Enhancement of erythroid differentiation in clones of human leukemic cell line K562 by fetal calf serum

Clone cells of K562 that are able to synthesize hemoglobin spontaneously on a relatively high level were obtained by cell-cloning technique. The clone cell proliferated 25 times by day 6 in culture, and the growth rate was not affected by changing the dose of fetal calf serum (FCS) from 5% to 30%. On the other hand, the erythroid differentiation could be linearly enhanced by increasing dosage of FCS, reaching a maximum after four days in culture. The wild-type K562 cells were also slightly stimulated to synthesize hemoglobin by adding FCS (30% final concentration). The enhancing effect of 30% FCS on the erythroid differentiation in the clone cells was greater than that of 12.5 microM hemin, while in the wild-type cells the relationship was reversed. There were no effects of erythropoietin (Epo) on the hemoglobin synthesis in either the clone cells or the wild-type cells. When various kinds of sera were added to the standard culture of the clone cells, only FCS had the enhancing effect. These results suggest that spontaneous erythroid differentiation is not induced by hemin or Epo in FCS but by FCS-specific substance(s).     

Characterization of human erythroid burst-promoting activity derived from bone marrow conditioned media

Bone marrow conditioned media (BMCM) increases burst number and the incorporation of 59Fe into heme by bursts when peripheral blood or bone marrow cells are cultured at limiting serum concentrations. Burst- promoting activity (BPA) has now been purified approximately 300-fold from this source by ion-exchange chromatography on DEAE-Sephadex and absorption chromatography on hydroxyapatite agarose gel. Marrow BPA increased burst number and hemoglobin (Hb) synthesis in a dose- dependent manner. A larger increase in Hb synthesis than in burst number was consistently observed, which was probably a consequence of the increase in the number of cells per burst that occurs in the presence of BPA. The role of BPA in culture could be distinguished from erythropoietin (Ep), since no bursts grew in the absence of Ep, whether or not BPA was present, and since it had no effect on the growth of erythroid colonies scored at day 5 of culture. Our purified fraction did not support the growth of CFU-C in culture. Activity was stable at temperatures of 70 degrees C or lower for 10 min; exposure to 80 degrees C resulted in approximately 50% loss of activity. BPA was completely inactivated by treatment at 100 degrees C for 10 min. Thus, human bone marrow cells produce a heat-sensitive factor that specifically promotes the growth of early erythroid progenitors in culture.     

Embryonic-fetal erythroid characteristics of a human leukemic cell line.

We have studied a number of cell surface, enzyme, and protein markers in the human leukemic K562 cell line. We have confirmed previous observations that these cells accumulate human embryonic hemoglobins after exposure to hemin. In addition, our results demonstrated that these cells possess in the "ininduced" state i surface antigen, lactate dehydrogenase isoenzymes characteristic of embryonic or fetal erythroid cells, fetal and embryonic globin chains, and globin mRNAs. The levels if i antigen, embryonic globin chains, and embryonic globin mRNA increased substantially after exposure of the cells to hemin in suspension culture. In contrast, K562 cells lacked several surface, enzymatic, and functional properties typical of granulocytes, lymphocytes, monocytes, or adult erythroblasts, including HLA surface antigens, surface immunoglobulins, sheep erythrocyte rosetting, phagocytosis, terminal deoxynucleotidyl transferase, carbonic anhydrase, ABO and Rh blood groups, and adult hemoglobins. The K562 cell line therefore exhibits phenotypic properties of embryonic erythroid progenitor cells and a quantitative increase in the expression of some of these properties can be achieved by exposure of the cells to hemin.     

Production of erythroid potentiating factor(s) by a human monocytic cell line

U-937 is a human monocytic cell line that has been to elaborate factors that affect normal human hematopoiesis in vitro. Studies on the effects of these factors demonstrated an erythroid potentiating factor (EPF) and a potent inhibitor of granulocyte-macrophage (CFU-GM) colony growth. The EPF was present in both serum-containing and serum-free U- 937 conditioned media, had a dose-dependent effect on erythroid colony formation and was remarkably heart stable. The CFU-GM inhibitory activity was also detected in serum-free conditioned medium, was dose- dependent, heart labile and its effect was reversed by Indomethacin. Indomethacin (Sigma, St. Louis, Mo.) did not alter the erythroid effects of the U-937 conditioned medium. No colony stimulating factor (CSF) or erythropoietin (Ep) could be detected in this medium. The existence of a human cell line capable of production EPF without simultaneous CSF production will permit further studies on the biochemical and biologic nature of these factors.     

Human spleen cell generation of factors stimulating human pluripotent stem cell, erythroid, and myeloid progenitor cell growth

Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.     

Murine erythroid colony-promoting factor derived from thymocyte conditioned media

It has been shown that murine thymocytes have some effects on the proliferation and differentiation of hemopoietic stem cells in mice. However, the precise mechanisms are not well known. We, therefore, investigated the effects of thymocytes on the proliferation of erythroid progenitors (CFUE) by using a normal murine syngeneic system. The addition of thymocytes to marrow cultures increased the number of CFUE colonies from marrow cells. Furthermore, conditioned medium prepared from thymocytes enhanced the in vitro colony growth of CFUE in the presence of erythropoietin. This enhancing factor(s) had a molecular weight of about 32,000 daltons and was heat stable at 70 degrees C for 30 min. We have concluded that murine thymocytes promote erythroid colony formation in vitro by producing an erythropoietic stimulating factor(s), although its detailed characteristics remain to be elucidated.     

Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum-free cell-culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum-free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL-3 was TPO able to stimulate a small increase (∼25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34⁺ cells were cultured for 7 d in serum-free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre-cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit-erythroid (BFU-E) colonies as cells which were pre-cultured in the absence of TPO. Further, when primitive CD34⁺, Kit⁺ MNC were cultured for 3–7 d under serum-free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.     

Origin and characterization of a human bipotent liver progenitor cell line

BACKGROUND & AIMS: Liver progenitor cells may be important in carcinogenesis resulting from human chronic liver diseases. The HepaRG cell line has been established from a liver tumor associated with chronic hepatitis C. We observed that these cells showed an evident morphological heterogeneity, displaying both hepatocyte-like and biliary-like epithelial phenotypes. Our goal was to determine whether they could share some features with liver progenitor cells. METHODS: Phenotypic studies using immunofluorescence, immunoblotting, and flow cytometry were performed at different culture stages. RESULTS: HepaRG cells progressively exhibited polarized and functional hepatocytes and bile duct-like cell features under defined conditions. Cytokeratin 18 and 19 coexpression was, however, observed all along the maturation process together with oval cell-specific markers (M2-PK, OV-1, OV-6, and CD34); kinetics and expression profiles were dependent on the cell population. In addition, a strong commitment toward the hepatocytic lineage could be observed in the presence of epidermal growth factor. Immunohistochemistry on the fibrotic liver showing the atypical ductular reaction from which HepaRG cells originated displayed a comparable immunophenotype specifically restricted to bile neoductules. CONCLUSIONS: HepaRG cells constitute the first described human hepatic bipotent progenitor cell line regarding phenotype and histological origin.     

Enhancement of erythroid colony growth in culture by hemin.

Hemin was found to enhance the growth of murine erythroid colonies in culture. In the presence of 100 mU/ml erythropoietin (EPO), the addition of hemin (0.05-0.2 mM) resulted in the growth of twice as many colonies as were obtained with EPO alone. Hemin also significantly increased erythroid colony formation in culture in the absence of added EPO. Hemoglobin synthesis as measured by the incorporation of 59Fe into cyclohexanone extractable heme was augmented in culture by hemin. Neither delta-aminolevulinic acid, a hemin precursor, nor FeCl3 increased colony number.     

Characterization of hemopoietic activities in media conditioned by a murine marrow-derived adherent cell line, B.Ad

The hemopoietic activities present in medium conditioned by a murine bone marrow-derived adherent cell line (B.Ad) have been studied. B.Ad-conditioned medium stimulated neutrophil, neutrophil-macrophage, and macrophage colonies in agar cultures of bone marrow cells and 90% of this activity was neutralized by antimacrophage colony-stimulating factor (anti-M-CSF). The conditioned medium supported the generation and/or maintenance of spleen colony-forming units (CFU-S) in liquid cultures and synergized with multilineage colony-stimulating factor (Multi-CSF; IL-3) to stimulate colony formation by day-3 post-5-fluorouracil (FU)-treated bone marrow cells. When used as feeder layers, B.Ad cells stimulated erythroid colony-forming units (CFU-E) and markedly enhanced erythroid burst-forming units (BFU-E) stimulation more than did maximal Multi-CSF (IL-3) and Epo stimulation. No CFU-E- or BFU-E-stimulating activities were detected in medium conditioned by B.Ad cells. Similarly, B.Ad-conditioned medium was unable to stimulate Multi-CSF (IL-3) or granulocyte-macrophage (GM)-CSF-dependent cell lines. The data suggest that medium conditioned by this bone marrow-derived adherent cell line contains M-CSF and other factors not detectable as CSFs that either directly or by means of a synergistic mechanism are able to stimulate CFU-S and colony-forming cells (CFC).     

Abnormal erythroid progenitor cells in human preleukemia

Ten patients with preleukemia were studied by the erythroid cell clonal culture technique. In nine of these patients, erythroid colonies derived from peripheral blood BFU-E were not observed, while the other patient had markedly decreased peripheral blood BFU-E-derived erythroid colonies in vitro. In three patients, marrow cells were also cultured and no BFU-E-derived erythroid colonies were detected. These studies indicate that immature erythroid progenitor cells, BFU-E, in patients with preleukemia are either markedly decreased in number or grossly defective in their proliferative or differentiative capacities.     

In vitro differentiation of a human erythroid cell line (KMOE) induced by some metabolic inhibitors

Summary KMOE-2/05, a continuous human erythroid cell line derived from a patient with acute erythremia was capable of differentiating into benzidine positive cells following exposure to cytosine arabinoside (CA), mitomycin C, or daunorubicin. Among the three substances, CA was the most effective inducer. Other compounds, reported as effective inducers on human and murine erythroid cells, were also tested but they were ineffective.Benzidine positive cells were counted to be approximately 500/1×10⁵ cells after 10 days incubation with CA at its optimal concentration of 1×10^–5 M. Under the same conditions, the hemoglobin (Hb) concentration quantitated by radioimmunoassay (RIA) was more than 500 ng/l×10⁶ cells. Quantitative kinetics of synthesized Hb and of benzidine-positive cell counts, after exposure to CA were closely correlated.This work was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Public Health and Welfare of Japan and also by a Research Grant from the Cancer Prevention Association in Fukuoka     

Further characterization of the hemin-induced enhancement of primitive erythroid progenitor cell growth in vitro

We have previously demonstrated that hemin specifically enhances the in vitro plating efficiency of primitive murine erythroid progenitors (day 7 BFUE), whereas it does not appear to affect more mature progenitors (mature BFUE or CFUE). In this report, we further characterize the effects of hemin on marrow-derived day 7 BFUE growth in vitro. BFUE were enhanced by hemin in a dose-dependent manner and to a greater extent in methyl cellulose than in plasma clot cultures. That hemin might increase the rate of cell division was suggested by the greater size of colonies grown in its presence as well as their earlier appearance in culture. In contrast, the addition of hemin to marrow cell cultures did not appear to affect the survival rate of BFUE or their progeny. While significantly augmenting the frequency of BFUE, hemin had no consistent stimulatory effect on CFUGM. Lastly, hemin was equally capable of augmenting burst growth in adherent cell-depleted as in whole marrow cell preparations. These experiments suggest that hemin augments directly and in a cell-specific manner the proliferation and/or differentiation of primitive marrow erythroid progenitors in vitro.     

Resveratrol inhibits cell growth in a human cholangiocarcinoma cell line

Background/Aims: Cholangiocarcinoma is a devastating tumour with a poor prognosis. An efficient therapy is unavailable in unoperable patients and new drugs are widely sought for and required. Resveratrol (RES) is a natural molecule with a reported anticancer effect, evaluated on different tumour cell lines. We tested the efficacy of RES on a cholangiocarcinoma cell line for the first time. Methods: We used the human SK‐ChA‐1 cell line, cultured in the classical two‐dimensional model and in the three‐dimensional spheroids. After RES exposure morphology, cell viability (colony‐forming assay), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and cancer antigen (CA) 19‐9 medium releases, cellular transglutaminase activity, karyotype and cell cycle were evaluated. Results: Resveratrol inhibited cell growth in both the cell culture systems used (from ?15 to ?80% vs untreated controls) and induced a 40‐fold increase of LDH and ALP activities in the culture medium. Also, transglutaminase (TG) activity increased in the cell lysates, together with a cell cycle perturbation characterised by an accumulation in the G₁/S phase. Karyotype and CA 19‐9 expression were not influenced by the treatment. Conclusions: The observed cytotoxic effect of RES on the human cholangiocarcinoma SK‐ChA‐1 cell line cultured two‐ and three‐dimensionally suggests to further analyse its chemotherapic/chemopreventive possibilities for this kind of cancer.