Two rare populations of mouse Thy-1lo bone marrow cells repopulate the thymus.

Two-color FACS analysis of mouse bone marrow reveals a rare population, comprising 0.1-0.3% of the total, that expresses low levels of the Thy-1 antigen but does not express any of five surface markers that characterize differentiated hematolymphoid cells. We demonstrate here that this fraction of mouse bone marrow is enormously enriched in cells that can home to the thymus and differentiate into mature T lymphocytes, subsequently migrating to peripheral lymphoid organs. Only a subset of the FACS-isolated fraction (1/90 after intrathymic injection) is capable of responding to the thymic microenvironment with a productive commitment to the T cell lineage. A second fraction of mouse bone marrow, which expresses how levels of Thy-1 but is also positive for at least one of five hematolymphoid lineage-specific markers, also contains cells that home to the thymus and establish colonies of thymocytes. The two fractions each contribute approximately equal amounts of thymic colony-forming units (CFUt) to the bone marrow, and together can account for at least half of the CFUt in whole bone marrow.

Functional heterogeneity is associated with the cell cycle status of murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are characterized by their ability to differentiate into all hematopoietic cell lineages while retaining their capacity for self renewal. One of the predictions of this model is the existence of a heterogeneous pool of HSCs, some members of which are destined to become lineage restricted progenitor cells while others function to renew the stem cell pool. To test whether HSCs are heterogeneous with respect to cell cycle status, we determined the fraction of phenotypically defined murine HSCs (Thy1.1lo Lin-/lo Sca-1+) that contain > 2n amount of DNA as measured by propidium iodide staining, Hoechst dye uptake and [3H]thymidine labeling; that fraction is 18-22%. In contrast, in the developing fetal liver, 40% of HSCs are in the S/G2/M phases of the cell cycle. Those HSCs which exhibit a low level of staining with rhodamine 123 are almost exclusively in G0/G1 (97%) whereas only 70% of HSCs which stain brightly for rhodamine 123 are in G0/G1. The injection of 100 G0/G1 HSCs rescued 90% of lethally irradiated mice in contrast to 100 S/G2/M HSCs, which protected only 25% of lethally irradiated recipients. Enhanced long-term donor-derived multilineage reconstitution of the peripheral blood was observed in recipients of 100 G0/G1 HSCs compared to recipients of 100 S/G2/M cells. These data indicate that a significant proportion of HSCs are actively proliferating during steady state hematopoiesis and that this subpopulation of cells exhibits reduced stem cell activity.

Purification and characterization of mouse hematopoietic stem cells.

Mouse bone marrow hematopoietic stem cells were isolated with the use of a variety of phenotypic markers. These cells can proliferate and differentiate with approximately unit efficiency into myelomonocytic cells, B cells, or T cells. Thirty of these cells are sufficient to save 50 percent of lethally irradiated mice, and to reconstitute all blood cell types in the survivors.

Hematopoietic stem cells: generation and self-renewal.

Adult stem cells hold great promise for future therapeutic applications. Hematopoietic stem cells (HSCs) are among the best-characterized adult stem cells. As such, these cells provide a conceptual framework for the study of adult stem cells from other organs. Here, we review the current knowledge of HSC generation during embryonic development and HSC maintenance in the bone marrow (BM) during adult life. Recent scientific progress has demonstrated that the development of HSCs involves many anatomical sites in the embryo, but the relative contribution of each of these sites to the adult HSC pool remains controversial. Specialized anatomical sites in the BM have been identified as stem cell niches, and these play essential roles in regulating the self-renewal and differentiation of HSCs through recently identified signaling pathways. Extracellular signaling from stem cell niches must integrate with the intracellular molecular machinery and/or genetic programs to regulate HSC fate choice. The exact cellular and/or molecular mechanisms defining stem cell niche and 'stemness' of HSC is largely unknown although substantial progress has been made recently. Hence, many questions remain to be answered even in this relatively well-defined model of stem cell biology.

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.

PGM-1: a transplantable murine leukemia of granulocyte-macrophage progenitor cells.

PGM-1 is a transplantable leukemia of C3H/HeJ mice growing as a population of undifferentiated blast cells with a predisposition to form subcutaneous tumors and to grow in lymphoid organs. Cell survival and proliferation in vitro are absolutely dependent on stimulation by hemopoietic growth factors, and up to 100% of tumor cells can form colonies of mature granulocytes and/or macrophages in semisolid cultures, the colonies containing no clonogenic cells. Most clonogenic cells in the leukemic population respond to stimulation by multi-colony-stimulating factor (IL-3) or GM-CSF, but some respond also to M-CSF, G-CSF, IL-4, IL-5, or IL-6. In their surface phenotype and proliferative characteristics in vitro, PGM-1 leukemic cells resemble normal granulocyte-macrophage progenitor cells, and the leukemia may be a useful model for human chronic myeloid leukemia.

Marrow engraftment of hematopoietic stem and progenitor cells is independent of Galphai-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.

A simplified method for enrichment of mouse hematopoietic stem cells.

A simplified method for enriching mouse hematopoietic stem cells using standard two-color fluorescence-activated cell sorting (FACS) has been developed. By eliminating one fluorescence parameter from a previously described four-parameter (three-color) method, FACS enrichment of mouse hematopoietic stem cells to a purity within twofold of that accomplished by the more complex method could be achieved using a single-laser, two-color FACS instrument. The method involves positive selection of mouse bone marrow cells expressing the Ly-6A.2 molecule (previously termed stem cell antigen-1, or Sca-1) and negative selection for expression of a number of cell surface markers characteristic of differentiated cells of hematolymphoid lineages (Lin-). Cell populations selected by this method contain 480 +/- 100 day-13 splenic colony-forming units (CFUs-13) per 10(4) cells, whereas the day-8 splenic colony-forming unit (CFUs-8) content is about tenfold lower. The frequency of thymic colony-forming units (CFUt) is about one in ten cells. Long-term hematopoietic repopulation of irradiated animals was observed following the transfer of 60-100 cells. However, as few as 20 cells could mediate radioprotection of lethally irradiated mice. An analysis of the cell surface phenotype of isolated Ly-6A.2+Lin- cells showed that 30%-50% expressed low levels of the Thy-1 antigen and that the CFUs-13 activity was predominantly associated with the Thy-1lo cells. The Ly-6A.2+Lin- cells expressed intermediate levels of phagocyte glycoprotein-1 (Pgp-1), low levels of heat-stable antigen (HSA), and high levels of class I major histocompatibility antigens (H2 K/D), leukocyte common antigen (Ly-5), and carbohydrate binding sites for the lectin wheat-germ agglutinin (WGA). By these criteria, Ly-6A.2+Lin- cells are phenotypically similar to mouse hematopoietic stem cells isolated by other methods.

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

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.

Regulation by the skin of lymphoid cell recirculation and localization properties.

The existence of epidermal Langerhans cells, Ia-positive dermal dendritic cells, lymphocytes which can demonstrate epidermitropism, and keratinocytes capable of secreting Interleukin-1-like molecules, each support the concept that skin can function as an immunologic organ. Such conclusions are further strengthened by the knowledge that both afferent and efferent immune responses can take place exclusively within the skin. The purpose of our study was to evaluate the ability of skin to regulate lymphoid cell recirculation and localization properties. The use of ultraviolet radiation as an exogenous stimulus resulted in a pronounced redistribution of antigen-presenting cells from central (spleen) to peripheral (skin and lymph node) lymphoid tissues as well as marked increase in the rate of lymphocyte entry into skin draining lymph nodes. This latter condition was due to elevations in the quantitative levels of high endothelial venules present within the peripheral lymph nodes. The ability of epidermal keratinocytes to express Class II molecules is known to be associated with a number of skin diseases. However, the functional significance of this phenomenon is unknown. The results of our studies, employing a nude mouse model, indicate that the expression of Class II molecules by keratinocytes facilitates the movement of Langerhans cell precursors into the epidermis and may also function to enhance lymphocyte entry into the skin. We conclude that nonlymphoid components resident within the skin can influence essential aspects of the adaptive immune response through the production of soluble factors (e.g., Interleukin-1 or through the cellular expression of Class II molecules.

Paradigm shifts in stem-cell biology.

Hematopoiesis is a physiologic process that can be transplanted by intravenous infusion of stem and progenitor cells. Because these cells contribute to blood production over a lifespan, they are attractive targets for cell-based therapies of hematologic malignancies and genetic defects. A more complete understanding of the basic biology of hematopoiesis will accelerate our progress toward the clinical goal of improved stem-cell-based therapies. Many advances in recent years have brought us closer to that goal and have, in addition, challenged a number of dogmatic notions about hematopoiesis. Three of these advances are briefly addressed here: (1) an emerging appreciation of the complex relationship between cell-cycle status, engraftment potential, and self-renewal in the hematopoietic system; (2) the demonstration of new progenitor populations and lineage relationships in early hematopoietic development; and (3) a reanalysis of the embryonic origins of hematopoiesis. These and other advances are allowing the mysteries of hematopoiesis to be unlocked at a pace that was unimaginable just a few years ago.

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.

(R)evolutionary considerations in hematopoietic development.

Evolutionary aspects of three characteristics of the mammalian hematopoietic system are considered in the context of both established and recent data. First, the lineage relationships among early members of the hematopoietic hierarchy are reconsidered in a tripartite model proposing lineage segregation based on vascular function, innate immunity, and acquired immunity on an evolutionary time scale. Second, the observation of two stem cell populations that differ in cell cycle status is considered as an evolved mechanism to enhance survival of the species in response to exposure to environmental toxins. Finally, the mobilization of hematopoietic stem cells into the peripheral circulation is proposed to be a mechanism for rapid dissemination of myeloid function during acute bacterial infections. These revolutionary hypotheses challenge some conventional concepts of stem cell biology, and provide an evolutionary context for considering mammalian hematopoiesis.

Observations of residual differentiation potential during lineage commitment.

We have recently described a subset of the multipotent progenitor pool that contains a common lymphoid progenitor. This subset of cells is lineage negative and expresses c-kit and Sca-1, but lacks expression of Thy 1.1 (Thyneg). Based on the observation that lethally irradiated mice transplanted with these cells die from anemia unless supported with competitor marrow, we hypothesized that these progenitors lacked erythroid potential. We analyzed the erythroid potential of these cells by transplanting them into mice allelic at the hemoglobin locus and compared their erythroid potential with the Thy-1.1low (Thylow) subset that contains hematopoietic stem cells. We also performed CFU-C assays in methylcellulose containing recombinant cytokines and determined erythroid contribution to colonies using in situ benzidine staining. Donor-derived hemoglobin was observed following transplant of Thyneg cells, even though 19 of 20 of these animals died from anemia. In contrast, recipients of Thylow cells showed complete donor-derived engraftment 30 days following transplant. While approximately 60% of day 4 colonies derived from Thyneg cells expressed hemoglobin, by day 11 less than 5% were hemoglobinized. In contrast, greater than 70% of the Thylow subset contained hemoglobinized cells at the end of the observation period. A similar transient appearance of myeloid progeny was also observed in colonies derived from c-kitlow Thyneg lymphoid progenitor cells. We conclude that these studies demonstrate commitment to the lymphoid lineage at the Thylow-to-Thyneg interface, and that the loss of erythroid and myeloid potential is gradual rather than abrupt. Hemoglobinized colonies may be undergoing apoptosis because of down-regulation of GATA-1 or because of a death signal from surrounding nonerythrocytic cells.

Direct effects of cyclosporin A on proliferation of hematopoietic stem and progenitor cells.

Cyclosporin A (Cy A) has been reported to both stimulate and inhibit bone marrow colony assays in a dose-dependent manner. The observation that anti-gamma-IFN antibodies stimulate hematopoiesis to the same degree as Cy A has led several groups to propose that the stimulatory effects of Cy A are due to inhibition of gamma-IFN production by T cells. In this study we observed that cultures of highly enriched hematopoietic stem/progenitor cells (HSPC), devoid of CD3/5/8+ T cells, also exhibit enhanced cloning efficiency when cultured in the presence of Cy A. Normal bone marrow cells or Thy-1.1lowSca-1+Lin(neg) HSPC were incubated in methylcellulose cultures and stimulated with various combinations of steel factor (SF), interleukin (IL)-3, IL-6, granulocyte colony stimulating factor (G-CSF), and erythropoietin (EPO) in the presence of increasing concentrations of Cy A. HSPC cultures with SF, IL-3, and IL-6 stimulation and low Cy A concentrations had from 24% to 78% higher cloning efficiencies than did parallel cultures without Cy A, and did not fall below control levels until the Cy A concentration was increased to more than 1.25 microg/ml. The addition of EPO and G-CSF abrogated the Cy A stimulation observed with SF, IL-3, and IL-6. These results were reflected in whole bone marrow, but with a higher range of variability. Cultures in which FK-506 replaced Cy A showed no consistent stimulation or inhibition of colony formation. These studies show that Cy A can stimulate hematopoietic stem cell growth independent of mediation by T cells. Consequently, these results argue for a direct positive effect of Cy A on the signal transduction pathways in HSPC.

Production and characterization of new monoclonal antibodies that distinguish subsets of mink lymphoid cells.

Several hybridoma clones that produce monoclonal antibodies (MAbs) reacting with subpopulations of mink lymphoid cells were established. Two of the MAbs, MTS-4.3 and MTS-9.3, reacted with relatively small populations of surface immunoglobulin (Ig)-negative (Ig-) lymphocytes. MTS-4.3+ and MTS-9.3+ cells were distributed in the thymic cortex and medulla, paracortical areas of lymph nodes, and periarterial lymphoid sheaths of the spleen, indicating that these MAbs identify T lymphocytes. Another MAb, MTB-5.6, reacted with a large proportion of surface Ig+ lymph node cells, but not with surface Ig- cells. In immunohistochemistry this MAb stained dendritic epithelial cells of thymic cortex, large polygonal cells of thymic medulla, a large proportion of lymphocytes in the mantle zone of lymphoid follicles, dendritic-shaped cells of paracortical area, and some lymphocytes and macrophage-like cells of medullary cords and sinuses of lymph nodes. The expression of the cell-surface antigen reacting with MTB-5.6 on Ig+ lymph node cells was increased after concanavalin A stimulation. These new reagents may be useful to analyze cellular basis of the abnormal immune responses observed in Aleutian mink disease, a classical model of human autoimmune diseases.

Using fluorescence-activated cell sorting followed by fluorescence in situ hybridization to study lineage relationships: the 8;21 translocation is present in neutrophils but not monocytes in a patient with severe congenital neutropenia and a granulocyte colony-stimulating factor-responsive clonal abnormality.

UNLABELLED: Severe congenital neutropenia (Kostmann syndrome) is a disorder that presents in the neonatal period, but predisposes to leukemia later in life. This report describes a 4-y-old female with a history of severe congenital neutropenia, who developed a clonal abnormality associated with the translocation (7;21;8) (q32;q22;q22) (AML-1/ETO). She had circulating peripheral blasts and bone marrow blast counts as high as 64% when she received recombinant granulocyte colony-stimulating factor (rG-CSF). Her marrow blasts decreased to 4-20% when rG-CSF was discontinued. Fluorescence in situ hybridization analysis was performed on bone marrow cell populations sorted by flow cytometry to determine which cell populations had the AML-1/ETO translocation. The translocation was found in mature neutrophils and blasts, but not in monocytes, lymphocytes or stem cells. CONCLUSION: These findings suggest that the translocation occurred in a neutrophil progenitor, past the point in ontogeny where monocytes and neutrophils separate. The techniques described may be useful in understanding lineage relationships and leukemogenesis in other clonal abnormalities associated with myelodysplasia and leukemia.

Biological and clinical aspects of hematopoietic stem cells.

Recent advances in cell isolation techniques have greatly enhanced our understanding of the phenotype and function of hematopoietic stem cells in mice and humans. Many clinical studies have established the efficacy of using peripheral blood stem cells to supplement or replace bone marrow transplantation as a therapeutic modality for several types of malignancies. This new approach to malignant disease management, perhaps in combination with posttransplantation cytokine therapy, promises to completely alter the clinical course of bone marrow transplantation.