Short-term effects of early-acting and multilineage hematopoietic growth factors on the repair and proliferation of irradiated pure cord blood (CB) CD34+ hematopoietic progenitor cells

PURPOSE: Hematopoietic growth factor(s) (GF) may exert positive effects in vitro or in vivo on the survival of hematopoietic stem and progenitor cells after accidental or therapeutic total body irradiation. METHODS AND MATERIALS: We studied the clonogenic survival and DNA repair of irradiated (0.36, 0.73, and 1.46 Gy) CD34+ cord blood (CB) cells after short-term incubation (24 h) with GFs. CD34+ cells were stimulated with basic fibroblast growth factor (bFGF), stem cell factor/c-kit ligand (SCF), interleukin-3 (IL-3), IL-6, leukemia inhibitory factor (LIF), and granulocyte-monocyte colony stimulating factor (GM-CSF) alone or in combination in short-term serum-free liquid suspension cultures (LSC) immediately after irradiation and then assayed for clonogenic progenitors. DNA repair was evaluated by analysis of DNA strand breaks using the comet assay. Survival of CFU-GM, BFU-E, and CFU-Mix was determined and dose-response curves were fitted to the data. RESULTS: The radiobiological parameters (D[0] and n) showed significant GF(s) effects. Combination of IL-3 with IL-6, SCF or GM-CSF resulted in best survival for CFU-GM BFU-E and CFU-Mix, respectively. Combinations of three or more GFs did not increase the survival of clonogenic CD34+ cells compared to optimal two-factor combinations. The D[0] values for CFU-GM, BFU-E, and CFU-Mix ranged between 0.56-1.15, 0.41-2.24, and 0.56-1.29 Gy, respectively. As for controls, the curves remained strictly exponential, i.e., all survival curves were strictly exponential without any shoulder (extrapolation numbers n=1 for all tested GF(s). DNA repair capacity of CD34+ cells determined by comet assay, was measured before, immediately after irradiation, as well as 30 and 120 min after irradiation at 1 Gy. Notably, after irradiation the 2-h repair of cytokine-stimulated and unstimulated CD34+ cells was similar. CONCLUSION: Our data indicate that increased survival of irradiated CB CD34+ cells after short-term GF treatment is mediated through proliferative GF effects on the surviving fraction but not through improved DNA repair capacity.     

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.     

Proliferation and survival of mammary carcinoma cells are influenced by culture conditions used for ex vivo expansion of CD34(+) blood progenitor cells

Malignant cell contamination in autologous transplants is a potential origin of tumor relapse. Ex vivo expansion of CD34(+) blood progenitor cells (BPC) has been proposed as a tool to eliminate tumor cells from autografts. To characterize the influence of culture conditions on survival, growth, and clonogenicity of malignant cells, we isolated primary mammary carcinoma cells from pleural effusions and ascites of patients with metastatic breast cancer and cultured them in the presence of stem cell factor (SCF), interleukin-1beta (IL-1beta), IL-3, IL-6, and erythropoietin (EPO), ie, conditions previously shown to allow efficient ex vivo expansion of CD34(+) BPC. In the presence of serum, tumor cells proliferated during a 7-day culture period and no significant growth-modulatory effect was attributable to the presence of hematopoietic growth factors. When transforming growth factor-beta1 (TGF-beta1) was added to these cultures, proliferation of breast cancer cells was reduced. Expansion of clonogenic tumor cells was seen in the presence of SCF + IL-1beta + IL-3 + IL-6 + EPO, but was suppressed by TGF-beta1. Cocultures of tumor cells in direct cellular contact with hematopoietic cells showed that tumor cell growth could be stimulated by ex vivo expanded hematopoietic cells at high cell densities (5 x 10(5)/mL). In contrast, culture under serum-free conditions resulted in death of greater than 90% of breast cancer cells within 7 days and a further decrease in tumor cell numbers thereafter. In the serum-free cultures, hematopoietic cytokines and cellular contact with CD34(+) BPC could not protect the tumor cells from death. Therefore, ex vivo expansion of CD34(+) BPC in serum-free medium provides an environment for efficient purging of contaminating mammary carcinoma cells. These results have clinical significance for future protocols in autologous progenitor cell transplantation in cancer patients.     

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.     

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.     

Enhancement of adenovirus-mediated gene transfer to human bone marrow cells

Adenovirus infection of CD34+ hematopoietic stem/progenitor cells is dependent on the multiplicity of infection (MOI), time of incubation, the volume in which the co-incubation occurs and the presence or absence of growth factors. Studies revealed that a brief co-incubation (1-8 hours), resulted in low levels of transgene expression, suggesting that adenovirus infection of CD34+ cells occurs slowly, and optimal transduction requires a 24 hour exposure to adenovirus. Infection by Ad/beta-gal or Ad/p53 at a MOI of 500:1 provided a high transduction efficiency but inhibited hematopoietic function. However, treatment at a MOI of 50-100 resulted in efficient transduction (10.7-15.7% positive) without detectable toxicity. Secondary proof of adenovirus transgene expression was demonstrated by detection of mRNA for p53 in Ad/p53 infected stem cells. We conclude that a 24 hour exposure to recombinant adenovirus encoding p53 or beta-gal, at a MOI of 50-100 is optimal for in vitro gene transfer to BM cells and has no significant effect on hematopoietic function. Adenovirus-mediated transduction of BM cells can also be modulated by growth factors (IL-3, GM-CSF and G-CSF) with improved gene delivery and maintenance of hematopoietic function. In summary, adenovirus vectors can be used to transiently transduce stem cells, and conditions have been defined to maximize expression and limit inhibitory effects on CD34+ cells. These data support continued investigation of this vector for local cytokine delivery and purging of stem cell products.     

Thrombopoietin directly and potently stimulates multilineage growth and progenitor cell expansion from primitive (CD34+ CD38-) human bone marrow progenitor cells: distinct and key interactions with the ligands for c-kit and flt3, and inhibitory effects of TGF-beta and TNF-alpha

Thrombopoietin (Tpo) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice suggest that Tpo might also play an important role in early hemopoiesis. Here, the direct ability of Tpo to stimulate stroma-independent growth, multilineage differentiation, and progenitor cell expansion from single primitive CD34+ CD38- human bone marrow cells was investigated. Tpo alone stimulated limited clonal growth, but synergized with c-kit ligand (KL), flt3 ligand (FL), or IL-3 to potently enhance clonogenic growth. Whereas KL and FL in combination stimulated the clonal growth of only 3% of CD34+ CD38- cells, 40% of CD34+ CD38- cells were recruited by KL+FL+Tpo, demonstrating that Tpo promotes the growth of a high fraction of CD34+ CD38- progenitor cells. Additional cytokines (IL-3, IL-6, and erythropoietin (Epo)) did not significantly enhance clonal growth above that observed in response to KL+FL+Tpo. In contrast, Tpo enhanced clonogenic growth in response to KL+FL+IL-3+IL-6+Epo by as much as 80%, implicating a key role for this cytokine in early hemopoiesis. Importantly, we also demonstrate that the majority of Tpo-recruited CD34+ CD38- progenitor cells have a multilineage differentiation potential, and that Tpo promotes prolonged expansion of multipotent progenitors. Specifically, whereas progenitor cells were reduced in cultures containing only KL+FL, addition of Tpo resulted in 40-fold expansion of multipotent progenitors following a 14-day incubation. Finally, we identified inhibitors of Tpo-induced progenitor cell growth, in that TGF-beta as well as TNF-alpha almost completely abrogated the growth of CD34+ CD38- progenitor cells in response to Tpo alone as well as KL+FL+Tpo.     

Interleukin-3 cooperates with tumor necrosis factor alpha for the development of human dendritic/Langerhans cells from cord blood CD34+ hematopoietic progenitor cells

We have previously shown that tumor necrosis factor (TNF)alpha strongly potentiates the granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin (IL)-3-dependent proliferation of CD34+ hematopoietic progenitor cells (HPC) through the recruitment of early progenitors with high proliferative potential. Furthermore, the combination of GM-CSF and TNFalpha allows the generation of large numbers of dendritic/Langerhans cells (D-Lc). Herein, we analyzed whether IL-3, when combined to TNFalpha would, as does GM-CSF, allow the generation of CD1a+ D-Lc. Accordingly, cultures of cord blood CD34+ HPC with IL-3 + TNFalpha yielded 20% to 60% CD14+ cells and 11% to 17% CD1a+ cells, while IL-3 alone did not generate significant numbers of CD1a+ cells. Although the percentage of CD1a+ cells detected in IL3 + TNFalpha was lower than that observed in GM-CSF + TNFalpha (42% to 78%), the strong growth induced by IL-3 + TNFalpha generated as many CD1a+ cells as did GM-CSF + TNFalpha. The CD14+ and CD1a+ cells generated with IL-3 + TNFalpha are similar to CD14+ and CD1a+ cells generated in GM-CSF alone and GM-CSF + TNFalpha, respectively. CD1a+ cells differed from CD14+ cells by (1) dendritic morphology, (2) higher expression of CD1a, CD1c, CD4, CD40, adhesion molecules (CD11c, CD54, CD58), major histocompatibility complex (MHC) class II molecules and CD28 ligands (CD80 and CD86), (3) lack of Fc receptor FcgammaRI (CD64) and complement receptor CR1 (CD35) expression, and (4) stronger induction of allogeneic T-cell proliferation. Thus, in combination with TNFalpha, IL-3 is as potent as GM-CSF for the generation of CD1a+ D-Lc from cord blood CD34+ HPC. The dendritic cell inducing ability of IL-3 may explain why mice with inactivated GM-CSF gene display dendritic cells.     

Bone marrow repopulation by human marrow stem cells after long-term expansion culture on a porcine endothelial cell line

In vitro exposure of murine hematopoietic stem cells (HSCs) to cell cycle-inducing cytokines has been shown to result in a defect in the ability of these cells to engraft. We used a porcine microvascular endothelial cell (PMVEC) line in conjunction with exogenous interleukin (IL)-3, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) to expand human HSCs that express the CD34 and Thy-1 antigens but lack lineage-associated markers (CD34+Thy-1+Lin- cells). Ex vivo expansion of hematopoietic cells was evaluated in comparison to stromal cell-free, cytokine-supplemented cultures. Cells expressing the CD34+Thy-1+Lin- phenotype were detectable in both culture systems for up to 3 weeks. These cells were reisolated from the cultures and their ability to engraft human fetal bones implanted into SCID mice (SCID-hu bone) was tested. HSCs expanded in PMVEC coculture were consistently capable of competitive marrow repopulation with multilineage (CD19+ B lymphoid, CD33+ myeloid, and CD34+ cells) progeny present 8 weeks postengraftment. In contrast, grafts composed of cells expanded in stroma-free cultures did not lead to multilineage SCID-hu bone repopulation. Proliferation analysis revealed that by 1 week of culture more than 80% of the cells in the PMVEC cocultures expressing the primitive CD34+CD38- phenotype had undergone cell division. Fewer than 1% of the cells that proliferated in the absence of stromal cells remained CD34+CD38-. These data suggest that the proliferation of HSCs in the presence of IL-3, IL-6, GM-CSF, and SCF without stromal cell support may result in impairment of engraftment capacity, which may be overcome by coculture with PMVECs.     

Retroviral transfer of the glucocerebrosidase gene into CD34+ cells from patients with Gaucher disease: in vivo detection of transduced cells without myeloablation

Retroviral gene transfer of the glucocerebrosidase gene to hematopoietic progenitor and stem cells has shown promising results in animal models and corrected the enzyme deficiency in cells from Gaucher patients in vitro. Therefore, a clinical protocol was initiated to explore the safety and feasibility of retroviral transduction of peripheral blood (PB) or bone marrow (BM) CD34+ cells with the G1Gc vector. This vector uses the viral LTR promoter to express the human glucocerebrosidase cDNA. Three adult patients have been entered with follow-up of 6-15 months. Target cells were G-CSF-mobilized and CD34-enriched PB cells or CD34-enriched steady state BM cells, and were transduced ex vivo for 72 hr. Patient 1 had PB cells transduced in the presence of autologous stromal marrow cells. Patient 2 had PB cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. Patient 3 had BM cells transduced in the presence of autologous stroma, IL-3, IL-6, and SCF. At the end of transduction, the cells were collected and infused immediately without any preparative treatment of the patients. The transduction efficiency of the CD34+ cells at the end of transduction was approximately 1, 10, and 1 for patients 1, 2, and 3, respectively, as estimated by semiquantitative PCR on bulk samples and PCR analysis of individual hematopoietic colonies. Gene marking in vivo was demonstrated in patients 2 and 3. Patient 2 had vector-positive PB granulocytes and mononuclear bone marrow cells at 1 month postinfusion and positive PB mononuclear cells at 2 and 3 months postinfusion. Patient 3 had a positive BM sample at 1 month postinfusion but was negative thereafter. These results indicate that gene-marked cells can engraft and persist for at least 3 months postinfusion, even without myeloablation. However, the level of corrected cells (<0.02%) is too low to result in any clinical benefit, and glucocerebrosidase enzyme activity did not increase in any patient following infusion of transduced cells. Modifications of vector systems and transduction conditions, along with partial myeloablation to allow higher levels of engraftment, may be necessary to achieve beneficial levels of correction in patients with Gaucher disease.     

Efficient retroviral mediated transfer of the glucocerebrosidase gene in CD34+ enriched umbilical cord blood human hematopoietic progenitors

Obtaining efficient transfer of a normal gene and its sustained expression in self-renewing hematopoietic stem cell populations is a central concern for gene therapy initiatives. Potentially, 10(8) to 10(9) CD34+ enriched cells per patient will be required for transduction and subsequent reimplantation. These studies present an efficient method for the transduction of human CD34+ cells that can be used in a clinical study of gene transfer. The method uses a centrifugation-enhanced technique for the retroviral-mediated transfer of the normal human glucocerebrosidase (GC) gene to human CD34+ enriched umbilical cord blood cells (CB). Previous studies had described high expression of GC in CD34+ enriched cells but had not reported transduction efficiency in the progenitor population specifically. The data demonstrate an average transduction efficiency in the progenitor cell population of 50% as measured by polymerase chain reaction (PCR) for the integrated GC-cDNA in clonogenic cells. Measurements of enzyme activity comparing transduced and nontransduced fractions at 6 days posttransduction indicate an average enzyme increase of six-fold over normal background levels. PCR of colony forming units-granulocyte/macrophage (CFU-GM) plated at 6 weeks from long-term culture-initiating cell (LTC-IC) cultures also indicates transfer of the transgene to early progenitor cells. Finally, experiments were carried out with the human erythroleukemia cell line, TF-1, to estimate the durable expression of the transgene. Enzymatic activities in transduced TF-1 cultures remained at 30-fold above the activity of nontransduced controls. The expression persisted for 6 weeks in culture. These studies demonstrate efficient transduction of early progenitor cells and sustained expression of the transgene in cell cultures.     

Characterization of peripheral blood steady-state progenitor cells preserved in liquid culture conditions with or without GM-CSF and IL2

The cellular characteristics of steady-state peripheral blood progenitor cell (PBPC) apheresis, including total number of lymphomononuclear cells, CD34 and CFUs, was evaluated in a group of 26 chemo-radiotherapy patients as well as in a group of 23 surgically resected cancer patients. Three-to seven-day incubation in standard liquid culture conditions with growth factors (IL2, GM-CSF or both) correlated with a statistically significant increase in CD34+ and CD56+ cell populations compared with incubation without growth factors, especially when both GM-CSF and IL2 were used. In addition, an increase in CD33+, CD13+ and HLA-DR+ cell populations was observed after 3-7 days incubation with GM-CSF. The basal culture control exhibited a decrease in CD33+ and CD13+ cell populations while CD34+ and CD56+ cell populations were maintained. These results were similar in the treated and untreated groups of patients. The infusion of GM-CSF and IL2 preincubated PBPC after intensive chemotherapy was associated with a rapid hematological recovery with a median time duration for WBC < 500/uL, WBC < 1.000/uL and platelets < 20.000/uL of 7.9 days, 14.9 days and 10.7 days respectively. We conclude that a short GM-CSF and IL2 preincubation of steady-state PBPC is associated with an increase in cell populations exhibiting the immune and progenitor cell phenotypes and correlates with an early hematological recovery after intensive chemotherapy.     

Generation and functional characterization of human dendritic cells derived from CD34 cells mobilized into peripheral blood: comparison with bone marrow CD34+ cells

Dendritic cells (DCs) are the most powerful professional antigen-presenting cells (APC), specializing in capturing antigens and stimulating T-cell-dependent immunity. In this study we report the generation and characterization of functional DCs derived from both steady-state bone marrow (BM) and circulating haemopoietic CD34+ cells from 14 individuals undergoing granulocyte colony-stimulating factor (G-CSF) treatment for peripheral blood stem cells (PBSC) mobilization and transplantation. Clonogenic assays in methylcellulose showed an increased frequency and proliferation of colony-forming unit-dendritic cells (CFU-DC) in circulating CD34+ cells, compared to that of BM CD34+ precursors in response to GM-CSF and TNF-alpha with or without SCF and FLT-3L. Moreover, peripheral blood (PB) CD34+ cells generated a significantly higher number of fully functional DCs, as determined by conventional mixed lymphocyte reactions (MLR), than their BM counterparts upon different culture conditions. DCs derived from mobilized stem cells were also capable of processing and presenting soluble antigens to autologous T cells for both primary and secondary immune response. Replacement of the early-acting growth factors SCF and FLT-3L with IL-4 at day 7 of culture of PB CD34+ cells enhanced both the percentage of total CD1a+ cells and CD1a+ CD14- cells and the yield of DCs after 14 d of incubation. In addition, the alloreactivity of IL-4-stimulated DCs was significantly higher than those generated in the absence of IL-4. Furthermore, autologous serum collected during G-CSF treatment was more efficient than fetal calf serum (FCS) or two different serum-free media for large-scale production of DCs. Thus, our comparative studies indicate that G-CSF mobilizes CD34+ DC precursors into PB and circulating CD34+ cells represent the optimal source for the massive generation of DCs. The sequential use of early-acting and intermediatelate-acting colony-stimulating factors (CSFs) as well as the use of autologous serum greatly enhanced the growth of DCs. These data may provide new insights for manipulating immunocompetent cells for cancer therapy.     

Ability of early acting cytokines to directly promote survival and suppress apoptosis of human primitive CD34+CD38- bone marrow cells with multilineage potential at the single-cell level: key role of thrombopoietin

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38- human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38- but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38- progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38- long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38- cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.     

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.     

Selective immunoaffinity-based enrichment of CD34+ cells transduced with retroviral vectors containing an intracytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (deltaLNGFR) gene

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.     

Use of flow cytometric CD34 analysis to quantify hematopoietic progenitor cells

This review summarizes our experiments on flow cytometric analysis of CD34 positive mononuclear cells (MNC) and on colony formation of myeloid hematopoietic progenitor cells in the clonogenic assay. We examined MNC isolated by density centrifugation of bone marrow, cord blood and peripheral blood. The latter samples originated either from patients recovering from myelosuppressive treatment who received no growth factors or from patients treated with G-CSF or GM-CSF. We attempted to correlate the results obtained by CD34 analysis with the cloning efficiency determined after a 14 day culture period in the methylcellulose-based clonogenic assay. The highest cloning efficacy (60%-100%) was observed in cord blood, however, a good correlation was found in both untreated and GM-CSF treated peripheral blood samples in which a mean of 50% and 20% of the number of CD34 positive MNC gave rise to myeloid colonies. In bone marrow, the cloning efficacy was generally lower and ranged between 5% and 15%. The lowest values were observed in G-CSF treated peripheral blood in which colonies were grown from only 1%-9% of the CD34+ MNC. Due to the variable numbers of CD34+ lymphoid and/or more committed myeloid precursors which form either no colonies or only clusters, there was a greater variation and a lower cloning efficiency in the latter two cell sources. In conclusion, one colour CD34 analysis of cord blood MNC and untreated or GM-CSF treated peripheral blood MNC provides reliable results with respect to the content of myeloid progenitors. Analysis of bone marrow MNC and G-CSF treated peripheral blood MNC requires two colour staining using CD34 and CD45RA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thiamine deficiency-induced disruptions in the diurnal rhythm and regulation of body temperature in the rat

A Moloney-derived retrovirus containing both LacZ and NeoR genes (G1BgSVNa from Genetic Therapy, Inc.), was used to transduce human and murine bone marrow stromal cells. Different kinds of stromal cells that were able to support hematopoiesis were transduced by incubation for 24 h in the presence of virus-containing supernatant. Semiconfluent layers of MRC-5 (human, myofibroblastic, fetal, pulmonary) and MS-5 (murine, myofibroblastic, medullary) cells were successfully transduced after one 24-h incubation, as demonstrated by G418 resistance and Escherichia coli beta-galactosidase staining. In contrast, human stromal cells, purified from primary confluent layers grown for 3-4 weeks, could not be transduced. However, stromal cells generated after 10-12 days in culture from Stro-1+ and 1B10+ stromal precursors were successfully transduced in the presence of basic fibroblast growth factor. Transduced stromal cells maintained a myofibroblastic phenotype, although with a decreased number of alpha-SM actin-positive microfilaments in MS-5 cells. The ability to support the generation of stroma-adherent colony-forming cells from cocultured cord blood CD34+ cells after 4 weeks in culture was similar before and after transduction and G418 selection. In conclusion, human primary stromal precursors can be efficiently transduced, and the stromal cell phenotype and function are not significantly altered after retroviral-mediated transfer of marker genes.     

Neurotrophin mRNA expression in the developing tooth suggests multiple roles in innervation and organogenesis

Dendritic cells (DCs), which are antigen presenting cells of potential use in human antitumor vaccination trials, are presently the subject of intense investigation. Many recent studies have reported the possibility of generating ex vivo large numbers of DCs with high antigen presenting capacity by the culture of bone marrow or blood progenitors. In this study, we examined the differentiation into DCs of CD34+ progenitors isolated from the G-CSF mobilized blood of 3 healthy donors and 5 patients with breast cancer and cultured in the presence of GM-CSF + IL-13. The characteristics of the cells were compared to those of cells obtained in the presence of GM-CSF + TNF alpha. By day 15, one third of the bulk cells cultured with IL-13 were CD1a+/CD14- and strongly expressed CD1c, CD40, CD80 and HLA-DR. In contrast, cells obtained with TNF alpha expressed CD1a on one in three cells but with a considerably lower fluorescence intensity than on IL-13-cultured cells and strongly expressed CD14 on more than 50% of cells. CD1a+/CD14- cells emerged in IL-13 cultures at day 5, while in TNF alpha cultures CD14+ cells appeared before CD1a+ cells. Cells grown in the presence of IL-13 had an increased capacity to present antigens to autologous lymphocytes and to stimulate allogeneic T-lymphocytes. This effect was greater than that of cells grown in the presence of TNF alpha. These cells should therefore have greater effector potential in any therapeutic applications in humans.     

Haemopoietic growth factor production by normal and aplastic anaemia stroma in long-term bone marrow culture

Defective marrow stroma, or microenvironment, have been proposed as one of several mechanisms to account for bone marrow failure in aplastic anaemia (AA). This could involve defects in positive- or negative-acting haemopoietic regulator expression by AA stroma, or alteration of normal stroma-stem cell interactions. We have used a sensitive bioassay to investigate production of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage-colony stimulating factor (GM-CSF), interleukin (IL)-3, IL-6 and stem cell growth factor (SCF), by normal and AA stroma in long-term bone marrow culture (LTBMC). LTBMC were grown to confluence, irradiated and harvested to yield a single cell suspension. These cells were cocultured with normal target bone marrow mononuclear cells (BMMC), or CD34+ cells, in clonogenic assays, in the absence of exogenous cytokines. Cytokines responsible for the colony-stimulating activity (CSA) and burst-promoting activity (BPA) produced by stromal cells were identified by neutralizing antibodies to specific cytokines. All normal stroma populations produced G-CSF and GM-CSF, 93% produced IL-3, 80% produced IL-6, and 70% produced SCF. Similarly, all AA stroma produced G-CSF and GM-CSF, and 71% produced SCF. In contrast, only 71% of AA stroma produced IL-3 and 36% produced IL-6. Target cell stimulation was not dependent on direct stroma-target cell contact, suggesting production of soluble cytokines. However, although both IL-6 and G-CSF were detected in LTBMC supernatants by enzyme-linked immunoassay (ELISA), IL-3 and GM-CSF were undetectable, perhaps indicating low-level local production of these factors.