Search PubMed⌕ Search

Biomedical subjects

C Peschle

Publications and source records attributed to C Peschle.

At least 37 records · Page 2Linked to original sources
PubMed1999

Expression and role of PML gene in normal adult hematopoiesis: functional interaction between PML and Rb proteins in erythropoiesis.

The expression of the PML gene was investigated in purified early hematopoietic progenitor cells (HPCs) induced to unilineage erythroid or granulocytic differentiation. PML mRNA and protein, while barely detectable in quiescent HPCs, are consistently induced by growth factor stimulation through the erythroid or granulocytic lineage. Thereafter, PML is downmodulated in late granulocytic maturation, whereas it is sustainably expressed through the erythroid pathway. In functional studies, PML expression was inhibited by addition of antisense oligomers targeting PML mRNA (alpha-PML). Interestingly, early treatment (day 0 HPCs) with alpha-PML reduced the number of both erythroid and granulocytic colonies, whereas late treatment (day 5 culture) reduced erythroid, but not granulocytic, clonogenesis. These findings suggest that PML is required for early hematopoiesis and erythroid, but not granulocytic maturation. The pattern of PML expression in normal hematopoiesis mimics that of retinoblastoma pRb 105. Combined treatment of HPCs with alpha-PML and alpha-Rb oligomers inhibited both PML and Rb protein expression and completely blocked erythroid colony development. Furthermore, PML and pRb 105 were co-immunoprecipitated in cellular lysates derived from erythroid precursors indicating that this functional interaction may have a biochemical basis. These results suggest a key functional role of PML in early hematopoiesis and late erythropoiesis: the latter phenomenon may be related to the molecular and functional interaction of PML with pRb 105.

Adult↗
PubMed1999

Unicellular-unilineage erythropoietic cultures: molecular analysis of regulatory gene expression at sibling cell level.

In vitro studies on hematopoietic control mechanisms have been hampered by the heterogeneity of the analyzed cell populations, ie, lack of lineage specificity and developmental stage homogeneity of progenitor/precursor cells growing in culture. We developed unicellular culture systems for unilineage differentiation of purified hematopoietic progenitor cells followed by daughter cell analysis at cellular and molecular level. In the culture system reported here, (1) the growth factor (GF) stimulus induces cord blood (CB) progenitor cells to proliferate and differentiate/mature exclusively along the erythroid lineage; (2) this erythropoietic wave is characterized by less than 4% apoptotic cells; (3) asymmetric divisions are virtually absent, ie, nonresponsive hematopoietic progenitors with no erythropoietic potential are forced into apoptosis; (4) the system is cell division controlled (cdc), ie, the number of divisions performed by each cell is monitored. Single-cell reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was applied to this culture system to investigate gene expression of diverse receptors, markers of differentiation, and transcription factors (EKLF, GATA-1, GATA-2, p45 NF-E2, PU.1, and SCL/Tal1) at discrete stages of erythropoietic development. Freshly isolated CD34(+) cells expressed CD34, c-kit, PU.1, and GATA-2 but did not express CD36, erythropoietin receptor (EpoR), SCL/Tal1, EKLF, NF-E2, GATA-1, or glyocophorin A (GPA). In early to intermediate stages of erythroid differentiation we monitored the induction of CD36, Tal1, EKLF, NF-E2, and GATA-1 that preceeded expression of EpoR. In late stages of erythroid maturation, GPA was upregulated, whereas CD34, c-kit, PU.1, and GATA-2 were barely or not detected. In addition, competitive single-cell RT-PCR was used to assay CD34 mRNA transcripts in sibling CD34(+)CD38(-) cells differentiating in unilineage erythroid cultures: this analysis allowed us to semiquantitate the gradual downmodulation of CD34 mRNA from progenitor cells through their differentiating erythroid progeny. It is concluded that this novel culture system, coupled with single-cell RT-PCR analysis, may eliminate the ambiguities intrinsic to molecular studies on heterogeneous populations of hematopoietic progenitors/precursors growing in culture, particularly in the initial stages of development.

Antigens, CD34↗
PubMed1999

Phorbol ester-induced disruption of the CD4-Lck complex occurs within a detergent-resistant microdomain of the plasma membrane. Involvement of the translocation of activated protein kinase C isoforms.

Recent studies have highlighted the existence of discrete microdomains at the cell surface that are distinct from caveolae. The function of these microdomains remains unknown. However, recent evidence suggests that they may participate in a subset of transmembrane signaling events. In hematopoietic cells, these low density Triton-insoluble (LDTI) microdomains (also called caveolae-related domains) are dramatically enriched in signaling molecules, such as cell surface receptors (CD4 and CD55), Src family tyrosine kinases (Lyn, Lck, Hck, and Fyn), heterotrimeric G proteins, and gangliosides (GM1 and GM3). Human T lymphocytes have become a well established model system for studying the process of phorbol ester-induced down-regulation of CD4. Here, we present evidence that phorbol 12-myristate 13-acetate (PMA)-induced down-regulation of the cell surface pool of CD4 occurs within the LDTI microdomains of T cells. Localization of CD4 in LDTI microdomains was confirmed by immunoelectron microscopy. PMA-induced disruption of the CD4-Lck complex was rapid (within 5 min), and this disruption occurred within LDTI microdomains. Because PMA is an activator of protein kinase C (PKC), we next evaluated the possible roles of different PKC isoforms in this process. Our results indicate that PMA induced the rapid translocation of cytosolic PKCs to LDTI microdomains. We identified PKCalpha as the major isoform involved in this translocation event. Taken together, our results support the hypothesis that LDTI microdomains represent a functionally important plasma membrane compartment in T cells.

Biological Transport↗
PubMed1999

Enforced expression of HOXB7 promotes hematopoietic stem cell proliferation and myeloid-restricted progenitor differentiation.

Hematopoietic progenitor/stem cells (HPCs/HSCs) purified from human adult peripheral blood (PB) were triggered into cycling, retrovirally transduced with HOXB7 and then functionally assayed in vitro. HPCs were assayed in multi- and unilineage differentiation cultures in either liquid phase or semisolid medium, primitive HPCs in the high proliferative potential colony-forming cell (HPP-CFC) evaluation system and putative HSCs in Dexter type long-term culture (LTC) as LTC initiating cells (LTC-ICs). Control experiments ensured that the exogenous HOXB7 gene was constantly expressed, while the endogenous one was barely or not transcribed. Enforced expression of the gene markedly modulated the proliferation/differentiation program of the entire HSC/HPC population. Enforced HOXB7 expression exerted a potent stimulatory effect on the proliferation of the primitive HPC and putative HSC subsets, assayed as HPP-CFCs and LTC-ICs respectively. While not modifying the total number of HPCs, exogenous HOXB7 induced an increase of the number of granulo-monocytic (GM) HPCs [colony-forming unit GM (CFU-GM) CFU-GM, CFU-G and CFU-M, as evaluated by clonogenic assays] and markedly amplified the progeny of both CFU-G and CFU-M, which showed a sustained proliferation through at least 1-2 months (as evaluated in liquid suspension culture). The prolonged proliferative stimulus induced by HOXB7 transfer into LTC, primitive and GM oriented HPC culture was characterized by persistent proliferation of a discrete population of blast cells and a large pool of differentiated myeloid precursors. Altogether, these results suggest the hypothesis that the proliferative stimulus exerted by exogenous HOXB7 in primitive and GM-oriented HPCs may represent a preleukemic immortalization step. Consistent with the functional role of HOXB7 in the initial ontogenetic phase, these studies indicate that ectopic HOXB7 expression in early HPCs and HSCs from adult PB stimulates their self renewal, sustained proliferation and myeloid differentiation.

Adult↗
PubMed1999

B-MYB transactivates its own promoter through SP1-binding sites.

B-MYB is an ubiquitous protein required for mammalian cell growth. In this report we show that B-MYB transactivates its own promoter through a 120 bp segment proximal to the transcription start site. The B-MYB-responsive element does not contain myb-binding sites and gel-shift analysis shows that SP1, but not B-MYB, protein contained in SAOS2 cell extracts binds to the 120 bp B-myb promoter fragment. B-MYB-dependent transactivation is cooperatively increased in the presence of SP1, but not SP3 overexpression. When the SP1 elements of the B-myb promoter are transferred in front of a heterologous promoter, an increased response to B-MYB results. In contrast, c-MYB, the prototype member of the Myb family, is not able to activate the luciferase construct containing the SP1 elements. With the use of an SP1-GAL4 fusion protein, we have determined that the cooperative activation occurs through the domain A of SP1. These observations suggest that B-MYB functions as a coactivator of SP1, and that diverse combinations of myb and SP1 sites may dictate the responsiveness of myb-target genes to the various members of the myb family.

Adenovirus E1A Proteins↗
PubMed1999

Apoptotic role of Fas/Fas ligand system in the regulation of erythropoiesis.

The possible involvement of Fas and Fas ligand (FasL) in the regulation of erythropoiesis was evaluated. Immunohistochemistry of normal bone marrow specimens revealed that several immature erythroblasts undergo apoptosis in vivo. Analysis of bone marrow erythroblasts and purified progenitors undergoing unilineage erythroid differentiation showed that Fas is rapidly upregulated in early erythroblasts and expressed at high levels through terminal maturation. However, Fas crosslinking was effective only in less mature erythroblasts, particularly at basophilic level, where it induced apoptosis antagonized by high levels of erythropoietin (Epo). In contrast, FasL was selectively induced in late differentiating Fas-insensitive erythroblasts, mostly at the orthochromatic stage. FasL is functional in mature erythroblasts, as it was able to kill Fas-sensitive lymphoblast targets in a Fas-dependent manner. Importantly, FasL-bearing mature erythroblasts displayed a Fas-based cytotoxicity against immature erythroblasts, which was abrogated by high levels of Epo. These findings suggest the existence of a negative regulatory feedback between mature and immature erythroid cells, whereby the former cell population might exert a cytotoxic effect on the latter one in the erythroblastic island. Hypothetically, this negative feedback operates at low Epo levels to moderate the erythropoietic rate; however, it is gradually inhibited at increasing Epo concentrations coupled with enhanced erythrocyte production. Thus, the interaction of Fas and FasL may represent an apoptotic control mechanism for erythropoiesis, contributing to the regulation of red blood cell homeostasis.

Adult↗
PubMed1999

Caspase activation without death.

Since molecular cloning of the C. elegans ced-3 gene revealed its homology with mammalian IL-1beta-converting enzyme,1 14 members of the caspase family have been identified, which have often been involved as mediators of one or more phases of the apoptotic process. 2,3 However, an over-simplified role of these proteases may be insufficient to explain the usually constitutive expression of such a large and complex family of enzymes, many of which display overlapping specificity. In addition to the well-established role of caspase-1 in the production of active IL-1beta and IL-18 in inflammation,4 an increasing number of reports has recently suggested that caspases may have a function outside of apoptosis. In this review, the situations in which cells survive despite the presence of activated caspases in their cytoplasm will be examined and discussed, with the intent to gather all recent advances in this new field that promises to be a focus for caspase research in the near future.

Animals↗
PubMed1998

Chromatin immunoselection defines a TAL-1 target gene.

Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.

Amino Acid Sequence↗
PubMed1998

Transduction of the SkBr3 breast carcinoma cell line with the HOXB7 gene induces bFGF expression, increases cell proliferation and reduces growth factor dependence.

Several melanomas, carcinomas, glioblastomas and leukemias showed coordinated expression of HOXB7 and bFGF with exception of the SkBr3 mammary carcinoma that was negative for both. Transduction of HOXB7 gene into SkBr3 cells, induced bFGF expression, increased growth rate, independence from serum withdrawal and ability to form colonies in semisolid medium. ELISA assay showed that most of bFGF was associated to cell lysate when cells were cultured at 1% serum whereas in cells kept to 10% serum bFGF was detected both within cell lysate or secreted into cell supernatants. Antisense oligos to bFGF inhibited the growth of cells cultured in 1%, indicating that beside the possible activation of additional genes other than bFGF by HOXB7 transduction, only bFGF induction accounts for the observed results. Moreover, since inhibition of cell proliferation occurred in cells kept in 1% but not 10% serum, a bFGF intracrine loop appears operative in serum starved SkBr3/HOXB7 cells. Also, these results further indicate bFGF as target of HOXB7.

Agar↗
PubMed1998

Oxidative stress leads to a rapid alteration of transferrin receptor intravesicular trafficking.

Several studies have demonstrated that perturbations of intracellular oxidative balance play a key role in numerous physiological as well as pathological conditions leading to various morbidity states. In previous studies we have shown that the free radical inducer menadione rapidly and specifically downmodulates the membrane transferrin receptor (TfR) by blocking receptor recycling. This modulation is due to receptor redistribution and not to receptor loss. Here we show that other oxidant compounds, such as hydrogen peroxide, also induce a rapid downmodulation of membrane TfR and that pretreatment of cells with the antioxidant, thiol supplier, N-acetylcysteine inhibits the downmodulation of these receptors elicited by either menadione or hydrogen peroxide. This observation suggests that intracellular thiol redox status may be a critical determinant of TfR downmodulation induced by oxidative stress. Furthermore, immunocytochemical results show that, in menadione-treated cells, TfRs are associated with the Golgi complex, where normally only 20% of total cellular TfRs is found and is mainly detected in the cytoplasm as scattered punctuations. Accordingly, menadione and hydrogen peroxide also elicited a downmodulation of low density lipoprotein receptor (LDLR) which mediates, like TfR, the transport of nutrients to the cell and is endocytosed through clathrin-coated pits. Finally, experiments carried out using okadaic acid, an inhibitor of phosphatases, suggest that H2O2 and menadione downmodulate surface TfR via different biochemical pathways. Taken together these results suggest the existence of a potentially important protective mechanism through which iron uptake is prevented in oxidatively imbalanced cells. Iron uptake can in fact give rise to the formation of highly toxic hydroxyl radicals reacting with hydrogen peroxide and leading to cytotoxicity. Downmodulation of surface TfR may thus represent the physiological control mechanism for reducing iron uptake in diverse pathological conditions including hypoxia-reperfusion injury, acquired immunodeficiency syndrome, and aging.

Acetylcysteine↗
PubMed1998

The PML/RARalpha fusion protein inhibits tumor necrosis factor-alpha-induced apoptosis in U937 cells and acute promyelocytic leukemia blasts.

We investigated the effect of the acute promyelocytic leukemia (APL) specific PML/RARalpha fusion protein on the sensitivity to TNF-alpha-mediated apoptosis. The U937 leukemia cell line was transduced with PML/RARalpha cDNA. PML/RARalpha expression caused a markedly reduced sensitivity to TNF-alpha, even if apoptosis was triggered by agonistic antibodies to TNF-alpha receptors I and II (TNF-alphaRI, II). PML/RARalpha induced a 10-20-fold decrease of the TNF-alpha-binding capacity via downmodulation of both TNF-alphaRI and TNF-alphaRII: this may mediate at least in part the reduced sensitivity to TNF-alpha. Furthermore, the fusion protein did not modify Fas expression (CD95) or sensitivity to Fas-mediated apoptosis. The pathophysiological significance of these findings is supported by two series of observations. (a) Fresh APL blasts exhibit no TNF-alpha binding and are resistant to TNF-alpha-mediated apoptosis. Conversely, normal myeloblasts-promyelocytes show marked TNF-alphaR expression and are moderately sensitive to TNF-alpha-mediated cytotoxicity. Similarly, blasts from other types of acute myeloid leukemia (AML M1, M2, and M4 FAB types) show an elevated TNF-alpha binding. (b) The NB4 APL cell line, which is PML/RARalpha+, shows low TNF-alphaR expression capacity and is resistant to TNF-alpha-triggered apoptosis; conversely a PML/RARalpha- NB4 subclone (NB4.306) exhibits detectable TNF-alpha-binding capacity and is sensitive to TNF-alpha-mediated cytotoxicity. These studies indicate that the PML/RARalpha fusion protein protects against TNF-alpha-induced apoptosis, at least in part via downmodulation of TNF-alphaRI/II: this phenomenon may play a significant role in APL, which is characterized by prolonged survival of leukemic blasts.

Antigens, CD↗
PubMed1998

Productive human immunodeficiency virus-1 infection of purified megakaryocytic progenitors/precursors and maturing megakaryocytes.

We have evaluated the susceptibility to human immunodeficiency virus (HIV)-1 infection of in vitro grown megakaryopoietic progenitors/precursors and maturing megakaryocytes (MKs), based on the following approach: (1) human hematopoietic progenitor cells (HPCs), stringently purified from peripheral blood and grown in serum-free liquid suspension culture supplemented with thrombopoietin (Tpo), generated a relatively large number of >/= 98% to 99% pure megakaryocytic precursors and then mature-terminal MKs; (2) at different days of culture (ie, 0, 5, 8, 10) the cells were inoculated with 0.1 to 1.0 multiplicity of infection (m.o.i.) of the lymphotropic NL4-3 or 0.1 m.o.i. of the monocytotropic BaL-1 HIV-1 strain; (3) finally, the presence of viral mRNA and proteins was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR)/in situ hybridization and antigen capture assays, respectively, on day 2 to 12 of culture. MKs derived from day 0 and day 5 BaL-1-challenged cells do not support viral replication as assessed by p24 enzyme-linked immunosorbent assay (ELISA) and RT-PCR. On the contrary, HIV transcripts and proteins were clearly detected in all NL4-3 infection experiments by RT-PCR and p24 assay, respectively, with the highest viral expression in day 5 to 8 challenged MKs. In situ hibridization studies indicate that the percentage of HIV+ MKs varies from at least 1% and 5% for day 0 and day 5 infected cells, respectively. Production of an infectious viral progeny, evaluated by the capability of culture supernatants from day 5 NL4-3-challenged MKs to infect C8166 T-lymphoblastoid cell line, was consistently observed (viral titer, approximately 5 x 10(3) tissue culture infectious dose50/mL/10(6) cells). Exposure of MKs to saturating concentration of anti-CD4 OKT4A monoclonal antibody (MoAb), which recognizes the CD4 region binding with the gp120 envelope glycoprotein, markedly inhibited HIV infection, as indicated by a reduction of p24 content in the supernatants: because the inhibitory effect was incomplete, it is apparent that the infection is only partially CD4-dependent, suggesting that an alternative mechanism of viral entry may exist. Morphologic analysis of day 12 MKs derived from HPCs infected at day 0 showed an impaired megakaryocytic differentiation/maturation: the percentage of mature MKs was markedly reduced, in that approximately 80% of cells showed only one nuclear lobe and a pale cytoplasm with few granules. Conversely, megakaryocytic precursors challenged at day 5 to 8 generated fully mature day 10 to 12 MKs showing multiple nuclear segmentation. Thus, the inhibitory effect of HIV on the megakaryopoietic gene program relates to the differentiation stage of cells subjected to the viral challenge. Finally, HPCs treated with 20 or 200 ng/mL of recombinant Tat protein, analyzed at different days of culture, showed an impaired megakaryocytopoiesis comparable to that observed in HIV-infected cells, thus suggesting that Tat is a major mediator in the above described phenomena. These results shed light on the pathogenesis of HIV-related thrombocytopenia; furthermore, they provide a model to investigate the effects of HIV on megakaryocytic differentiation and function.

Adult↗
PubMed1998

Enforced TAL-1 expression stimulates primitive, erythroid and megakaryocytic progenitors but blocks the granulopoietic differentiation program.

In human adult hematopoiesis, the TAL-1 gene is up- and down-modulated in erythropoiesis and granulopoiesis, respectively [G. L. Condorelli et al., Blood, 86: 164-175, 19951. Here, it is shown that, in a hematopoietic progenitor cell (HPC) unilineage differentiation culture, tal-1 is induced and then expressed, in a sustained manner, in the megakaryopoietic lineage, whereas it is barely or not detected in the monocytopoietic series. We have investigated the role of enforced tal-1 expression by retroviral transfer into HPCs [erythroid burst-forming units and megakaryocytic and granulomonocytic colony-forming units (CFUs)], primitive HPCs (high proliferative potential colony-forming cells), and putative hematopoietic stem cells (HSCs), assayed as long-term culture initiating cells. TAL-1 overexpression induces an increase of erythroid burst-forming unit colony number and size and megakaryocytic CFU colony number and an inhibition of granulomonocytic CFU and granulocytic CFU (CFU-G) but not monocytic CFU colony number; conversely, TAL-1 mutants with defective heterodimerizing or DNA-binding domains do not exert these effects at a significant level. Although it does not affect long-term culture initiating cells, exogenous TAL-1 causes a significant proliferative stimulus on primary and secondary high proliferative potential colony-forming cells. In conclusion, exogenous tal-1 exerts differential and stage- and lineage-specific effects on the HPC/HSC differentiation/proliferation gene programs. Thus, it induces a stimulatory effect at the level of erythroid and megakaryocytic HPCs, while exerting a selective proliferative action on downstream erythropoiesis. Furthermore, it induces differential effects on the myeloid series: the partial blockade of CFU-G differentiation is possibly linked to the sharp down-modulation of endogenous TAL-1 expression at the level of the CFU-G-to-granulopoietic precursor differentiation step; in contrast, no significant effect is observed on monocytic CFU colony formation. Finally, the stimulatory effect on primitive HPCs but not putative stem cells suggests subtle differences in the effects exerted by tal-1 overexpression on primitive HPC/HSC subsets in adult life.

Adult↗
PubMed1998

Arsenic trioxide as an inducer of apoptosis and loss of PML/RAR alpha protein in acute promyelocytic leukemia cells.

BACKGROUND: Retinoids, which are derivatives of vitamin A, induce differentiation of acute promyelocytic leukemia (APL) cells in vitro and in patients. However, APL cells develop resistance to retinoic acid treatment. Arsenic trioxide (As2O3) can induce clinical remission in patients with APL, including those who have relapsed after retinoic acid treatment, by inducing apoptosis (programmed cell death) of the leukemia cells. In this study, we investigated the molecular mechanisms by which As2O3 induces apoptosis in retinoic acid-sensitive NB4 APL cells, in retinoic acid-resistant derivatives of these cells, and in fresh leukemia cells from patients. METHODS: Apoptosis was assessed by means of DNA fragmentation analyses, TUNEL assays (i.e., deoxyuridine triphosphate labeling of DNA nicks with terminal deoxynucleotidyl transferase), and flow cytometry. Expression of the PML/RAR alpha fusion protein in leukemia cells was assessed by means of western blotting, ligand binding, and immunohistochemistry. Northern blotting and ribonuclease protection assays were used to evaluate changes in gene expression in response to retinoic acid and As2O3 treatment. RESULTS AND CONCLUSIONS: As2O3 induces apoptosis without differentiation in retinoic acid-sensitive and retinoic acid-resistant APL cells at concentrations that are achievable in patients. As2O3 induces loss of the PML/RAR alpha fusion protein in NB4 cells, in retinoic-acid resistant cells derived from them, in fresh APL cells from patients, and in non-APL cells transfected to express this protein. As2O3 and retinoic acid induce different patterns of gene regulation, and they inhibit the phenotypes induced by each other. Understanding the molecular basis of these differences in the effects of As2O3 and retinoic acid may guide the clinical use of arsenic compounds and provide insights into the management of leukemias that do not respond to retinoic acid.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

High-efficiency gene transfer and selection of human hematopoietic progenitor cells with a hybrid EBV/retroviral vector expressing the green fluorescence protein.

We report a retroviral expression vector (PINCO) that allows high-efficiency gene transfer and selection of hemopoietic progenitor cells (HPCs). The main characteristics of this vector are the presence outside the two long terminal repeats of the EBV origin of replication and the EBNA-1 gene and the presence in the retrovirus of the cDNA that encodes for the enhanced green fluorescence protein (GFP), controlled by a cytomegalovirus promoter. Transient transfection of PINCO in Phoenix packaging cells results in episomal propagation of the plasmid and generates viral titers as high as 10(7) colony-forming units/ml. Infection of established cell lines with the PINCO retrovirus yields more than 95% GFP-expressing cells. GFP expression remains stable for months in infected cell cultures and can easily be monitored by fluorescent microscopy or fluorescence-activated cell-sorting (FACS) analysis of living cells. The PINCO vector allows efficient expression of a second gene (thymidine kinase, Shc, and PML), and there is strict correlation between GFP and second gene expression levels in the infected cells. PINCO was used to infect human HPCs; infection efficiency was about 50%. GFP-positive cells can be FACS sorted to yield a homogeneous population of infected cells. FACS-sorted GFP-positive HPC cells have, with respect to unfractionated HPC cells, the same frequency of long-term culture initiating cells and an identical capacity to undergo multilineage and unilineage differentiation. The entire gene transfer procedure, from the transfection of the packaging cell line to the infection of target cells, requires less than a week. The high viral titer and the easy obtainment of homogeneously infected cell populations without drug selection procedures make PINCO an ideal vector for gene transfer of human primary hemopoietic cells.

3T3 Cells↗
PubMed1998

Unilineage hematopoietic differentiation in bulk and single cell culture.

The rarity of hematopoietic stem and progenitor cells (HSCs, HPCs) has hampered the analysis of cellular and molecular mechanisms underlying early hematopoiesis. Methodology for HPC purification has partially offset this limitation. A further hurdle has been represented by the heterogeneity of the analyzed HPC/precursor populations: recently, development of unilineage HPC differentiation cultures has provided homogeneous populations of hematopoietic cells, particularly in the early differentiation state, i.e., populations pertaining to a single lineage and a restricted stage of differentiation/maturation, but sufficiently large for cellular/molecular analysis. This report focuses on the development and characterization of the unilineage HPC differentiation culture systems. A section is devoted to selected cellular and molecular mechanisms underlying hematopoiesis, which have been investigated by the HPC unilineage culture approach. Finally, recent advances in the development of HPC unilineage cultures at single cell level are discussed.

Animals↗