Murine bone marrow expressing the neomycin resistance gene has no competitive disadvantage assessed in vivo

The neomycin phosphotransferase (neo) gene is one of the most common marker genes used in gene transfer experimentation, but potential effects of neo gene expression in vivo have not been systematically investigated. Several early clinical retroviral gene transfer studies have suggested that neo gene expression could have deleterious effects on hematopoiesis, owing to a discrepancy between the level of neo-marked transduced marrow progenitor cells compared with mature circulating progeny cells posttransplantation (Brenner et al., 1993; Kohn et al., 1995; Brenner, 1996b). We examined the long-term in vivo repopulating ability of bone marrow from transgenic mice expressing neo from a strong constitutive promoter using a competitive repopulation assay. Different ratios of neo transgenic and wild-type congenic marrow cells were cotransplanted into W/Wv recipient mice. The percentages of blood cells containing the neo transgene in each group of recipient mice monitored for 4 months posttransplantation closely matched the input ratios of neo transgenic to congenic control marrow cells. Similar concordances of engraftment with input ratios of neo transgenic cells were also found in spleen, thymus, and whole marrow of recipient mice at 4 months posttransplantation. Analysis of the beta-hemoglobin phenotype (beta(single) for the neo transgenic and C57 control cells and beta(diffuse) for the congenic competitor HW80 cells) in recipients confirmed erythroid repopulation from neo transgenic marrow cells at levels matching the input ratios. We conclude that hematopoietic cells expressing neo had no engraftment or maturation defects detectable in vivo. These results suggest that the low-level contribution of vector-marked cells to circulating populations in clinical trials is not due to direct deleterious effects of neo gene expression on hematopoiesis.     

Intracellular immunization of rhesus CD34+ hematopoietic progenitor cells with a hairpin ribozyme protects T cells and macrophages from simian immunodeficiency virus infection

Evaluation of candidate genes for stem cell gene therapy for acquired immunodeficiency syndrome (AIDS) has been limited by the difficulty of supporting in vitro T-cell differentiation of genetically modified hematopoietic progenitor cells. Using a novel thymic stromal culture technique, we evaluated the ability of a hairpin ribozyme specific for simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) to inhibit viral replication in T lymphocytes derived from transduced CD34+ progenitor cells. Retroviral transduction of rhesus macaque CD34+ progenitor cells with a retroviral vector (p9456t) encoding the SIV-specific ribozyme and the selectable marker neomycin phosphotransferase in the presence of bone marrow stroma and in the absence of exogenous cytokines resulted in efficient transduction of both colony-forming units and long-term culture-initiating cells, with transduction efficiencies ranging between 21% and 56%. After transduction, CD34+ cells were cultured on rhesus thymic stromal culture (to support in vitro differentiation of T cells) or in the presence of cytokines (to support differentiation of macrophage-like cells). After expansion and selection with the neomycin analog G418, cells derived from transduced progenitor cells were challenged with SIV. CD4+ T cells derived from CD34+ hematopoietic cells transduced with the ribozyme vector p9456t were highly resistant to challenge with SIV, exhibiting up to a 500-fold decrease in SIV replication, even after high multiplicities of infection. Macrophages derived from CD34+ cells transduced with the 9456 ribozyme exhibited a comparable level of inhibition of SIV replication. These results show that a hairpin ribozyme introduced into CD34+ hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication after T-cell differentiation and support the feasibility of intracellular immunization of hematopoietic stem cells against infection with HIV and SIV. Protection of multiple hematopoietic lineages with the SIV-specific ribozyme should permit analysis of stem cell gene therapy for AIDS in the SIV/macaque model.     

Occupational Stress and Dysmenorrhea in Women Working in Cotton Textile Mills

Investigation of the adeno-associated virus (AAV) life cycle has enabled the establishment of methodology and identification of critical cis-acting sequences required for recombinant AAV production. Vectors derived from the defective human parvovirus (AAV) have been used for successful gene transfer and expression in many diverse mammalian cell types, such as erythroid, airway epithelium, and neuronal cells. One of the crucial steps in the continued case of AAV as a vector is the development of packaging systems that will allow efficient encapsidation of foreign genes into AAV virions. For this reason, the focus of this article will be generation of recombinant AAV vectors.     

Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.     

Preradiation chemotherapy of children and young adults with malignant brain tumors: results of the German pilot trial HIT''88/''89

Murine fibroblast NIH3T3 cells engineered to secrete interleukin-6 (NIH3T3-IL-6) were implanted intraperitoneally into mice and observed for their hematopoietic effects with or without 5-fluorouracil (5-FU) administration. In normal mice, the platelet and neutrophil counts in peripheral blood increased significantly after implantation of NIH3T3-IL-6 cells, but the total white blood cell numbers showed no obvious elevation. The granulocyte-macrophage colony-forming unit (CFU-GM) and megakaryocyte colony-forming unit (CFU-MK) numbers formed by stem cells in bone marrow and spleen were also found to be significantly increased after implantation of NIH3T3-IL-6 cells when compared with those in mice after implantation of NIH3T3 cells transduced with neomycin gene (NIH3T3-Neo). To observe the protective effects of NIH3T3-IL-6 cells on hematopoietic depression in chemotherapy-treated mice, the mice were preinjected with 5-FU at a dosage of 150 mg/kg before implantation of NIH3T3-IL-6 cells. The platelet and neutrophil counts showed accelerated recovery after implantation of NIH3T3-IL-6 cells. The numbers of CFU-GM and CFU-MK in bone marrow and spleen were also found to be markedly increased in hematopoietic-depressed mice when compared with those in mice implanted with NIH3T3-Neo cells. These data suggest that fibroblast-mediated IL-6 gene therapy, which can augment hematopoiesis in mice with or without chemotherapy, will be of great help in the recovery from hematopoietic depression after chemotherapy.     

Thrombopoietin does not induce lineage-restricted commitment of Mpl-R expressing pluripotent progenitors but permits their complete erythroid and megakaryocytic differentiation

In this study, we examined the in vitro and in vivo effects of forced expression of Mpl-R (the thrombopoietin receptor) on the progeny of murine hematopoietic stem cells. Bone marrow cells from 5-FU-treated mice were transduced with retroviral vectors containing the human Mpl-R cDNA, or the neomycine gene as a control. After 7 days cocultivation on virus-producer cells, GpE86-Mpl-R or Gp86-Neo, the types of hematopoietic progenitor cells responding to thrombopoietin (TPO) were studied by clonogenic assays. Mpl-R-infected cells gave rise to CFU-GEMM, BFU-E, CFU-MK, but not CFU-GM while Neo-infected cells produced only megakaryocytic colonies. In addition, when nonadherent cells from GpE86-Mpl-R cocultures were grown with TPO as the only stimulus for 7 days, a marked expansion of CFU-GEMM, BFU-E, and CFU-MK was observed, while no change in CFU-GM number was seen. Erythroid and megakaryocytic maturation occurred in the presence of TPO while a block in granulocytic differentiation was observed at the myeloblast stage. The direct effects of TPO on Mpl-R-transduced progenitor cells were demonstrated by single cell cloning experiments. To analyze the effects of the constitutive expression of Mpl-R on the determination of multipotent progenitors (CFU-S) and long-term repopulating stem cells, Mpl-R- or Neo-infected cells were injected into lethally irradiated recipient mice. No difference was seen in (1) the number of committed progenitor cells contained in individual CFU-S12 whether colonies arose from noninfected or Mpl-R-infected CFU-S; (2) the mean numbers of progenitor cells per leg or spleen of mice reconstituted with Mpl-R- or Neo-infected cells, 1 or 7 months after the graft; and (3) the blood parameters of the two groups of animals, with the exception of a 50% reduction in circulating platelet counts after 7 months in mice repopulated with Mpl-R-infected bone marrow cells. These results indicate that retrovirus-mediated expression of Mpl-R in murine stem cells does not modify their ability to reconstitute all myeloid lineages of differentiation and does not result in a preferential commitment toward the megakaryocytic lineage.     

Characterization and screening for mutations of the growth arrest-specific 11 (GAS11) and C16orf3 genes at 16q24.3 in breast cancer

BACKGROUND: Both fibroblast-mediated cytokine gene therapy and bone marrow transplantation (BMT) have proven to be efficient protocols for the recovery of bone marrow depression. In this report, the effects of fibroblast-mediated interleukin (IL)-6 gene therapy, in combination with BMT, on the recovery of irradiation-induced bone marrow depression were investigated. METHODS: NIH3T3 fibroblast cells engineered to secrete IL-6 (NIH3T3-IL-6) or NIH3T3 cells transduced with the neomycin gene (NIH3T3-Neo), in combination with 10(7), 10(6), or 10(5) syngeneic bone marrow cells, were implanted into irradiated mice. RESULTS: The platelets and white blood cells in the peripheral blood of the irradiated mice increased greatly 12 days after implantation of NIH3T3-IL-6 cells and BMT, the white blood cell counts were restored to a normal level 32 days after the combined therapy, and the platelet number was obviously higher than that in mice implanted with NIH3T3-Neo and BMT. Twenty and 25 days after the combined therapy, the mice showed accelerated recovery of colony-forming unit (CFU)-granulocyte/macrophages and CFU-megakaryocytes when compared with the mice implanted with NIH3T3-Neo cells and BMT. Ten days after lethal irradiation with gamma rays, the spleens formed more CFU-spleen in mice implanted with NIH3T3-IL-6 cells and BMT than in mice injected with phosphate-buffered saline or NIH3T3-Neo cells. Combined therapy with NIH3T3-IL-6 cell implantation and BMT delayed the survival period of the hematopoietic-depressed mice significantly when compared with therapy with NIH3T3-Neo cell implantation and BMT. CONCLUSIONS: These data demonstrated that the combined therapy of fibroblast-mediated IL-6 gene therapy and BMT could significantly promote the recovery of irradiation-induced hematopoietic depression.     

Retroviral-mediated gene transduction of human alkyltransferase complementary DNA confers nitrosourea resistance to human hematopoietic progenitors

Myelosuppression is the dose-limiting toxicity for nitrosourea chemotherapy due to low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase in myeloid precursors. We have shown that high-efficiency myeloproliferative sarcoma virus (vM5MGMT)-mediated transduction of the human MGMT cDNA into murine bone marrow (BM) cells leads to high MGMT expression and increased progenitor resistance to 1,3-bis-(2-chloroethyl) nitrosourea (BCNU) in vitro immediately after infection and after BM transplantation. These experiments were designed to increase MGMT expression in human hematopoietic progenitors. CD34(+) BM cells were isolated over an immunoaffinity column (CEPRATE, CellPro, Inc.), resulting in a mean 66-fold enrichment in clonogenic progenitors (colony-forming unit granulocyte-macrophage + burst-forming unit erythroid + colony-forming unit granulocyte erythroid macrophage = megakaryocyte), with an average progenitor yield of 58 +/- 11.5% and a final population that was 54% CD34(+). Seventy % of progenitors derived from CD34(+) cells were transduced after coculture with AM12-vM5MGMT retroviral producers. vM5MGMT-transduced progenitors were over 2-fold more resistant to concentrations of BCNU between 30 and 50 micrometer than were concurrently LacZ-transduced progenitors (P < 0.003). In vitro selection of transduced, cytokine-stimulated CD34(+) cells with 20 micrometer BCNU resulted in survival of 4.7% of MGMT+ clonogenic progenitors compared to 0.05% of LacZ+ progenitors. These studies indicate that MGMT-transduced human hematopoietic progenitors have increased resistance to nitrosoureas, and in a clinical transplant setting, this strategy may reduce alkylating agent myelosuppression.     

Preservation of mucosal and systemic adjuvant properties of ISCOMS in the absence of functional interleukin-4 or interferon-gamma

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34(+)-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34(+)-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP cells led to 83-100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in longterm culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.     

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed lambdaSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.     

Correction of Fanconi anemia type C phenotypic abnormalities using a clinically suitable retroviral vector infection protocol

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.     

Transduction of CD34+ hematopoietic progenitor cells with an antitat gene protects T-cell and macrophage progeny from AIDS virus infection

Transduction of hematopoietic stem cells with genes that inhibit human immunodeficiency virus (HIV) replication has the potential to reconstitute immune function in individuals with AIDS. We evaluated the ability of an autoregulated gene, antitat, to inhibit replication of simian immunodeficiency virus (SIV) and HIV type 1 (HIV-1) in hematopoietic cells derived from transduced progenitor cells. The antitat gene expresses an antiviral RNA encoding polymeric Tat activation response elements in combination with an antisense tat moiety under the control of the HIV-1 long terminal repeat. CD34+ hematopoietic progenitor cells were transduced with a retroviral vector containing the antitat gene and then cultured under conditions that support in vitro differentiation of T cells or macrophage-like cells. Rhesus macaque CD4+ T cells and macrophage-like cells derived from CD34+ bone marrow cells transduced with the antitat gene were highly resistant to challenge with SIV, reflecting a 2- to 3-log reduction in peak SIV replication compared with controls. Similarly, human CD4+ T cells derived from CD34+ cord blood cells transduced with antitat were also resistant to infection with HIV-1. No evidence for toxicity of the antitat gene was observed in any of five different lineages derived from transduced hematopoietic cells. These results demonstrate that a candidate therapeutic gene introduced into hematopoietic progenitor cells can retain the ability to inhibit AIDS virus replication following T-cell differentiation and support the potential use of the antitat gene for stem cell gene therapy.     

Development of animal models for adeno-associated virus site-specific integration

The adeno-associated virus (AAV) is unique in its ability to target viral DNA integration to a defined region of human chromosome 19 (AAVS1). Since AAVS1 sequences are not conserved in a rodent's genome, no animal model is currently available to study AAV-mediated site-specific integration. We describe here the generation of transgenic rats and mice that carry the AAVS1 3.5-kb DNA fragment. To test the response of the transgenic animals to Rep-mediated targeting, primary cultures of mouse fibroblasts, rat hepatocytes, and fibroblasts were infected with wild-type wt AAV. PCR amplification of the inverted terminal repeat (ITR)-AAVS1 junction revealed that the AAV genome integrated into the AAVS1 site in fibroblasts and hepatocytes. Integration in rat fibroblasts was also observed upon transfection of a plasmid containing the rep gene under the control of the p5 and p19 promoters and a dicistronic cassette carrying the green fluorescent protein (GFP) and neomycin (neo) resistance gene between the ITRs of AAV. The localization of the GFP-Neo sequence in the AAVS1 region was determined by Southern blot and FISH analysis. Lastly, AAV genomic DNA integration into the AAVS1 site in vivo was assessed by virus injection into the quadriceps muscle of transgenic rats and mice. Rep-mediated targeting to the AAVS1 site was detected in several injected animals. These results indicate that the transgenic lines are proficient for Rep-mediated targeting. These animals should allow further characterization of the molecular aspects of site-specific integration and testing of the efficacy of targeted integration of AAV recombinant vectors designed for human gene therapy.     

Rescue and autonomous replication of adeno-associated virus type 2 genomes containing Rep-binding site mutations in the viral p5 promoter

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) play a crucial role in the rescue, replication, and integration of the viral genome. In the absence of a helper virus, little expression of the AAV Rep proteins occurs, and the AAV genome fails to undergo DNA replication. Since previous studies have established that expression of the Rep78 and Rep68 proteins from the viral p5 promoter is controlled by the Rep-binding site (RBS) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RBS and the YBS in the p5 promoter. These plasmids were transfected in HeLa or 293 cells and analyzed for the potential to undergo AAV DNA rescue and replication. Our studies revealed that (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in the RBS resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 but not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gene products was insufficient to mediate rescue and/or replication of the AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions that were biologically active in secondary infection of HeLa cells in the presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantly affected stable integration only in 293 cells, presumably because rescue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or KB cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RBS interaction plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficient to confer autonomous replication competence to AAV in 293 cells.     

Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector

Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clinical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38lo Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primitive progenitor cells was successful. Equal to fresh CD34+/CD38lo cells, >90% of reselected CD34+/CD38lo cells were in G0/G1. CD34+/CD38lo reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38lo cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38lo population) and recovery (62% of initial LTC-IC number in CD34+/CD38lo cells were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38lo cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.     

Successful replication of parvovirus B19 in the human megakaryocytic leukemia cell line MB-02

The pathogenic human parvovirus B19 has been shown to undergo productive replication in the erythroid lineage in primary normal human hematopoietic progenitor cells. However, none of the established erythroleukemia cell lines has allowed B19 virus replication in vitro. The remarkable erythroid tissue tropism of B19 virus was evaluated with a human megakaryocytic leukemia cell line, MB-02, which is dependent on the growth factor granulocyte-macrophage colony-stimulating factor but can be induced to undergo erythroid differentiation following treatment with erythropoietin (Epo). Whereas these cells did not support B19 virus DNA replication in the presence of granulocyte-macrophage colony-stimulating factor alone, active viral DNA replication was observed if the cells were exposed to Epo for 5 to 10 days prior to B19 virus infection, as detected by the presence of the characteristic B19 virus DNA replicative intermediates on Southern blots. No replication occurred if the cells were treated with Epo for 3 days or less. In addition, complete expression of the B19 virus genome also occurred in Epo-treated MB-02 cells, as detected by Northern blot analysis. B19 progeny virions were released into culture supernatants that were biologically active in secondary infection of normal human bone marrow cells. The availability of the only homogeneous permanent cell line in which induction of erythroid differentiation leads to a permissive state for B19 virus replication in vitro promises to yield new and useful information on the molecular basis of the erythroid tissue tropism as well as parvovirus B19-induced pathogenesis.