Long-term culture of aplastic anaemia bone marrow

Long-term bone marrow cultures (LTBMC) were established with marrow from 11 patients with aplastic anaemia (AA). Bone marrow from five patients, with low numbers of committed progenitor cells, exhibited an increase in committed progenitor cell production to normal levels in the first week of LTBMC. None of 44 haematologically normal marrow cultures showed this increase. Mature and committed progenitor cell production in all cultures from aplastic anaemia bone marrow, declined faster than in normal cultures. This study indicates that short-term culture for committed progenitor cells is an underestimate of the proliferative capacity of bone marrow from some patients with AA. LTBMC may provide a useful system for further studies into the mechanisms responsible for this increased growth in some patients with AA.     

Enhanced myelopoiesis in long-term cultures of human bone marrow pretreated with recombinant granulocyte-macrophage colony-stimulating factor

Long-term bone marrow culture (LTBMC) for human hemopoiesis supports continuous proliferation and differentiation within the myeloid progenitor population by the formation of an adherent stromal monolayer. LTBMC represents the most suitable in vitro model for the study of regulatory mechanisms in human hemopoiesis. We investigated the effect of recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF) on bone marrow of normal donors in LTBMC. The cells (2 x 10(6)/ml) were incubated with 100 ng/ml rhuGM-CSF for 24 h in culture medium supplemented with 10% fetal calf serum. After the preincubation, LTBMCs were started and maintained over a period of 10 weeks. After 1 week in culture we observed a statistically significant difference with a 1.5-fold higher number of nonadherent cells in the LTBMCs containing the bone marrow preincubated with rhuGM-CSF (p less than 0.05). This increase was due to an expansion of the mature myeloid cells. At the same time point the number of GM colony-forming units (CFU-GM)/ml in the LTBMCs with rhuGM-CSF-preincubated bone marrow was slightly increased compared to the controls without reaching a statistically significant level. We conclude that rhuGM-CSF at a saturation dose is a potent stimulator of in vitro myelopoiesis stem cell pool. This in vitro result is of relevance for the clinical use of rhuGM-CSF in patients undergoing bone marrow transplantation. The incubation of donor bone marrow prior to transplantation might be a new approach to facilitate the engraftment and to shorten the phase of pancytopenia.     

Functional studies of bone marrow haemopoietic and stromal cells in the myelodysplastic syndrome (MDS)

Long-term bone marrow culture (LTBMC) was used to investigate the proliferative behaviour of marrow cells from a spectrum of cases of the myelodysplastic syndrome (MDS), and the results compared with those obtained in the conventional short-term clonal assay. Two broad patterns of growth were revealed in LTBMC. In one group the incidence of haemopoietic progenitor cells steadily declined to abnormally low levels at 4 weeks, while in a second group they were maintained near normal levels for periods of up to 7 weeks. These growth patterns, which were not predictable from clonogenic assays on the marrow cells prior to LTBMC, or from the morphology of the bone marrow, may reflect the stage of evolution of the disease. Further studies of clonality are required to establish whether or not patients exhibiting the second pattern have a potentiality to harbour residual normal haemopoiesis. LTBMC was also used to study the function of MDS marrow stroma in terms of its ability to sustain the growth of normal haemopoietic progenitor cells. Although the phenotype of the cultured adherent cell layer, obtained from some patients, was atypical, no consistent functional defect of MDS stroma could be identified by studying the level of haemopoiesis reached by normal cells seeded into MDS stroma.     

Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01)

A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.     

Characteristics of megakaryocyte colony formation in normal individuals and in primary thrombocythemia: studies using an optimal cloning system

Colony formation by megakaryocyte (MK) progenitors was studied in 36 normal individuals and in 26 patients with primary thrombocythemia (PT) using an improved plasma clot cloning system. MK colonies were identified by immunoperoxidase staining using a monoclonal antibody against human platelet glycoprotein IIb/IIIa complex. In normal individuals, a frequency of 194 +/- 23 MK colony-forming units (CFU-MK) per 5 X 10(5) bone marrow nonadherent mononuclear cells and 11 +/- 4 CFU-MK per 5 X 10(5) blood mononuclear cells was found. These CFU-MK grew as large (greater than 20 cells), mean (11-20 cells), small (3-10 cells), or mix-MK colonies (at least two MK) that comprised 15%, 23%, 62%, or 5% of all MK colonies in bone marrow and 0%, 8%, 92%, or 1% in peripheral blood, respectively. In PT, several abnormalities of MK colony formation were observed: 1) increased circulating CFU-MK numbers, 2) increased mix-MK colony formation, 3) spontaneous MK colony formation without phytohemagglutinin-stimulated leukocyte-conditioned medium and normal serum, 4) decreased proportion of larger MK colonies (greater than 11 cells) in PT bone marrow, and 5) failure of PT plasma or serum to stimulate MK colony formation by normal marrow cells in a normal fashion. These results indicate some of the characteristics of quantitative and qualitative abnormalities of in vitro megakaryocytopoiesis in PT.     

Role of different medium and growth factors on placental blood stem cell expansion: an in vitro and in vivo study

Expansion of haemopoietic stem cells from placental blood has been obtained with a combination of flt3 ligand (FL), thrombopoietin (TPO), kit-ligand (KL) with or without interleukin-6 (IL6) in serum-replete medium. For clinical use, cell expansion in the absence of serum is a clear advantage. Therefore, stem cell expansion in serum-free (SF) medium with a combination of three (FL, TPO, KL) or four (FL, TPO, KL, IL6) growth factors was compared with the results obtained using fetal calf serum (FCS) or human serum (HS). Human CD34(+) placental blood cells were cultured in the presence of FL, TPO, KL +/- IL6 with SF medium, HS and FCS for up to 8 weeks. CD34(+), CFC, LTC-IC content was measured at intervals. To determine the in vivo repopulating capacity of expanded cells, CD34(+) expanded cells were transplanted in sublethally irradiated NOD/SCID mice. With the three growth factor combination the CD34(+) cell number increased steadily up to the 8 weeks of culture. CD34(+) cells were expanded 67.5-fold with SF, 11.7 with HS and 49.2 with FCS. However, when CFCs and LTC-ICs were considered, a continuous expansion was observed only with HS and FCS, whereas in SF medium after 6 weeks their number started to decline. The addition of IL-6 did not change the expansion significantly. Cells grown ex vivo for 14 days were transplanted into NOD/SCID mice. The engraftment of human cells in mice was higher for serum-replete than for SF expanded cells. Nevertheless, SF cultured cells were also able to engraft both marrow and spleen in all animals. In addition, engrafted human cells still maintained clonogenic ability. With KL, FL, TPO +/- IL6 it is possible to expand haemopoietic progenitor cells in a SF medium. Compared with serum-replete cultures, the absolute number of clonogenic cells and in vivo repopulating cells is lower. Although the degree of expansion remains significant, a clinical trial still needs to be carried out to address the question of whether this expansion might be useful in reducing post-transplant aplasia.     

Parvovirus-induced aplastic crisis in a child with hereditary spherocytosis: studies on the mechanisms of hemopoietic suppression

A 4 4/12-year-old girl with hereditary spherocytosis (HS) who presented with an aplastic crisis during a human parvovirus (HPV) B19 infection is reported. IgM and IgG antibodies to the HPVB19 and HPV19 DNA were detected. Each of Leu 7+, Leu 11+ and HLA-DR+ cells increased. OKT4/OKT8 ratio decreased to 0.71. In order to investigate the mechanism of aplastic crisis, we used an in vitro culture technique for erythroid and granulocyte-macrophage progenitor cells (BFU-E, CFU-E and CFU-C). The patient's HPV19 DNA-containing aplastic phase serum inhibited the formation of BFU-E, CFU-E and CFU-C. After removal of the adherent cells from the aplastic phase bone marrow, the numbers of BFU-E significantly increased. These results suggest that aplastic crisis of the patient with HS was caused by HPVB19, and that monocytes-macrophages and NK cells played an important role in the pathogenesis of aplastic crisis.     

Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro.

An in vitro liquid suspension culture system was used to determine the role of cytokines in sustaining long-term human megakaryocytopoiesis. Bone marrow cells expressing CD34 but not HLA-DR (CD34+DR-) were used as the inoculum of cells to initiate long-term bone marrow cultures (LTBMC). CD34+DR- cells (5 x 10(3)/mL) initially contained 0.0 +/- 0.0 assayable colony-forming unit-megakaryocytes (CFU-MK), 6.2 +/- 0.4 assayable burst-forming unit-megakaryocytes (BFU-MK), and 0.0 +/- 0.0 megakaryocytes (MK). LTBMCs were recharged every 48 hours with granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-1 alpha (IL-1 alpha), IL-3, and/or IL-6, alone or in combination. LTBMCs were demidepopulated weekly or biweekly, the number of cells and MK enumerated, and then assayed for CFU-MK and BFU-MK. LTBMCs receiving no cytokine(s) contained no assayable CFU-MK or BFU-MK and no observable MK. LTBMCs receiving GM-CSF, IL-1 alpha, and/or IL-3 contained assayable CFU-MK and MK but no BFU-MK for 10 weeks of culture. The effects of GM-CSF and IL-3, IL-1 alpha and IL-3, but not GM-CSF and IL-1 alpha were additive with regards to their ability to augment the numbers of assayable CFU-MK during LTBMC. LTBMCs supplemented with IL-6 contained modest numbers of assayable CFU-MK for only 4 weeks; this effect was not additive to that of GM-CSF, IL-1 alpha, or IL-3. The addition of GM-CSF, IL-1 alpha, and IL-3 alone or in combination each led to the appearance of significant numbers of MKs during LTBMC. By contrast, IL-6 supplemented cultures contained relatively few MK. These studies suggest that CD34+DR- cells are capable of initiating long-term megakaryocytopoiesis in vitro and that a hierarchy of cytokines exists capable of sustaining this process.     

Proliferation of myeloid progenitor cells in human long-term bone marrow cultures is stimulated by interleukin-1 beta

To study the effect of interleukin-1 (IL-1) beta on the proliferation of hematopoietic progenitor cells (HPC) in long-term bone marrow cultures (LTBMC), stromal cell layers were established from normal human bone marrow. Autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells were reinoculated on the stromal layers in fresh culture medium, with or without the addition of human IL-1 beta (30 U/mL). Once a week, half of the culture supernatant was replaced with fresh culture medium with or without IL-1, and all nonadherent cells were returned to the flasks. At weekly intervals during a period of 5 weeks, one culture was sacrificed to determine the total number of cells and hematopoietic progenitor cells, present in the adherent and the nonadherent cell fractions. In IL-1-stimulated cultures, the number of cells recovered during a period of 5 weeks exceeded the number of cells in unstimulated control cultures by 1.5 times. This difference was attributed to a twofold increase in the number of adherent cells. The number of HPC recovered from IL-1- stimulated cultures was not different from that recovered from controls. The levels of colony-stimulating activity (CSA) in supernatants from IL-1-stimulated cultures were significantly higher than those in supernatants from control cultures. These results indicate that IL-1 enhances the recovery of cells in LTBMC by stimulating the proliferation of HPC with the concurrent release of CSA from stromal cells, without diminishing the number of HPC.     

In vitro effects of hematopoietic growth factors on the proliferation, endoreplication, and maturation of human megakaryocytes

A liquid culture technique was used to study regulation of human megakaryocytopoiesis in vitro. Low-density cells from adult bone marrow were cultured in the presence of normal plasma, plasma from patients with aplastic marrows (AP), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and interleukin-3 (IL-3). Megakaryocytes (MK) were studied at day 10 of culture by a two-color staining technique using a pool of monoclonal antibodies for their identification and propidium iodide to label DNA. Their ploidy distribution was analyzed by flow cytometry. In some experiments cytoplasmic maturation was also studied by ultrastructural techniques. Normal plasma provides a low number of MK with a ploidy distribution including 8 N and 16 N MK. AP promoted in a dose-dependent manner proliferation of MK and some batches favored endoreplication. This effect was clearly demonstrated when ploidy distribution was compared between normal plasma and AP on parallel marrow cultures. However, ploidy distribution was shifted toward low values compared with uncultured MK. rhGM-CSF had no significant effect on these two parameters. In contrast, rhIL-3 from 0.1 U/mL to 100 U/mL had a proliferative effect but was unable to induce endoreplication. Furthermore, when associated with AP it totally abrogated the effect of AP on endoreplication because in most experiments more than 90% of MK were 2 N and 4 N. This effect was also observed when rhIL-3 was added after 7 days of culture (when it has little proliferative effects). Studies of the maturation of MK grown with rhIL-3 indicate that the majority were small mature cells synthesizing alpha-granules and demarcation membranes. The effect of AP on MK proliferation and endoreplication was not related to IL-6 because its IL-6 content was identical to that of normal plasma and its neutralization did not modify these parameters. In conclusion, this study indicates that liquid culture technique in association with flow cytometry could be a powerful tool in identifying the humoral regulators of human megakaryocytopoiesis.     

Differential effects of recombinant human interleukin-13 on the in vitro growth of human haemopoietic progenitor cells

Summary. Effects of recombinant human interleukin (IL)-13 on in vitro haemopoiesis from non-adherent mononuclear cells (NAMC) or highly enriched CD34⁺ cells of human cord blood (CB) were studied. IL-13 significantly increased megakaryocyte (MK) colony formation from either NAMC or CD34⁺ cells cultured in a plasma clot system supplemented with aplastic anaemia serum (AAS) and phytohaemag-glutinin-stimulated human peripheral blood leucocyte-conditioned medium (PHA-LCM) in a dose-dependent manner. Experiments using a modified plasma clot culture, in which normal AB serum and various cytokines were added to replace AAS and PHA-LCM, demonstrated an increased MK colony number in the presence of IL-13, especially in combination with IL-3. However, IL-13 had no stimulatory effect, but rather a slight inhibitory effect in some cases on granulocyte-macrophage (GM) colony formation in both plasma clot cultures. Furthermore, the growth of GM progenitor cells in a methylcellulose culture system in the presence of IL-3, GM-CSF, Epo, G-CSF or in combination was significantly inhibited by the addition of IL-13. On the other hand, high concentrations (lOOng/ml) of IL-13 were needed to cause a slight inhibition on the growth of BFU-E-derived colonies under the same methylcellulose culture. These results indicate that IL-13, alone and synergistically with the effect of IL-3, promotes MK colony formation, but it inhibits the growth of GM and erythroid progenitor cells in vitro.     

Spontaneous formation of megakaryocyte progenitors (CFU-MK) in primary thrombocythaemia

An improved plasma clot culture method for CFU megakaryocytes (MK) has been developed with a higher plating efficiency and easier identification and enumeration of MK colonies by an indirect immunoperoxidase staining using a monoclonal antibody specific to the platelet glycoprotein IIb/IIIa complex. This technique has been used to study megakaryocytopoiesis in 20 normal individuals, 4 recently diagnosed patients with untreated primary thrombocythaemia (PT) and 2 patients with secondary thrombocytosis (ST). An increased number of MK colonies was evident in peripheral blood (mean 115 +/- 31 CFU-MK/5 X 10(5) seeded cells or 206 +/- 91/ml) and to a lesser extent in bone marrow (mean 188 +/- 26 CFU-MK/5 X 10(5) seeded cells or 696 +/- 103/ml) of PT patients as compared to controls (mean 11 +/- 4/5 X 10(5) seeded cells or 13 +/- 3/ml of peripheral blood and 153 +/- 15/5 X 10(5) seeded cells or 319 +/- 43/ml of bone marrow). There was a very obvious difference between PT patients and the others (controls plus ST patients) because CFU-MK growth with no added stimulus (PHA-LCM or PHA-LCM and normal serum) could be seen in PT patients only.     

Two antigenically different types of colony-stimulating activities in sera of patients with aplastic anemia

Sera obtained from seven normal volunteers and 11 patients with aplastic anemia were assayed for two types of colony-stimulating activity (CSA) using monolayer agar cultures containing human unfractionated and monocyte-depleted bone marrow cells. Sera obtained from normal volunteers had low CSA for unfractionated bone marrow cells and no CSA for monocyte-depleted bone marrow cells. On the other hand, sera obtained from patients with aplastic anemia (patients' sera) had moderate CSA for both unfractionated and monocyte-depleted bone marrow cells. The patients' serum CSA titer for unfractionated bone marrow cells rose markedly with the addition of a small amount of diluted control rabbit serum (control serum) into the CSA assay system, while it did not rise with addition of the same amount of diluted rabbit antiserum against partially purified human urinary colony-stimulating factor (CSF) (antiserum). The patients' serum CSA titer for monocyte-depleted human bone marrow cells rose with the addition of control serum as well as antiserum. These findings indicate that the addition of rabbit serum enhances colony formation by human bone marrow cells in the presence of aplastic anemia sera as a source of CSA, and that sera of patients with aplastic anemia contain two antigenically different types of CSA: one that is active on human unfractionated bone marrow cells and partially neutralized by the addition of antiserum, and another that is active on human monocyte-depleted bone marrow cells and is not neutralized by the addition of antiserum.     

Interleukin-6 and its receptor are expressed by human megakaryocytes: in vitro effects on proliferation and endoreplication

Interleukin-6 (IL-6) is a pleiotropic cytokine that plays an important role in the megakaryocytic differentiation. Recently, we have observed that IL-6 is synthesized by several human cell lines with megakaryocytic features. In this study, we have investigated whether a similar phenomenon occurs during normal megakaryocytic differentiation. Human megakaryocytes (MK) were obtained by culturing normal marrow in liquid culture with aplastic plasma (AP). First, an IL-6 secretion in bone marrow culture enriched in MK as well as in purified MK populations was demonstrated by a biologic assay. Second, IL-6 mRNA was detected in a purified population of MK by the polymerase chain reaction and dot blot analysis. IL-6 mRNA and protein were undetectable in platelets. Third, in situ hybridization procedure demonstrated the presence of IL-6 mRNA in individual immature MK. Fourth, IL-6 protein was detected in MK at the unicellular level by an immunoalkaline phosphatase technique using a monoclonal antibody against IL-6. Furthermore, the presence of IL-6 receptor (IL-6-R) on MK was demonstrated by in situ hybridization using an IL-6-R probe and in situ autoradiography after binding with [125I]-labeled recombinant IL-6. The IL-6 endogenously produced in liquid cultures containing normal human plasma or AP was subsequently neutralized. This resulted in a 50% decrease of the MK growth with a minor shift in the ploidy distribution toward lower values. In semisolid cultures the addition of anti-IL-6 antibodies led to a 42% decrease in colony number in cultures stimulated by IL-3 but not in other conditions of culture. These results suggest that normal human megakaryocytopoiesis might be regulated in part by an IL-6 autocrine loop.     

Human interleukin for DA cells (HILDA) does not affect the proliferation and differentiation of hematopoietic progenitor cells in human long-term bone marrow cultures.

Human Interleukin for DA cells (HILDA), a cytokine also known as leukemia inhibitory factor (LIF), induces proliferation without concurrent differentiation of murine embryonic stem cells. Therefore, we investigated the effects of recombinant HILDA/LIF on the proliferation and differentiation of human hematopoietic progenitor cells (HPC) grown in long-term bone marrow cultures (LTBMC). Pre-established stromal cell layers were reinoculated with autologous cryopreserved mononuclear phagocyte- and T-lymphocyte-depleted bone marrow cells in the presence or absence of HILDA/LIF (200 U/ml). At weekly intervals cultures were sacrificed, and the cells in the adherent and the nonadherent cell fractions were counted. The numbers of HPC were determined by culturing these cells in semisolid medium stimulated with phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM), and LTBMC supernatants were assayed in semisolid cultures for the presence of colony-stimulating activity (CSA). The total number of cells, their differential counts, the number of HPC, and the concentrations of CSA in culture supernatants were similar for long-term cultures containing HILDA/LIF and for controls. These data suggest that HILDA/LIF may not play a role in the proliferation and differentiation of normal human (early) HPC in LTBMC. Moreover, HILDA/LIF did not stimulate the proliferation of relatively mature progenitor cells in semisolid cultures, not did it influence the colony formation induced by other colony-stimulating factors (CSF). Finally, using a [3H]thymidine suicide test we could not find an effect of HILDA/LIF on the cell-cycle status of HPC.     

Developmental changes in human megakaryocyte ploidy

Megakaryocytes (MK) obtained from the differentiation of MK colony-forming units (CFU-MK) were grown from fetal liver, cord blood, and adult marrow in liquid culture containing aplastic plasma. Ploidy distribution was studied by a double-staining technique and flow cytometry and MK maturation by ultrastructural techniques. Cultured MK from fetuses and neonates were small sized (about 10 microns) in comparison to adult MK. They were mature cells that contained large membrane complexes as previously found in vivo. Only 2N and 4N MK were usually present in 8- to 10-week-old fetus cultures; 8N MK were detected at 20 weeks of gestation and in neonates. Higher ploidy classes were present in culture from adults but with a much lower frequency than in marrow. Therefore, a progressive shift to higher ploidy and an increase in MK size were observed simultaneously during development. Interleukin 3 (IL-3) increased MK proliferation as in adults but abrogated MK ploidization of 20-week-old fetus culture. The present results suggest that the changes occurring during ontogenesis are related to intrinsic MK modifications because no inhibitor of MK ploidization could be detected in fetal cultures.     

Immunocytochemical study of a Ca(2+)-ATPase enzyme in the human megakaryocytic lineage

We report the presence of a Ca(2+)-ATPase in human megakaryocytes (MK) using an immunofluorescence technique on bone marrow smears and especially on normal MK progenitors in culture. This finding is based on the comparative staining of MK with 1) a well-characterized antibody raised against purified rabbit skeletal sarcoplasmic reticulum Ca(2+)-ATPase, 2) antibody P2 raised against the glycoprotein IIb-IIIa complex as a marker of megakaryocytic lineage, and 3) anti-glycophorin A as a marker of erythroid lineage. On bone marrow smears, all cells recognized by P2 were also labeled with the anti-Ca(2+)-ATPase antibody. In culture, a maximum number of MK colonies was observed at day 11. From days 2-4, some MK precursors appeared stained both with the anti-Ca(2+)-ATPase and P2 antibodies; other cells were reactive with both anti-Ca(2+)-ATPase and anti-glycophorin A antibodies. From day 5 of culture, cells were either simultaneously stained with P2 and anti-Ca(2+)-ATPase antibodies or with anti-glycophorin A antibody, but not with the anti-Ca(2+)-ATPase antibody. Besides this first evidence of an early expression of a Ca(2+)-ATPase in MK, this work provides a useful tool for identification of MK by immunofluorescence.     

Growth of erythroid burst-forming units (BFU-E) in cultures of canine bone marrow and peripheral blood cells: effect of serum from irradiated dogs

Erythroid burst-forming units (BFU-E) from canine bone marrow and peripheral blood could be grown in methylcellulose in the presence of an appropriate batch of fetal calf serum (FCS), transferrin, and erythropoietin (Epo). However, improved colony formation (size and number of bursts) was obtained when serum from total body irradiated dogs was present in the culture. This serum, obtained from dogs at day 9 after total body irradiation with a dose of 3.9 Gy, reduced markedly the Epo requirement of BFU-E. Furthermore, it allowed the omission of FCS from the culture medium if cholesterol and bovine serum albumin (BSA) were used as FCS substitutes. BFU-E concentrations were found to be rather different in the peripheral blood and in bone marrow samples from different sites (i.e., iliac crest, sternum, and humerus) of normal beagles. The studies further show that canine bone marrow BFU-E can be cryopreserved in liquid nitrogen.     

In vitro assessment of marrow ''stem cell''and stromal cell function in aplastic anaemia

An in vitro model system is described that allows separate assessment of 'stem cell' and stromal cell function in aplastic anaemia (AA). Seven patients with non-severe AA, who had responded to immunosuppressive therapy and had haematological evidence of residual marrow function, were studied. Of these, three with otherwise typical AA had an acquired clonal cytogenetic marker. Purified bone marrow haemopoietic progenitors labelled with CD34 monoclonal antibody were positively selected using the fluorescence activated cell sorter (FACS) from both normal subjects and from patients with AA. The generative capacity of the CD34 positive cells was assessed by monitoring the output of granulocyte/macrophage colony forming cells (CFU-GM) in the non-adherent layer after inoculation onto irradiated performed long-term marrow culture (LTBMC) stromas. Stromal function in AA was assessed by inoculating CD34 positive cells from normal bone marrow onto performed irradiated stromas from patients with AA. Haemopoietic cell ('stem cell') function in AA was assessed by inoculating CD34 positive cells from AA patients onto confluent irradiated normal marrow stromas. Using these crossover/LTBMC experiments, all patients exhibited severe defects in haemopoietic cell function with normal functioning stroma. The proportion of CD34 positive cells present in bone marrow from these patients was reduced compared with controls, they comprised fewer small primitive 'blast-like' cells which in normal bone marrow are known to possess marrow repopulating ability, and demonstrated reduced clonogenic potential in short-term colony assays.     

Uncoordinated expression of fibrinogen compared with thrombospondin and von Willebrand factor in maturing human megakaryocytes

The localization of three known alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fg) has been studied in human megakaryocytes (MK) by immunofluorescence and immunoelectron microscopy. For this study, highly purified populations of MK were prepared from human bone marrow either by counterflow centrifugal elutriation or by cell culture from normal subjects and from two patients with megakaryoblastic leukemia. In normal bone marrow immature MK, TSP, and vWF were observed in the Golgi-associated vesicles and in small immature alpha-granules; in mature MK, they were found in the matrix of the mature large alpha-granules. Surprisingly, Fg was detected neither in the Golgi area, nor in the small precursors of alpha-granules; it was only found in the mature alpha-granules but this labeling was generally weaker than in blood platelets. In order to confirm these differences between the expression of Fg and vWF or TSP additional studies were performed on cultured maturing MK: immunofluorescent and ultrastructural immunogold labeling confirmed that vWF appeared early in the maturation while the same immature MK were negative for Fg. In the late maturation stage, the three proteins were detected in the alpha-granules. In order to know whether Fg was lately synthesized or endocytosed from the outside medium, normal MK were grown in the presence of either normal or afibrinogenemic plasma, and normal serum. Fg was detected only in the alpha-granules of MK grown in normal plasma. Similar results were observed with malignant MK, whose maturation was independent of the culture conditions. In conclusion, this study brings immunocytochemical evidence that vWF and TSP are synthesized by immature MK, whereas Fg appears later in the MK alpha-granules and its expression is dependent of the presence of an exogenous Fg source.