Hematopoiesis and stem cell renewal in long-term bone marrow cultures containing catalase

Culturing mouse bone marrow in the presence of catalase dramatically alters hematopoiesis. Granulocyte output is initially increased 4- to 5-fold. This increase is transient and granulocyte production declines as immature (Sca-1+/LIN-) cells accumulate. One third of these immature cells have a phenotype (Sca-1+/c-Kit+) characteristic of hematopoietic stem cells. At 2 to 3 weeks there are greater than 200-fold more Sca-1+/c-Kit+/LIN- cells in treated cultures than in controls. This population contains functional stem cells with both short-term and long-term bone marrow repopulating activity. In addition to myeloid progenitors, this Sca-1+/LIN- population contains a large number of cells that express CD31 and CD34 and have an active Tie-2 promoter, indicating that they are in the endothelial lineage. After 3 to 4 weeks hematopoiesis in treated cultures wanes but if catalase is removed, hematopoiesis resumes. After 7 to 10 days the cultures are indistinguishable from untreated controls. Thus, protected from H2O2, hematopoietic progenitors multiply and become quiescent. This sequence resembles in vivo development in normal marrow. These results make it clear that peroxide-sensitive regulatory mechanisms play an important role in controlling hematopoiesis ex vivo and presumably in vivo as well. They also indicate that manipulation of the peroxide levels can be used to enhance the growth of hematopoietic stem cells in culture.     

Hemin stimulation of hemopoiesis in murine long-term bone marrow culture.

The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 microM, was added weekly to LTBMC and was found to produce a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 microM) were more effective for sustained cellularity in older cultures (10-12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 microM) was found to have a beneficial effect on the support of newly seeded cultures; however, the effect of lower hemin concentrations (0.1-1 microM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of granulocyte-macrophage colony-forming units (CFU-GM), and longevity of hemopoiesis in LTBMC was significantly increased with 0.1 microM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, whereas there was a less noticeable effect on the early erythroid progenitors (erythroid burst-forming units, BFU-E). Hemin, at 0.1 microM, increased spleen colony-forming units (CFU-S) to numbers several-fold higher than those of the control. Results suggest that hemin may produce mobilization of hemopoietic cells and committed precursors from adherent cells into suspension. Further, supplementation with hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.     

Characterization of engraftable hematopoietic stem cells in murine long-term bone marrow cultures

OBJECTIVE: Long-term bone marrow cultures (LTBMC) are a potential source of hematopoietic stem cells (HSC) for transplantation. Previous reports indicate that feeding LTBMCs induces hematopoietic progenitor cycling, and other studies link HSC cycle phase with engraftability. Our study was initiated to further characterize LTBMC engraftability and determine if a cycle phase-related engraftment defect affects HSC from LTBMCs. MATERIALS AND METHODS: Competitive repopulation of lethally irradiated BALB/c females was used to examine engraftability of LTBMCs under "fed" or "unfed" conditions at 3 to 5 weeks culture. Tritiated thymidine suicide was used to determine the cycle status of HPP-CFC and CFU-S from LTBMCs. RESULTS: Total cell number in LTBMCs decreases from input. Quantitatively, both fed and unfed 3-, 4-, or 5-week cultures compete strongly with fresh marrow for 2 and 8 weeks, but not 6 months, after transplantation. Short-term engraftable HSCs expand between 3 and 5 weeks of culture. Clonal assays indicate no peak in S-phase of CFU-S at 24 and 48 hours after feeding, and fluctuation in both content and cycle status of HPP-CFC after feeding. CONCLUSIONS: Our LTBMCs engraft in all conditions, and the level of engraftment capability does not correlate with cell-cycle phase of CFU-S or HPP-CFC, or with time from feeding. Although the total cell number decreases from input, the proportion of short- and intermediate-term engrafting HSC in whole LTBMCs approximates that of fresh marrow and expands from 3 to 5 weeks in culture, whereas long-term engraftable HSCs are decreased in culture.     

Stimulation of mucosal mast cell growth in normal and nude rat bone marrow cultures.

Mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) appear and proliferate to form the predominant cell type in rat bone marrow cultures stimulated with factors from antigen- or mitogen-activated lymphocytes. Conditioned media causing a selective proliferation of MMC were derived from mesenteric lymph node cells of Nippostrongylus brasiliensis-infected rats restimulated in vitro with specific antigen or from normal or infected rat mesenteric lymph node cells stimulated with concanavalin A. MMC growth factor is not produced by T-cell-depleted mesenteric lymph node cells or by the mesenteric lymph node cells of athymic rats. By contrast, MMC precursors are present in the bone marrow of athymic rats and are normally receptive to the growth factor produced by the lymphocytes of thymus-intact rats. The thymus dependence of MMC hyperplasia is thus based on the requirement of a thymus-independent precursor for a T-cell-derived growth promoter.     

IL-2 abolishes fibroblast proliferation in long-term bone marrow culture by inhibition of an accessory cell

Addition of interleukin-2 (IL-2 (greater than 250 U/ml) during the first 3 d of long-term bone marrow culture (LTBMC) permanently abolished the fibroblast component of the stromal layer, even when IL-2 was removed after the 3 d culture period. When IL-2 was added at more than 72 h after initiation of culture, no effect was observed. Stromal growth in IL-2-treated culture was restored by addition of irradiated bone marrow, indicating that the IL-2 inhibited an accessory cell rather than the fibroblast directly. Accessory cells were shown to be necessary for fibroblast proliferation at low cell densities and were also inhibited by IL-2. The accessory cell effect could not be replaced by LTBMC supernatants or extracellular matrix. It is suggested that these observations are relevant to the suppression of haemopoiesis observed in patients receiving IL-2.     

Haemopoiesis in long-term bone marrow cultures. A review.

Bone marrow-derived adherent cell layers, containing endothelial cells, fat cells and macrophages will support prolonged haemopoiesis in vitro. Evidence suggests that the adherent layer is acting as an in vitro haemopoietic inductive environment for stem cell proliferation and differentiation into the variety of committed progenitor cells of the granulocytic, erythroid, megakaryocytic and lymphoid lineages. Using this system we have analysed the factors controlling proliferation of stem cells (CFU-S), differentiation and maturation of granulocyte/macrophage precursor cells (CFU-C), leukaemic transformation by chemicals and viruses, the role of environment versus stem cells in the aetiology of haemopoietic aplasias and the possible usefulness of long-term cultures for bone marrow transplantation.     

Molecular and cell biologic aspects of erythropoiesis in long-term bone marrow cultures

In long-term marrow cultures, proliferation and differentiation of hemopoietic stem cells occurs for several months. Normally, only the most primitive erythroid progenitor cells are produced (the BFU-E). Following treatment with anemic mouse serum (AMS) or normal mouse serum plus erythropoietin, the BFU-E mature into CFU-E, which then go to produce mature nonnucleated red cells. This development is associated with the production of adult type hemoglobin. Furthermore, erythropoiesis and granulopoiesis occur in association with discrete cellular elements of the adherent cell layer in the long-term culture. Following treatment with AMS, erythropoiesis is enhanced while granulopoiesis is depressed, with no apparent competition at the stem cell or progenitor cell level.     

Indirect stimulation of hemopoiesis by hemoregulatory peptide (HP5b) dimer in murine long-term bone marrow cultures.

The hemoregulatory dipeptide (pEEDCK)2 was shown to stimulate production of a synergistic activity (SA) in 6- to 8-week-old primary stromal cell cultures. The SA increased colony formation by murine bone marrow cells (approximately 50% above control levels) in cultures stimulated by optimal concentrations of L-cell-derived macrophage colony-stimulating factor (M-CSF). An increased number of granulocyte-macrophage colony-forming cells (GM-CFC) was also observed in long-term bone marrow cell cultures following daily administration of dipeptide for 5 days. The increase in GM-CFC was approximately 90% above control as assessed by colony formation in soft agar and coincided with SA production. It appears that the dipeptide augments the production of myeloid progenitor cells through an indirect mechanism mediated by accessory cells.     

In vitro hemopoiesis in human micro long-term bone marrow cultures recharged with either allogeneic,T-cell-depleted allogeneic,or syngeneic bone marrow cells

Summary The production of granulocyte-macrophage colony-forming cells (GM-CFC) and the proliferation period in human long-term bone marrow cultures are inferior to murine cultures. There is also evidence that recharge of the cultures after establishing confluent stromal layers will not greatly improve myelopoiesis. Data in the literature indicate that PHA-responsive T lymphocytes persist for up to 5 weeks in human but not in murine long-term marrow cultures. We therefore analyzed the effects of recharging micro long-term bone marrow cultures with bone marrow cell samples depleted by T lymphocytes. Depletion was performed in a complement-mediated cytotoxicity assay by applying the monoclonal antibody CAMPATH-1. Our data show that regardless of whether T cells were removed only at recharge, at both initiation and recharge, or only at initiation, obvious enhancement could neither be achieved in the GM-CFC production nor in the proliferation period. Furthermore, no advantage was seen when using syngeneic marrow cells. We conclude that in allogeneic long-term marrow cultures hemopoiesis is not limited by immunological incompatibilities.     

Adherent cell growth from murine bone marrow in liquid cultures. Inbred strain variability and effects of mutations affecting hemopoiesis

The marrow cells of mice from seven hematologically normal strains (AKR/J, AKR/Cum, CBA/J, C3H/HeJ, C57B1/6J, DBA/2J, and RF/J) and of mice with five mutations in loci affecting hemopoiesis (W, Sl, nu, xid, NZB) were tested. The following parameters of adherent cell growth in 14 day liquid culture were analyzed: number and diameter of macroscopic adherent colonies, cellular composition with particular reference to the number of macrophage-like cells attached to a single fibroblast-like cell on peripheries of adherent colonies and the number of macrophage-like cells per eyefield of intercolony spaces. No qualitative differences were observed between tested genotypes in the cellular composition of adherent layers. These layers were uniformly made of colonies of fibroblast-like cells that were overgrown by macrophage-like cells. Macrophage-like cells grew also in intercolony spaces but this never occurred to fibroblast-like cells. At least using light microscopy and Wright's staining no morphological differences could be observed between either fibroblast-like cells or macrophage-like cells derived from marrow of various murine strains. This is of particular significance for Sl/Sld mice that suffer from the functional defect of hemopoietic microenvironment. On the other hand, large strain variability was observed in the number (5-16/10(6) marrow cells, 10.6 on the average) and diameter (1.9-3.7 mm, 2.9 mm on the average) of macroscopic adherent colonies. Among mutant genotypes, only NZB mice and animals with xid mutation formed significantly increased numbers of macroscopic colonies, while values for other mutant genotypes, Sl/Sld included, did not differ from normal littermate controls.     

Studies on the role of serum in long-term bone marrow cell cultures

The effect of fetal calf serum (FCS) concentration on hemopoiesis in long-term bone marrow cell cultures (supplemented with 10(-6) M hydrocortisone) was investigated. The duration of hemopoiesis was directly related to the concentration of serum in the cultures. At each concentration, marked differences were seen between different batches of FCS. The longevity of cultures established with a suboptimum concentration of serum was increased by the addition of HEPES buffer. Removal of all non-adherent cells and growth medium for the weekly feedings increased the overall production of hemopoietic cells when compared to the standard feeding procedure of replacing one-half of the growth medium.     

Maintenance of hemopoiesis in long-term bone marrow cultures from S1/S1d and W/Wv mice

Although phenotypically similar, the cellular defects in congenitally anemic mice of genotype W/Wv and S1/S1d are quite different. W/Wv mice have defective hemopoietic stem cells; in contrast, S1/S1d mice have normal stem cells, but their hemopoietic microenvironment cannot support normal differentiation of the stem cells. We also observed defective hemopoiesis as measured by granulocyte-macrophage colony-forming units (GM-CFU) in long-term bone marrow cultures (LTBMC) established with marrow from these mutants, but in contrast to an earlier report we obtained long-term maintenance of hemopoiesis (up to 20 weeks) albeit at a lower level than the control (28% of control for S1/S1d and 23% for W/Wv). These levels probably reflect more accurately the in vivo effects of the mutations than the severe defect reported previously. However, this level of hemopoiesis and the high variability observed in replicate flasks in LTBMC make it difficult to study these mutants in tissue culture.     

Role of stromal populations in hemopoietic stem cell proliferation. I. Physically distinct subpopulations of hemopoietic stem cells and stromal progenitors determine long-term culture hemopoiesis

The in vitro study of stem-cell-stromal-cell interactions has previously been made possible by the existence of a technique for long-term murine marrow culture, but the occurrence of both stem cells and stromal cells in fresh bone marrow (BM) explants and the heterogeneity of stromal cells have delayed functional categorization. Therefore, single-cell suspensions of murine BM were passed over nylon wool columns to allow fractionation of cells having distinctive function for in vitro hemopoiesis. A subpopulation of nylon-column-nonadherent (NNA) cells (20% +/- 1% total cells) is devoid of stromal progenitors that form the in vitro microenvironment, but the NNA subpopulation has control numbers of hemopoietic colony-forming cells, GM-CFU-C, and high-proliferative-potential colony forming cells (HPP-CFC). This subpopulation also produces control numbers of in vivo spleen colony-forming cells, CFU-S, and has control numbers of primitive, noncycling colony-forming cells that resist in vitro treatment with 5-fluorouracil (5-FU). By contrast, nylon-adherent populations, when eluted by mechanical agitation (MA) (41% +/- 1%) or by subsequent EDTA treatment (CA) (6% +/- 1%) could reform the hemopoietic microenvironment in vitro. Stromal progenitor cells were negative for surface markers Thy-1 and Mac-1. When NNA stem cells were added to culture with nylon-adherent stromal fractions, lodgement of stem cells occurred, resulting in stem cell proliferation for up to three months.     

Use of scid mice to identify and quantitate lymphoid-restricted stem cells in long-term bone marrow cultures

Mice homozygous for an autosomal recessive scid (severe combined immune deficiency) mutation on chromosome 16 exhibit a defect that specifically impairs lymphoid differentiation but not myelopoiesis. Consequently such mice are deficient in both humoral and cell-mediated immune functions. Despite their defect, scid mice survive under pathogen-free conditions and are fertile. The mutation does not impair the hematopoietic microenvironment necessary for lymphoid differentiation, since these mice can be cured with grafts of normal bone marrow (BM) or cells from long-term BM cultures (LTBMC); however, reconstitution requires sublethal (400 cGy) irradiation of recipients. Engraftment with cells from LTBMC gave near-normal levels of colony-forming B cells (CFU-B) in spleen and BM of the recipients by 6 weeks postgrafting. Since LTBMC are devoid of all mature B and pre-B cells but contain stem cells that restore lymphoid function in scid mice, we used a limiting-dilution assay to characterize and enumerate the number of stem cells in LTBMC capable of restoring lymphoid function. Curing was determined by the CFU-B-cell assay, since CFU-B are not detectable in normal scid mice. The results indicate that fewer cells from LTBMC than from fresh BM are required to obtain lymphoid reconstitution. As few as 10(3) LTBMC cells can repopulate significant B- and T-cell function in scid recipients. From these results we conclude that scid mice can be used as recipients to quantify lymphoid-restricted stem cells and that there is a functional separation of lymphoid- and myeloid-restricted stem cells in LTBMC with an enrichment for lymphoid-restricted stem cells in these cultures.     

Regulation of megakaryopoiesis in long-term murine bone marrow cultures

Megakaryocytes and their precursor cells were sustained in mouse bone marrow suspension cultures for over 4-6 wk. Megakaryocyte precursor cells were detected by their capacity to form colonies of megakaryocytes in semisolid agar cultures. Colony formation was dependent on the presence of medium conditioned by a myelomonocytic leukemic cell line (WEHI-3CM). Megakaryocytes from the liquid and semisolid cultures were identified by cytoplasmic acetylcholine esterase and by ultrastructural analysis. The suspension medium from the bone marrow liquid cultures which sustained megakaryopoiesis was not directly acitive in stimulating megakaryocyte colony formation in the semisolid agar cultures, but potentiated the number of colonies detected when WEHI-3CM was present. Bone marrow-conditioned medium increased the sensitivity of megakaryocyte progenitor cells to the stimulus in WEHI-3CM. Addition of the activities present in the two sources produced a quantitative assay for the detection of mouse megakaryocyte progenitor cells. These studies showed: (1) that no inductive regulator of in vitro clones of megakaryocytes was present in the supernatants from the long-term marrow cultures and, (2) that at least two factors were necessary for the induction of megakaryocyte progenitors to proliferate and differentiate in semisolid cultures in vitro.     

Stimulation of fetal hemoglobin synthesis in bone marrow cultures from adult individuals.

The regulation of fetal hemoglobin in adult erythroid cells was investigated with bone marrow cultures. Fetal hemoglobin (Hb F) was identified in individual erythroid colonies with fluorescent antibodies against Hb F and synthesis of gamma chains was determined with analyses of radioactive globins. The appearance of fetal hemoglobin in erythroid colonies was clonal. All the cells of the Hb F synthesizing colonies contained fetal hemoglobin. The frequency of erythroid colonies showing Hb F was higher than expected compared to the frequency of Hb F containing cells in the blood. Production of Hb F in culture, as shown by analysis of the radioactive globins, was 5 to 14 times higher than baseline Hb F synthesis. These results suggest that the ability for gamma chain synthesis in erythroid cells is determined at or above the level of the precursor cell from which the erythroid colonies, in vitro, derive (probably an erythropoietin responsive stem cell), and that stimulation of fetal hemoglobin synthesis in adult erythroid cells is possible.     

The continuum model of marrow stem cell regulation

PURPOSE OF REVIEW: We review the continuum model of stem cell regulation. A series of studies on purified lineage negative rhodamine low Hoechst low murine stem cells driven through cell cycle by cytokine exposure have shown that many phenotypic features show reversible changes with cycle progression. RECENT FINDINGS: We and others have shown that purified murine marrow stem cells are a cycling population. Features that are labile with cycle progression are in-vivo engraftment, progenitor numbers, expression of adhesion proteins and cytokine receptors, global gene expression and differentiation into granulocytes and megakaryocytes. These observations have led to a theory that regulation of hematopoietic stem cells is on a continuum and not in a hierarchy. Out-of-tissue plasticity in which marrow cells show a capacity to produce nonhematopoietic cells in non-marrow tissues also exists. We have shown 'robust' production of lung and skeletal muscle cells by marrow cells in the presence of appropriate tissue injury and demonstrated that the capacity of marrow cells to produce nonhematopoietic cells in the lung also varies reversibly with cell cycle status. SUMMARY: Thus, stem cells show a plastic plasticity and the continuum appears to hold for both nonhematopoietic and hematopoietic lineages.     

HLA-class II antigens on hemopoietic and stromal cells in human micro long-term bone marrow cultures

Using a complement-dependent cytotoxicity assay (CDC), we analyzed the presence of HLA-class II antigens on both stromal and hemopoietic cells in a miniaturized human long-term bone marrow culture system. 4-Hydroperoxycyclophosphamide (4-HC)-resistant hemopoietic stem cells capable of restoring in vitro hemopoiesis on irradiated stromal cell layers were HLA-DR, -DP, and -DQ negative. In addition, these cells failed to bind the monoclonal antibody (mAb) Tü 39, previously proposed as a candidate for the recognition of a novel class II antigen, "-DY." On the other hand, the formation of confluent stromal cell layers was inhibited by HLA-DR- or -DP-specific mAbs, but not by the HLA-DQ-specific mAb Tü 22. This suggests the presence of HLA-DR- and/or HLA-DP-positive, but HLA-DQ-negative stromal precursor cells.     

The radiation response and recovery of bone marrow stroma with particular reference to long-term bone marrow cultures

There is evidence for long-term haematopoietic dysfunction in some patients treated with radiotherapy. Although the underlying mechanisms are unclear, both stem cell and environmental defects have been implicated. In the present article we review the evidence concerning the role of stromal cells. According to the endpoints used, a wide range of radiosensitivities for the stroma have been reported. Long-term bone marrow cultures provide a system in which both functional and regenerative aspects of the stroma can be studied. A dose of 5 Gy applied prior to the establishment of long-term bone marrow cultures decreases both the formation of a confluent adherent stromal layer and its capacity to support haematopoiesis. In contrast, in its fully established phase, the adherent layer displays a high radioresistance due to the low proliferative stress applied to its stromal populations. A dose of 10 Gy given to a fully established adherent layer does not prevent haematopoietic engraftment and sustained haematopoiesis. At doses above 100 Gy a macrophage-like and epithelioid cell-type become dominant, which preserve their ability of producing growth regulatory molecules at doses as high as 500 Gy. These data suggest that the main effect on the stroma is a delayed expression of irradiation damage due to the slow rate of turnover of stromal cells. So far, there is little evidence for persistent deficiencies in the functional roles of stromal cell populations.