Gerald J. Spangrude

Major Age‐Related Changes Of Mouse Hematopoietic Stem/Progenitor Cells

Abstract: To study age‐related changes of mouse bone marrow (BM) cells and hematopoietic stem cells (HSCs), we isolated rhodamine‐123low (Rhlow) Thy1.1low Lin−Sca‐1+ (TLS) HSCs from the BM of old mice and compared their functional characteristics to cells of the same phenotype isolated from young mice. We observed impaired recovery of B lymphocytes and decreased self‐renewal in recipients of old Rhlow cells compared to young Rhlow cells. Blockade of Rh efflux using verapamil improved lymphoid reconstitution by enriched HSCs, and isolation of aged HSCs based on efflux of a fluorescent multi‐drug resistance (MDR) substrate (Bodipy‐verapamil) resulted in enrichment of HSC activity equivalent to that obtained with Rh. These observations suggest a complex relationship between MDR activity and HSC function during aging. To address whether the difference between young and aged donors was intrinsic to the HSC compartment or was due to a shift in HSC phenotype, we co‐transplanted normal BM derived from young or old donors and followed repopulation simultaneously in the same recipient animals. In a parallel experiment, we co‐transplanted HSCs purified from old donors with BM derived from young donors. In both experiments, transplants were given to both young and old recipients. The results show a clear defect in B‐cell engraftment from either BM or HSCs of old donors, irrespective of the age of the recipient. In contrast, myeloid engraftment was predominantly derived from BM or HSCs derived from aged donors, again irrespective of recipient age. These data suggest a stem cell basis for B‐cell immuno‐senescence and the increased incidence of myelocytic leukemia in elderly people.

Expression of CD27 on murine hematopoietic stem and progenitor cells

Hematopoietic stem cells (HSC) are defined by self-renewal and multilineage differentiation potentials. In order to uncover the genetic program of HSC, we utilized high-density arrays to compare gene expression in highly purified mouse HSC and their mature progeny. One molecule specifically expressed in immature cells is CD27, a member of the TNF receptor family previously shown to play roles in lymphoid proliferation, differentiation, and apoptosis. We show here that the CD27 protein is expressed by about 90% of cells in a purified HSC population. Interestingly, the CD27pos cells are enriched for cells with short-term hematopoietic activities (colony forming potential in vivo and in vitro), while the minority CD27neg population is more effective in clonal long-term transplantation.

Primitive stem cells alone mediate rapid marrow recovery and multilineage engraftment after transplantation.

The engraftment of hematopoietic stem and progenitor cells in lethally irradiated mice was evaluated following transplants of enriched hematopoietic cell populations which were defined by surface antigen and rhodamine-123 staining. Phenotypically defined long-term repopulating stem cells, short-term pluripotent progenitors, and committed myeloerythroid progenitors all rapidly reconstituted splenic cellularity and peripheral red blood cells after transplant into myeloablated animals. In contrast, marrow cellularity was reconstituted only after transplant of long-term repopulating stem cells. In addition, peripheral blood platelet and lymphocyte counts increased only after transplantation of the long-term repopulating population. Transplantation of highly enriched multipotent progenitors resulted in a transient increase in peripheral blood myeloid cells that occurred with kinetics similar to that seen after transplant of the primitive stem cell population. Erythroid reconstitution was similar in all groups, suggesting that the effect of myeloerythroid progenitor cells in mouse marrow transplants is primarily on reconstitution of the erythroid lineage due to splenic hematopoiesis. Collectively, these results suggest that the cells which function to rapidly reconstitute the nucleated blood cells in a transplant setting are more closely related to primitive, marrow-homing stem cells than to committed progenitor cells.

Enrichment of murine haemopoietic stem cells: diverging roads

The cellular elements of the peripheral blood must be constantly replenished by the process of haemopoiesis, since most blood cells have a limited life span of only days or weeks. Although the developmental lineages of haemopoietic differentiation have been depicted in textbooks for decades, the actual details of the early stages of haemopoiesis are relatively unknown due to the very low numbers of haemopoietic stem cells in bone marrow or spleen. Only by isolating these rare stem cells and developing in-vitro culture systems to maintain them can a complete understanding of the early stages of haemopoiesis be achieved. This approach has already been successfully applied to the study of the later stages of haemopoiesis. In this review, Gerald Spangrude examines several experimental approaches that have been used to enrich murine haemopoietic stem cells.

Ascorbate regulates haematopoietic stem cell function and leukaemogenesis

Stem-cell fate can be influenced by metabolite levels in culture, but it is not known whether physiological variations in metabolite levels in normal tissues regulate stem-cell function in vivo. Here we describe a metabolomics method for the analysis of rare cell populations isolated directly from tissues and use it to compare mouse haematopoietic stem cells (HSCs) to restricted haematopoietic progenitors. Each haematopoietic cell type had a distinct metabolic signature. Human and mouse HSCs had unusually high levels of ascorbate, which decreased with differentiation. Systemic ascorbate depletion in mice increased HSC frequency and function, in part by reducing the function of Tet2, a dioxygenase tumour suppressor. Ascorbate depletion cooperated with Flt3 internal tandem duplication (Flt3ITD) leukaemic mutations to accelerate leukaemogenesis, through cell-autonomous and possibly non-cell-autonomous mechanisms, in a manner that was reversed by dietary ascorbate. Ascorbate acted cell-autonomously to negatively regulate HSC function and myelopoiesis through Tet2-dependent and Tet2-independent mechanisms. Ascorbate therefore accumulates within HSCs to promote Tet activity in vivo, limiting HSC frequency and suppressing leukaemogenesis.

Characterization of Thymic Progenitors in Adult Mouse Bone Marrow

Thymic cellularity is maintained throughout life by progenitor cells originating in the bone marrow. In this study, we describe adult mouse bone cells that exhibit several features characteristic of prothymocytes. These include 1) rapid thymic engraftment kinetics following i.v. transplantation, 2) dramatic expansion of thymic progeny, and 3) limited production of hemopoietic progeny other than thymocytes. The adult mouse bone marrow population that is depleted of cells expressing any of a panel of lineage-specific Ags, stem cell Ag-1 positive, and not expressing the Thy1.1 Ag (Thy1.1−) (Thy1.1− progenitors) can repopulate the thymus 9 days more rapidly than can hemopoietic stem cells, a rate of thymic repopulation approaching that observed with transplanted thymocytes. Additionally, Thy1.1− progenitors expand prolifically to generate thymocyte progeny comparable in absolute numbers to those observed from parallel hemopoietic stem cell transplants, and provide a source of progenitors that spans multiple waves of thymic seeding. Nevertheless, the Thy1.1− population yields relatively few B cells and rare myeloid progeny posttransplant. These observations describe the phenotype of an adult mouse bone marrow population highly enriched for rapidly engrafting, long-term thymocyte progenitors. Furthermore, they note disparity in B and T cell expansion from this lymphoid progenitor population and suggest that it contains the progenitor primarily responsible for seeding the thymus throughout life.

Marrow engraftment of hematopoietic stem and progenitor cells is independent of Gαi-coupled chemokine receptors

The mechanism of hematopoietic stem and progenitor cell (HSPC) homing to hematopoietic organs after transplantation is still poorly understood. There is evidence that HSPC homing is a multistep process involving integrins and other adhesion molecules as well as stimulation of cytokine and chemokine receptors, similar to the process of lymphocyte recirculation and leukocyte emigration. This study examined the effect of pertussis toxin (PT), an inhibitor of signaling by many Galphai protein-coupled chemokine receptors, on engraftment of HSPC. An in vitro incubation of total bone marrow cells in PT-supplemented media prior to transplantation into lethally irradiated syngeneic mice resulted in an increase in marrow repopulation and a parallel decrease of colony-forming unit-spleen (CFU-S) on day 13. PT treatment of Rh(low)Lineage(neg)Sca-1pos cells prior to transplant resulted in delayed spleen cell engraftment, but no observable difference in the bone marrow cellularity compared to animals transplanted with untreated cells. FACS analysis of hematopoietic organs revealed that myeloid cell recovery in the bone marrow was unaffected by PT treatment of HSPC. However, a reduced myeloid cell recovery in the spleen and an increased B lymphoid recovery in both the spleen and the bone marrow were observed in recipients of PT-treated grafts relative to untreated grafts. To test the hypothesis that PT inhibits proliferation rather than engraftment of HSPC in the spleen, the effect of PT on cytokine-stimulated proliferation of HSPC was tested. Although an inhibition of the growth of microcolonies in response to interleukin 6 as a single cytokine could be observed after PT treatment, colony growth of HSPC after steel factor or steel factor + interleukin 6 stimulation was unaffected by PT. This study demonstrates that bone marrow, but not splenic, recovery after HSPC transplantation is independent of PT-sensitive mechanisms. It is likely that PT inhibits spleen cell recovery by disrupting a Galphai-coupled homing receptor expressed by HSPC. These studies support the hypothesis that distinct mechanisms regulate splenic vs bone marrow engraftment of HSPC, and that B lymphocyte progenitors and HSPC can utilize a PT-resistant homing mechanism to localize in hematopoietic tissues after transplantation.

Hematopoietic stem-cell differentiation

Hematopoietic stem cells can be identified and isolated from hematopoietic tissues of mammalian hosts. Assay systems that solely reflect hematopoietic stem cell activity are being developed, and new cytokines that influence hematopoietic stem-cell proliferation and differentiation have been described. Differentiation pathways that lead to lymphoid stages of hematopoiesis have also been suggested.

Localized Production of IL-10 Suppresses Early Inflammatory Cell Infiltration and Subsequent Development of IFN-γ–Mediated Lyme Arthritis

IL-10 is a nonredundant inflammatory modulator that suppresses arthritis development in Borrelia burgdorferi-infected mice. Infected C57BL/6 (B6) IL-10−/− mice were previously found to have a prolonged IFN-inducible response in joint tissue. Infection of B6 IL-10 reporter mice identified macrophages and CD4+ T cells as the primary sources of IL-10 in the infected joint tissue, suggesting that early local production of IL-10 dampened the proarthritic IFN response. Treatment of B6 IL-10−/− mice with anti–IFN-γ reduced the increase in arthritis severity and suppressed IFN-inducible transcripts to wild-type levels, thereby linking dysregulation of IFN-γ to disease in the B6 IL-10−/− mouse. Arthritis in B6 IL-10−/− mice was associated with elevated numbers of NK cell, NKT cell, α/β T cell, and macrophage infiltration of the infected joint. FACS lineage sorting revealed NK cells and CD4+ T cells as sources of IFN-γ in the joint tissue of B6 IL-10−/− mice. These findings suggest the presence of a positive-feedback loop in the joint tissue of infected B6 IL-10−/− mice, in which production of inflammatory chemokines, infiltration of IFN-γ–producing cells, and additional production of inflammatory cytokines result in arthritis. This mechanism of arthritis is in contrast to that seen in C3H/He mice, in which arthritis development is linked to transient production of type I IFN and develops independently of IFN-γ. Due to the sustained IFN response driven by NK cells and T cells, we propose the B6 IL-10−/− mouse as a potential model to study the persistent arthritis observed in some human Lyme disease patients.

Modulation of hematopoietic stem/progenitor cell engraftment by transforming growth factor β

OBJECTIVE To investigate if cell cycle progression plays a role in modulating the engraftment potential of mouse hematopoietic stem and progenitor cells (HSPC). MATERIALS AND METHODS HSPC were isolated from adult mouse bone marrow, cultured in vitro under conditions promoting cell cycle arrest, and subsequently were evaluated for cell cycle status, clonogenic activity, and transplant potential. RESULTS In the presence of steel factor (STL) as a survival cytokine, transforming growth factor beta (TGF-beta) increased the G0/G1 fraction of cycling progenitor cells (Rh(high)) after a 20-hour culture. Clonogenic activity of quiescent long-term repopulating (Rh(low)) HSPC was unaffected by this culture, whereas clonogenic potential of Rh(high) cells decreased by about 30%. In competitive repopulation assays, Rh(low) cells cultured in STL + TGF-beta engrafted better than cells cultured in STL alone. However, culture in STL + TGF-beta did not overcome the failure of Rh(high) cells to engraft after transplant. We also utilized a two-stage culture system to first induce proliferation of Rh(low) HSPC by a 48-hour culture in STL + interleukin 6 + Flt-3 ligand, followed by shifting the culture to STL + TGF-beta for 24 hours to induce cycle arrest. A competitive repopulation assay demonstrated a relative decrease in repopulating potential in cultures that were cycle arrested compared to those that were not. CONCLUSION Cell cycle progression by itself cannot account for the decrease in repopulating potential that is observed after ex vivo expansion. Other determinants of engraftment must be identified to facilitate the transplantation of cultured HSPC.