In vitro photoinhibition by psoralen and ultraviolet A radiation of human hematopoietic progenitors

The in vitro sensitivity of human hematopoietic progenitors to PUVA, 8-MOP and UVA alone was investigated. 8-MOP alone at final concentrations of 150, 200, 600 and 1,000 ng/ml did not modify colony growth of circulating and bone marrow erythroid (BFU-E), myeloid (CFU-GM) and immature (CFU-GEMM) hematopoietic progenitors obtained from normal controls. The exposure of the same progenitors to increasing doses of UVA, up to 12 J/cm2, progressively decreased hematopoietic colony growth (with estimated 50% inhibition occurring at about 5 J/cm2). In vitro PUVA treatment (8-MOP 200 ng/ml followed by UVA 5 J/cm2) caused 90% growth inhibition of circulating and bone marrow hematopoietic progenitors. In addition, the treatment completely inhibited the formation of spontaneous erythroid colonies, obtained from 5 polycythemic patients, that are considered to be a marker of this neoplastic disease. PUVA cytotoxicity was assessed by the colorimetric MTT assay. The percentage of cell death after PUVA exposure was 29 +/- 10% for both peripheral and bone marrow mononuclear cells. Our findings indicate that 8-MOP alone is not toxic to hematopoietic progenitors whereas UVA treatment determines in vitro a dose-dependent inhibition of the clonogenic capacity of normal hematopoietic cells. PUVA treatment enhances this effect, causing a quite complete inhibition of hematopoietic progenitors colony formation from normal donors and spontaneous BFU-E colony formation from polycythemic patients.     

Differential sensitivity of human myeloma cell lines and normal bone marrow colony forming cells to a recombinant diphtheria toxin-interleukin 6 fusion protein

The cytotoxicity of a recombinant interleukin 6 (IL-6)-diphtheria toxin (DT) fusion protein towards human myeloma cell lines was investigated. DAB389-IL-6 inhibited protein synthesis and methylcellulose colony formation by U266 myeloma cells. In the clonogenic assay, the fusion protein approached the level of cytotoxicity achieved by native DT. The specificity of killing by DAB389-IL-6 was demonstrated by inhibition of cytotoxicity by a molar excess of free rhIL-6. The effect of DAB389-IL-6 on colony formation by six OCI-My cell lines was assessed. Similar to U266 cells, colony growth by the OCI-My 5 and -My 2 cell lines was inhibited in a simple dose dependent manner. However, a biphasic effect was observed for the IL-6 dependent OCI-My 4 cells; DAB389-IL-6 stimulated colony formation at low (< or = 10(-11) M) concentrations, yet was inhibitory at higher doses. Three other cell lines whose growth was not altered by IL-6 were relatively unaffected by DAB389-IL-6, despite their sensitivity to native DT. Flow cytometric analysis for IL-6 receptor expression using phycoerythrin-conjugated IL-6 demonstrated specific binding sites on both DAB389-IL-6 sensitive and certain insensitive cell lines, suggesting that other factors in addition to the expression of IL-6 receptors are involved in killing by the fusion toxin. Despite evidence for a role of IL-6 in myeloid cell development, normal bone marrow was insensitive to the IL-6 fusion toxin. In cultures containing both normal bone marrow and U266 cells DAB389-IL-6 effectively inhibited the growth of U266 myeloma colonies but had little effect on normal bone marrow erythroid, granulocyte and mixed erythroid/granulocyte colony growth. From these experiments we conclude that DAB389-IL-6 is specifically cytotoxic towards a subset of IL-6-responsive human myeloma cell lines and may be useful, in some cases, in the selective elimination of tumour cells from mixed populations of normal and malignant cells.     

Hemoglobin switching in sheep and goats. VI. Commitment of erythroid colony-forming cells to the synthesis of betaC globin

Bone marrow from mature goats and sheep was cultured in plasma clots, and three erythropoietin (ESF)-dependent responses-growth (colony formation), differentiation (globin production), and initiation of hemoglobin C (alpha2beta2C) synthesis--were quantitated. ESF concentrations below 0.01 U/ml supported colony growth and adult hemoglobin production in cultures of goat marrow, while maximal hemoglobin C synthesis (70%), as measured between 72 and 96 h in culture, required a 100-fold higher ESF concentration. Sheep marrow was cultured in a medium enriched to enhance growth and to permit complete maturation of colonies. These colonies active in hemoglobin synthesis between 24 and 96 h produced mainly adult hemoglobin, and only between 96 and 120 h did sheep colonies develop which produced mainly hemoglobin C (up to 70%). A similar heterogeneity may exist among goat colonies. Thus, when goat bone marrow was fractionated by unit gravity sedimentation, more hemoglobin C synthesis was observed in colonies derived from cells of intermediate sedimentation velocity than in colonies derived from the most rapidly sedimenting cells. Brief exposure of sheep (in vivo) and goat (in vitro) bone marrow to a high ESF concentration committed precursor cells to the generation of colonies which, even at low ESF concentration, produced hemoglobin C. Committment to hemoglobin phenotype appears to be an early and probably irreversible event in the development of an erythroid cell.     

Decline in the growth potential of spleen-colonizing bone marrow stem cells of long-lived aging mice

The growth capacity of femoral bone marrow stem cells from young and old long-lived mice was assessed in the spleen of X-irradiated young and old syngeneic recpients by determining: (a) the number of stem cells colonizing the spleen, (b) the rate of incorporation of 125I-labeled iododeoxyuridine by proliferating colony cells, and (c) the number of cells present in the largest colonies at the end of the growth phase.We found that the growth capacity of stem cells declined with age. We further found that the spleen-seeking and spleen colony growth capacities of old stem cells remained characteristically old even after they were allowed to self-replicate in the bone marrow of young recipients for an extended period of time. On the other hand, the spleen colony growth capacity of young stem cells could be reduced by allowing them to self-replicate in old recipients. These results suggest that the growth capacity of old stem cells is an intrinsic characteristic which cannot be readily altered, but that of young stem cells can be aged in an accelerated manner by allowing them to self-replicate in old recipients. An additional reduction was noted in the frequency of both young and old stem cells colonizing the spleen of old recipients and in the cell density of the largest colonies produced. These results indicate that factors extrinsic to the stem cells are also responsible for the decline with age in their spleen colony growth capacity.Thus, the growth capacity of old stem cells in old recipients could be as low as 10% that of young stem cells in young recipients.     

Circulating erythropoietic precursors assessed in culture: characterization in normal men and patients with hemoglobinopathies

Circulating erythropoietic precursors in normal men and patients with hemoglobinopathies were characterized in culture. Blood mononuclear cells harvested with a modification of the Ficoll-Isopaque technique were cultured in methylcellulose for 14 days. The majority of erythropoietic colonies consisted of several subcolonies assuming the morphology of erythropoietic "bursts" described in murine marrow cultures. Time course studied of colony formation from marrow and blood nucleated cells confirmed that the circulating erythropoietic precursors represented only early stages of development. Peak sedimentation velocity of the circulating precursors analyzed using a Staput apparatus averaged 5.31 mm/hr and corresponded with that of the early erythropoietic precursors in human marrow. One ml of blood yielded an average of 153 colonies in normal men and 785 colonies in patients with hemoglobinopathies. No correlation was observed between colony formation and reticulocyte indices of individual patients. Examination of the proliferative state of the erythropoietic precursors using high specific activity tritium-labeled thymidine revealed that almost none of the cells in normal men or patients with hemoglobinopathies were in the DNA synthetic phase.     

The effect of DLE fractions on GM-progenitors of haematopoietic stem cells in vitro

Dialysable leucocyte extract (DLE) prepared from buffy coats of human blood, potentiates the effect of Colony-stimulating factor (CSF) on the growth of granulocyte-macrophage colony forming cell (GM-CFC) colonies in vitro. This relative increase of the number of colonies is apparent when diluted CSF (present in lung conditioning medium) as a control, and DLE, in a wide range of concentrations are added to the culture of mouse bone marrow cells. Fractionation of DLE on Amicon membranes revealed that the activity resides in molecules of 0-5 kD. Molecules 5-10 kD have no potentiating effect. DLE and its fractions (0-5 kD, 0-1 kD), except fractions 0-500 D and 5-10 kD, when added undiluted i.e. at the initial concentration, exerted a suppressive effect: colonies are not formed despite the presence of CSF. In a pilot experiment, it was shown that DLE is able to stimulate colony-forming activity of earlier progenitors of erythroid cells (BFUe), under the influence of erythropoietin.     

Chronic pancreatitis: terminology and classification

Rubidazone, the new semi-synthetic benzol hydrazone hydrochloride derivative of dauorubicin, has proved on a molecular weight basis to be less toxic than adriamycin and similar to daunorubicin in cardiac toxicity studies in the hamster as well as in other in vivo and in vitro test systems. It has proven effectiveness against several animal tumours and human acute leukaemias. We have compared the inhibitory effect of rubidazone to that of adriamycin on P388 leukaemia and normal bone marrow colony-forming units (CFU) using the spleen colony assay system in male DBA2 mice. The efficacy ratios (i.e., the ratio of the slopes of the normal bone marrow CFU to leukaemic CFU dose-survival curves) in the spleen colony assay system for rubidazone and adriamycin were 7-8 and 7-5 respectively. This near identity of efficacy ratios fro rubidazone and adriamycin correlated with the results of median survival time studies in the leukaemic mice. Their dose-median survival time curves were almost parallel, having nearly identical slopes. Rubidazone's equal therapeutic index as compared to adriamycin in the spleen colony assay system together with its known decreased toxicity to cardiac muscle cells makes it an extremely promising new anthracycline derivative to study in comparison to adriamycin in human malignancies.     

Use of flow cytometric CD34 analysis to quantify hematopoietic progenitor cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mononuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only 1%-9% of the CD34+ MNC. Due to the variable numbers of CD34+ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Normal human cementum-derived cells: isolation, clonal expansion, and in vitro and in vivo characterization

Cultures of primary human cementum-derived cells (HCDCs) were established from healthy premolar teeth extracted for orthodontic reasons. Cementum was manually dissected, fragmented, and digested twice with collagenase. Following a thorough wash to remove liberated cells, the remaining cementum fragments were plated in Dulbecco's modified Eagle's medium/F12 medium containing 10% fetal bovine serum. Discrete colonies that contained cells exhibiting fibroblast-like morphology were visible after 14-21 days of culture. When the colonies became sufficiently large, cells from individual colonies were isolated and subcultured. Cementum-derived cells exhibited low levels or no alkaline phosphatase activity and mineralized in vitro to a lesser degree than human periodontal ligament (PDL) cells and human bone marrow stromal cell (BMSC) cultures. To study differentiation capacities of HCDCs, cells were attached to hydroxyapatite/tricalcium phosphate ceramic and transplanted subcutaneously into immunodeficient mice. The transplants were harvested 3, 6, and 8 weeks after transplantation and evaluated histologically. In human BMSC transplants, new bone tissue was formed with a prominent osteoblastic layer and osteocytes embedded in mineralized bone matrix. No osseous tissue was formed by PDL cells. Of six single colony-derived strains of HCDCs tested, three formed a bone-like tissue that featured osteocyte/cementocyte-like cells embedded within a mineralized matrix and which was lined with a layer of cells, although they were somewhat more elongated than osteoblasts. These results show that cells from normal human cementum can be isolated and expanded in vitro. Furthermore, these cells are capable of differentiating and forming mineralized tissue when transplanted into immunodeficient mice.     

Leptin stimulates the proliferation of murine myelocytic and primitive hematopoietic progenitor cells

Leptin, the product of obese gene, was originally identified as a factor regulating body-weight homeostasis and energy balance. The present study has shown that leptin acts on murine hematopoiesis in vitro. In the culture of bone marrow cells (BMC) of normal mice, leptin induced only granulocyte-macrophage (GM) colony formation in a dose-dependent manner, and no other types of colonies were detected even in the presence of erythropoietin (Epo). Leptin also induced GM colony formation from BMC of db/db mutant mice whose leptin receptors were incomplete, but the responsiveness was significantly reduced. The effect of leptin on GM colony formation from BMC of normal mice was also observed in serum-free culture, and comparable with that of GM-colony-stimulating factor (CSF ). Although leptin alone supported few colonies from BMC of 5-fluorouracil (5-FU)-treated mice in serum-free culture, remarkable synergism between leptin and stem cell factor (SCF ) was obtained in the colony formation. The addition of leptin to SCF enhanced the SCF-dependent GM colony formation and induced the generation of a number of multilineage colonies in the presence of Epo. When lineage (Lin)-Sca-1(+) cells sorted from BMC of 5-FU-treated mice were incubated in serum-free culture, leptin synergized with SCF in the formation of blast cell colonies, which efficiently produced secondary colonies including a large proportion of multilineage colonies in the replating experiment. In serum-free cultures of clone-sorted Lin-c-Kit+Sca-1(+) and Lin-c-Kit+Sca-1(-) cells, although synergism of leptin and SCF was observed in the colony formation from both cells, leptin alone induced the colony formation from Lin-c-Kit+Sca-1(-), but not Lin-c-Kit+Sca-1(+) cells. These results have shown that leptin stimulates the proliferation of murine myelocytic progenitor cells and synergizes with SCF in the proliferation of primitive hematopoietic progenitors in vitro.     

Human hematopoietic progenitors express erythropoietin

Erythropoietin (EPO) is a factor essential for erythroid cell proliferation, differentiation, and survival. The production of EPO by the kidneys in response to hypoxia and anemia is well documented. To determine whether EPO is also produced by hematopoietic cells, we analyzed the expression of EPO in normal human hematopoietic progenitors and in their progeny. Undifferentiated CD34(+)lin- hematopoietic progenitors do not have detectable EPO mRNA. Differentiating CD34(+) cells that are stimulated with recombinant human EPO in serum-free liquid cultures express both EPO and EPO receptor (EPOR). Because CD34(+) cells represent a heterogeneous cell population, we analyzed individual burst-forming units-erythroid (BFU-E) and nonerythroid colony-forming unit-granulocyte-macrophage colonies for EPO mRNA. Only BFU-E colonies were positive for EPO mRNA. Lysates from pooled BFU-E colonies stained positively for EPO by immunoblotting. To further confirm the intrinsic nature of erythroid EPO, we replaced extrinsic EPO in erythroid colony cultures with EPO-mimicking peptide (EMP). We show EPO expression in the EMP-stimulated BFU-Es at both mRNA and protein levels. Stimulation of bone marrow mononuclear cells (BMMCs) with EMP upregulated EPO expression. Furthermore, we found EPO and EPOR mRNAs as well as EPO protein in K562 cells, a human erythroleukemia cell line. Stimulation of K562 cells with EMP upregulated EPO expression. We suggest that EPO of erythroid origin may have a role in the regulation of erythropoiesis.     

Removal of carcinoma cells from contaminated bone marrow using the lipophilic cation rhodamine 123

Autologous bone marrow transplants for solid tumor treatment are severely limited by the potential presence of residual cancer cells in the reinfused bone marrow and can lead to future tumor recurrence. This article presents a novel method of removing carcinoma cells from bone marrow with contaminating cancer cells. This method is based on our previous studies demonstrating that carcinoma cells have a higher uptake of lipophilic cations such as rhodamine 123 than their normal epithelial counterparts. When the relative differences in rhodamine 123 uptake are quantified, carcinoma cell lines demonstrated a 7.4-21 times greater uptake of rhodamine 123 than normal mouse bone marrow cells. More important, when normal bone marrow cells and carcinoma cell lines are mixed to simulate carcinoma-contaminated bone marrow, individual cell populations continue to exhibit characteristic and identifiable relative differences (10-20 times) in rhodamine 123 uptake. Differential sorting of bone marrow/carcinoma cell mixtures with respect to rhodamine 123 fluorescence intensity resulted in the removal of 95-99% of the "contaminating carcinoma cells." The recovered bone marrow cells were fully viable as ascertained by their ability to form splenic colonies. In our preliminary experiments, sorted bone marrow cells transplanted into lethally irradiated C57BL6 mice allowed the mice to survive for more than 8 months. In light of these promising results, we propose that lipophilic cations may play a role in the purification of autologous bone marrow used in transplants for patients with advanced solid tumors.     

A complement independent erythropoietic inhibitor acting on the progenitor cell in refractory anemia

An erythropoietic inhibitor was detected in the serum of a patient with refractory anemia. Using an in vitro heme synthesis method, the patient's serum produced tenfold inhibition of erythropoietin-stimulated radioactive iron (Fe59) incorporation into heme of normal human marrow at 72 hours, as compared with AB serum. In a separate experiment the patient's serum produced threefold inhibition, whereas immunoglobulin G (IgG) prepared from the same serum sample produced 12-fold inhibition. To identify the site of action of the inhibitor, serum was tested in a cell culture system whereby human marrow cells, grown in a plasma clot, respond to exogenous erythropoietin with the appearance of nucleated erythroid colonies. Each colony arises from a committed erythroid progenitor. The patient's serum produced a two- or tenfold reduction in the number of colonies from normal human marrow. The effect was also demonstrated on autologous marrow obtained when the patient was in "partial clinical remission". Serum samples obtained at various times during the course of the patient's illness all demonstrated a suppressive effect on colony growth. All serums were heat-inactivated, and total hemolytic complement could not be detected in either culture system. It is concluded that the anemia is due to an inhibitor, probably of IgG class, that acts on the erythroid progenitor cell. The absence of heat-labile complement components in the culture systems suggests that the mechanism is not due to immune cytolysis.     

Fibroblast precursors in normal and irradiated mouse hematopoietic organs

Using the in vitro colony assay, clonogenic fibroblast precursor cells (CFU-F) were detected in the bone marrow, spleen and thymus from adult mice. The survival curve for CFU-F of mouse bone marrow irradiated in vitro has a D0 of 220 r. Regeneration of bone marrow CFU-F after whole-body irradiation with 150 r is characterized by a marked secondary loss and post-irradiation lag and dip, lasting 6 days, followed by return to normal values by about the 25th day. This pattern of post-radiation recovery of CFU-F is similar to that of the CFU-s. In addition, during the first 6 hours following irradiation the number of CFU-F increased approximately twofold.     

Effect of serum on vascular smooth muscle function

We investigated the in vitro and in vivo effects of KT6352, a derivative of indolocarbazole compound, on murine megakaryocytopoiesis. When serum-free megakaryocyte (Meg) colony assay was performed with 100 U/mL of recombinant mouse interleukin-3 (rmIL-3), the addition of 1x10(-11)M to 1x10(-9)M of KT6352 increased the number of Meg colonies. An additional increase of Meg colonies by KT6352 was observed in the serum-free culture containing rmIL-3 plus recombinant mouse interleukin-6 or rmIL-3 plus recombinant mouse stem cell factor. KT6352 did not stimulate Meg colony formation without rmIL-3. When KT6352 was administered intraperitoneally to normal BALB/c male mice at a dose of 10 mg/kg daily for 5 consecutive days, a 2.1-fold increase in the platelet count was observed on day 14, and the prolonged thrombocytopoiesis was detectable from 9 to 27 days after KT6352 administration. A marked increase in the white blood cell count was also observed from 5 to 14 days after KT6352 treatment. Before the gradual increase of platelet counts, 8 days after KT6352 administration, a marked increase in the number of colony-forming units of megakaryocytes (CFU-Megs) in bone marrow and spleen was observed, and a substantial increase in the number of splenic CFU-Megs was observed 14 and 23 days after KT6352 administration. Bone marrow Meg ploidy analysis by two-color flow cytometry showed a shift in the modal ploidy class from 16 to 32 and an increase in the frequency of 64 cells in KT6352-treated mice. These results suggest a possible therapeutic benefit of KT6352 in the management of thrombocytopenia.     

Cause analysis of pain following vitreous surgery and nursing strategies]

OBJECTIVE: To assess the efficacy of "method of activating blood circulation and removing stasis (ABCRS)" in treating polycythemia vera. METHODS: 28 cases of polycythemia vera were treated with ABCRS and observed with bone marrow colonies. RESULTS: 15 casses were responsibe, of them 4 cases reached clinical remission, 11 cases were improved. Both bone marrow colonies of colony forming unit-erythroid dependent of erythropoietin (EPO + CFU-E) and colonies of CFU-E independent of EPO. (EPO-CFU-E) from these cases decreased significantly, especially the latter. ABCRS mainly suppressed the proliferation and differentiation of EPO-CFU-E. In addition, bone marrow colonies of colony formng unit-fibroblastoids (CFU-F) from these cases also changed obviously. CONCLUSION: ABCRS is effective in treating polycythemia vera.     

In vitro sensitivity of post-bone marrow transplantation CFU-GM and BFU-E to TNF-alpha and IFN-gamma

Graft failure remains one of the limitations of successful marrow transplantation. T cell-depleted (TCD) bone marrow transplantation (BMT) is reported to have a higher incidence of graft failure than unmodified (UM) BMT. In most cases of secondary graft failure, no cellular immune mechanism has been identified and etiology remains unclear. In an effort to delineate a cytokine-mediated mechanism of secondary graft failure, we investigated colony-forming unit-granulocyte/macrophage (CFU-GM) and burst-forming unit-erythroid (BFU-E) growth and pattern of inhibition by tumor necrosis factor-alpha (TNF-alpha) and gamma-interferon (IFN-gamma) in the early posttransplant period (day 28). Gradient-separated bone marrow mononuclear cells (BMMNC) from 38 recipients of TCD BMT, 15 recipients of UM BMT, and 23 normal donors (NLD) were plated in cultures of semisolid, serum-containing medium with the addition of stem cell factor (SCF), erythropoietin (Epo), and granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Three to seven times more CFU-GM and BFU-E colonies were cultures from NLD BM-derived BMMNC than from BMMNC of recipients of TCD or UM BMT (p = 0.0001). There was no difference in colony number between recipients of UM and TCD BMT on day 28 posttransplant, however. Under G-CSF culture conditions, CFU-GM colonies from recipients of UM and TCD BMT were more susceptible (p < or = 0.05) to suppression by IFN-gamma at concentrations of 1 and 100 U/mL than NLD BMMNC-derived colonies. No other difference in IFN-gamma inhibition was detected among the three groups. Under G-CSF and GM-CSF culture conditions, maximal inhibition was obtained at TNF-alpha concentrations > 10 ng/mL. Although early posttransplant BMMNC was more sensitive to inhibition than NLD-derived BMMNC, overall, no difference in colony growth or percent of inhibition induced by TNF-alpha or IFN-gamma was observed between recipients of unmodified and T. cell-depleted transplants. In this series, two recipients of TCD BM and one recipient of UM BMT developed graft failure; no distinct pattern of colony growth or colony inhibition was evident for those patients. The optimized in vitro conditions and specific cytokines used in this study do not indicate any quantitative or qualitative differences in the hematopoietic progenitors present in recipients of unmodified and T cell-depleted bone marrow early posttransplant to explain an increased risk of graft failure following a T cell-depleted BMT compared to an unmodified BMT.     

Effect of notoginsenoside R1 on the synthesis of components of the fibrinolytic system in cultured smooth muscle cells of human pulmonary artery

Manipulation of autologous bone marrow cells (BM) for transplantation in chronic myeloid leukemia (CML) to enrich for normal cells is a novel approach that may improve survival for patients not suitable for allogeneic transplantation. Limitations of this technique include the reported low frequency of normal stem cells in CML and the difficulties in obtaining sufficient BM for manipulation. To address these problems we compared the apheresis product with the diagnostic bone marrow at diagnosis as a source of primitive BCR/ABL-negative progenitors. We analyzed the CD34+ HLA-DR- and CD34+CD38(-) populations in five CML patients to evaluate the frequency of BCR-ABL-negative progenitors and pre-progenitors in these populations. Progenitor analysis was performed by RT-PCR of individual hemopoietic colonies from a standard CFU-GM assay. Analysis of pre-progenitors involved RT-PCR of secondary colonies derived from a stroma-free pre-CFU assay. Our results show variable levels of BCR-ABL-negative progenitors in the 34+DR- population but very low levels of BCR-ABL-negative progenitors in the 34+38- population in blood. Analysis of pre-progenitors from the 34+DR- fraction of peripheral blood (PB) and BM showed 80-100% and 85-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. Analysis of pre-progenitors from the 34+38- fraction of PB and BM showed 23-100% and 42-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. In summary, pre-progenitors from the 34+DR- and 34+38- populations are predominantly BCR-ABL negative in both marrow and blood at diagnosis. Apheresis product collected at diagnosis is a more abundant sources of BCR-ABL-negative pre-progenitors than BM. Thus, apheresis product could potentially be utilized as a source of BCR-ABL-negative stem cells in CML.     

Effects of stem cell factor (SCF) on human marrow neutrophil, neutrophil/macrophage mixed, macrophage and eosinophil progenitor cell growth

The effects of stem cell factor (SCF) on the subpopulations of granulocyte/macrophage colony-forming units (CFU-GM) were examined. Hematopoietic progenitor cells were enriched from normal adult bone marrow specimens by immunomagnetic beads using an anti-CD34 antibody and lineage marker antibodies for positive selection and negative selection, respectively. SCF enabled neutrophil and neutrophil/macrophage mixed progenitors to respond to granulocyte/macrophage colony-stimulating factor (GM-CSF) or interleukin 3 (IL-3) and to develop the colony and further cluster formation. The neutrophil colonies stimulated by GM-CSF or IL-3 consisted mainly of immature cells, while the colonies stimulated by granulocyte colony-stimulating factor (G-CSF) consisted of mature neutrophils irrespective of the addition of SCF. In macrophage and eosinophil lineages, SCF augmented the colony formation in the presence of GM-CSF or IL-3, whereas the enhancement of total progenitor cell growth (colonies plus clusters) was not so marked as compared with the neutrophil lineage. Time-course observation revealed that SCF could stimulate macrophage and eosinophil progenitors to form colonies rapidly. These findings indicate that SCF acts on late myeloid progenitor cells in manners different from the lineages of commitment.