Effect of erythropoietic stimulation of the chamber host on the growth of haemopoietic colonies in plasma clot diffusion chambers (PCDC)

Murine marrow cells were cultured in Millipore diffusion chambers implanted into the peritoneal cavity of variously conditioned murine hosts. Preirradiation (350 cGy), bleeding (0.5 ml) and phenylhydrazine injection (75 mg/kg i.v.) when performed together on the chamber host, induced better growth of erythropoietic and granulopoietic colonies inside the PCDCs than either of these manoeuvres alone. Small erythrocytic colonies (CFU-E derived) and small granulocytic colonies were observed at day 3 of marrow culture. Erythropoietic bursts and large granulocytic colonies were observed at day 8 of chamber culture. Colonies of macrophage-like cells, fibroblast-like cells, mixed erythro-granulopoietic colonies and megakaryoblasts were observed less regularly in chambers incubated in these conditions. The study provides a standardized, relatively reproducible PCDC culture system for studies of both erythro- and granulopoiesis, and does not require a hypoxic chamber.     

Diffusion chamber and spleen colony assay of murine haematopoietic stem cells

Mouse bone marrow cells have been cultured in diffusion chambers and their capacity to form spleen colonies in irradiated mice investigated after different culture periods. The number of spleen colony-forming units (CFU) in the chambers decreased during the first day of culture. The number then increased rapidly to a level significantly above the original chamber value on the third to fifth day of culture. By that time large numbers of granulocytes and macrophages had also appeared. Histological examination of spleen colonies showed that prior culturing did not alter the ratio between the different types of colonies. Cultured bone marrow cells which were transferred to new chambers retained granulopoietic capacity. This capacity increased between the first and second day of primary culturing. At this time hydroxyurea injections to chamber hosts revealed that the progenitor cells were proliferating. The results show that the granulopoietic progenitor cells of the chambers are stem cells, and that one progenitor cell type is identical with the CFU.     

Colony-forming units in diffusion chambers (CFU-d) and colony-forming units in agar culture (CFU-c) obtained from normal human bone marrow: a possible parent-progeny relationship.

An series of experiments was performed to elucidate the relationship between cells that form granulocytic colonies in fibrin clot diffusion chambers implanted into the peritoneum (i.p.) of irradiated mice (CFU-d) and day 7 and day 14 CFU-U which give rise to colonies after 7 and 14 days in agar cultures in vitro, respectively. Normal human bone marrow cells were cultured in suspension in vitro or in diffusion chambers implanted into irradiated or non-irradiated mice. During these culture conditions there was an initial decrease in the number of CFU-c per culture. This was followed by an increase between day 2 and day 7 of culture. No similar increase of neutrophilic CFU-d was observed. When CFU-d, day 14 and day 7 CFU-c in normal marrow were separated by velocity sedimentation and cultured in suspension culture or in diffusion chambers for 7 days, the maximum increase of day 7 and day 14 CFU-c was observed in slowly sedimenting cell fractions which contained the majority of CFU-d. After 3 days in suspension culture, the maximum increase of day 14 CFU-c was found in fractions which also gave rise to maximum numbers of CFU-c after 7 days. However, day 7 CFU-c were found in fractions which initially contained the majority of day 14 CFU-c. No increase in CFU-d was found in fractions initially containing peak numbers of CFU-c. Between 53 and 71% of CFU-c harvested from diffusion chambers in irradiated mice or from suspension cultures were sensitive to pulse incubation with tritiated thymidine, suggesting that the cells were proliferating during these culture conditions. In diffusion chambers implanted into non-irradiated mice, however, CFU-c were found to be relatively resistant to this treatment (3-11% sensitive to tritiated thymidine). Thus marked increases in CFU-c were also observed during experimental conditions, where no significant DNA synthesis was detected. A reproducible time sequence of increase in CFU-c populations in culture was observed. Day 14 CFU-c and cells that gave rise to clusters on day 7 in agar increased between day 2 and day 4, whereas day 7 CFU-c increased between day 4 and day 7. The results suggested that CFU-d gave rise to CFU-c in culture and that day 14 CFU-c were precursors of day 7 CFU-c.     

COLONY-FORMING UNITS IN DIFFUSION CHAMBERS (CFU-d) AND COLONY-FORMING UNITS IN AGAR CULTURE (CFU-c) OBTAINED FROM NORMAL HUMAN BONE MARROW: A POSSIBLE PARENT–PROGENY RELATIONSHIP

A series of experiments was performed to elucidate the relationship between cells that form granulocytic colonies in fibrin clot diffusion chambers implanted into the peritoneum (i.p.) of irradiated mice (CFU-d) and day 7 and day 14 CFU-c which give rise to colonies after 7 and 14 days in agar cultures in vitro, respectively. Normal human bone marrow cells were cultured in suspension in vitro or in diffusion chambers implanted into irradiated or non-irradiated mice. During these culture conditions there was an initial decrease in the number of CFU-c per culture. This was followed by an increase between day 2 and day 7 of culture. No similar increase of neutrophilic CFU-d was observed. When CFU-d, day 14 and day 7 CFU-c in normal marrow were separated by velocity sedimentation and cultured in suspension culture or in diffusion chambers for 7 days, the maximum increase of day 7 and day 14 CFU-c was observed in slowly sedimenting cell fractions which contained the majority of CFU-d. After 3 days in suspension culture, the maximum increase of day 14 CFU-c was found in fractions which also gave rise to maximum numbers of CFU-c after 7 days. However, day 7 CFU-c were found in fractions which initially contained the majority of day 14 CFU-c. No increase in CFU-d was found in fractions initially containing peak numbers of CFU-c. Between 53 and 71% of CFU-c harvested from diffusion chambers in irradiated mice or from suspension cultures were sensitive to pulse incubation with tritiated thymidine, suggesting that the cells were proliferating during these culture conditions. In diffusion chambers implanted into non-irradiated mice, however, CFU-c were found to be relatively resistant to this treatment (3–11% sensitive to tritiated thymidine). Thus marked increases in CFU-c were also observed during experimental conditions, where no significant DNA synthesis was detected. A reproducible time sequence of increase in CFU-c populations in culture was observed. Day 14 CFU-c and cells that gave rise to clusters on day 7 in agar increased between day 2 and day 4, whereas day 7 CFU-c increased between day 4 and day 7. The results suggested that CFU-d gave rise to CFU-c in culture and that day 14 CFU-c were precursors of day 7 CFU-c.     

Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

T-cell depletion of bone marrow does not inhibit in vitro hematopoiesis

One approach to overcome the problem of histoincompatibility in bone marrow transplantation is to use T cell depleted marrow from a haploidentical donor in an attempt to ameliorate graft-versus-host disease. Since the T cell requirements for normal hematopoiesis are uncertain, experiments were performed to study the effects of E rosette-T cell depletion on in vitro growth of hematopoietic progenitor cells. Marrow mononuclear cells were cultured in a modified CFU-GEMM assay before and after T cell depletion. The number of 7 day granulocytic and erythrocytic colonies, and 14 day granulocytic, erythrocytic and mixed colonies were enumerated and expressed in terms of colonies per 10(5) non T cells plated. T cell depletion did not result in decreased proliferation of any of these progenitors save possibly for 14 day granulocytic colonies in one of four experiments. In two cases, T cell depletion resulted in increased growth of progenitor cells. Three of four patients transplanted with T cell depleted haploidentical marrow cells engrafted. It is concluded that E rosette depletion of T cells from marrow does not decrease the potential of these cells to establish hematopoiesis in vitro or in vivo.     

In vivo regulation of granulocyte protluction in acute inflammation

In order to study in vivo the enhanced granulopoiesis that occurs during acute inflammation, 1-3 sterile metallic copper rods were inserted subcutaneously into mice either at the same place (one abscess) or at different sites (multiple abscesses). Diffusion chambers filled with bone marrow cells were implanted intraperitoneally for 3 days. When a single abscess was created, the granulocytic content of the diffusion chamber increased similarly whatever the number of inserted copper rods. However, there was a direct relationship between the number of abscesses and the number of granulocytic cells harvested from the diffusion chambers. In order to investigate the role of T-lymphocytes in the production of diffusible stimulating factors that act on diffusion chamber granulopoiesis, cyclosporin A (CyA) was given to the mice with implanted copper rods. CyA abrogated the induced enhancement of CFU-S, CFU-GM and mature granulocyte numbers inside the diffusion chamber. The stimulatory effect of inflammation on diffusion chamber granulopoiesis was not observed in T-lymphocyte-deficient nude mice. These data suggest that in vivo stimulation of granulopoiesis is related to the level of inflammation, and that this effect requires the functional integrity of T-lymphocytes.     

FETAL HEMOPOIESIS IN DIFFUSION CHAMBER CULTURES

The growth pattern of fetal liver (FL), normal adult bone marrow (NABM) and regenerating (post Velban treatment) adult bone marrow (RABM) colony forming units (CFU) cultured in diffusion chambers (DC) was studied. When twenty CFU were implanted into DC the recovery of CFU after 4 days with FL, NABM or RABM was 133 ± 7, 19 + 2 and 34 ± 2 CFU, respectively. The transplantation fraction of CFU from NABM decreased from 10-4% on day 0 to 6–9 % on day 4; that of FL did not change from the initial 6-2%. The growth rate of CFU derived from FL was substantially greater than that from NABM. The relative growth of FL and RABM CFU was clearly inhibited when the concentration of cells cultured was increased. Spleen colonies from FL cells before culture were larger (P < 0–005) than colonies from NABM but after 7 days of culture there was no difference between the two groups. Histological examination of spleen colonies showed that after DC culture FL and NABM CFU were differentiating along the three normal pathways. These data suggest that intrinsic differences exist between fetal and adult stem cells in the in vivo diffusion chamber culture system.     

The effects of incubation of marrow in tissue fragments in vitro on the growth of adherent cells from this marrow

Femoral marrow was either cultured as a single cell suspension immediately following collection from the donor mouse or following 4 day incubation in vitro of the whole marrow shaft. Several parameters of growth of adherent, i.e. composed of fibroblastoid cells and macrophages, colonies were determined following 14 day culture in Dulbecco medium at 37 degrees C. These included: number and diameter of macroscopic colonies, number of macrophages per fibroblastoid cell inside the colonies and per eyefield in intercolony spaces, number of cells in supernatant from the culture. The 4 day incubation of marrow fragments in vitro (Dulbecco medium, 37 degrees C) doubled the number of adherent colonies grown from this marrow and, moreover, the colonies formed were increased in size. Other parameters of cell growth in these cultures were unchanged. These data suggest that under conditions of in vitro incubation of marrow shaft (close cell-to-cell contact) marrow fibroblastoid colony forming units (MF-CFU) are stimulated to self-renewal.     

Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum

An analysis has been made of cell colonies developing in agar cultures from mouse bone marrow cells following stimulation either by neonatal kidney cell feeder layers or AKR lymphoid leukemia serum. Colonies arose by cell proliferation and were mixtures of granulocytic and mononuclear cells. Colonies stimulated by kidney feeder layers reached a mean size of 2000 cells by day 10 of incubation and remained predominantly granulocytic in nature. When bovine serum was substituted for fetal calf serum, cell colonies grew to a smaller size and lost their granulocytic nature, finally becoming almost pure populations of mononuclear cells. Colonies stimulated by AKR leukemic serum reached a mean size of 350 cells by day 10 of incubation. Although these colonies initially were granulocytic in nature, they finally became almost pure populations of mononuclear cells. The colony mononuclear cells actively phagocytosed carbon, and contained metachromatic granules probably derived from ingestion of agar. The mononuclear cells in these colonies may not have been members of the original colony, but may have been incorporated in the colony as it expanded in size, subsequently proliferating in the favourable environment of the colony.     

The influence of histamine on precursors of granulocytic leukocytes in murine bone marrow

The effect of histamine on granulocytic progenitor cells in murine bone marrow was studied in vitro. When bone marrow cells were cultured for three days with the drug, 10(-8) M to 10(-5) M of histamine stimulated differentiation and proliferation of myeloid precursor cells. Subsequently, the number of descendant cells, such as metamyelocytes and neutrophils, increased dose-dependently. Co-existence of equimolar H2 blockers such as cimetidine and ranitidine completely suppressed this effect of histamine, though this was not the case with an H1 blocker/histamine combination. Significant increase in 3H-thymidine incorporation was observed almost exclusively in myeloblasts, promyelocytes and myelocytes after exposure to histamine at concentrations higher than 10(-8) M. Also, selective incorporation of 3H-histamine into bone marrow cells was observed in myeloblasts and promyelocytes, but histamine incorporation was not influenced by the presence of either of histamine agonists or antagonists. While histamine, via H2 receptors, selectively increased the number of granulocytic colony forming units in culture (CFU-C), it had no such effect on macrophage colonies. Considering these findings, it was concluded that histamine promotes proliferation and differentiation of granulocytic myeloid cells via 1) H2 receptors in the CFU-C stage and 2) histamine receptors which are neither H1 nor H2 in the stages of myeloblast and promyelocyte differentiation.     

Microencapsulated human bone marrow cultures: a potential culture system for the clonal outgrowth of hematopoietic progenitor cells

Currently the most successful methods for culturing human hematopoietic cells employ some form of perfused bioreactor system. However, these systems do not permit the clonal outgrowth of single progenitor cells. Therefore, we have investigated the use of alginate-poly-L-lysine microencapsulation of human bone marrow, combined with rapid medium exchange, as a system that may overcome this limitation for the purpose of studying the kinetics of progenitor cell growth. We report that a 12 to 24-fold multilineage expansion of adult human bone marow cells was achieved in about 16 to 19 days with this system and that visually identifiable colonies within the capsules were responsible for the increase in cell number. The colonies that represented the majority of cell growth originated from cells that appeared to be present in a frequency of about 1 in 4000 in the encapsulated cell population. These colonies were predominantly granulocytic and contained greater than 40,000 cells each. Large erythroid colonies were also present in the capsules, and they often contained over 10,000 cells each. Time profiles of the erythroid progenitor cell density over time were obtained. Burst-forming units erythroid (BFU-E) peaked around day 5, and the number of morphologically identifiable erythroid cells (erythroblasts through reticulocytes) peaked on day 12. We also report the existence of a critical inoculum density and how growth was improved with the use of conditioned medium derived from a microcapsule culture initiated above the critical inoculum density. Taken together, these results suggest that microencapsulation of human hematopoietic cells allows for outgrowth of progenitor, and possible preprogenitor, cells and could serve as a novel culture system for monitoring the growth and differentiation kinetics of these cells.     

Separation by velocity sedimentation of human haemopoietic precursors forming colonies in vivo and in vitro cultures

Cells which give rise to granulocyte-macrophage colonies under the influence of peripheral blood white cells (CFU-c (WBC] and Mo T cell conditioned medium (CFU-c (Mo] sedimented at a faster rate than the cells which form mixed erythroid-granulocytic colonies in methylcellulose in vitro (CFU-mix) and granulocytic (CFU-dg) and megakaryocytic (CFU-dm) colonies in diffusion chambers in mice. Despite identical peak sedimentation rate for the two CFU-c populations, sedimentation profiles suggest that they are heterogeneous with respect to size. A proportion of CFU-c (Mo) may be identical with CFU-dg and CFU-mix. Sedimentation profiles for cells which give rise to mixed colonies in vitro (CFU-mix) and to granulocytic colonies in diffusion chambers in cyclophosphamide pretreated mice (CFU-dg (CY] and in Mo conditioned medium treated mice (CFU-dg (Mo] were similar. On the average CFU-dm sedimented somewhat slower than CFU-dg. These and other observations suggesting a close relationship between CFU-dg and multipotential haemopoietic precursors are discussed.     

Growth of in vitro colony-forming cells from normal human peripheral blood leukocytes cultured in diffusion chambers

The growth of granulopoietic progenitor cells (CFU-C) in diffusion chambers during culture of peripheral blood leukocytes from 10 normal subjects has been studied. At various times after initiation of diffusion chamber culture, cells harvested from the chambers were transferred to agar culture for measurement of CFU-C concentration. Under these conditions colonies could be grown successfully in agar culture provided pronase, necessary for the chamber harvesting procedure, was first removed by careful washing. A marked increase in the number of CFU-C, up to 25-fold the initial value, was observed in 8 out of 10 subjects. Here the growth pattern was similar, independent of the initial CFU-C values, with an immediate rise to a maximum between 6 and 13 days of culture followed by a decrease. In the other two subjects the growth of CFU-C throughout the diffusion chamber culture period was very poor. The growth of CFU-C from a given individual's blood was shown to be reproducible in repeated studies in 2 subjects, one of whom showed a proliferative and the other a non-proliferative pattern. Evidence suggests that the increase in CFU-C in diffusion chambers is the result of both self-renewal of these cells and influx from a more primitive compartment, although the present data do not allow an estimate of the relative magnitude of each.     

Separation By Velocity Sedimentation of Human Haemopoietic Precursors Forming Colonies in vivo and in vitro Cultures

Cells which give rise to granulocyte-macrophage colonies under the influence of peripheral blood white cells (CFU-c (WBC)) and Mo T cell conditioned medium (CFU-c (Mo)) sedimented at a faster rate than the cells which form mixed erythroid-granulocytic colonies in methylcellulose in vitro (CFU-mix) and granulocytic (CFU-dg) and megakaryocytic (CFU-dm) colonies in diffusion chambers in mice. Despite identical peak sedimentation rate for the two CFU-c populations, sedimentation profiles suggest that they are heterogeneous with respect to size. A proportion of CFU-c (Mo) may be identical with CFU-dg and CFU-mix. Sedimentation profiles for cells which give rise to mixed colonies in vitro (CFU-mix) and to granulocytic colonies in diffusion chambers in cyclophosphamide pretreated mice (CFU-dg (CY)) and in Mo conditioned medium treated mice (CFU-dg (Mo)) were similar. On the average CFU-dm sedimented somewhat slower than CFU-dg. These and other observations suggesting a close relationship between CFU-dg and multipotential haemopoietic precursors are discussed.     

THE DEVELOPMENT OF FIBROBLAST COLONIES IN MONOLAYER CULTURES OF GUINEA-PIG BONE MARROW AND SPLEEN CELLS

In monolayer cultures of guinea-pig bone marrow and spleen the development of discrete fibroblast colonies takes place on days 9–12. The linear increase in the number of colonies with increasing numbers of explanted cells and the distribution of male and female cells in mixed cultures support the view that fibroblast colonies are clones. The concentration of colony-forming cells in bone marrow and spleen is approximately 10^-5. Bone marrow culture (but not spleen culture) fibroblasts are capable of spontaneous bone formation in diffusion chambers. Fibroblasts from both bone marrow and spleen cultures are inducible to osteogenesis in diffusion chambers in the presence of transitional epithelium.     

Growth kinetics by scanning of granulocytic cell colonies in glass capillaries.

Pure granulocytic colonies were cultivated from mouse bone marrow cells in agar contained in glass capillary tubes using mouse embryo conditioned medium as colony stimulating activity. A random distribution of colonies along the agar gels was achieved under controlled conditions. Only 3 capillaries were needed for a coefficient of variation around 5% provided at least 104 cells were seeded per capillary. The daily growth of single colonies within an agar capillary was followed, using the light scattering properties of the colonies for automatic scanning. The position of the colonies in the capillary greatly affected the scan signal; the consequences of positional changes were studied in detail. Using the mean peak height as growth parameter, the onset of measureable granulocytic colony growth was found between day 2 and 3, the maximum colony size was reached between day 7 and 9, after which the colonies decayed. Other parameters such as colony count and total peak are were determined and their sinificance discussed.     

Two-step growth curve from undisturbed cultures of Tetrahymena pyriformis

Single cells from developing two day granulocytic bone marrow colonies were transfered in agar cultures. After three to five days, 48 of 239 transfered single cells had transformed to single macrophages or proliferated to form aggregates of pure macrophages or mixed macrophage-granulocyte aggregates. Some granulocytes in colonies developing in vitro from bone marrow cells appear to have the capacity to transform to macrophages.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Chicken myeloid stem cells infected by retroviruses carrying the v-fps oncogene do not require exogenous growth factors to differentiate in vitro

To determine the function of c-fps in chicken macrophages and granulocytic cells we have infected chicken bone marrow cells with retroviruses containing the v-fps oncogene. Normal chicken macrophage progenitors, M-CFCs, give rise to macrophage colonies in semisolid cultures when macrophage colony stimulating factor (M-CSF) is added into the culture medium. Upon infection with v-fps bearing retroviruses, we observed that M-CFCs were induced to develop macrophage colonies in vitro without exogenous M-CSF. This activation results from a direct effect of v-fps on the M-CFCs. No leukemic transformation was observed in the infected colonies. By comparing the effects of several retroviruses, we showed that the induction of M-CFC development is specific to v-fps containing viruses and mediated by the v-fps protein. These observations support the hypothesis that the c-fps gene is involved in the control of proliferation and/or differentiation of myeloid cells.