In vitro stimulation of human granulopoiesis by purified protein derivative (PPD)

Summary The effect of purified protein derivative (PPD) on human granulopoiesis was studied in an in vitro semisolid culture system of human bone marrow in which PPD was incorporated into the leukocyte feeder layers. We observed that preincubation of the feeder layers with PPD was necessary to induce a significant rise of agar culture colony-forming units (CFU-c) with a maximum of 3 days' preincubation and a dose of 200 g for 10 ⁶ leukocytes. A similar effect was obtained when a conditioned medium from PPD-stimulated leukocytes was used instead of feeder layers. We have found a significant correlation between the skin test response of the leukocyte donors to PPD and the colony-stimulating activity of their leukocytes exposed to PPD: these results suggest that PPD could stimulate human granulopoiesis by an indirect effect on CSF-producing mononuclear 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

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.     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.     

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.     

Remission of acute myeloid leukemia: studies of in vitro colony formation by bone marrow cells

Human bone marrow contains cells which form leukocyte colonies in semisolid culture media. Each leukocyte colony arises from a single colony-forming cell which is thought to be a unipotential stem cell, and which is subject to regulation in vitro by colony-stimulating factor. In acute myelogenous leukemia variable abnormalities in colony formation by marrow cells occur. Usually colony formation either fails to occur or the colonies that are formed are small and contain fewer than 50 cells. Similar abnormalities have been described in bone marrow dysfunction preceding overt leukemia. Usually remission of leukemia is accompanied by improved cloning by marrow cells. In this study three patients are reported in whom remission was associated with impaired cloning, and one of these patients has remained in continuous remission for a further 18 months. These observations suggest that remission status is not necessarily associated with repopulation of the bone marrow by normal hematopoietic cells.     

A monoclonal antibody to antigens expressed on MLR-activated T cells inhibits granulocyte macrophage colony formation

A monoclonal antibody specifically reactive with MLR-activated T cells (MLR2) was added to light density normal marrow cells, depleted of adherent cells and T lymphocytes, and plated in soft agar for granulocyte macrophage colony formation. Colonies from MLR2-treated marrow cells were reduced to less than 10% of expected growth. The inhibition was not complement dependent, did not require the continuous presence of MLR2 in culture, and could not be detected also when human placenta-conditioned medium was used in the place of leukocyte feeder layers as a source of colony-stimulating factor (CSF). Co-culture experiments with MLR2 treated and untreated marrow cells further excluded the possibility of an indirect effect of MLR2 on CFU-c via auxiliary cells. The results of this study suggest that myeloid progenitor cells express a lymphoid antigen that is absent on resting or activated B cells and on resting T cells, but is expressed on activated T cells.     

Haemopoietic Inductive Capacity of Irradiated Stromal Cell Layers In Human Micro Long-Term Bone Marrow Cultures

Abstract. In a micro long-term bone marrow culture (LTBMC) system the effects of irradiation on confluent stromal cell layers were studied. In order to individually analyse the number of granulocyte-macrophage colony-forming cells (GM-CFC) per LTBMC a miniaturized human GM-CFC assay was established. the normalized GM-CFC numbers in the micro-assay compared well with data by the conventional GM-CFC assay. Pre-formed stromal cell layers were irradiated with doses up to 20 Gy and subsequently recharged with allogeneic bone marrow cells (BMC). Immediately before recharge the BMC were stromal cell-depleted by nylon wool filtration. When stromal cell-depleted BMC were inoculated on empty culture dishes, in vitro haemopoiesis rapidly declined. Sustained GM-CFC production, however, was seen when these cells were used as a second inoculum. It is concluded that irradiation doses of up to 20 Gy do not cause alteration of the haemopoietic inductive capacity of confluent stromal cell layers.     

Defective lymphopoiesis in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mutant mice. II. Description of a microenvironmental defect for the generation of terminal deoxynucleotidyltransferase-positive bone marrow cells in vitro

We have presented evidence in a previous paper that the development of prothymocytes, pre-B cells, and TdT+ lymphoid precursor cells in the bone marrow of motheaten (me/me) and viable motheaten (mev/mev) mice is defective. In the present study, we have used a selective culture system that supports the generation of rat- and mouse-origin TdT+ bone marrow lymphoid cells in vitro to further investigate the early stages of lymphopoiesis in me/me and mev/mev mice. The results demonstrate that bone marrow stromal cell feeder layers derived from me/me and mev/mev mice do not support the growth of rat TdT+ cells in vitro, whereas stromal cell feeder layers from heterozygous (+/-) littermates and wild type (+/+) control mice do. Moreover, composite feeder layers formed by mixing as few as one part me/me and mev/mev bone marrow cells with 7 to 9 parts +/- littermate bone marrow cells also fail to effectively support the generation of TdT+ cells in vitro. In contrast to me/me and mev/mev mice, other mutant mouse models of autoimmune (NZB, NZB/W), immunodeficient (nu/nu), and hemopoietic (W/Wv, Sl/Sld) disorders form feeder layers that support normal to elevated levels of TdT+ cell growth in vitro. Thus, to date, only the me/me and mev/mev mutant mice have been found to lack the appropriate microenvironment for the generation of TdT+ bone marrow cells. Histologic analysis of the stromal cell feeder layers that are formed in our culture system shows that multilayered cellular patches, which normally are the most active sites of TdT+ cell development in vitro, are absent in feeder layers of me/me and mev/mev cells. Moreover, feeder layers from mev/mev mice contain a population of MAC 1+, basophilic, nonvacuolated, macrophage-like cells; whereas feeder layers from control mice contain MAC 1+, eosinophilic, vacuolated macrophage-like cells. Stromal cell feeder layers formed by mixtures of me/me or mev/mev and control mouse bone marrow cells contain numerous multilayered cellular patches and vacuolated mononuclear cells, but also contain large numbers of basophilic mononuclear cells. These composite feeder layers have a disproportionately reduced capacity to support the generation of TdT+ cells in vitro. Although the stromal microenvironment of me/me and mev/mev bone marrow does not support the growth of TdT+ cells in vivo or in vitro, the bone marrow from these mutant mice contains detectable numbers of pre-TdT+ cells. Thus, when cultured on normal mouse feeder layers, mutant mouse bone marrow rapidly generates TdT+ cells in vitro, albeit at significantly reduced levels as compared to +/- littermate controls.(ABSTRACT TRUNCATED AT 400 WORDS)     

The non-equivalence of mouse and human marrow culture in the assay of granulopoietic stimulatory factors

Mouse bone marrow forms colonies of granulocytes and monocytic phagocytes when cultured in the presence of human plasma, urine or “feeder layers” prepared from human leukocytes. By contrast, human marrow produces colonies in the presence of leukocyte feeder layers but not in the presence of plasma or urine. It has been tacitly assumed that the response of mouse marrow to human blood leukocyte feeder layers is a measure of physiological substances released by those leukocytes which might control human granulopoiesis. This assumption however, has never been put to the test by comparing the response of mouse and human marrow to stimulation by leukocytes from the same individual. This has been done in the present study by using leukocytes from normal and leukemic subjects. Different human marrows responded similarly to stimulation by the same normal feeder layers, but there was no quantitative or qualitative correlation between the response of human and mouse marrows. Feeder layers from patients with acute granulocytic leukemia did not stimulate colony growth in normal human marrow but were as potent in stimulating mouse marrow colony growth as were feeder layers of normal leukocytes. We conclude that different factors may stimulate human and mouse marrows and that assays of granulopoietic factors of human origin should in future be carried out in human rather than mouse marrows.     

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.     

Factors influencing in vitro production of colony-stimulating factor by mononuclear leukocytes from humans.

The purpose of this study was to identify factors that influence the production of colony-stimulating factor by leukocytes of humans. The use of nonadherent light-density bone marrow cells is semisolid agar cultures to assay the concentrations of colony-stimulating factor in the supernatant of monocyte and mononuclear leukocyte cultures made it possible to distinguish between colony-stimulating factor, which stimulates colony-forming cells directly, and monocyte-dependent stimulating activity, which acts indirectly, by increasing the monocyte production of colony-stimulating factor. Colony-stimulating factor was not detectable in the cytosol of monocytes; that detected in culture must, therefore, have been newly synthesized. Synthesis was enhanced independently by heat-inactivated human serum and by semipurified serum fractions enriched with monocyte-dependent stimulating activity. The kinetics of the production of colony-stimulating factor in the presence and absence of monocyte-dependent stimulating activity indicated that the latter facilitated monocyte production of the former. Factors released from neutrophils were shown to reduce the production of colony-stimulating factor and thr proliferation of colony-forming cells and thus may provide a feedback control mechanism limiting the proliferation of neutrophils.     

Haemopoietic inductive capacity of irradiated stromal cell layers in human micro long-term bone marrow cultures

In a micro long-term bone marrow culture (LTBMC) system the effects of irradiation on confluent stromal cell layers were studied. In order to individually analyse the number of granulocyte-macrophage colony-forming cells (GM-CFC) per LTBMC a miniaturized human GM-CFC assay was established. The normalized GM-CFC numbers in the micro-assay compared well with data by the conventional GM-CFC assay. Pre-formed stromal cell layers were irradiated with doses up to 20 Gy and subsequently recharged with allogeneic bone marrow cells (BMC). Immediately before recharge the BMC were stromal cell-depleted by nylon wool filtration. When stromal cell-depleted BMC were inoculated on empty culture dishes, in vitro haemopoiesis rapidly declined. Sustained GM-CFC production, however, was seen when these cells were used as a second inoculum. It is concluded that irradiation doses of up to 20 Gy do not cause alteration of the haemopoietic inductive capacity of confluent stromal cell layers.     

Human placenta-conditioned medium for stimulation of human granulopoietic precursor cell (CFU-C) colony growth in vitro.

Human placenta-conditioned medium (HPCM) has been reported to stimulate colony formation by human granulopoietic stem cells (CFU-C) in vitro. The present work was performed to further characterize this colony formation. The majority of HPCM batches tested stimulated colony growth equivalent to recombined human leukocyte feeder layers with optimal cellular composition. A broad plateau of the dose-response curve of HPCM was found. A linear correlation exists between the number of marrow cells plated and the number of colonies grown. Optimal duration of culture is between 9 and 11 days. Colonies are large and tend to be compact. Admixture of mature granulocytes does not affect the colony growth pattern under optimal culture conditions. These data document that HPCM is a suitable source of colony-stimulating activity for the routine assay of human CFU-C. Due to the constant colony stimulation, HPCM appears particularly valuable for longitudinal studies of human CFU-C.     

Clonal analysis of progenitor cell commitment to granulocyte or macrophage production

Granulocyte-macrophage colony formation by C57BL bone marrow cells was initiated in agar cultures either by the granulocyte-macrophage stimulus, GM-CSF, or by the predominantly macrophage stimulus, M-CSF. After 24 hours, paired daughter cells of granulocyte-macrophage colony-forming cells (GM-CFC) were separated by micromanipulation and one cultured in GM-CSF, the other in M-CSF. From the differentiation pattern of the resulting colonies, irreversible commitment of some cells occurred during the first 24 hours and completion of the first cell division. A similar result was obtained using granddaughter cells present after 24 hours of incubation. However, when intact developing day 2 and days 3 clones were cross-transferred to GM-CSF or M-CSF recipient cultures, irreversible commitment was more obvious. Most M-CSF-initiated clones exhibited irreversible commitment to macrophage formation in GM-CSF cultures and a high proportion of GM-CSF-initiated clones continued to produce granulocyte progeny after transfer to M-CSF. The results indicated that GM-CSF and M-CSF can irreversibly commit the progeny of GM-CFC respectively to granulocyte or macrophage production. While for some GM-CFC this occurs within 24 hours and one cell division, for many cells, the process is slower and requires an incubation period of up to 48 hours and/or several cell divisions. Calculations from the data indicated that two-thirds of GM-CFC in adult C57BL marrow are biresponsive and respond to stimulation both by GM-CSF and M-CSF.     

Human bone marrow colony growth in agar-gel

A technique for growing human bone marrow cell colonies in agar-gel medium is described. “Feeder layers” containing 1 × 10⁶ normal human peripheral white blood cells are used as the stimulus for colony growth. Human bone marrow aspirates are collected in heparinized syringes and plated as 2 × 10⁵ cells on “feeder layers.” Normal human bone marrow yields 32–102 colonies per 2 × 10⁵ cells plated. Colonies are almost exclusively granulocytic. Growth rate of colonies is slower than with mouse bone marrow but colonies reach a comparable size (500–1500 cells) at days 12–16.     

Radiosensitivity increases with differentiation status of murine hemopoietic progenitor cells selected using enriched marrow subpopulations and recombinant growth factors

The radiosensitivity of populations of colony-forming cells (CFC) in murine bone marrow was investigated using different recombinant colony-stimulating factors (CSFs; murine IL-3 and granulocyte-macrophage CSF and human granulocyte CSF), or purified murine macrophage CSF. With unfractionated normal bone marrow the CFC increased in radiosensitivity as they progressed through the granulocyte lineage. The D0 values ranged from 129 +/- 12 cGy for CFC stimulated with GM-CSF down to 42 +/- 2 cGy after stimulation with G-CSF. IL-3 stimulated a CFC population which gave the only survival curve with a shoulder (n = 1.9 +/- 0.3). With semipurified populations of primitive or bipotential CFC, D0 values were generally lower with respect to the equivalent values for unpurified bone marrow (range 62 +/- 7 cGy to 135 +/- 7 cGy). Changes in cluster/colony ratio and colony morphology together possibly with products of accessory cells influence the interpretation of the radiosensitivity parameters.     

Chronopharmacologic Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on Hematopoiesis in Mice

Chronopharmacologic effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating white blood cell and differential counts as well as bone marrow granulocyte-macrophage colony-forming units (CFU-GM) counts were investigated in B6D2F1 mice. The animals were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD 12:12) for 3 weeks prior to study, then received a daily subcutaneous injection of rhG-CSF (400 µg/kg/day) for 4 consecutive days at 3, 9, 15 or 21 h ours a fter l ight o nset (HALO). Samples were obtained on the fifth day at the same circadian stage as that of rhG-CSF injection. rhG-CSF significantly increased the 24-h mean of leukocyte, neutrophil, lymphocyte and CFU-GM counts. Maximum increase in leukocyte and neutrophil counts was observed when rhG-CSF was administered in the middle of the dark span, while maximum stimulatory effect on circulating lymphocytes or on CFU-GM counts was obtained with rhG-CSF administration near the middle of the light span. The results indicate that choosing the dosing time of this cytokine may selectively orient its pharmacologic action. Appropriate chronomodulated delivery schemes of rhG-CSF may further reduce hematological toxicity following chemotherapy.     

Chronopharmacologic Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on Hematopoiesis in Mice

Chronopharmacologic effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating white blood cell and differential counts as well as bone marrow granulocyte-macrophage colony-forming units (CFU-GM) counts were investigated in B6D2F1 mice. The animals were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD 12:12) for 3 weeks prior to study, then received a daily subcutaneous injection of rhG-CSF (400 µg/kg/day) for 4 consecutive days at 3, 9, 15 or 21 h ours a fter l ight o nset (HALO). Samples were obtained on the fifth day at the same circadian stage as that of rhG-CSF injection. rhG-CSF significantly increased the 24-h mean of leukocyte, neutrophil, lymphocyte and CFU-GM counts. Maximum increase in leukocyte and neutrophil counts was observed when rhG-CSF was administered in the middle of the dark span, while maximum stimulatory effect on circulating lymphocytes or on CFU-GM counts was obtained with rhG-CSF administration near the middle of the light span. The results indicate that choosing the dosing time of this cytokine may selectively orient its pharmacologic action. Appropriate chronomodulated delivery schemes of rhG-CSF may further reduce hematological toxicity following chemotherapy.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.