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Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.

The power of flow cytometry in field plant research may be greatly enhanced by analysis of nonfresh tissues. Previous attempts to use chemical fixatives, however, received only little attention because of protocol complexity and limited time, after which successful assays were achieved. This unit describes simple and rapid procedures for relative nuclear DNA content estimation in dehydrated tissues of vascular plants by DAPI flow cytometry. Histograms with reasonable resolution can be obtained in several-year-old specimens. The approach here is particularly suitable for reliable determination of DNA ploidy level, although detection of small variation in nuclear DNA content is also possible in many cases. Retrospective ploidy inference in silica-dry or herbarium vouchers and simplified transport of plant material from remote areas are among the principal benefits for plant biosystematics, ecology, and population biology.

Background: Only fresh plant material is generally used for rapid DNA ploidy estimation by flow cytometry (FCM). This requirement, however, substantially limits convenient FCM application in plant biosystematics, population biology, and ecology. As desiccation is a routine way for sample preservation in field botany, potential utilization of dehydrated tissues of vascular plants in FCM research was examined., Methods: Standard DAPI protocol was employed to evaluate the performance of 60 air-dried species, spanning more than 100-fold range of nuclear DNA amounts. Multiploid Vaccinium subg. Oxycoccus was selected as model taxon for detailed investigation and cytotype comparison., Results: A majority of analyzed plants yielded distinct peaks with reasonable coefficients of variation after several months of storage at room temperature. Fluorescence intensity of nuclei isolated from desiccated tissues was highly comparable with that for fresh material, allowing reliable DNA ploidy estimation. Deep-freezer preservation substantially extended Vaccinium samples lifetime (at least to 3 years) and maintained high histogram resolution., Conclusions: The introduced approach eliminates the need for fresh material in many vascular plants and thus opens new prospects for plant FCM. Convenient cytotype investigation in field research and retrospective ploidy determination in already herbarized samples are among the principal advantages.

Background and Aims: Nuclear DNA amounts of 12 diploid and one tetraploid taxa and 12 natural interspecific hybrids of Cirsium from 102 populations in the Czech Republic, Austria, Slovakia and Hungary were estimated., Methods: DAPI and PI flow cytometry were used., Key Results: 2C-values of diploid (2n = 34) species varied from 2.14 pg in C. heterophyllum to 3.60 pg in C. eriophorum (1.68-fold difference); the 2C value for the tetraploid C. vulgare was estimated at 5.54 pg. The DNA contents of hybrids were located between the values of their putative parents, although usually closer to the species with the smaller genome. Biennial species of Cirsium possessed larger nuclear DNA amounts than their perennial relatives. Genome size was negatively correlated with Ellenberg's indicator values for continentality and moisture and with eastern limits of distribution. A negative relationship was also detected between the genome size and the tendency to form natural interspecific hybrids. On the contrary, C-values positively corresponded with the spinyness (degree of spinosity). AT frequency ranged from 48.38 % in C. eriophorum to 51.75 % in C. arvense. Significant intraspecific DNA content variation in DAPI sessions was detected in C. acaule (probably due to the presence of B-chromosomes), and in tetraploid C. vulgare. Only the diploid level was confirmed for the Pannonian C. brachycephalum, generally considered to be tetraploid. In addition, triploidy was discovered for the first time in C. rivulare., Conclusions: Considerable differences in nuclear DNA content exist among Central European species of Cirsium on the diploid level. Perennial soft spiny Cirsium species of wet habitats and continental distributions generally have smaller genomes. The hybrids of diploid species remain diploid, and their DNA content is smaller than the mean of the parents. Species with smaller genomes produce interspecific hybrids more frequently.

The genus Reynoutria is represented by four taxa in the Czech Republic: Reynoutria japonica var. japonica, R. japonica var. compacta, R. sachalinensis and R. xbohemica. By using flow cytometry, cytological variability within the genus is described based on 257 Reynoutria samples. The varieties of R. japonica are cytologically uniform, var. japonica is exclusively octoploid (2n = 8x = 88) and var. compacta occurs only as a tetraploid (2n = 4x = 44), but R. sachalinensis and R. xbohemica exhibit some variation in chromosome numbers. Reynoutria sachalinensis is predominantly tetraploid (2n = 4 x = 44), but also occurs occasionally as hexaploid and octoploid cytotypes. The most common ploidy level in R. xbohemica is hexaploid (2n = 6x = 66), but tetraploid and octoploid clones were also found. The four taxa occurring in the Czech Republic are described briefly and the possible origins of the cytotypes discussed.

Hepatocyte apoptosis occurs during involution of hyperplastic liver induced by administration of xenobiotic compounds in rats. With this hyperplasia and involution, hepatic transforming growth factor (TGF)-beta1 is reported to be expressed to stimulate hepatocyte apoptosis. In regenerating liver after partial resection showing no hyperplasia, such expression of TGF-beta1 is also seen. However, no hepatocyte apoptosis develops despite the high levels of TGF-beta1. When rats received an intravenous injection of human hepatocyte growth factor at 12 h intervals for 14 days, the hepatic DNA content was increased 12 h after the last injection to 140% of control. This DNA content was significantly decreased at 108 and 180 h after discontinuation of treatment. At 60 h after the last injection, the number of apoptotic bodies positive for nick end-labelling of DNA in hepatocytes was significantly greater in treated rats than in control rats. Hepatocyte apoptosis was also identified electron micrographically. Hepatic TGF-beta1 mRNA levels in treated rats were significantly lower than in control rats at 12 h and then gradually increased towards control levels. We conclude that hyperplastic liver induced in normal rats by hepatocyte growth factor regresses with hepatocyte apoptosis and suppressed hepatic TGF-beta1 mRNA levels.

Two highly related receptor tyrosine kinases, TIE and TEK, comprise a family of endothelial cell-specific kinase. We established monoclonal antibodies against them and performed detailed analyses on their expression and function in murine hematopoietic stem cells (HSCs). TIE and TEK were expressed on 23.7% and 33.3% of lineage marker-negative, c-Kit+ and Sca-1+ (Lin- c-Kit+ Sca-1+) HSCs that contain the majority of day-12 colony-forming units-spleen (CFU-S) and long-term reconstituting cells, but not committed progenitor cells. Lin- c-Kit+ Sca-1+ cells were further divided by the expression of TIE and TEK. TIE+ and TEK+ HSCs as well as each negative counterpart contained high proliferative potential colony-forming cells and differentiated into lymphoid and myeloid progenies both in vitro and in vivo. However, day-12 CFU-S were enriched in TIE+ and TEK+ HSCs. Our findings define TIE and TEK as novel stem cell marker antigens that segregate day-12 CFU-S, and provide evidence of novel signaling pathways that are involved in the functional regulation of HSCs at a specific stage of differentiation, particularly of day-12 CFU-S.

The involvement of focal adhesion kinase (FAK) in myeloid differentiation was investigated in primary murine bone marrow (BM) cells. In unstimulated BM, FAK mRNA was detected in myeloid and lymphoid cells, but not in erythroid precursors. When the BM cells were incubated with granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), FAK expression showed a remarkable difference depending on the cytokine. Although FAK was upregulated in the cells stimulated by GM-CSF (GM-treated cells), the kinase was barely detectable in the cells cultured with IL-3 (IL-3-treated cells). Morphology and flow cytometry analysis showed GM-CSF promoted the growth and differentiation of monocyte/macrophage lineage stronger than IL-3. In addition, motility of the cytokine-differentiated cells showed an overt distinction between the cultures, which was closely correlated with FAK expression. After 7 days of stimulation, GM-treated cells showed active migration and chemoattractant-induced morphologic polarization. In contrast, IL-3-treated cells showed minimal migration and polarization. These results suggest an important role of GM-CSF in the terminal differentiation of monocytes/macrophages, and possible involvement of FAK in functional maturity of this lineage.

CHK/HYL is a non-receptor tyrosine kinase that belongs to CSK (C-terminal Src kinase) family. Northern blotting and RT-PCR analyses showed that CHK/HYL was expressed in large spectrum of hematopoietic cells except for erythroid cells and brain. To explore the function of CHK/HYL in hematopoietic cells, we generated CHK/HYL deficient mice. The mutant mice were apparently normal and fertile, while CSK knockout mice died until E11.5 from a defect in the neural tube formation. Hematological observations including blood counts and FACS analysis showed no significant abnormalities in CHK/HYL mutant mice. CHK/HYL did not affect the activity of Src, Hck, and Fgr in cultured bone marrow cells, although CSK negatively regulates Src family kinases. These results suggest that CHK/HYL might not have the same function as CSK.

The authors investigated the long-term effect of antiarrhythmic treatment on the maintenance of the sinus rhythm in a group of 32 patients with paroxysmal atrial fibrillation and flutter where the antiarrhythmic drug was selected because of negative results of tests by programmed atrial stimulation, using the method of oesophageal stimulation. After a one-year follow up 56% of the patients had permanently a sinus rhythm without paroxysms of supraventricular arrhythmia. Comparison with results of work evaluating the effectiveness of different antiarrhythmic drugs as regards maintenance of the sinus rhythm without testing suggests that selection of an antiarrhythmic drug by programmed stimulation of the atria by oesophageal stimulation has no predictive value for the effect of antiarrhythmic treatment.

Background: Immunohistochemical studies have demonstrated that CD34 antigen is present on the surface of vascular endothelial cells and hematopoietic cells. Roles of CD34 for angiogenesis and its function as a homophilic adhesion molecule between endothelial and hematopoietic cells have been speculated., Experimental Design: Northern blotting was used to examine the expression levels of CD34 mRNA, and in situ hybridization was used to localize CD34 mRNA. First, we investigated changes in CD34 mRNA expression in developmental processes of mice. Second, we investigated the changes in skin tissues of adult mice in the process of wound healing and tumor growth., Results: CD34 mRNA was strongly expressed by most of the vascular endothelial cells in developing organs. The magnitude of the expression decreased after birth but increased again in the process of wound healing and tumor growth. Although CD34 mRNA signals were observed in hematopoietic cells in the yolk sac and fetal liver, the endothelial cells of these tissues did not express CD34 mRNA signals. CD34 mRNA signals were detectable in neither hematopoietic nor endothelial cells of the bone marrow. In tissues other than hematopoietic ones, however, blood cells within the vessels did not express CD34 mRNA, although the endothelial cells expressed CD34 mRNA., Conclusions: The expression pattern of CD34 mRNA suggested its significant role in development of blood vessels not only in embryos but also in adults. Although CD34 mRNA was expressed both by endothelial cells and hematopoietic cells, the expression did not occur within the same organ, suggesting that the CD34 molecule may not be used as a homophilic adhesion molecule between endothelial and hematopoietic cells.

The first stages of the pathway by which lymphocytes differentiate from hemopoietic stem cells were studied at a clonal level. When 211 interleukin 3 (IL-3)-induced blast colonies shown to be capable of differentiating into a variety of hemopoietic cells were individually transferred into wells containing a monolayer of stromal cells, growth in granulocyte, macrophage, megakaryocyte, or mast cell lineages was observed in 192 wells. In seven of these 192 wells, lymphoid cell growth also was seen. The lymphoid cells were proved to be B lymphocytes by phenotype and immunoglobulin gene rearrangement analyses and by demonstration of surface expression of IgM. The clonal origin of myeloid and B lymphocyte lineage cells was further confirmed by the generation of both myeloid and B lymphoid cells in the same well following FACS clone-sorting of IL-3 induced blast cells. These results provide in vitro evidence that cells of B lymphoid and myeloid lineage can originate clonally from single primitive hemopoietic stem cells.

In order to examine the effect of recombinant growth factors on hemopoietic stem cells, these cells were enriched using wheat germ agglutinin (WGA) and monoclonal antibodies for lineage markers (Lin) such as B220, L3T4, Lyt-2, asialo GM1, Mac-1, and AL-21. Spleen colony-forming units (CFU-S) and in vitro colony-forming units were highly enriched in the fraction of WGA+Lin- spleen cells. To eliminate committed progenitor cells, spleen cells of 5-fluorouracil (5-FU)-treated mice were used. By this treatment, day-8 CFU-S disappeared but day-14 CFU-S were preserved. Day-14 CFU-S were also contained in the fraction of WGA+Lin- cells, which made up about 0.5% of total nucleated spleen cells. Moreover, this fraction contained primitive stem cells that could reconstitute the hemopoiesis of irradiated mice. Sorted WGA+Lin- spleen cells obtained from male 5-FU-treated mice were injected into lethally irradiated female mice. Southern hybridization using a mouse Y chromosome-specific probe showed that the bone marrow, spleen, and thymus of the recipients was reconstituted by male mouse-derived cells. When sorted WGA+Lin- spleen cells of the 5-FU-treated mice were cultured in vitro in the presence of recombinant interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), colony formation was observed only in wells with IL-3, whereas unfractionated spleen cells formed colonies in the presence of IL-3, IL-6, or G-CSF. However, IL-6 but not G-CSF acted synergistically on enriched hemopoietic stem cells in the presence of IL-3. These data suggest that G-CSF or IL-6 did not affect primitive stem cells independently but showed the effect on these cells indirectly or synergistically with IL-3.

We studied the effects of interleukin-3 (IL-3) on colony formation by hemopoietic progenitors in methylcellulose cultures of spleen cells from 5-fluorouracil (FU)-treated mice. Purified IL-3 supported the growth of various types of multilineage colonies including blast cell colonies. The types of colonies were similar to those supported by pokeweed-mitogen spleen cell conditioned medium (PWM-SCM), except that IL-3 supported eosinophil and neutrophil expression better. Delayed addition of IL-3 to cultures 7 days after cell plating decreased the number of colonies to one-half the number in cultures with IL-3 added on day 0. It did not alter the proliferative and differentiation characteristics of late emerging multipotential blast cell colonies. These observations suggest that IL-3 does not trigger hemopoietic progenitors into active cell proliferation but is necessary for their continued proliferation. This permissive role of IL-3 is consistent with a stochastic model of stem cell proliferation which features random entry into cell cycle. IL-3 also supported the growth of multilineage colonies from single cells isolated from blast cell colonies by micromanipulation. This result shows that IL-3 acts directly on multipotential progenitors. Analysis of colonies derived from paired progenitors revealed disparate lineage expression and was in accordance with the stochastic model of stem cell differentiation.

To clarify whether in vitro-cultured basophils have features of mast cells, basophils grown in semisolid and liquid culture were studied ultrastructurally and cytochemically and were compared with freshly isolated basophils and mast cells. Ultrastructurally, the cultured basophils had pleomorphic cytoplasmic granules, some of which were similar to mast cell granules. However, scroll formation, characteristic of mast cell granules, was not observed in cells grown in semisolid and liquid culture. Cytochemically, the reactivity patterns of acidic glycoconjugates in the cultured cells more closely resembled those of the freshly isolated basophils than of the mast cells. In addition, the basophils grown in liquid culture showed a more matured form and had stronger reactivity to glycoconjugates, acid phosphatase and peroxidase than did the cells grown in semisolid culture, suggesting that the liquid culture system was better for the basophil.

Murine embryonic cells including yolk sac prepared from 8-day embryos were co-infected with Abelson murine leukemia virus (A-MuLV) and/or a recombinant retrovirus containing large T and small t antigens, and early region of simian virus 40 (M-SV40). By coinfection with A-MuLV and M-SV40, megakaryoblastic cells were obtained in addition to mast cells and fibroblastic cells. However, following infection with A-MuLV or M-SV40 alone, no megakaryoblastic cells were detected, although mast cells and/or fibroblastic cells developed. The same results were obtained in several experiments. By single-cell transfer, 6 acetyl-cholinesterase (AchE)-positive clonal cell lines were established. Characteristics of megakaryocytes, such as AchE, glycoproteins IIb and IIIa, and platelet peroxidase were detected in two representative cells (C1 and C8). More significant changes expressing differentiation were observed following treatment with phorbol myristate acetate or pokeweed mitogen-stimulated murine spleen cell conditioned medium, although release of platelets was not observed. This is the first report showing development of megakaryocytic cells as the result of coinfection with retroviruses.

An instance of the rare occurrence of neonatal hepatitis and extrahepatic biliary atresia in the same sibship is reported. The older brother with neonatal hepatitis developed jaundice at the age of 4 days and had clay-colored stools from early infancy. Cholangiography by exploratory laparotomy at the age of 3 months showed a normal bile duct pattern. After laparotomy, jaundice rapidly disappeared, and stools became yellow. His liver function has been normal since age 6 months to the present (6 years old). The younger brother developed jaundice and clay-colored stools at the age of 1 month. The diagnosis of extrahepatic biliary atresia was made at laparotomy at the age of 4 1/2 months. Hepatojejunostomy was performed with successful bile drainage, although he had frequent attacks of ascending cholangitis since operation. These cases support a recent hypothesis that neonatal hepatitis and extrahepatic biliary atresia may be produced by the same disease process.

Cell-lineage involvement in a typical case of chronic myelomonocytic leukemia (CMML) was examined by simultaneous analysis of morphology and chromosomes on the same single colonies. Cytogenetic analysis of patient's bone marrow cells showed two clones: 46,X,-Y,+M1 (63/68 cells) and 47,X,-Y,+M1,+M2 (5/68 cells). Bone marrow or peripheral blood mononuclear cells were plated at 1 X 10(4)/ml or 2 X 10(5)/ml (if thawed) in methylcellulose medium containing phytohemagglutinin-stimulated, leukocyte-conditioned medium and erythropoietin. On days 9-14 of culture, 68 single colonies were lifted and each colony served for both morphological and chromosome examination. Of the 68 colonies, 23 had two or more analyzable metaphases, yielding a total of 79 metaphases. Morphological examination revealed that 10 colonies contained macrophages (m), 10 had erythroblasts (E) and blasts (bl), and three had E, respectively. All of the single colonies were derived from abnormal clones, i.e., 22 (9m, 10b1E,3E) were from 46,X,-Y,+M1, and one (1m) was from 47,X,-Y,+M1,+M2. These findings demonstrate that erythroid and myeloid lineages are involved in CMML.

Ultrastructural and ultracytochemical studies were performed on blast cells from 12 Down's syndrome neonates with transient myeloproliferative disorder (TMD) and 13 Down's syndrome patients with megakaryoblastic leukaemia (MKL), in order to clarify the cytological characteristics of these cells. Average platelet peroxidase-positivity in blast cells of TMD patients was similar to that found in cases of MKL. Blast cells from subjects with TMD contained a number of different granules, namely, alpha granules, those that were myeloperoxidase (MPO)-positive, electron-lucent or basophil-like, and those containing membrane components or ferritin particles. On the other hand, granules found in the blast cells of MKL patients with Down's syndrome included the electron-lucent variety, those with membrane components and a few that were basophil-like, but not alpha and MPO-positive granules nor those containing ferritin particles. A demarcation membrane system was observed in blasts from the TMD group, but not in the MKL group. These findings suggest that blast cells in TMD patients differentiate to megakaryocytes, neutrophils, basphils and erythroblasts, while those in cases of MKL show limited differentiation to immature megakaryocytes, erythroblasts and, sometime, basophils. Such results correspond well with those of culture studies, in which TMD blasts were found to be precursors of various types of blood cells.

Transient myeloproliferative disorder accompanied by Down's syndrome has been characterized as exhibiting self-limiting haematological abnormalities. We studied six patients suffering from this disorder in order to clarify the biological nature of their blast cells. Metaphases of leucocytes stimulated with phytohaemagglutinin (PHA) showed trisomy 21 in all patients except one. The exception was constitutionally trisomy 21 mosaic (46,XY = 89/47,XY,+21 = 11). However, metaphases from the peripheral blood cells (blast cells: 70%) without PHA stimulation showed exclusively trisomy 21. Simultaneous examination for morphology and chromosomal analysis on single colonies revealed that granulocyte-macrophage (GM) colonies and an erythroid colony contained only cells with the trisomy 21 karyotype. The blast cells showed positive reactions for platelet peroxidase (PPO) and with monoclonal antibodies against platelet-megakaryocyte antigen (TP 80 and TP 82). In methylcellulose and liquid culture systems, high plating efficiencies were observed, and mainly mature basophils containing histamine developed in the presence of PHA-stimulated leucocyte conditioned medium (PHA-LCM). In vivo, mature neutrophils, basophils, eosinophils or megakaryocytes coexisted with PPO-positive blast cells in the peripheral blood of some patients with this disorder. These findings suggest that transient myeloproliferative disorder is characterized by a proliferation of PPO-positive blast cells with trisomy 21, although some heterogeneity may be seen.

To clarify the mode of erythropoiesis, the in vitro proliferation of single human erythroblasts was recorded continuously for 80 hours by time-lapse, phase-contrast cinemicrography. Progenies from single erythroblasts were followed, their pedigrees were delineated, and their generation times were measured by counting the frames of the film. In one erythroblast pedigree, a daughter cell continued to divide three times to yield eight smaller erythroblasts; another daughter cell yielded four progenies, three of which abruptly lost most of their cytoplasm and became immobile. The generation time ranged from 15.8-30.0 hours (mean +/- SD: 23.3 +/- 4.8 hours), which corresponded to generation times calculated from in vivo data, such as the mitotic index, isotope labeling, or red cell turnover. Paired daughter cells showed very similar generation times. During succeeding mitoses, erythroblast size and nucleus/cytoplasma ratio decreased, cytoplasm darkened, and cell movement became more prominent. These studies on clonal cell proliferation using cinemicrography provide considerable information on the mechanism of hemopoiesis.

Several investigators have described hemopoietic colonies expressing multi-lineage differentiation in culture. We recently identified a class of murine hemopoietic progenitors which form blast cell colonies with very high replating efficiencies. In order to clarify further the relationship between progenitors for blast cell colonies and progenitors for the multilineage hemopoietic colonies in culture, we carried out analyses of kinetic and differentiation properties of murine blast cell colonies. Serial observations of the development of blast cell colonies into multilineage (and single lineage) colonies in cultures of spleen cells obtained from 5-fluorouracil (5-FU)-treated mice confirmed the transitional nature of the murine blast cell colonies. The data also suggested that the early pluripotent progenitors are in G0 for variable periods, and that when triggered into cell cycle, they proliferate at relatively constant doubling rates during the early stages of differentiation. The notion that some of the pluripotent progenitors are in G0 was also supported by long-term thymidine suicide studies in which spleen cells were exposed to 3H-thymidine with high specific activity for 5 days in culture, washed, and assayed for surviving progenitors. Comparison of replating abilities of day-7 and day-16 blast cell colonies from normal as well as 5-FU-treated mice indicated that some of the day-7 blast cell colonies are derived from maturer populations of progenitors which are sensitive to 5-FU. In contrast, progenitors for the day-16 blast cell colonies are dormant in cell cycle and were not affected by 5-FU treatment. Previously we reported that progenitors for day-16 blast cell colonies have a significant capacity for self-renewal. These observations suggest the hypothesis that the capability for self-renewal is accompanied by long periods of G0, and that once commitment to differentiation takes place, then active cell division occurs.

We performed the present study to define the in vitro hemopoietic activity of murine recombinant (r) granulocyte colony-stimulating factor (G-CSF) using murine hemopoietic culture systems of normal bone marrow cells, fetal liver cells, and spleen cells of 5-fluorouracil (FU)-treated mice. Recombinant G-CSF supported only neutrophil and/or macrophage colony formation by normal bone marrow cells. It did not enhance the formation of erythroid bursts in the fetal liver cell assay, but interleukin-3 (IL-3) did. Paradoxically, rG-CSF could support the colony formation of multilineage colonies as well as blast colonies from the spleen cells of 5-FU-treated mice, while r-granulocyte-macrophage colony-stimulating factor (GM-CSF) and r-erythropoietin (Ep) did not. When blast colonies, formed in the presence of G-CSF, were replated to dishes containing IL-3, they were able to differentiate along multilineage pathways. However, when they were replated to dishes containing rG-CSF, they could differentiate only into neutrophils and macrophages. Single cells transferred from blast colonies formed only neutrophil-macrophage colonies. These data indicate that rG-CSF had a direct effect on the growth and development of GM progenitors at a late stage and a significant effect on multipotential hemopoietic precursors. Although it remains to be clarified how G-CSF acts on multipotential stem cells, this unique effect is important in the understanding of its pluripotent hemopoietic activity in vivo.

We examined the effect of human recombinant (r) interleukin 6 (IL-6) on the differentiation of murine and human hemopoietic progenitors. Human IL-6 supported colony formation by murine bone marrow cells. These colonies consisted of neutrophils and macrophages. Recombinant IL-6 was able to support multilineage colony formation by spleen cells from 5-fluorouracil (5-FU)-treated mice. These colonies consisted of greater than 1 x 10(4) cells. Differential counts revealed large colonies exhibiting different combinations of cell lineages: neutrophils, macrophages, eosinophils, mast cells, and megakaryocytes. However, when blast cell colonies supported by interleukin 3 were replated into secondary dishes containing IL-6, they could differentiate into only neutrophils and macrophages. Single cells transferred from blast cell colonies formed only neutrophil/macrophage colonies. These results indicate that IL-6 had a direct effect on the growth and development of murine granulocyte-macrophage progenitors at a late stage and a significant effect on multipotential hemopoietic precursors that might be indirect through other cells. By contrast, human rIL-6 did not support colony formation by human bone marrow mononuclear cells. IL-6 may not show an independent activity for human hemopoiesis of myeloid lineage. However, the synergistic activity of IL-6 remains to be clarified.

We investigated the in vitro effects of 4-hydroperoxycyclophosphamide (4-HC) on human hemopoietic stem cells. Marrow cells were exposed to 4-HC and then assayed for mixed (CFU-GEMM), erythroid (BFU-E), megakaryocyte (CFU-M), and granulocyte-macrophage (CFU-GM) colony forming cells. We found that highly proliferative colony forming cells, especially CFU-GEMM and BFU-E, were relatively spared by 4-HC treatment. One third of the surviving progenitors formed large colonies, some of which contained more than 50,000 cells. By sequential examination of the formation of these large colonies, we found immature colonies consisting of blasts at the early stage of culture. The morphology of these "blast cell colonies" in situ was arbitrarily classified into four types. Among them were the blast cell colonies consisting of the individual cells that were dispersed and had a few granules within the cytoplasm (type A); these cells finally formed very large colonies on day 22 of culture. Approximately 70% of the single cells derived from type A blast cell colonies produced secondary colonies consisting of erythroblasts, macrophages, eosinophils, and/or basophils. These results show that the blast cells in type A colonies have a highly proliferative capacity. The availability of a highly enriched population of primitive hemopoietic progenitors will provide us with a unique opportunity to study the interaction between a single stem cell and purified hemopoietic factors.

To clarify the lineage involvement of haemopoietic progenitor cells in erythroleukaemia, the morphology and chromosomes of single colonies from a patient with erythroleukaemia were analysed simultaneously. The cytogenetic analysis of bone marrow cells revealed two clones; 44,XY,-7,-12,-17, del (5)(q31), +Mar and 43,XY,-7,-12,-17,-19,del(5)(q31),+Mar. Of 40 metaphases examined, there were 34 and six of these clones, respectively. Bone marrow mononuclear cells were plated at 5 X 10(4)/ml in methylcellulose medium containing phytohaemagglutinin-stimulated leucocyte conditioned medium and erythropoietin. Seventeen colonies, i.e. nine blast cell colonies, four myeloid (Sudan black B-positive) colonies, and four erythroid (benzidine-positive) colonies contained analysable metaphases, yielding 102 metaphases in total. Except for chromosome random loss, the karyotype within a colony remained constant. All three types of colonies showed an abnormal clone; 44,XY,-7,-12,-17,del(5)(q31),+Mar. From these findings, it is concluded that myeloid and erythroid lineages in erythroleukaemia were derived from the same abnormal clone.

Mice of genotype W/Wv have less than 1% of normal mast cells in the skin, stomach, and cecum. In order to further clarify the mechanism of this deficiency, we studied committed mast cell progenitors and multipotent progenitors, which are capable of mast cell differentiation in clonal culture. The relative concentration of mast cell progenitors in the bone marrow, spleen, and peripheral blood of W/Wv mice was similar to that of +/+ mice. However, the cellularity of the marrows of W/Wv mice was 54% of that of their normal littermates. Identification of mast cells was established by metachromatic staining with toluidine blue, transmission electron microscopy, and demonstration of membrane receptors for immunoglobulin E. The time course of colony formation and the morphology of W/Wv mast cell colonies in culture was identical to that of normal littermates. The percentages of mast cells in individual multi-lineage colonies were extremely variable. The histamine content of mast cells derived from W/Wv mice was similar to that of mast cells from +/+ mice. These studies demonstrated the normal capacity for differentiation and proliferation in culture of mast cell progenitors from W/Wv mice.

NFS-60 cells were previously obtained from leukemia cells that were infected with the Cas-Br-M murine leukemia virus in vivo. We examined the proliferation and differentiation capacity of NFS-60 cells in the presence of native and recombinant (r) interleukin 3 (IL-3), recombinant granulocyte colony-stimulating factor (rG-CSF), recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and r-erythropoietin (Ep) using methylcellulose culture methods. This cell line was able to form colonies in response to each hemopoietic factor, but colony formation was rarely seen in their absence. Some populations of NFS-60 cells could differentiate into neutrophils and macrophages in the presence of IL-3 and GM-CSF. Moreover, in the presence of Ep, this cell line formed well-hemoglobinized colonies as well as nonerythroid colonies. In the presence of G-CSF, NFS-60 cells remained in the promyelocytic state. Electron microscopic studies confirmed these morphologies. A single-cell transfer experiment demonstrated that neutrophils, macrophages, and erythroblasts were derived from a single cell. It is concluded that the NFS-60 cell line is a factor-dependent, bipotential hemopoietic cell line.

To clarify the lineage involvement of haemopoietic progenitor cells in idiopathic myelofibrosis (MF), simultaneous analysis of morphology and chromosomes were performed on single colonies from a 70-year-old Japanese woman with typical presentation of MF. The cytogenetic analysis of unstimulated peripheral blood cells revealed three cell lines 47,XX,-6,+9,+der(6)t (1;6)(1qter----1q21::6p21----6qter), 46,XX,-6,+der(6) and 46,XX. Of 91 metaphases examined, the numbers of each cell line were 43, 41 and 7, respectively. Blood mononuclear cells were plated at 2 X 10(3) or 1 X 10(4)/ml in methylcellulose medium containing phytohaemagglutinin-stimulated leucocyte conditioned medium and erythropoietin. On days 10, 12 and 14 of culture, 47 individual colonies were lifted and served for morphological and cytogenetic examination. Morphological examination revealed that the colonies contained neutrophils (n), macrophages (m), basophils (b), erythrocytes (E) and megakaryocytes (M). Twenty-two of the 47 colonies had analysable metaphases, yielding totally 158 metaphases. Twelve colonies contained three or more metaphases. Eight colonies (3bE, 1E, 2b, 1nEM, 1bEM) were of the abnormal cell line 46,XX,-6,+der(6), and four colonies (2E, 1bE, 1mE) were of the karyotypically normal cell line 46,XX. The other abnormal cell line 47,XX,-6,+9,+der(6) was not detected in any of the metaphases obtained from single colonies. These results provided direct evidence that the pluripotent stem cells were involved in MF.