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G Migliaccio

Publications and source records attributed to G Migliaccio.

At least 37 records · Page 2Linked to original sources
PubMed1998

The making of an erythroid cell. Molecular control of hematopoiesis.

The number of circulating red cells is regulated by the daily balance between two processes: the destruction of the old red cells in the liver and the generation of new cells in the bone marrow. The process during which hematopoietic stem cells generate new red cells is called erythropoiesis. This manuscript will describe the molecular mechanisms involved in the process of erythroid differentiation as we understand them today. In particular it will review how erythroid specific growth factor-receptor interactions activate specific transcription factors to turn on the expression of the genes responsible for the establishment of the erythroid phenotype.

Animals↗
PubMed1998

Stem cell factor induces proliferation and differentiation of fetal progenitor cells in the mouse.

We have investigated the kinetics of the amplification of the progenitor cell compartments (CFC) in haemopoietic organs during murine ontogenesis and compared the growth requirements of fetal and adult CFC. Two haemopoietic phases were recognized in the fetal liver (FL): an exponential growth phase, from 11.5 to 15.5 d post conception (p.c.), during which the mean number of nucleated cells and of CFC in the FL increased from 4.9 x 10(5) to 7.0 x 10(7) and from 4.5 x 10(3) to 2.7 x 10(5), respectively, and a recessive phase after 15.5 d p.c., during which the CFC number in the FL gradually decreased, although some CFC were still detectable in the liver after birth. In serum-deprived cultures, FL and adult marrow (AM) CFC had similar responses to GM-CSF, and did not respond to G-CSF or IL-3. In contrast, FL, but not AM, erythroid colonies grew Epo-independently whereas SCF alone induced formation of maximal numbers of erythroid bursts from FL, but not from AM cells. The proliferative and differentiative effect of SCF alone on fetal cells was confirmed in serum-deprived cultures of purified early progenitor cells isolated by cell sorting on the basis of multiple parameters from FL and AM light-density cells. In culture of purified FL cells, SCF alone induced a similar amplification of total cells (maximal amplification at day 12: 800-300-fold) and total CFC (11-38-fold of maximal amplification at day 6) to the combination of SCF plus IL-3 (1300-800-fold amplification of total cells and 31-88-fold amplification of CFC). In contrast, SCF alone allowed only survival of purified AM early progenitor cells. Therefore FL early progenitor cells have an intrinsic higher potential than their adult counterpart to respond to SCF, confirming the potent role of this growth factor in the development of the murine haemopoietic system.

Animals↗
PubMed1998

Thymus-independent T-cell differentiation in vitro.

The generation of large quantities of novel human T-cell clones ex vivo would make a wide range of gene- and immuno-therapies for tumours and viral infections possible. Several techniques have been described to generate, in vitro and in vivo (using xenogenic hosts), mature T cells from fetal-neonatal and adult human CD34+ cells. All these techniques are cumbersome and cannot be easily translated into clinical protocols because they involve co-cultivation of CD34+ cells with thymic fragments from either human or murine fetuses. We report that the mononuclear cells of human cord blood contain a cell population that supports the differentiation of CD34+ cells into CD4+ or CD8+ naive T cells in serum-deprived cultures stimulated with stem cell factor and interleukin 7. CD4+ or CD8+ CD45RA+ TCRalphabeta+ T cells were continuously produced in vitro over a period of 20 d under these conditions. The generation of T cells in these cultures was a dynamic process and clones of T cells expressing new T-cell receptor beta-chain rearrangements were generated over time. These results pave the way for the development of very simple culture conditions for ex-vivo production of naive helper or cytotoxic T cells which could be very useful for gene- and immuno-therapy of human diseases.

Adult↗
PubMed1997

Expression in hematopoietic cells of GATA-1 transcripts from the alternative "testis" promoter during development and cell differentiation.

GATA-1 is a transcription factor expressed both in the hematopoietic system and in the Sertoli cells of the testis, and is essential for correct erythropoiesis. Hematopoietic and Sertoli cells transcribe GATA-1 from two different promoters: the proximal (erythroid) is active in hematopoietic cells; the distal (testis) is active in Sertoli cells. We investigated by RT-PCR the possibility that GATA-1 might be transcribed from the testis promoter also in hematopoietic cells. Testis promoter-derived transcripts are present at low levels in vivo at all stages of hematopoietic development. Purified multipotent progenitors, fractionated into populations expressing low or high levels of GATA-1, do not contain any "testis" transcripts. However, when grown in vitro, they rapidly express GATA-1 from the testis promoter in the presence of Erythropoietin (Epo) but not in that of other growth factors. This result reflects an Epo-dependent differentiation event, rather than a direct effect of Epo. Indeed, immortalized progenitor cell lines which respond to both Epo and SCF, continue to express testis-derived transcripts when switched from Epo to SCF.

Alternative Splicing↗
PubMed1997

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 alpha and beta 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 alpha- and beta-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 (> or = 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 alpha 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 beta subunits of the IL-3 and GM-CSF receptor, the alpha 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, alpha- and beta-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 alpha 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 (alpha- and beta-globin positive and in myeloid (alpha- and beta-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.

Animals↗
PubMed1997

Chimeric Sindbis viruses dependent on the NS3 protease of hepatitis C virus.

The hepatitis C virus (HCV) NS3 protease cleaves the viral polyprotein at specific sites to release the putative components of the HCV replication machinery. Selective inhibition of this enzyme is predicted to block virus replication, and NS3 is thus considered an attractive candidate for development of anti-HCV therapeutics. To set up a system for analysis of NS3 protease activity in cultured cells, we constructed a family of chimeric Sindbis viruses which carry sequences coding for NS3 and its activator, NS4A, in their genomes. HCV sequences were fused to the gene coding for the Sindbis virus structural polyprotein via an NS3-specific cleavage site, with the expectation that processing of the chimeric polyprotein, nucleocapsid assembly, and generation of viable viral particles would occur only upon NS3-dependent proteolysis. Indeed, the chimeric genomes encoding an active NS3 protease produced infectious viruses in mammalian cells, while those encoding NS3 inactivated by alanine substitution of the catalytic serine did not. However, in infected cells chimeric genomes recombined, splicing out HCV sequences and reverting to pseudo-wild-type Sindbis virus. To force retention of HCV sequences, we modified one of the initial chimeras by introducing a second NS3 cleavage site in the Sindbis virus portion of the recombinant polyprotein, anticipating that revertants not encoding an active NS3 protease would not be viable. The resulting chimera produced infectious viruses which replicated at a lower rate than the parental construct and displayed a marked temperature dependence in the formation of lysis plaques yet stably expressed NS3.

Animals↗
PubMed1997

In vitro study of the NS2-3 protease of hepatitis C virus.

Processing at the C terminus of the NS2 protein of hepatitis C virus (HCV) is mediated by a virus-encoded protease which spans most of the NS2 protein and part of the NS3 polypeptide. In vitro cotranslational cleavage at the 2-3 junction is stimulated by the presence of microsomal membranes and ultimately results in the membrane insertion of the NS2 polypeptide. To characterize the biochemical properties of this viral protease, we have established an in vitro assay whereby the NS2-3 protease of HCV BK can be activated posttranslationally by the addition of detergents. The cleavage proficiency of several deletion and single point mutants was the same as that observed with microsomal membranes, indicating that the overall sequence requirements for proper cleavage at this site are maintained even under these artificial conditions. The processing efficiency of the NS2-3 protease varied according to the type of detergent used and its concentration. Also, the incubation temperature affected the cleavage at the 2-3 junction. The autoproteolytic activity of the NS2-3 protease could be inhibited by alkylating agents such as iodoacetamide and N-ethylmaleimide. Metal chelators such as EDTA and phenanthroline also inhibited the viral enzyme. The EDTA inhibition of NS2-3 cleavage could be reversed, at least in part, by the addition of ZnCl2 and CdCl2. Among the common protease inhibitors tested, tosyl phenylalanyl chloromethyl ketone and soybean trypsin inhibitor inactivated the NS2-3 protease. By means of gel filtration analysis, it was observed that the redox state of the reaction mixture greatly influenced the processing efficiency at the 2-3 site and that factors present in the rabbit reticulocyte lysate, wheat germ extract, and HeLa cell extract were required for efficient processing at this site. Thus, the in vitro assay should allow further characterization of the biochemical properties of the NS2-3 protease of HCV and the identification of host components that contribute to the efficient processing at the 2-3 junction.

Cysteine Endopeptidases↗
PubMed1996

Nonrheumatoid IgM in human hepatitis C virus-associated type II cryoglobulinemia recognize mimotopes of the CD4-like LAG-3 protein.

Type II mixed cryoglobulinemia (CryoII) is an autoimmune disorder frequently associated to human hepatitis C virus (HCV) infection and characterized by the presence of cold-insoluble immunocomplexes containing IgM with rheumatoid activity. To identify disease-related epitopes, we screened a phage-displayed random peptide library using purified IgM from patients with HCV-associated CryoII(CryoII/HCV). A dominant population of phage isolates bearing the HPLAP pentapeptide consensus motif was identified and shown to be recognized by a nonrheumatoid IgM species strongly associated to CryoII/HCV. The phage-borne mimotopes (phagotopes) displayed a strong homology with an exposed extra-loop region of human lymphocyte activation 3 gene (LAG-3) product. Consistently, rabbit sera raised against a synthetic LAG-3 peptide efficiently recognized the selected phagotopes. Furthermore, one such phagotope was revealed to be a good immunogenic mimic of LAG-3 when injected into rabbits. IgM purified from CryoII/HCV patients' sera specifically reacted with the LAG-3 peptide in ELISA, and this binding was inhibited by the selected phagotopes. These results provide experimental support for a general strategy to identify novel autoantigens.

Amino Acid Sequence↗
PubMed1996

Isolation and biological characterization of two classes of blast-cell colony-forming cells from normal murine marrow.

In this study, a primitive progenitor cell, the blast-cell colony-forming cell (BC-CFC), which is thought by some to be equivalent to the hematopoietic stem cell (HSC), those cells capable of long-term marrow repopulation, has been isolated from normal murine marrow. The cell separation method we employed has, as its final step, the purification of marrow cells based on their ability to take up (bright) or exclude (dull) the mitochondrial dye, Rhodamine (Rho)-123. Rho-bright and Rho-dull cells are enriched for multipotential progenitor cells or for HSC, respectively. It was found that Rho-bright cells contain more BC-CFC than Rho-dull cells (310 +/- 50 v 120 +/- 40 per 10(5) purified cells, respectively). However, the BC-CFC that copurified with the Rho-bright and the Rho-dull cells were different in terms of replating efficiency and response to interleukin-3 (IL-3) and stem cell factor (SCF). In fact, on replating, the blast-cell colonies cultured from the Rho-dull population give rise to many more secondary colonies and had a greater replating efficiency than those obtained from Rho-bright cells (replating efficiency: 29.0 +/- 6.3 v 19.5 +/- 3.7, respectively, P < .01). Furthermore, while the same numbers of blast-cell colonies were detected in culture of Rho-bright cells stimulated with IL-3 alone or in combination with SCF, blast-cell colonies were generated in cultures of Rho-dull cells only in the presence of both IL-3 and SCF. After 5 days in suspension culture stimulated with IL-3 and SCF, Rho-dull cells generated BC-CFC whose replating potential was similar to the replating potential of BC-CFC contained in the Rho-bright population. These results indicate that BC-CFC contained in the Rho-bright and -dull populations are qualitatively different. Because the Rho-dull population contains HSC, the results indicate that few, if any, BC-CFC are HSC.

Animals↗
PubMed1996

Molecular control of erythroid differentiation.

The number of circulating red cells is regulated by the daily balance between two processes: the destruction of the old red cells in the liver and the generation of new cells in the bone marrow. The process during which hematopoietic stem cells generate new red cells is called erythropoiesis. This article describes the most recent advances in molecular and cellular biology which have allowed the identification of the molecular mechanisms involved in the process of erythroid differentiation. It reviews the cellular compartments involved in the process, what is known on how these cells respond to erythroid specific growth factors and how the cells progressively activate specific transcription factors in order to express genes involved in the establishment of the erythroid phenotype.

Cell Differentiation↗
PubMed1996

Circulating progenitor cells in human ontogenesis: response to growth factors and replating potential.

We have measured the number of progenitor cells circulating in fetal (17-32 weeks of gestation), perinatal (36 weeks of gestation), and adult (30-50 years old) blood. The progenitor cells at each ontogenetic stage were also characterized in terms both of the minimal combinations of growth factors they required to form maximal numbers of colonies in vitro and of their self-replication potential, as measured by the number of secondary and tertiary progenitor cells each could generate. The number of progenitor cells circulating in fetal and perinatal blood can be measured by directly plating the unfractionated blood. In this assay, fetal blood contains half the number of progenitor cells detected in perinatal blood (18.0 +/- 16.4 versus 40.88 +/- 0.63, p < 0.01), and the number of progenitor cells in adult blood is below the level of detection of the assay (< 1/8 microliter of blood). To compare the number of progenitor cells in all three stages of human development, progenitor cell counts were performed on blood mononuclear cells enriched by density separation. In this case, the light density cell fractions from fetal and neonatal blood contained the same number of progenitor cells (300/10(5) cells), numbers that were 10-fold higher than those observed with adult blood (30/10(5) cells). Circulating fetal-neonatal erythroid and multipotential progenitor cells were found to differ from their adult counterparts in terms of their response to growth factors and their self-renewal ability. In fact, the number of cytokines required to observe maximal colony formation increased with the ontogenetic stage of the cells. No differences were found in the frequency of primary colonies containing progenitor cells or in the mean number of secondary progenitor cells per primary colony in cultures of fetal, neonatal, or adult blood. Differences between the three ontogenetic stages, however, were found with respect to the number of sequential replatings that were possible. In fact, although both secondary granulocyte-macrophage (GM) and mixed-cell colonies derived from fetal cells gave rise to tertiary colonies, only perinatal secondary mixed-cell colonies grew in tertiary cultures, and no growth was observed in tertiary cultures of adult cells. These results suggest that the greater amplification of progenitor cells observed in liquid culture of fetal/neonatal versus adult blood is due both to a higher proliferative capacity of neonatal progenitor cells (up to two replatings versus one) and to a higher frequency in these samples of mixed-cell colony-forming cells (CFC) (37.7 +/- 7.3 versus 2.0 +/- 0.7/10(5) light density cells, respectively). Because of the high numbers of progenitor cells circulating in the fetus, as well as their high proliferative capacity, it is predicted that if blood could be harvested directly in utero, fetal blood would be as good a source of stem cells for transplantation as perinatal placental/cord blood. Circulating fetal stem cells would, therefore, represent an ideal target for gene therapy and in utero autologous transplantation.

Adult↗
PubMed1996

Erythroid-specific activation of the distal (testis) promoter of GATA1 during differentiation of purified normal murine hematopoietic stem cells.

To understand the molecular mechanisms of erythroid differentiation, we analyzed by semiquantitative RT-PCR the expression of the transcription factor GATA1, the erythropoietin receptor (EpoR), and erythroid (beta-globin) differentiation markers in purified hematopoietic stem cells (HSCs) after in-vitro-induced differentiation. Whether GATA1 transcription was from the proximal (with respect to the AUG, also known as erythroid) or the distal (also known as testis) promoter was analyzed as well. Low-density marrow cells which bind to wheat germ agglutinin, but not to the antibody 15.1.1, and which either do or do not retain the dye rhodamine-123 (Rho-bright and Rho-dull, respectively), were purified. Rho-dull, but not Rho-bright cells permanently reconstitute lymphomyelopoiesis in W/Wv and severe-combined-immunodeficiency mice and, therefore, contain HSCs. Both Rho-dull and Rho-bright cells give rise to progenitor and differentiated cells (peak values at days 15 and 5, respectively) in liquid culture. Multilineage, erythroid-restricted or myeloid-restricted differentiation is observed when the cultures are stimulated with stem cell factor (SCF) + interleukin (IL)-3, SCF + IL-3 + Epo, or SCF + IL-3 + granulocyte-colony-stimulating factor, respectively. Rho-dull cells have barely detectable reconstitution potential at day 5 of culture. None of the genes examined were expressed in purified Rho-bright or Rho-dull cells. The only exception was GATA1 which was expressed at maximal levels in Rho-bright cells at the onset of culture. Rho-dull cells did not express GATA1 before day 3 of culture (maximal expression at days 10-15). Activation of GATA1 and EpoR was observed in all growth of mRNA for the two genes expressed by the cells. In contrast, beta-globin mRNA was detected only in the presence of Epo. The transcription of GATA1 was exclusively from the proximal promoter in the absence of Epo but both proximal and distal transcripts were observed in its presence. Maximum transcription from the distal promoter (approximately equal to 0.2% of total GATA1 mRNA) coincided with maximal globin mRNA levels (day 5 or day 15 for Rho-bright and Rho-dull cells, respectively). These results indicate that GATA1 is activated at the transition point between HSCs and pluripotent progenitor cells and erythroid-specific GATA1 regulation involves activation of the distal GATA1 promoter.

Actins↗
PubMed1995

Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution.

Clinical evidence of hematopoietic restoration with placental/umbilical cord blood (PCB) grafts indicates that PCB can be a useful source of hematopoietic stem cells for routine bone marrow reconstitution. In the unrelated setting, human leukocyte antigen (HLA)-matched donors must be obtained for candidate patients and, hence, large panels of frozen HLA-typed PCB units must be established. The large volume of unprocessed units, consisting mostly of red blood cells, plasma, and cryopreservation medium, poses a serious difficulty in this effort because storage space in liquid nitrogen is limited and costly. We report here that almost all the hematopoietic colony-forming cells present in PCB units can be recovered in a uniform volume of 20 ml by using rouleaux formation induced by hydroxyethyl starch and centrifugation to reduce the bulk of erythrocytes and plasma and, thus, concentrate leukocytes. This method multiples the number of units that can be stored in the same freezer space as much as 10-fold depending on the format of the storage system. We have also investigated the proportion of functional stem/progenitor cells initially present that are actually available to the recipient when thawed cryopreserved PCB units are infused. Progenitor cell viability is measurably decreased when thawed cells, still suspended in hypertonic cryopreservative solutions, are rapidly mixed with large volumes of isotonic solutions or plasma. The osmotic damage inflicted by the severe solute concentration gradient, however, can be averted by a simple 2-fold dilution after thawing, providing almost total recovery of viable hematopoietic progenitor cells.

Blood Cell Count↗
PubMed1995

Functional characterization of lymphoid cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice.

We have investigated the phenotypic and functional characteristics of murine pre-B cells obtained in semisolid and liquid culture with stem cell factor (SCF) and interleukin 7 (IL-7). Both serum-supplemented and serum-deprived culture conditions were used. The source of bone marrow cells was either normal mice (CD1 and C3H) or the lupus strain of mice MRL/lpr and its congenic strain MRL/+. SCF (100 ng/ml) and IL-7 (250 ng/ml) supported murine B cell proliferation in vitro from all the murine strains analyzed both in serum-supplemented and serum-deprived conditions. Maximal colony growth was observed in both cases when the factors were used in combination. The growth factors alone induced some colony growth in serum-supplemented cultures but were either ineffective or had modest activity in serum-deprived cultures. Cells harvested from the colonies or generated in liquid cultures and stimulated with SCF + IL-7 in the absence of serum had almost exclusively a pre-B cell phenotype (BP-1+, B220+, slg-, CD4-, CD8-, Mac-1-, RB-6-). Both the maximal colony growth in semisolid culture and the maximal number of cells in liquid culture were observed at day 12-14. At this time, the pre-B cells failed to differentiate further and started to die. Pre-B cells generated in vitro were, however, capable of differentiating in vivo. SCID mice injected with 2 x 10(6) pre-B cells had readily detectable serum levels of IgM (54 +/- 26 micrograms/ml) and IgG (60 +/- 95 micrograms/ml) at 4 weeks and 6 weeks posttransplantation, respectively. Mature B and T cells of the donor major histocompatibility complex type were detected in the SCID mice at sacrifice 14 weeks posttransplantation. These data indicate that purified (> 80% BP-1+) populations of functional pre-B cells can be grown from murine bone marrow of normal mice as well as of lupus mice in serum-deprived cultures stimulated with SCF and IL-7. These cultures, therefore, provide a highly enriched source of pre-B cells but also contain T cell precursors that differentiate upon adoptive transfer into SCID mice.

Animals↗
PubMed1995

The NS2 protein of hepatitis C virus is a transmembrane polypeptide.

The NS2 protein of hepatitis C virus (HCV) is released from its polyprotein precursor by two proteolytic cleavages. The N terminus of this protein is separated from the E2/p7 polypeptide by a cleavage thought to be mediated by signal peptidase, whereas the NS2-3 junction located at the C terminus is processed by a viral protease. To characterize the biogenesis of NS2 encoded by the BK strain of HCV, we have defined the minimal region of the polyprotein required for efficient cleavage at the NS2-3 site and analyzed the interaction of the mature polypeptide with the membrane of the endoplasmic reticulum (ER). We have observed that although cleavage can occur in vitro in the absence of microsomal membranes, synthesis of the polyprotein precursor in the presence of membranes greatly increases processing at this site. Furthermore, we show that the membrane dependency for efficient in vitro processing varies among different HCV strains and that host proteins located on the ER membrane, and in particular the signal recognition particle receptor, are required to sustain efficient proteolysis. By means of sedimentation analysis, protease protection assay, and site-directed mutagenesis, we also demonstrate that the NS2 protein derived from processing at the NS2-3 site is a transmembrane polypeptide, with the C terminus translocated in the lumen of the ER and the N terminus located in the cytosol.

Animals↗
PubMed1994

Long-term generation of human mast cells in serum-free cultures of CD34+ cord blood cells stimulated with stem cell factor and interleukin-3.

The generation of murine mast cells is supported by several cytokines, and mast cell lines are frequently established in long-term cultures of normal murine marrow cells. In contrast, growth of human mast cells was initially dependent on coculture with murine fibroblasts. The growth factor produced by murine fibroblasts and required to observe differentiation of human mast cells is attributable in part to stem cell factor (SCF). However, other factors are likely involved. We have previously shown that the combination of SCF and interleukin-3 (IL-3) efficiently sustains proliferation and differentiation of colony-forming cells (CFCs) from pre-CFC enriched from human umbilical cord blood by CD34+ selection. With periodic medium changes and the addition of fresh growth factors, five consecutive cultures of different cord blood samples gave rise to differentiated cells and CFCs for more than 2 months. Although differentiated cells continued to be generated for more than 5 months, CFCs were no longer detectable by day 50 of culture. The cells have the morphology of immature mast cells, are Toluidine blue positive, are karyotypically normal, are CD33+, CD34-, CD45+, c-kit-, and c-fms-, and die in the absence of either SCF or IL-3. These cells do not form colonies in semisolid culture and are propagated in liquid culture stimulated with SCF and IL-3 at a seeding concentration of no less than 10(4) cells/mL. At refeedings, the cultures contain a high number (> 50%) of dead cells and have a doubling time ranging from 5 to 12 days. This suggests that subsets of the cell population die because of a requirement for a growth factor other than SCF or IL-3. These results indicate that the combination of cord blood progenitor and stem cells, plus a cocktail of growth factors including SCF and IL-3, is capable with high efficiency of giving rise in serum-deprived culture to human mast cells that behave like factor-dependent cell lines. These cells may represent a useful tool for studies of human mast cell differentiation and leukemia.

Antigens, CD↗
PubMed1994

Alternatively spliced mRNAs encoding soluble isoforms of the erythropoietin receptor in murine cell lines and bone marrow.

32D Epo and 32D GM cells are subclones of the murine 32D cell line which are selectively dependent for proliferation and survival on erythropoietin (Epo) or granulocyte/macrophage colony-stimulating factor (GM-CSF), respectively. 32D GM cells were previously shown to express significant levels of the Epo receptor mRNA and protein which was retained intracellularly and did not appear on the cell surface. We have now analyzed the EpoR mRNA from the 32D GM line, using PCR followed by direct sequencing. Several alternatively spliced products were detected. In some molecules, intron 5 (I5) or part of I6 or both were retained. Retention of I5 results in a mRNA potentially encoding an almost complete extracellular domain, while retention of I6 gives rise to a mRNA encoding the complete extracellular and transmembrane domains. A different type of splicing results in the loss of exon 5 (E5), giving rise to a sequence encoding a truncated extracellular domain. These alternatively spliced sequences are differentially represented in 32D Epo versus 32D GM cells. All are additionally present in normal bone marrow cells. Apart from these alternatively spliced EpoR RNAs, no other abnormalities were detected in EpoR RNA from 32D GM cells that could account for the intracellular retention of EpoR in the non-erythroid subclones of 32D.

Alternative Splicing↗