Selection of lineage-restricted cell lines immortalized at different stages of hematopoietic differentiation from the murine cell line 32D

Erythropoietin (Epo), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor- (G-CSF) dependent cell lines have been derived from the murine hematopoietic cell line 32D with a selection strategy involving the culture of the cells in FBS-deprived medium supplemented only with pure recombinant Epo, GM-CSF, or G-CSF. The cells retain the diploid karyotype of the original 32D clone, do not grow in the absence of exogenous growth factor, and do not induce tumors when injected into syngeneic recipients. The morphology of the Epo-dependent cell lines (32D Epo1, -2, and -3) was heterogeneous and evolved with passage. The percent of differentiated cells also was a function of the cell line investigated. Benzidine-positive cells ranged from 1-2% (32D Epo3) to 50-60% (32D Epo1). These erythroid cells expressed carbonic anhydrase I and/or globin mRNA but not carbonic anhydrase II. The GM-CSF- and G-CSF-dependent cell lines had predominantly the morphology of undifferentiated myeloblasts or metamyelocytes, respectively. The GM-CSF-dependent cell lines were sensitive to either GM-CSF or interleukin-3 (IL-3) but did not respond to G-CSF. The G-CSF-dependent cell lines grew to a limited extent in IL-3 but did not respond to GM-CSF. These results indicate that the cell line 32D, originally described as predominantly a basophil/mast cell line, has retained the capacity to give rise to cells which proliferate and differentiate in response to Epo, GM-CSF, and/or G-CSF. These cells represent the first nontransformed cell lines which can be maintained in growth factors other than IL-3 and which differentiate in the presence of physiologic signals. As such, they may represent a model to study the molecular mechanisms underlying the process of hematopoietic differentiation, as well as sensitive targets for bioassays of specific growth factors.     

Erythropoietin-dependent autocrine secretion of tumor necrosis factor-alpha in hematopoietic cells modulates proliferation via MAP kinase--ERK-1/2 and does not require tyrosine docking sites in the EPO receptor

Primary erythroid cells and erythroid cell lines may synthesize and secrete tumor necrosis factor-alpha (TNF-alpha) following stimulation with erythropoietin (EPO). The effect of triggering TNF-alpha synthesis and secretion was investigated in erythroleukemia and myeloid cell lines: HCD57, DA3-EPOR, and BAF3-EPOR. The EPO-induced, membrane-bound form of autocrine TNF-alpha seemed to enhance proliferation of HCD57 and DA3-EPOR cells; however, the concentration of secreted autocrine/paracrine TNF-alpha was never sufficient to have an effect. Autocrine TNF-alpha acts through TNFRII receptors to stimulate proliferation. Modulation of mitogen-activated protein kinase (MAPK)/extracellular signal-related kinase (ERK-1/2) activity by the membrane-bound form of autocrine TNF-alpha apparently played a central role in the control of EPO-dependent proliferation of HCD57 and DA3-EPOR cells. Primary erythroid cells and DA3-EPOR cells were found to express similar, high levels of both TNFRI and TNFRII, showing that differential expression of TNF-alpha receptors does not explain why primary cells are inhibited and DA3-EPOR cells are stimulated by autocrine TNF-alpha. BAF3 cells expressing a mutant EPOR with no cytoplasmic tyrosine residues were capable of triggering EPO-dependent TNF-alpha synthesis and secretion, indicating that tyrosine-docking sites in the EPOR were not required for EPO-dependent TNF-alpha secretion.     

Growth factor receptor expression during in vitro differentiation of partially purified populations containing murine stem cells

We have investigated, by semiquantitative RT-PCR, the kinetics of activation of hematopoietic receptors and differentiation markers in partially purified murine hematopoietic stem cells (HSC) induced to differentiate in serum-free culture with combinations of growth factor (GF). The combinations of GF used sustained either multilineage [stem cell factor (SCF) + interleukin 3 (IL-3)], or erythroid [SCF + IL-3 + erythropoietin (Epo)] or myeloid [SCF + IL-3 + granulocyte colony-stimulating factor (G-CSF)] differentiation. The GF receptor genes investigated were the α and β subunits of the IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor, the erythropoietin receptor, the G-CSF receptor, and c-Fms, the receptor for macrophage colony-stimulating factor (M-CSF). The expression of Gata1 and α- and β-globin was investigated at the same time as a marker of erythroid differentiation. HSC were purified according to standard protocols, which include partitioning of lineage-negative bone marrow cells with the mitochondrial dye Rhodamine 123 (Rho) into Rho-dull (≥17% of which reconstitute long-term hematopoiesis in recipient mice) and into Rho-bright (which are as capable as Rho-dull of multilineage differentiation but do not permanently reconstitute the host). The following pattern of expression was observed: the α subunit of the IL-3 receptor clearly was expressed in both Rho-bright and Rho-dull cells at the outset, and its expression did not change over time in culture. The β subunits of the IL-3 and GM-CSF receptor, the α subunit of the GM-CSF receptor, the Epo and G-CSF receptors and Fms barely were expressed in purified Rho-bright and Rho-dull cells, but their expression increased in cells cultured both in erythroid and in myeloid GF combinations. Gata1 was expressed maximally in Rho-bright cells but was below the level of detection in Rho-dull cells. Rho-dull cells expressed Gata1 when cultured both in erythroid and in myeloid GF combinations. In contrast, α- and β-globin, which also were not expressed in the purified cells, were induced only in cells stimulated with Epo. These results indicate that the genes for all the GF receptors investigated (with the exception of the α subunit of the IL-3 receptor) are expressed at low levels, if any, in purified Rho-bright or Rho-dull cells, but are expressed in their progeny cultured either in erythroid or myeloid GF combinations. The expression of the Epo receptor,in particular, is activated both in erythroid (α- and β-globin positive) and in myeloid (α- and β-globin negative) cells. Therefore, activation of the expression of the Epo receptor gene and activation of the erythroid differentiation program are two independent events in normal hematopoiesis. J. Cell. Physiol. 171:343–356, 1997. © 1997 Wiley-Liss, Inc.     

Direct effects of IL-4 on the in vitro differentiation and proliferation of hematopoietic progenitor cells

The purpose of this study was to analyze the effects of recombinant human interleukin 4 (IL-4) on the differentiation and proliferation in vitro of human granulocyte/macrophage (GM) and erythroid progenitors. IL-4 was added to either fetal bovine serum (FBS)-supplemented or to FBS-deprived cultures of unfractionated human marrow cells or marrow cells depleted of adherent and/or T cells. Paradoxical effects similar to those reported in the murine system were detected in these experiments. In FBS-supplemented cultures, IL-4, which had no effect on the growth or erythroid bursts (from burst-forming cells; BFU-E) detected in the presence of Epo alone, decreased by 46% the number of erythroid bursts detected in the presence of Epo and phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM). In contrast, in FBS-deprived cultures, IL-4 increased by 30-700% the number of erythroid bursts in cultures containing Epo alone or containing Epo, IL-3, and GM-CSF. The stimulatory effect of IL-4 on erythroid burst growth under FBS-deprived conditions was particularly evident when adherent cells were removed. Under the conditions investigated, IL-4 had little effect on the growth of GM colonies. In FBS-deprived suspension cultures of nonadherent, T-cell-depleted marrow cells, IL-4 maintained both the number of BFU-E and CFU-GM for at least 8 days. In these cultures, IL-4 antagonized the capacity of IL-3 to increase the number of BFU-E but IL-4 and IL-3 acted together to maintain the number of CFU-GM. To determine if IL-4 acted directly or indirectly, its effects on the growth of factor-dependent subclones of the murine progenitor cell line 32D were analyzed. Three subclones were studied: the original IL-3-dependent clone 32D cl.3, the Epo-dependent erythroid clone 32D Epo-1, and the G-CSF-dependent myeloid clone 32D G-1. IL-4 alone failed to induce colony growth from these cell lines. However, IL-4 inhibited by 25% the number of colonies formed by 32D cl.3 in the presence of IL-3 while increasing by 25% and 25-50% the number of colonies formed by 32D Epo-1 and 32D G-1 in the presence of Epo or G-CSF, respectively. These results indicate that human IL-4, as its murine counterpart, is a multilineage growth factor with paradoxical effects which are mediated by the direct action of IL-4 on progenitor cells.     

Bone marrow stromal cells selectively stimulate the rapid expansion of lineage-restricted myeloid progenitors

Bone marrow stromal cells serve hematopoietic microenvironments where different blood cells are controlled in their growth and differentiation. To characterize functions of stromal cells, 33 bone marrow stromal cells including preadipocytes, endothelial cells, and fibroblasts were established from transgenic mice harboring temperature-sensitive SV40 T-antigen gene and their selective stimulatory abilities to support large colony formation of lineage-specific hematopoietic progenitor cells (erythroid, monocyte/macrophage, granulocyte, and monocyte-granulocyte) were examined. Among established stromal cells, 27 clones showed erythropoietic stimulatory activity in the presence of erythropoietin. On myeloid progenitors, the stromal cells showed lineage-restricted stimulatory activity and a reciprocal relationship was observed between granulocyte formation and macrophage formation, but these activities were not dependent on the amount of produced colony-stimulating factors (CSFs). Our present study with many stromal cells established from bone marrow indicated that each stromal cell in the bone marrow may provide the preferable microenvironment for a rapid expansion of the lineage-restricted progenitor cells in combination with CSFs. © 1995 Wiley-Liss, Inc.     

Distinct cytoplasmic regions of the human granulocyte colony-stimulating factor receptor involved in induction of proliferation and maturation.

The granulocyte colony-stimulating factor receptor (G-CSF-R) transduces signals important for the proliferation and maturation of myeloid progenitor cells. To identify functionally important regions in the cytoplasmic domain of the G-CSF-R, we compared the actions of the wild-type receptor, two mutants, and a natural splice variant in transfectants of the mouse pro-B cell line BAF3 and two myeloid cell lines, 32D and L-GM. A region of 55 amino acids adjacent to the transmembrane domain was found to be sufficient for generating a growth signal. The immediate downstream sequence of 30 amino acids substantially enhanced the growth signaling in the three cell lines. In contrast, the carboxy-terminal part of 98 amino acids strongly inhibited growth signaling in the two myeloid cell lines but not in BAF3 cells. Truncation of this region lead to an inability of the G-CSF-R to transduce maturation signals in L-GM cells. An alternative carboxy tail present in a splice variant of the G-CSF-R also inhibited growth signaling, notably in both the myeloid cells and BAF3 cells, but appeared not to be involved in maturation.     

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.     

5-azacytidine induces transgene silencing by DNA methylation in Chinese hamster cells

The cytosine analog 5-azacytidine (5-AzaC) is a demethylating agent that is also known to induce mutagenesis in mammalian cells. In this study, the mutagenic potential of this drug was tested in the G10 and G12 transgenic Chinese hamster cell lines, which have a single bacterial gpt gene integrated into the genome at different sites, with its expression driven by a simian virus 40 (SV40) promoter. We show that the mutation frequencies following a 48-h exposure to different concentrations of 5-AzaC were 10 to 20 times higher than those of any of the other numerous mutagens that have been tested in the G10-G12 system. Moreover, the mutation frequencies were much higher in the G10 cell line than in the G12 cells. Detailed molecular analysis of the 6-thioguanine (6-TG)-resistant variants demonstrated that transgene silencing by de novo DNA methylation and increased chromatin condensation in the SV40 promoter was the major factor responsible for this high level of 6-TG resistance. As would be expected, exposure to 5-AzaC lowered the overall genomic DNA methylation levels, but it unexpectedly caused hypermethylation and increased chromatin condensation of the transgene in both the G10 and G12 cell lines. These results provide the first evidence that 5-AzaC may also induce transgene-specific DNA methylation, a phenomenon that can further be used for the elucidation of the mechanism that controls silencing of foreign DNA.     

Bone morphogenetic proteins act synergistically with haematopoietic cytokines in the differentiation of haematopoietic progenitors

We examined the effects of bone morphogenetic protein-2 (BMP-2), -3, -4, -5, -6, and -7 on the proliferation and differentiation of bone marrow CD34+ haematopoietic progenitors in semi-solid medium. The BMPs had no effect on haematopoietic colony development when added to medium containing erythropoietin (Epo) or Interleukin-3 plus Epo. Synergistic effects with the haematopoietic cytokines stem cell factor (SCF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were observed. In conjunction with GM-CSF and Epo, BMP-4 increased the number of both erythroid and granulocyte/monocyte colonies formed in semi-solid medium (P<0.01). No other BMP stimulated erythroid colony development under these conditions, while BMP-3, BMP-7 (P<0.01), BMP-5, and BMP-6 (P<0.05) stimulated granulocyte/monocyte colony formation. BMP-7 acted synergistically with stem cell factor to increase granulocyte/monocyte colony formation but not erythroid colony formation. The other BMPs did not affect either erythroid or granulocyte/monocyte colony development under these conditions. These results suggest that individual BMPs form part of the complement of cytokines regulating the development of haematopoietic progenitors, and in particular, point to a role for BMP-4 in the control of definitive, as well as embryonic erythropoiesis.     

5-Azacytidine up regulates the expression of Epstein-Barr virus nuclear antigen 2 (EBNA-2) through EBNA-6 and latent membrane protein in the Burkitt''s lymphoma line rael.

Nonproductive infection of B lymphocytes by Epstein-Barr virus (EBV) is associated with a highly restricted expression of viral genes. In growth-transformed lymphoblastoid cell lines, the products of these genes include a complex of at least six EBV nuclear antigens (EBNAs) (EBNA-1 through EBNA-6) and one membrane protein (latent membrane protein [LMP]). EBV-carrying Burkitt's lymphoma (BL) biopsies and derived cell lines that have retained a representative phenotype (group I BL lines) express only EBNA-1 (M. Rowe, D. T. Rowe, C. D. Gregory, L. S. Young, P. J. Farrell, H. Rupani, and A. B. Rickinson, EMBO J. 6:2743-2751, 1987). We have found that EBNA-2 through EBNA-6 and LMP can be up regulated by treating the group I BL line Rael with the DNA-demethylating agent 5-azacytidine (5-AzaC). The drug acted in a time- and dose-dependent manner. EBNA-2-positive cells were detected by anti-complement immunofluorescence staining just 12 h after addition of 4 microM 5-AzaC and reached a maximum number at 72 h, when up to 75% of the population was positive. EBNA-2, EBNA-3, EBNA-4, EBNA-4, EBNA-6, and LMP were demonstrated immunoblots starting at 48 h. The EBV-encoded early antigens and viral capsid antigens were also induced but at a lower level. EBNA-2 and the lytic cycle-associated antigens appeared with a different time course and in largely nonoverlapping cell subpopulations, as demonstrated by double fluorescence staining. Thus, EBNA-2 expression was not restricted to lytically infected cells, nor was EBNA-2 required for entry into the lytic cycle. The coding and regulatory sequences of EBNA-2 and LMP were found to be highly methylated in Rael cells and were, as expected, demethylated after 5-AzaC treatment. These findings suggest that DNA methylation may participate in the regulation of growth transformation-associated viral genes in BL cells.     

Effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on single CD34-positive hemopoietic progenitors from human bone marrow

To determine the extent accessory cells mediate the effects of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) on human hemopoietic progenitors in vitro, we added this hemopoietin to liquid cultures of single CD34-positive marrow cells. These were selected on a fluorescence-activated cell sorter using the HPCA-1 (My10) antibody. Myeloid, erythroid and a few mixed clones developed in 13% of wells in the apparent absence of accessory cells at the beginning of culture. Although accessory cells were generated quickly from the myeloid progenitors and could have mediated the action of rhGM-CSF, this was not the case in the majority of the erythroid clones in which no other cell types were recorded. We conclude that rhGM-CSF can act directly on a subset of erythroid progenitors and probably induces a substantial number of myeloid clones directly.     

Slashing the timelines: Opting to generate high‐titer clonal lines faster via viability‐based single cell sorting

Chinese hamster ovary (CHO) cell line development (CLD) is a long and laborious process, which requires up to 5 ? 6 months in order to generate and bank CHO lines capable of stably expressing therapeutic molecules. Additionally, single cell cloning of these production lines is also necessary to confirm clonality of the production lines. Here we introduce the utilization of viability staining dye in combination with flow cytometer to isolate high titer clones from a pool of selected cells and single cell deposit them into the wells of culture plates. Our data suggests that a stringent selection procedure along with viability dye staining and flow cytometry‐based sorting can be used to isolate high expressing clones with titers comparable to that of traditional CLD methods. This approach not only requires less labor and consumables, but it also shortens CLD timelines by at least 3 weeks. Furthermore, single cell deposition of selected cells by a flow sorter can be regarded as an additional clonality assurance factor that in combination with Day 0 imaging can ensure clonality of the production lines. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:198–207, 2016     

Release of hemopoietic factors by normal human T cell lines with either suppressor or helper activity

We analyzed the release of activities capable of stimulating the in vitro growth of human hemopoietic progenitor cells by long-term cultured T cell growth factor (TCGF)-dependent human T lymphocytes. Seven cell lines tested produced colony-stimulating activity (CSA) as well as burst-promoting activity (BPA). The CSA stimulated primarily the growth of the cells forming colonies after 14 days of incubation. In addition the supernatants from these seven T-cell lines showed the ability to induce the in vitro growth of mixed granulocyte, erythroid, megakaryocyte, macrophage colonies (CFU-GEMM). The release of hemopoietic factors did not depend on the presence of accessory cells or phytohemagglutinin or serum during the incubation for factor production. In six of the T cell lines the majority of the cells were reactive to the OKT 8 monoclonal antibody (MoAb), whereas one cell line contained mostly OKT 4+ cells. Suppressor activity was detected in three tested OKT 8+ cell lines, while the one OKT 4+ displayed helper activity. All cell lines produced hemopoietic factors with equal efficiency. These results indicate that factors affecting human hematopoiesis are produced by normal T lymphocytes in long-term culture and this property is not related to the helper or suppressor activity of the cultured cells.     

Binding of iodinated multipotential colony-stimulating factor (interleukin-3) to murine bone marrow cells

Multipotential colony-stimulating factor (Multi-CSF or interleukin-3) was radioiodinated to high specific radioactivity (1-4 X 10(5) cpm/ng) with no detectable loss of biological activity and its binding to murine bone marrow cells and factor-dependent cell lines studied. Both the native glycosylated molecule purified from a cloned T-cell line (LB-3) and the purified non-glycosylated recombinant molecule produced by E. coli could be radioiodinated. Comparative binding studies with these derivatives demonstrated equal binding affinities and equal numbers of binding sites on various cell types indicating that carbohydrate moieties are not involved in the binding interactions. Binding of 125I-Multi-CSF to several factor-dependent continuous hemopoietic cell lines showed the presence of specific receptors on all cell lines, the receptor number per cell varying from 700 to 13,000 and the apparent dissociation constant from 400 pM to 1 nM. Specific binding of 125I-Multi-CSF was also observed to normal murine hemopoietic cells and the binding to murine bone marrow cells was studied in detail. Bone marrow cells showed 117-130 receptors per cell on average and an apparent dissociation constant of 126-233 pM. However, quantitative autoradiographic analysis indicated that receptors for 125I-Multi-CSF were not distributed randomly on bone marrow cells--nucleated erythroid and lymphoid cells were not labeled while essentially all neutrophilic granulocyte, eosinophilic granulocyte and monocytic cells were labeled. Moreover, in each of the labeled cell lineages grain counts (reflecting receptor number) decreased with increasing maturation and a small subpopulation of marrow cells (0.4-1.5% and including blast cells, monocytes, promyelocytes, and myelocytes) exhibited very high grain counts. The existence of such a subset of marrow cells raises the possibility of functional heterogeneity among marrow cells in their response to Multi-CSF.     

13q14断裂重排与非小细胞肺癌转移潜能关系的研究

肿瘤转移的细胞经常存在染色体数目异常和结构畸变,在多种有转移潜能的肿瘤细胞中都涉及到13q14的异常。以往研究表明在同一组织来源但转移潜能不同的肺腺癌细胞系AGZY83-a和Anip973中存在13q14的断裂重排。采用mRNA差异展示技术（mRNA DD）分析这一对细胞系得到的差异表达基因BRI基因位于13q14。为了进一步分析肺癌细胞的转移潜能与13q14断裂重排间的关系,采用13q涂染探针对具有不同转移潜能的非小细胞肺癌细胞系PAa、SPC-1-A和95D中期分裂相进行G显带后的荧光原位杂交分析。结果发现在3个肺癌细胞系中有多种13号染色体长臂的结构异常,其中此3个细胞系均涉及13q32-33的频发断裂。但是低转移肺癌细胞系PAa、SPC-1-A均未涉及13q14的断裂,而高转移肺癌细胞系95D的两种细胞克隆均可见13q14的断裂。提示13q14断裂点与肺癌细胞的转移能力有一定的相关性,两者之间的遗传学意义需要进一步研究探索。