice, suggesting that Tax1 expression is sufficient for lymphomagenesis. HTLV-1 infection of humanized NOD/SCID mice represents a novel in vivo model which recapitulates viral lymphomagenesis and provides a compelling system to investigate and characterize molecular events in human stem cells in the initiation and progression to ATLL.

Regulation of Adult Hematopoietic Stem Cells Fate for Enhanced Tissue-specific Repair

The ability to control the differentiation of adult hematopoietic stem cells (HSCs) would promote development of new cell-based therapies to treat multiple degenerative diseases. Systemic injection of NaIO(3) was used to ablate the retinal pigment epithelial (RPE) layer in C57Bl6 mice and initiate neural retinal degeneration. HSCs infected ex vivo with lentiviral vector expressing the RPE-specific gene RPE65 restored a functional RPE layer, with typical RPE phenotype including coexpression of another RPE-specific marker, CRALBP, and photoreceptor outer segment phagocytosis. Retinal degeneration was prevented and visual function, as measured by electroretinography (ERG), was restored to levels similar to that found in normal animals. None of the controls (no HSCs, HSCs alone and HSCs infected with lentiviral vector expressing LacZ) showed these effects. In vitro gene array studies demonstrated that infection of HSC with RPE65 increased adenylate cyclase mRNA. In vitro exposure of HSCs to a pharmacological agonist of adenylate cyclase also led to in vitro differentiation of HSCs to RPE-like cells expressing pigment granules and the RPE-specific marker, CRALBP. Our data confirm that expression of the cell-specific gene RPE65 promoted fate determination of HSCs toward RPE for targeted tissue repair, and did so in part by activation of adenylate cyclase signaling pathways. Expression by HSCs of single genes unique to a differentiated cell may represent a novel experimental paradigm to influence HSC plasticity, force selective differentiation, and ultimately lead to identification of pharmacological alternatives to viral gene delivery.

Simian immunodeficiency virus infection of hematopoietic stem cells and bone marrow stromal cells.

Using a well-characterized fetal rhesus monkey model, simian immunodeficiency virus (SIV) infection of hematopoietic stem cells (HSCs) and stromal cells was investigated to characterize bone marrow abnormalities in SIV-infected animals in vivo. Fetuses (n = 4) were inoculated in utero with pathogenic SIVmac251 using established techniques at 65 days; gestation (early second trimester), then harvested for tissues 65 days after inoculation (late third trimester), and compared with findings from controls of a comparable age (n = 11). Sorted bone marrow CD34CD38, CD34CD38, and marrow stromal cells were analyzed for the SIV genome. Results indicate that CD34CD38 HSCs were not infected, whereas the SIV genome was detected in the CD34CD38 hematopoietic cell population by PCR analysis. The SIV proviral sequence was not detected in stromal cells from SIV-infected fetuses, although SIVmac251 was found to readily infect these cells when assessed in vitro. Flow cytometric analysis revealed that stromal cells express low levels (1 in 600 total cells) of CD4 and CCR5 viral receptors, whereas CXCR4 expression was not observed. These data suggest the following: (1) SIV infection of HSCs requires some degree of differentiation for viral entry similar to findings for humans infected with HIV type 1, and (2) SIV can infect bone marrow stromal cells that express CD4 and CCR5 in vitro, but infected stromal cells are not readily found in vivo.

Bacterial infection of human hematopoietic stem cells induces monocytic differentiation

Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-α were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34 + hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response.

Pathogenesis and pathophysiology of anemia in HIV infection.

Anemia is common in human immunodeficiency virus infection, particularly in patients with acquired immunodeficiency syndrome. Anemia is multifactorial. HIV infection itself causes anemia, probably as a consequence of HIV infection of stromal cells rather than HIV infection of hematopoietic stem cells. Other common causes of anemia in AIDS are anemia of chronic disease consequent on opportunistic infections, bone marrow suppression by antiretroviral therapy, and hemolytic anemia induced by oxidant drugs. Thrombotic microangiopathy is a significant complication of HIV infection and may respond to plasmapheresis.

Loss of retroviral gene expression in bone marrow reconstituted mice correlates with down-regulation of gene expression in long-term culture initiating cells.

The question of whether a retroviral vector is expressed in vivo after infection of hematopoietic stem cells remains unpredictable. In this study, we show that a switch off of retroviral sequences can be tested in vitro using the long-term bone marrow culture system. The time kinetics of the down-regulation of retroviral expression in vitro and in vivo have been demonstrated to be similar. The correlation of switch off events in both systems allows for evaluation of retroviral expression in vitro provided the silencing is caused by viral sequences. This approach could be useful for testing vectors for gene therapeutical applications of the hematopoietic system.

High levels of human glucocerebrosidase activity in macrophages of long- term reconstituted mice after retroviral infection of hematopoietic stem cells

Gaucher disease is a leading candidate for somatic gene therapy using bone marrow (BM) cells as target tissue. Towards this end, we have constructed a retroviral vector (LG) in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). Day 12 to 14 colony-forming unit-spleen progenitor cells were infected by the LG virus with a 100% efficiency, and GC messenger RNA (mRNA) and protein were detected in the progeny of these cells. Tissues from long-term reconstituted mice analyzed 8 months posttransplantation with LG-infected BM contained the intact provirus at greater than 1 copy per cell, indicating effective infection of hematopoietic stem cells. Human GC mRNA generated by the viral LTR was detected in macrophages as well as other hematopoietic cells. Enzyme activity was increased fivefold and twofold in macrophages from BM and spleen, respectively, and could be precipitated with an antibody specific for human GC. Immunohistochemical analysis detected the human GC protein in 81% of the macrophages from five recipient mice. These data indicate that, after transduction of hematopoietic stem cells, the LG vector is capable of directing expression of human GC in the majority of macrophages from long-term reconstituted mice and producing enzyme levels comparable with endogenous mouse activity, suggesting that this virus may be useful in the treatment of Gaucher disease.

High Levels of Human Glucocerebrosidase Activity in Macrophages of Long-Term Reconstituted Mice After Retroviral Infection of Hematopoietic Stem Cells

Abstract Gaucher disease is a leading candidate for somatic gene therapy using bone marrow (BM) cells as target tissue. Towards this end, we have constructed a retroviral vector (LG) in which the human glucocerebrosidase (GC) cDNA is driven by the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR). Day 12 to 14 colony-forming unit-spleen progenitor cells were infected by the LG virus with a 100% efficiency, and GC messenger RNA (mRNA) and protein were detected in the progeny of these cells. Tissues from long-term reconstituted mice analyzed 8 months posttransplantation with LG-infected BM contained the intact provirus at greater than 1 copy per cell, indicating effective infection of hematopoietic stem cells. Human GC mRNA generated by the viral LTR was detected in macrophages as well as other hematopoietic cells. Enzyme activity was increased fivefold and twofold in macrophages from BM and spleen, respectively, and could be precipitated with an antibody specific for human GC. Immunohistochemical analysis detected the human GC protein in 81% of the macrophages from five recipient mice. These data indicate that, after transduction of hematopoietic stem cells, the LG vector is capable of directing expression of human GC in the majority of macrophages from long-term reconstituted mice and producing enzyme levels comparable with endogenous mouse activity, suggesting that this virus may be useful in the treatment of Gaucher disease.

In vitro myelosuppressive effects of the parvovirus minute virus of mice (MVMi) on hematopoietic stem and committed progenitor cells

The interaction of two strains of the parvovirus minute virus of mice (MVM) with the mouse hematopoietic system has been studied. The immunosuppressive strain MVMi, but not the prototype virus MVMp, inhibited hematopoiesis in vitro, as judged by colony-forming assays of the erythroid burst-forming unit and granulocyte-monocyte colony- forming unit (CFU-GM) progenitors. Interestingly, primitive hematopoietic cells of the stem compartment (CFU-S12d), were equally susceptible to the MVMi cytotoxic infection, unravelling an unprecedented feature of virus-hematopoiesis interactions. The replication of both strains of MVM virus was evaluated in primary myeloid cells of long-term bone marrow cultures. A high viral DNA synthesis and maturation was observed in MVMi-infected myeloid cells, but it was undetectable in MVMp infections; moreover, the expression of the cytotoxic nonstructural NS-1 protein, a more reliable parameter of cell permissiveness to MVM infection, was only detected in MVMi- infected cells. Correspondingly, MVMi was propagated to high titers of infectious virus and it mediated an acute myelosuppression in these cultures. We conclude that MVMi has a wider tropism than was previously suspected and it is proposed that cytotoxic infection of hematopoietic stem cells, besides that of committed progenitors, may provide an additional basis to understand the pathogenesis of certain animal and human bone marrow failures of viral etiology.

In Vitro Myelosuppressive Effects of the Parvovirus Minute Virus of Mice (MVMi) on Hematopoietic Stem and Committed Progenitor Cells

The interaction of two strains of the parvovirus minute virus of mice (MVM) with the mouse hematopoietic system has been studied. The immunosuppressive strain MVMi, but not the prototype virus MVMp, inhibited hematopoiesis in vitro, as judged by colony-forming assays of the erythroid burst-forming unit and granulocyte-monocyte colony-forming unit (CFU-GM) progenitors. Interestingly, primitive hematopoietic cells of the stem compartment (CFU-S12d), were equally susceptible to the MVMi cytotoxic infection, unravelling an unprecedented feature of virus-hematopoiesis interactions. The replication of both strains of MVM virus was evaluated in primary myeloid cells of long-term bone marrow cultures. A high viral DNA synthesis and maturation was observed in MVMi-infected myeloid cells, but it was undetectable in MVMp infections; moreover, the expression of the cytotoxic nonstructural NS-1 protein, a more reliable parameter of cell permissiveness to MVM infection, was only detected in MVMi-infected cells. Correspondingly, MVMi was propagated to high titers of infectious virus and it mediated an acute myelosuppression in these cultures. We conclude that MVMi has a wider tropism than was previously suspected and it is proposed that cytotoxic infection of hematopoietic stem cells, besides that of committed progenitors, may provide an additional basis to understand the pathogenesis of certain animal and human bone marrow failures of viral etiology.

Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells.

Long-term stable expression of foreign genetic sequences transferred into hematopoietic stem cells by using retroviral vectors constitutes a relevant model for somatic gene therapy. Such stability of expression may depend on vector design, including the presence or absence of specific sequences within the vector, in combination with the nature and efficiency of infection of the hematopoietic target cells. We have previously reported successful transfer of human DNA encoding adenosine deaminase (ADA) into CFU-S (colony-forming unit-spleen) stem cells using simplified recombinant retroviral vectors. Human ADA was expressed in CFU-S-derived spleen colonies at levels near to endogenous enzyme. However, because of the lack of an efficient dominant selectable marker and low recombinant viral titers, stability of long-term expression of human ADA was not examined. We report here the development of an efficient method of infection of hematopoietic stem cells (HSC) without reliance on in vitro selection. Peripheral blood samples of 100% of mice transplanted with HSC infected by this protocol exhibit expression of human ADA 30 days after transplantation. Some mice (6 of 13) continue to express human ADA in all lineages after complete hematopoietic reconstitution (4 months). The use of recombinant retroviral vectors that efficiently transfer human ADA cDNA into HSC leading to stable expression of functional ADA in reconstituted mice, provides an experimental framework for future development of approaches to somatic gene therapy.