Human granulocyte colony stimulating factor: effects on human long-term bone marrow cultures

Human granulocyte colony stimulating factor (G-CSF) was previously shown to support the survival and proliferation of early myeloid progenitors (pre-CFU) that are capable of generating more mature CFU-GM progenitor cells. To evaluate the scope of action of G-CSF in the hierarchy of hematopoietic stem cells, we studied the effects of recombinant G-CSF (rhG-CSF) on long-term cultures of normal human bone marrow cells (LTBMC). We found that rhG-CSF predominantly influenced initial cell proliferation and expansion of CFU-GM progenitor cells in LTBMC before establishment of a confluent adherent layer. In rhG-CSF-treated LTBMC, the stromal cell layer was associated with a higher proliferative capacity and progenitor cell content as compared to control cultures. This effect was pronounced early after layer confluence and was gradually lost with culture time. rhG-CSF did not alter the duration of the productive phase of LTBMC, suggesting that it may not be active on the hematopoietic stem cells responsible for LTBMC propagation. Alternatively, stromal cells may exert tight regulatory control over progenitor cells, even in the presence of rhG-CSF.     

Promotion of hematopoietic stem cell differentiation in vitro by a soluble mediator,allogeneic effect factor

This study was designed to investigate the effects of allogeneic effect factor (AEF), a soluble mediator derived from short-term mixed lymphocyte cultures (MLC) of in vitro alloantigen-primed T cells, on cultures of murine bone marrow cells. Cultures established under suboptimal conditions namely, in the absence of a pre-established adherent cell layer as required in conventional Dextertype cultures–declined and lost their stem cell activity rapidly. In contrast, supplementation of these cultures, at initiation and thereafter, with AEF, but not with T cell growth factor (TCGF), induced cell growth and proliferation for several weeks. Such AEF-supplemented cultures exhibited cellular heterogeneity and stem cell activity for significantly longer periods than the control cultures. Even in conventional Dexter cultures, established under optimal conditions, AEF had a beneficial effect on cellular growth and proliferation and myeloid progenitor cell (CFU-C) activity. Furthermore, cells capable of synergizing with suboptimal numbers of mature T cells in con A-induced mitogenic responses, shown by others to be pre-T cells, were detected in the AEF-supplemented cultures for several weeks.     

Radiosensitivity of human bone marrow granulocyte-macrophage progenitor cells and stromal colony-forming cells: effect of dose rate

Study of the radiation biology of human bone marrow hematopoietic cells has been difficult since unseparated bone marrow cell preparations also contain other nonhematopoietic stromal cells. We tested the clonogenic survival after 0.05 or 2 Gy/min X irradiation using as target cells either fresh human bone marrow or nonadherent hematopoietic cells separated from stromal cells by the method of long-term bone marrow culture (LTBMC). Sequential nonadherent cell populations removed from LTBMC were enriched for hematopoietic progenitors forming granulocyte-macrophage colony-forming unit culture (GM-CFUc) that form colonies at Day 7, termed GM-CFUc7, or Day 14 termed GM-CFUc14. The results demonstrated no effect of dose rate on the D0 or n of fresh marrow GM-CFUc (colonies greater than or equal to 50 cells) after plating in a source of their obligatory growth factor, colony-stimulating factor (CSF) (GM-CFUc7 irradiated at 2 Gy/min, D0 = 1.02 +/- 0.05, n = 1.59 +/- 0.21; at 0.05 Gy/min, D0 = 1.07 +/- 0.03, n = 1.50 +/- 0.04; GM-CFUc14 at 2 Gy/min, D0 = 1.13 +/- 0.03, n = 1.43 +/- 0.03; at 0.05 Gy/min, D0 = 1.16 +/- 0.04, n = 1.34 +/- 0.05). There was a decrease in the radiosensitivity of GM-CFUc7 and GM-CFUc14 derived from nonadherent cells of long-term bone marrow cultures compared to fresh marrow that was observed at both dose rates. In contrast, adherent stromal cells irradiated at low compared to high dose rate showed a significantly greater radioresistance (Day 19 colonies of greater than or equal to 50 cells; at 2 Gy/min, D0 = 0.99 Gy, n = 1.03; at 0.05 Gy/min D0 = 1.46 Gy, n = 2.00). These data provide strong evidence for a difference in the radiosensitivity of human marrow hematopoietic progenitor compared to adherent stromal cells.     

IL-1 inhibits B cell differentiation in long term bone marrow cultures

There is evidence that stromal cells are responsive to changes in their external milieu and that this can affect their function. IL-1 has been identified as one mediator that can affect stromal cells by increasing their secretion of CSF. The monokine has also been reported to be a B cell differentiation factor. The purpose of this study was to test the effects of IL-1 on the pattern of hemopoietic cell differentiation by adding IL-1 alpha to myeloid long term bone marrow cultures (MBMC) at the time of their transfer to lymphoid bone marrow culture conditions. This usually results in the cessation of myelopoiesis and the induction of B lymphopoiesis. The addition of 50 U/ml of rIL-1 alpha, but not 10 U/ml, to MBMC at the time of their transfer to lymphoid conditions resulted in a complete inhibition of B cell differentiation and sustained myelopoiesis. To determine whether adherent layer cells contributed to this effect, conditioned medium (CM) was collected from adherent layers treated previously with the antibiotic mycophenolic acid. This depletes the hemopoietic cells from the cultures and retains a purified population of stromal cells. CM from mycophenolic acid- treated adherent layers exposed for 24 h to 50 U/ml of IL-1 was added at volume concentrations of 5, 10, and 25% to MBMC at the time of transfer to lymphoid bone marrow culture conditions and at each feeding thereafter. Expression of the B lineage associated 14.8 Ag and IgM was inhibited on a dose dependent basis, and myelopoiesis was sustained in cultures to which 25% CM had been added. Induction of B lymphopoiesis occurred in cultures to which adherent cell CM not exposed to IL-1 had been added. The CM from the IL-1-treated adherent cells contained CSF, because it promoted the growth of myeloid colonies from fresh marrow or MBMC cells and stimulated the granulocyte-macrophage-CSF sensitive FDC-P1 cell line to proliferate. IL-3 was not present in the CM, because stimulation of the IL-3 sensitive 32D cell line was not observed. The CM from the IL-1-treated adherent cells stimulated thymocytes to proliferate in the presence of PHA. This raised the possibility that the induced CSF may have required IL-1 to mediate their effects in the cultures. However, B lymphopoiesis was inhibited and myelopoiesis maintained upon addition of recombinant granulocyte-, macrophage-, and granulocyte-macrophage-CSF to cultures, indicating that IL-1 or other non-CSF molecules induced by it need not be present.(ABSTRACT TRUNCATED AT 400 WORDS)     

IL-3 and stromal cell-derived factor synergistically stimulate the growth of pre-B cell lines cloned from long-term lymphoid bone marrow cultures

The addition of IL-3 to modified Whitlock-Witte long-term lymphocyte cultures was found to enhance the growth of a small but significant number of B cell precursors supported by an adherent stromal cell monolayer. Several pre-B cell lines were cloned from IL-3-treated long-term lymphocyte cultures. The growth requirements and physical properties of one representative clone, BL/3, are described. BL/3 cells were shown to be unresponsive to IL-3 except when it is used at very high concentrations. In contrast, significant growth was stimulated by stromal cell conditioned medium previously shown to contain a pre-B cell growth factor. Optimal growth of the pre-B cell clone was stimulated by stromal cell conditioned medium plus IL-3. Synergy between the stromal cell-derived factor and IL-3 occurred when IL-3 was used over a wide range of concentrations including a relatively low amount that was ineffective as a growth stimulus by itself. The finding that more than one factor is required to sustain optimal growth of some pre-B cells parallels the complex growth requirements reported for some primitive myeloid/erythroid progenitors.     

Continuous activation of primitive hematopoietic cells in long-term human marrow cultures containing irradiated tumor cells.

Human hematopoietic cells can be maintained in vitro for many weeks in the absence of exogenously provided hematopoietic growth factors if an adequate stromal cell containing adherent layer is present. We have now extended the use of this type of long-term culture (LTC) system to create a model of perturbed hematopoiesis in which human tumor cells that constitutively produce a variety of factors are co-cultured together with normal human marrow cells. In the present study, we used the human bladder carcinoma cell line (5637) because these cells were known to produce not only a variety of factors active directly on hematopoietic cells but also factors that can stimulate hematopoietic growth factor production by human marrow stromal cells. Analysis of mRNA extracted from the adherent layer and measurement of growth factor bioactivity in the medium of established LTC of human marrow containing irradiated 5637 cells, showed increased levels of interleukin-1 and -6, as well as granulocyte and granulocyte-macrophage colony-stimulating factor production by comparison to control cultures. As in normal cultures, high proliferative potential clonogenic hematopoietic cells were found almost exclusively in the adherent layer of these co-cultures, but these primitive cells were maintained in a state of continuous turnover, in contrast to control cultures where the same cell types showed the expected oscillation between a quiescent and a proliferating state following each weekly change of the medium. A similar perturbation of primitive progenitor cycling was achieved by adding medium conditioned by 5637 cells twice a week to otherwise normal LTC. The presence of irradiated 5637 cells in the LTC or the addition of 5637 conditioned medium also resulted in modest (2- to 3-fold) but sustained increases in the total hematopoietic progenitor population, as well as in the final output of terminally differentiated granulocytes and macrophages. These findings indicate that primitive hematopoietic cells in LTC can be kept in a state of continuous activation for many weeks by appropriate endogenous or exogenous hematopoietic growth factor provision and that this does not necessarily lead either to their rapid exhaustion or to a large amplification in output of mature progeny.     

Ex vivo expansion of primitive hematopoietic cells for cellular therapies: An overview

Sources of hematopoietic cells for bone marrow transplantation are limited by the supply of compatible donors, the possibility of viral infection, and autologous (patient) marrow that is depleted from prior chemo- or radiotherapy or has cancerous involvement. Anex vivo system to amplify hematopoietic progenitor cells could increase the number of patients eligible for autologous transplant, allow use of cord blood hematopoietic cells to repopulate an adult, reduce the amount of bone marrow and/or mobilized peripheral blood stem and progenitor cells required for transplantation, and reduce the time to white cell and platelet engraftment. The cloning of hematopoietic growth factors and the identification of appropriate conditions has enabled the development of successfulex vivo hematopoietic cell cultures. Purification systems based on the CD34 marker (which is expressed by the most primitive hematopoietic cells) have proven an essential tool for research and clinical applications. Present methods for hematopoietic cultures (HC) on stromal (i.e. accessory cells that support hematopoiesis) layers in flasks lack a well-controlled growth environment. Several bioreactor configurations have been investigated, and a first generation of reactors and cultures has reached the clinical trial stage. Our research suggests that perfusion conditions improve substantially the performance of hematopoietic reactors. We have designed and tested a perfusion bioreactor system which is suitable for the culture of non-adherent cells (without stromal cells) and readily scaleable for clinical therapies. Eliminating the stromal layer eliminates the need for a stromal cell donor, reduces culture time, and simplifies the culture system. In addition, we have compared the expansion characteristics of both mononuclear and CD34⁺ cells, since the latter are frequently assumed to give a superior performance for likely transplantation therapies.Abbreviations BFU0-E burst forming unit-erythroid - BM bone marrow - CB cord blood - CFU-C colony forming unit-culture - CFU-E colony forming unit-erythroid - CFU-F colony forming unit-fibroblast - CFU-GEMM colony forming unit-granulocyte, erythroid, macrophage, megakaryocyte - CFU-GM colony forming unit-granulocyte, macrophage - CFU-Mix colony forming unit-mixed (also known as CFU-GEMM) - CML chronic myeloid leukemia - CSF colony stimulating factor - DMSO dimethyl sulfoxide - ECM extracellular matrix - EPO erythropoietin - FL fetal liver - HC hematopoietic culture - LTBMC long-term bone marrow culture - LTC-IC long-term culture initiating cell - LTHC long-term hematopoietic culture - MNC mononuclear cells - PB peripheral blood     

Colony-stimulating factor and the proliferation of X-irradiated myeloid stem cells

Mouse bone marrow cells irradiated in vitro with X-rays (100R or 200R) were cultured for a week in semi-solid agar containing nutrients, horse serum and various amounts of colony-stimulating factor (CSF), and the number of the colonies was counted microscopically. The result showed that the diminution of proliferative activity of myeloid stem cells (CFU-C) induced by X-rays was partly recoverable by increasing the concentration of CSF, and that the irradiated CFU-C required a higher concentration of CSF than did the control CFU-C to produce colonies in the culture. The increased CSF-requirement was not due to the increased liberation of CSF-inactivator or CSF-antagonist in the culture.     

Comparative studies of the granulopoietic enhancing effects of biosynthetic human insulin-like growth factors I and II

The effect of biosynthetic human insulin-like growth factor I (IGF-I) and IGF-II on the in vitro growth of human marrow myeloid progenitors in the presence of recombinant human granulocyte colony stimulating factor (rhG-CSF), granulocyte-macrophage CSF (rhGM-CSF), or interleukin-3 (rhIL-3), was investigated. IGF-I and IGF-II similarly enhanced the growth of myeloid progenitors in cultures stimulated with any of the above hemopoietic regulators. Analysis of colony composition showed an increase in the numbers of granulocyte colonies, but no alteration in the numbers of macrophage or granulocyte/macrophage colonies. IGF-I induced an increase of 62 ± 16%, 84 ± 13%, and 107 ± 18% in granulocyte colony numbers in the presence of G-CSF, GM-CSF, or IL-3, respectively. The values for IGF-II were 66 ± 13%, 96 ± 12%, and 91 ± 12%. Similar enhancement of myeloid colony formation by both peptides was also detected in G-CSF and GM-CSF-stimulated cultures of marrow cells that had been depleted of accessory cells, while neither peptide exerted any effect in the presence of IL-3 in such cultures. The growth-promoting effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against the IGF-I (Type I) membrane receptor. IGF-I and IGF-II thus appear to exert their effects on human marrow myeloid progenitors via a direct mechanism involving the Type I receptor. © 1993 Wiley-Liss, Inc.     

Maintenance and proliferation control of primitive hemopoietic progenitors in long-term cultures of human marrow cells

Primitive, high-proliferative potential hemopoietic progenitors can be routinely maintained for many weeks in long-term marrow cultures (LTC) in the absence of added hemopoietic growth factors. Nevertheless, these progenitors are clearly responsive to both positive and negative regulatory control mechanisms that operate within the adherent layer as evidenced by cyclic changes in their proliferative activity each time the medium is replaced. The key event appears to be the addition of a constituent of fresh horse serum that is not found in fetal calf serum. Analogous primitive neoplastic progenitor cell types from CML or PV patients are insensitive to the negative arm of this proliferation control mechanism both in vitro and in vivo. A model to explain the progenitor cell cycle changes normally observed in the LTC system is proposed. This model suggests that perturbations of nonhemopoietic mesenchymal cells determine the net positive or negative influence that these regulatory cells exert on adjacent primitive hemopoietic cells, possibly by a mechanism involving direct cell contact. Recently, we have identified a number of cytokines that can simulate the transient positive effect of fresh horse serum, as well as another cytokine, that is, tumor growth factor-beta (TGF-beta), that can mimic the negative but reversible effect exerted by mesenchymal cells. These studies demonstrating the effects of positive and negative regulatory cytokines on the control of hemopoiesis in the adherent layer of LTC suggest new approaches for analyzing the basis of both normal and abnormal stem cell regulation by marrow stromal elements.     

Persistent production of colony-stimulating factor (CSF-1) by cloned bone marrow stromal cell line D2XRII after X-irradiation

The adherent stromal layer in long-term bone marrow cultures (LTBMC) provides the cellular environment necessary for the in vitro proliferation and differentiation of pluripotential hematopoietic stem cells. The role of humoral hematopoietic growth factors, colony-stimulating factors (CSF) in the regulation of hematopoietic cell production in this system is poorly understood. We have recently isolated and cloned an adherent cell line, D2XRII, derived from murine LTBMC. Plateau phase 25 cm2 cultures of 2 X 10(6) D2XRII cells in 8.0 ml produced CSF-1 (M-CSF) at around 100-150 units/0.1 ml medium. Following X-irradiation there was a dose-dependent decrease in the production of CSF-1 to a plateau of 50% of control levels at 10,000 rad. Higher doses did not produce a further decrease. The X-ray dose reducing CSF-1 production to 50% was 100-fold above the lethal dose as measured by clonagenic survival following trypsinization and replating. Trypsinized replated viable adherent but nondividing X-irradiated D2XRII cells were maintained for up to 8 weeks after irradiation and demonstrated continuous production of CSF-1. The data indicate significant divergence of two biologic effects of X-irradiation on plateau-phase marrow stromal cells: physiologic function of adherence and CSF-1 production, versus proliferative integrity. This divergence of effects may be very relevant to understanding the mechanism of X-irradiation-associated marrow suppression and leukemogenesis.     

Direct effects of IL-4 on the in vitro differentiation and proliferation of hematopoietic progenitor cells

The purpose of this study was to analyze the effects of recombinant human interleukin 4 (IL-4) on the differentiation and proliferation in vitro of human granulocyte/macrophage (GM) and erythroid progenitors. IL-4 was added to either fetal bovine serum (FBS)-supplemented or to FBS-deprived cultures of unfractionated human marrow cells or marrow cells depleted of adherent and/or T cells. Paradoxical effects similar to those reported in the murine system were detected in these experiments. In FBS-supplemented cultures, IL-4, which had no effect on the growth or erythroid bursts (from burst-forming cells; BFU-E) detected in the presence of Epo alone, decreased by 46% the number of erythroid bursts detected in the presence of Epo and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). In contrast, in FBS-deprived cultures, IL-4 increased by 30-700% the number of erythroid bursts in cultures containing Epo alone or containing Epo, IL-3, and GM-CSF. The stimulatory effect of IL-4 on erythroid burst growth under FBS-deprived conditions was particularly evident when adherent cells were removed. Under the conditions investigated, IL-4 had little effect on the growth of GM colonies. In FBS-deprived suspension cultures of nonadherent, T-cell-depleted marrow cells, IL-4 maintained both the number of BFU-E and CFU-GM for at least 8 days. In these cultures, IL-4 antagonized the capacity of IL-3 to increase the number of BFU-E but IL-4 and IL-3 acted together to maintain the number of CFU-GM. To determine if IL-4 acted directly or indirectly, its effects on the growth of factor-dependent subclones of the murine progenitor cell line 32D were analyzed. Three subclones were studied: the original IL-3-dependent clone 32D cl.3, the Epo-dependent erythroid clone 32D Epo-1, and the G-CSF-dependent myeloid clone 32D G-1. IL-4 alone failed to induce colony growth from these cell lines. However, IL-4 inhibited by 25% the number of colonies formed by 32D cl.3 in the presence of IL-3 while increasing by 25% and 25-50% the number of colonies formed by 32D Epo-1 and 32D G-1 in the presence of Epo or G-CSF, respectively. These results indicate that human IL-4, as its murine counterpart, is a multilineage growth factor with paradoxical effects which are mediated by the direct action of IL-4 on progenitor cells.     

Modulation of normal myelopoiesis invitro by retinoic acid

The effects of retinoic acid (RA) on the proliferation and differentiation of normal myeloid progenitor cells (CFU-C) were studied. In general, RA at 10^?10 to 10^?6 M enhanced primary myeloid colony formation in the presence of colony-stimulating factor(s). However, macrophage colony formation was strongly inhibited by RA. This may be related to the finding that RA is able to differentiate bipotential HL-60 cells into granulocytes but not into macrophages. Moreover, secondary colony formation was always suppressed by the addition of RA to the primary cultures. It means that self-renewal capacity of CFU-C was suppressed by RA. This finding suggests that normal myelopoiesis will be suppressed eventually by RA.     

Divergent regulation of 1,25-dihydroxyvitamin D3 on human bone marrow osteoclastogenesis and myelopoiesis

The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has influence over osteoclastogenesis and myelopoiesis, but the regulational mechanism is not well-defined. In this report, formation of osteoclast-like (OCL) cells from primitive myeloid colony-forming cells (PM-CFC) as mediated by 1,25(OH)2D3 was examined. Our results present in this report clearly show that 1,25(OH)2D3 dose-dependently stimulated OCL cell formation when added to suspension cultures of individually replated PM-CFC colonies. Marrow cells were plated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or the human bladder carcinoma cell line 5637 conditioned medium (5637 CM) as the source of colony-stimulating activity. The 1,25(OH)2D3 effect of osteoclast differentiation was associated with a concomitant decrease in clonogenic growth of myelopoietic progenitors in response to colony-stimulating activity. Secondly, the effect of adding the known stimulator of hematopoiesis, interleukin-1beta (IL-1beta) and/or 1,25(OH)2D3 on human myeloid colony growth was assessed. IL-1beta enhanced the formation of primitive myeloid colonies in response to GM-CSF by 160%. On the other hand, 1,25(OH)2D3 dose-dependently inhibited both GM-CSF- and 5637 CM-driven myeloid colony formation by as much as 90% at 100 nM. Addition of IL-1beta to GM-CSF-stimulated cultures dampened the inhibitory effect of 1,25(OH)2D3. The inhibition of myeloid clonogenic growth by 1,25(OH)2D3 was almost abolished (89%) by simultaneously adding anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha MoAb) to the culture medium. These results collectively suggest divergent roles for 1,25(OH)2D3 in osteoclastogenesis and myelopoiesis, promoting the differentiation of OCL cells from primitive myeloid cells but inhibiting the proliferation of later myeloid progenitor cells. This inhibition of myeloid progenitors may be mediated by TNF-alpha.     

Blast colony-forming cell binding from CML bone marrow, or blood, on stromal layers pretreated with G-CSF or SCF

Blast colony-forming cells (CFU-BL) represent a specific subpopulation of special primitive progenitors characterized by colony formation only in close contact with a preformed stromal layer. CFU-BL derived from bone marrow of chronic myeloid leukaemia (CML) patients have been proved to adhere poorly to bone marrow derived stromal layers suggesting that the appearance of progenitors and precursors in the circulation is due to a defective adhesion of these cells to the bone marrow microenvironment. In the present experiments the effect of short-term incubation of preformed normal bone marrow stroma on the adherence of CML derived CFU-BL was studied. For stroma cultures bone marrow cells were cultured in microplates in the presence of hydrocortisone. Cultures were used when stromal layers became confluent and no sign of haemopoiesis could be observed. CFU-BL were studied by panning plastic non-adherent mononuclear (PNAMNC) bone marrow or blood cells. 8.9 +/- 2.4 colonies/103 PNAMNC (six experiments) were formed from normal bone marrow on stromal layers and 4.8 +/- 2.1 colonies/103 PNAMNC (five experiments) from CML bone marrow. Colony formation from normal bone marrow was not increased if stromal layers were incubated with 100 ng/mL granulocyte colony-stimulating factor (G-CSF) or stem cell factor (SCF). Incubation of stroma with G-CSF or SCF, however, increased the colony formation of PNAMNC from CML bone marrow or blood significantly. These findings suggest that local concentration of haemopoietic growth factors at the time of panning may influence the attachment of CML progenitors to the stroma.     

A fractionation procedure of mouse bone marrow cells yielding exclusively pluripotent stem cells and committed progenitors

The cell surface phenotype of pluripotent hemopoietic stem cells (CFU-S) and committed progenitors (CFU-C1, CFU-C2, BFU-E) of mouse bone marrow was analyzed with respect to their binding of wheat germ agglutinin (WGA) and two monoclonal antibodies, anti-GM-1.2 and anti-PGP-1. Stained cells were fractionated on the basis of differences in fluorescence and light scatter intensity using a light-activated cell sorter. The 6% of the cells that bound most WGA and that also had a relatively high forward light scatter (FLS) and low perpendicular light scatter (PLS) contained nearly all stem cells (CFU-S) and progenitors. Anti-GM-1.2 stained only mature myeloid cells, not CFU-S or the in vitro colony-forming cells. Anti-PGP-1 stained all bone marrow cells in varying intensities: lymphoid cells were dull, CFU-S were intermediate, CFU-C2 were brighter, and mature myeloid cells very bright. Enrichment of progenitor cells was performed by a two-step sorting procedure. First, the 6% most WGA-binding cells with high FLS and low PLS were sorted out. A 10-15-fold enrichment of progenitors and CFU-S was obtained. Next, these cells were restained with anti-GM-1.2 or anti-PGP-1 and again fractionated on the FACS. The GM-1.2-negative cells were then another four- to sevenfold more enriched for stem cells and progenitors. Of the cells in this fraction, 95% could be assigned to a colony-forming unit. With anti-PGP-1, CFU-C2 could be partly separated from more early cells such as CFU-S and BFU-E.     

A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis

The production of B lymphocytes and myeloid cells occurs in the bone marrow in association with a supporting population of stromal cells. To determine whether these processes are dependent upon the same or different populations of stromal cells, stromal cell lines were generated from the adherent layer of a Dexter type long-term bone marrow culture. These cultures support myeloid cells and their precursors, a B cell precursor, and the adherent layer cells with support B cell differentiation under appropriate conditions. Two of the lines examined, S10 and S17, express class I histocompatibility antigens but not other hemopoietic cell surface determinants such as Thy-1, Lyt-1, Ig, Ia, Mac-1, or BP-1. Both lines could support myelopoiesis under Dexter conditions upon seeding with nylon wool-passed bone marrow. The nylon wool passage depletes stromal cells capable of forming adherent layers in vitro but retains hemopoietic precursors. The number of cells and colony-forming units-granulocytes/macrophages in the nonadherent cell population recovered 3 wk post-seeding had increased 19-fold and 10-fold, respectively, in the reseeded cultures of S10 and S17. After 3 wk of growth in Dexter conditions, the reseeded cultures were transferred to conditions optimal for B cell differentiation described by Whitlock and Witte. After 4 wk of growth, hemopoietic cells were consistently recovered from S17 cultures but not those of S10. A proportion of these cells from S17 cultures expressed the 14.8 antigen and were surface IgM positive. Surviving hemopoietic cells present in cultures of S10 were primarily macrophages. These findings indicate that S17 but not S10 can support both myelopoiesis and B lymphopoiesis and suggest that one stromal cell population has the capacity to form a hemopoietic microenvironment for both lineages.     

Effect of myleran on murine hemopoiesis. I. Granulocytic cell line specificity of action on progenitor cells.

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. The severity and duration was greatest at high dose levels of MY (800 microgram). The action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. The peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Effect of Myleran On Murine Hemopoiesis

Time- and dose-dependent patterns of depletion and regeneration of hemopoietic progenitor cells in mouse femora and spleens following treatment with the antileukemic agent Myleran (Busulphan, MY) were studied using the murine spleen colony system and the agar gel in vitro colony system. MY was found to depress granulopoiesis selectively, as manifested by the development of marked prolonged neutropenia, hypoplasia of the bone marrow and (to a lesser degree) of the spleen, reduction of the incidence of multipotential hemopoietic progenitor cells (CFU-S) and of granulocytic progenitor cells (CFU-C) in both femora and spleens, and impairment of the capacity of CFU-S from either tissue to generate granulocytic colonies in the spleens of irradiated hosts. the severity and duration was greatest at high dose levels of MY (800 μ). the action of MY on CFU-S was more pronounced than that on CFU-C, suggesting that MY is a cycle-independent agent. Repopulation of the CFU-C pool preceded that of the CFU-S pool. Development of neutropenia and maximal marrow hypoplasia followed the onset of depression of CFU-S and CFU-C incidence, while recovery of normal nucleated cellularity in the blood, femur and spleen preceded repopulation of the CFU-S and CFU-C pools. MY treatment resulted in transitory stimulation of colony stimulating factor (CSF) generation by the femur but had no effect on serum CSF levels. the peak of femoral CSF generation coincided with the nadir of CFU-C depression. These findings indicated that the prolonged neutropenia following MY treatment was secondary to depletion of the progenitor cell pools, that during recovery granulopoietic repopulation took precedence over self-maintenance of the hemopoietic progenitor cell pools, and that increased generation of CSF may play a role in the early phase of granulopoietic recovery.     

Differential effects of drugs upon hematopoiesis can be assessed in long-term bone marrow cultures established on nylon screens.

Hematotoxicity is associated with exposure to chemotherapeutic drugs and numerous other agents. Most measurements of the hematopoietic effects of prospective therapeutic drugs and environmental agents have been made in animal models. We tested the influence of various drugs on hematopoiesis in long-term cultures of Long-Evans rat bone marrow cells. These cultures were established on nylon screen-bone marrow stromal cell templates that were suspended in liquid medium. Previous phenotypic analyses of adherent zone cells of suspended nylon screen bone marrow cultures (NSBMC) using monoclonal antibodies and flow cytometry indicated that they maintain a multilineage character for extended periods in culture and display continuous proliferation of hematopoietic progenitors (colony-forming unit culture [CFU-C]). NSBMC of various ages were incubated for 21 hr with several concentrations of beta-D-cytosine arabinofuranoside, 5-fluorouracil, cyclophosphamide, or methotrexate. Adherent zone cells were dissociated enzymatically, phenotyped by flow cytometry, and assayed for colony-forming unit culture content. beta-D-cytosine arabinofuranoside, 5-fluorouracil, and methotrexate treatment of bone marrow cultures resulted in a dose-related diminution in colony-forming unit culture numbers in the adherent zones of NSBMC. Phenotypic analyses revealed similar trends but certain of these drugs manifested lineage specificities. Toxicity was also related to cyclophosphamide dose, but the presence of bone marrow stroma was necessary to demonstrate this effect in vitro. A subpopulation of these cells was found to metabolize ethoxyfluorescein ethyl ester to fluorescein after induction with 2,3,7,8-tetrachlorodibenzo-p-dioxin, an effect which was quantified by flow cytometry. NSBMC may be used to ascertain lineage-specific toxicities and evaluate the effects of drugs on the proliferation of hematopoietic progenitor cells.