The effect of thrombopoietin on the proliferation and differentiation of murine hematopoietic stem cells

In this study, we explored whether thrombopoietin (Tpo) has a direct in vitro effect on the proliferation and differentiation of long-term repopulating hematopoietic stem cells (LTR-HSC). We previously reported a cell separation method that uses the fluorescence-activated cell sorter selection of low Hoescht 33342/low Rhodamine 123 (low Ho/low Rh) fluorescence cell fractions that are highly enriched for LTR-HSC and can reconstitute lethally irradiated recipients with fewer than 20 cells. Low Ho/low Rh cells clone with high proliferative potential in vitro in the presence of stem cell factor (SCF) + interleukin-3 (IL-3) + IL-6 (90% to 100% HPP-CFC). Tpo alone did not induce proliferation of these low Ho/low Rh cells. However, in combination with SCF or IL-3, Tpo had several synergistic effects on cell proliferation. When Tpo was added to single growth factors (either SCF or IL-3 or the combination of both), the time required for the first cell division of low Ho/low Rh cells was significantly shortened and their cloning efficiency increased substantially. Moreover, the subsequent clonal expansion at the early time points of culture was significantly augmented by Tpo. Low Ho/low Rh cells, when assayed in agar directly after sorting, did not form megakaryocyte colonies in any growth condition tested. Several days of culture in the presence of multiple cytokines were required to obtain colony-forming units-megakaryocyte (CFU-Mk). In contrast, more differentiated, low Ho/high Rh cells, previously shown to contain short- term repopulating hematopoietic stem cells (STR-HSC), were able to form megakaryocyte colonies in agar when cultured in Tpo alone directly after sorting. These data establish that Tpo acts directly on primitive hematopoietic stem cells selected using the Ho/Rh method, but this effect is dependent on the presence of pluripotent cytokines. These cells subsequently differentiate into CFU-Mk, which are capable of responding to Tpo alone. Together with the results of previous reports of its effects on erythroid progenitors, these results suggest that the effects of Tpo on hematopoiesis are greater than initially anticipated.     

Inducer-mediated commitment of murine erythroleukemia cells to terminal cell division: the expression of commitment.

Murine erythroleukemia cells (MELC) are transformed cells that can be induced to differentiate by a variety of agents, such as hexamethylenebisacetamide (HMBA) and dimethyl sulfoxide. Dexamethasone suppresses HMBA-mediated MELC differentiation, but MELC retain a memory for their exposure to HMBA since, on transfer from culture with HMBA and dexamethasone to medium without additions, a portion of the cells express characteristics of terminal differentiation. This study characterizes the steroid suppressed steps in the multi-step process of inducer-mediated MELC terminal differentiation. MELC in culture with HMBA and dexamethasone show low levels of commitment to terminal cell division; upon transfer to culture with inducer alone there is a rapid increase in the proportion of committed cells. The magnitude of this rapid or "step-up" expression of commitment increased with the length of prior culture with inducer and steroid. This step-up expression is not inhibited by actinomycin D or cordycepin but is blocked by cycloheximide. HMBA is required for step-up expression of commitment. In the absence of inducer, there is a rapid decay in the capacity for step-up expression. Thus, HMBA initiates a series of changes leading to the accumulation of factors--which may be mRNAs--whose expression is blocked by dexamethasone. Hemin, which induces MELC accumulation of globin mRNA but not commitment to terminal cell division, cannot, as does HMBA or dimethyl sulfoxide, cause step-up expression of commitment.     

Regulation of hematopoietic stem cells by their mature progeny

Thrombopoietin (TPO), acting through its receptor Mpl, has two major physiological roles: ensuring production of sufficient platelets via stimulation of megakaryocyte production and maintaining hematopoietic stem cell (HSC) quiescence. Mpl also controls circulating TPO concentration via receptor-mediated internalization and degradation. Here, we demonstrate that the megakaryocytosis and increased platelet mass in mice with mutations in the Myb or p300 genes causes reduced circulating TPO concentration and TPO starvation of the stem-cell compartment, which is exacerbated because these cells additionally exhibit impaired responsiveness to TPO. HSCs from Myb(Plt4/Plt4) mice show altered expression of TPO-responsive genes and, like HSCs from Tpo and Mpl mutant mice, exhibit increased cycling and a decline in the number of HSCs with age. These studies suggest that disorders of platelet number can have profound effects on the HSC compartment via effects on the feedback regulation of circulating TPO concentration.     

T-cell-replacing factor (interleukin 5) induces expression of interleukin 2 receptors on murine splenic B cells.

Small, resting B lymphocytes express few, if any, interleukin 2 (IL-2) receptors, but activated B cells may express such receptors. This paper examines the requirements for receptor expression. Normal murine splenocyte populations were enriched for B cells and cultured at relatively low density. IL-2 receptor expression was studied by measuring the binding of the anti-IL-2 receptor monoclonal antibody PC61. Lymphoblasts arising through stimulation by Escherichia coli lipopolysaccharide failed to express IL-2 receptors. B cells cultured with conditioned medium from concanavalin A-stimulated EL4 thymoma cells, with or without LPS, displayed IL-2 receptors. This bioactivity of EL4 conditioned medium could not be replaced by any concentration of B-cell-stimulatory factor 1 (IL-4), IL-1, IL-2, or IL-3 tested. However, the recently cloned lymphokine T-cell-replacing factor (IL-5) was a potent inducer of IL-2 receptor expression, as was the probably identical material known as eosinophil differentiation factor. The receptors so induced appeared to be functional, as receptor-expressing (but not control) lymphoblasts, responded to IL-2 by proliferation, indicative of high-affinity-receptor expression.     

Sustained ex vivo expansion of hematopoietic stem cells mediated by thrombopoietin

The hematopoietic stem cell (HSC) is defined as a cell that can either self-replicate or generate daughter cells that are destined to commit to mature cells of different specific lineages. Self-replication of the most primitive HSC produces daughter cells that possess a long (possibly unlimited) clonal lifespan, whereas differentiation of HSC produces daughter cells that demonstrate a progressive reduction of their clonal lifespan, a loss of multilineage potential, and lineage commitment. Previous studies indicated that the proliferation of HSC ex vivo favors differentiation at the expense of self-replication, eventually resulting in a complete loss of HSC. In contrast, transplantation studies have shown that a single HSC can repopulate the marrow of a lethally irradiated mouse, demonstrating that self-renewal of HSC occurs in vivo. Thrombopoietin (TPO) has been shown to function both as a proliferative and differentiative factor for megakaryocytes and as a survival and weakly proliferative factor for HSC. Our studies focused on the effects of exogenous TPO on HSC in mouse long-term bone marrow cultures (LTBMC). Previous results indicate that HSC decline in LTBMC in the absence of TPO. In contrast, the continuous presence of TPO resulted in the generation of both long- and short-term repopulating HSC as detected by an in vivo competitive repopulation assay. HSC were generated over a 4-month period at concentrations similar to normal bone marrow. Our results demonstrate that TPO can mediate the self-replication of HSC in LTBMC, and provide proof that HSC can self-replicate ex vivo.     

Lineage commitment of hemopoietic progenitor cells in developing blast cell colonies: influence of colony-stimulating factors.

In clonal cultures of normal mouse marrow cells, combination of granulocyte, granulocyte-macrophage, or multipotential colony-stimulating factor (G-CSF, GM-CSF, or multi-CSF, respectively) with stem cell factor (SCF) did not alter the number of blast colonies stimulated to develop compared with SCF alone but induced an up to 25-fold increase in their mean cell content and an up to 6-fold increase in their mean progenitor cell content. Costimulation of blast colony formation by SCF plus G-CSF did not change the relative frequency of progenitor cells of different types within the colonies compared with colonies stimulated by SCF alone. However, combination of GM-CSF or multi-CSF with SCF significantly increased the relative frequency of granulocytic progenitors and, for multi-CSF, also of eosinophil progenitor cells. These changes in the relative frequencies of progenitor cells committed to the various lineages support the hypothesis that hemopoietic regulators have some ability to induce selective lineage commitment in the progeny of multipotential cells.     

Osteoblasts support B-lymphocyte commitment and differentiation from hematopoietic stem cells

Early B lymphopoiesis in mammals is induced within the bone marrow (BM) microenvironment, but which cells constitute this niche is not known. Previous studies had shown that osteoblasts (OBs) support hematopoietic stem cell (HSC) proliferation and myeloid differentiation. We now find that purified primary murine OBs also support the differentiation of primitive hematopoietic stem cells through lymphoid commitment and subsequent differentiation to all stages of B-cell precursors and mature B cells. Lin(-)Sca-1(+)Rag-2(-) BM cell differentiation to B cells requires their attachment to OBs in vitro, and this developmental process is mediated via VCAM-1, SDF-1, and IL-7 signaling induced by parathyroid hormone (PTH). Addition of cytokines produced by nonosteoblastic stromal cells (c-Kit ligand, IL-6, and IL-3) shifted the cultures toward myelopoiesis. Confirming the role of OBs in B lymphopoiesis, we found that selective elimination of osteoblasts in Col2.3Delta-TK transgenic mice severely depleted pre-pro-B and pro-B cells from BM, preceding any decline in HSCs. Taken together, these results demonstrate that osteoblasts are both necessary and sufficient for murine B-cell commitment and maturation, and thereby constitute the cellular homolog of the avian bursa of Fabricius.     

Induction of murine interleukin 1: stimuli and responsive primary cells.

An interleukin 1 alpha (IL-1 alpha) cDNA probe and an IL-1 responsive T-cell clone (D10.G4; half-maximal stimulation, 0.1-1 pM) have been used to study the production of IL-1 by primary murine cell populations, particularly macrophages and dendritic cells. Spleen and peritoneal macrophages produced IL-1 mRNA and released biologically active IL-1 when challenged with lipopolysaccharide (LPS). Induction of IL-1 was evident over a dose range of 0.01-10 micrograms of LPS per ml, and maximal mRNA levels were maintained from 4 to 20 hr. Several other stimuli did not induce IL-1 in cultured macrophages, including phorbol 12-myristate 13-acetate, gamma-interferon, Con A, macrophage colony-stimulating factor, IL-3, cachectin, and activated T cells. Activated T cells could markedly reduce the response of peritoneal macrophages to LPS. When other cell types were compared with macrophages, keratinocytes had high levels of IL-1 mRNA, apparently in response to endogenous LPS. However B and T lymphocytes did not yield detectable IL-1 during proliferative responses to LPS and Con A, respectively, while dendritic cells produced little or no IL-1 when challenged with a battery of stimuli. Therefore, IL-1 may not be required for the potent accessory function of dendritic cells in lymphocyte mitogenesis. The results indicate that macrophages and dendritic cells have different secretory capacities. The macrophage is the principal leukocyte that synthesizes IL-1, and select stimuli increase and decrease the levels of macrophage IL-1 mRNA.     

Multipotential hematopoietic blast colony-forming cells exhibit delays in self-generation and lineage commitment

Murine hematopoietic blast colony-forming cells (BL-CFCs) are able to generate up to 30,000 progeny blast cells within 10 d in agar cultures. Contained in these populations are large numbers of lineage-committed progenitor cells in the granulocytic and macrophage lineages. Sequential analyses of blast colonies revealed that self-generation of BL-CFCs occurs but is surprisingly late in clonal expansion, as is the emergence of progenitor cells committed to megakaryocytic and eosinophil lineages. Self-generating BL-CFCs were highly enriched in lineage⁻ Kit⁺ Sca1⁺ CD34⁻ Flt3R⁻ populations, and colonies generated by such cells contained colony-forming units–spleen and formed erythroid and lymphoid progeny in vivo. The data suggest the existence of a hierarchical structure in BL-CFC populations with at least a subset being cells assayable as colony-forming units–spleen. Because BL-CFCs can self-generate and are able to generate lymphoid and myeloid populations, BL-CFCs appear to be ideal cells in which to analyze the processes of self-generation and lineage commitment in clonal in vitro cultures.     

Survival and retrovirus infection of murine hematopoietic stem cells in vitro: effects of 5-FU and method of infection.

Gene replacement therapy for diseases of the hematopoietic system requires efficient gene transfer to pluripotent hematopoietic stem cells. We have systematically compared a number of protocols for retrovirus-mediated gene transfer into murine repopulating hematopoietic stem cells. Recipients of infected bone marrow cells were analyzed for the presence of the transduced provirus 4 months after transplantation. Our results show that 5-fluorouracil (5-FU) pretreatment of donor animals was required for efficient gene transfer and that 5-FU-treated bone marrow retained more repopulating activity in culture than untreated bone marrow. A comparison of retrovirus-mediated gene transfer by co-cultivation of bone marrow cells with retrovirus producer cells as opposed to gene transfer by culturing bone marrow cells in retrovirus-containing supernatant revealed that gene transfer by cocultivation was more efficient than supernatant infection. However, the repopulating ability of bone marrow cells cocultured with retrovirus producer cells was reduced compared to bone marrow cells cultured in virus-containing medium.     

5-Fluorouracil effect on cultured murine stem cell progeny and peripheral leukocytes

Pretreatment of mice with 5-fluorouracil (5-FU) depletes total marrow cellularity but leaves a residual population of cells with enhanced regenerative capability. Using the long-term Dexter liquid culture system, we studied the effects of 5-FU on murine marrow cells and their production of pluripotent stem cells (CFU-S) and monocyte-granulocyte precursors (CFU-C). We also examined oxidative and bactericidal activity of neutrophil progeny of marrow cells in culture to determine the effect of 5-FU on effector cell activity. As an in vivo comparison, effector cell activity of neutrophils from peritoneal exudates of 5-FU treated animals was examined. C57B1/6J mice were treated with 5-FU, 100 mg/kg or 150 mg/kg, 4-7 days prior to marrow cell harvest and culture. Total cell counts, CFU-S, and CFU-C were all reduced compared with values from saline-treated controls. Over time, cell production from 5-FU marrow increased, reaching supranormal levels by 2-3 weeks of culture. The neutrophil progeny obtained from these marrow cultures showed normal reduction of nitroblue tetrazolium dye (NBT), but abnormally low chemiluminescence. In contrast, neutrophils from peritoneal exudate of 5-FU-treated animals showed normal chemiluminescence, but abnormally low reduction of NBT. Normal bactericidal activity was exhibited by both neutrophil progeny from marrow cultures and by neutrophils from peritoneal exudates of 5-FU-treated animals. The present data indicate that mouse marrow cells surviving 5-FU have an enhanced proliferative capacity in vitro and are capable of producing neutrophil progeny that, despite some abnormalities of oxidative function, have normal bactericidal capability.     

Role of thrombopoietin in hematopoietic stem cell and progenitor regulation

Thrombopoietin performs an essential role during hematopoiesis by regulating the expansion and maturation of megakaryocytes. In keeping with this function, megakaryocytes, platelets, and their precursors all express the thrombopoietin receptor, Mpl, on their cell surface. However, Mpl is also expressed on primitive, pluripotent hematopoietic progenitors and plays an important role in the regulation of lineages other than megakaryocytes as well as primitive progenitors. Recently, the ability of thrombopoietin to maintain and expand repopulating stem cells has been demonstrated. Thus, thrombopoietin is unique among the hematopoietic cytokines because it is necessary both for terminal maturation and regulation of lineage-specific megakaryocytes and also for maintenance of the most primitive hematopoietic stem cells. Many new strategies are evolving to exploit the activity of thrombopoietin on primitive progenitors. This may lead to faster hematopoietic recovery from marrow-suppressive therapy, effective methods of ex vivo expansion of hematopoietic stem cells, and retroviral transduction of stem cells to facilitate gene therapy.     

Proliferative capacity of murine hematopoietic stem cells.

The present study demonstrates a decrease in self-renewal capacity with serial transfer of murine hematopoietic stem cells. Production of differentiated cell progeny is maintained longer than stem cell self-renewal. In normal animals the capacity for self-renewal is not decreased with increasing donor age. The stem cell compartment in normal animals, both young and old, appears to be proliferative quiescent. After apparent recovery from the alkylating agent busulfan, the probability of stem cell self-renewal is decreased, there is a permanent defect in the capacity of the bone marrow for serial transplantation, and the stem cells are proliferatively active. These findings support a model of the hematopoietic stem cell compartment as a continuum of cells with decreasing capacities for self-renewal, increasing likelihood for differentiation, and increasing proliferative activity. Cell progress in the continuum in one direction and such progression is not reversible.     

Interferons and interleukin 6 suppress phosphorylation of the retinoblastoma protein in growth-sensitive hematopoietic cells.

One approach to identify postreceptor molecular events that transduce the negative-growth signals of inhibitory cytokines is to analyze the cytokine-induced modifications in the expression of cell-cycle-controlling genes. Here we report that suppression of phosphorylation of the retinoblastoma gene product (pRb) is a receptor-generated event triggered by interferons and interleukin 6 (IL-6) in hematopoietic cell lines. The conversion of pRb to the underphosphorylated forms occurs concomitantly with the decline in c-myc protein expression and both events precede the G0/G1-phase arrest induced by the cytokines. Loss of IL-6-induced c-myc responses in cells that have been stably transfected with constitutive versions of the c-myc gene abrogates the typical G0/G1-phase arrest but does not prevent the specific dephosphorylation of pRb. Conversely, depletion of protein kinase C from cells interferes with part of the interferon-induced suppression of pRb phosphorylation and relieves the G0/G1-phase cell-cycle block without affecting the extent of c-myc inhibition. None of the cytokines, including transforming growth factor beta, reduce the phosphorylation of pRb in S-phase-blocked cells. In contrast, the other IL-6-induced molecular responses, including the decline in c-myc mRNA levels, are not phase-specific and develop normally in S-phase-blocked cells that are depleted of the underphosphorylated functional forms of pRb. These and the suppression of pRb phosphorylation, which occur independently of each other, and suggest that the development of the interferon- or IL-6-induced G0/G1-specific arrest requires at least these two receptor-generated events.