Chiara Corsini

Retrovirus‐mediated transfer of the multidrug resistance gene into human haemopoietic progenitor cells

Summary. We report the utilization of cord blood (CB) or bone marrow (BM) derived low density or purified CD34+ cells as a target for human multidrug resistance (MDR1) gene transfer, Cells were cocultivated for 48 h with an irradiated MDR1 retroviral producer line. Since some degree of MDR1 gene expression has been reported to occur in haemopoietic progenitor cells and in peripheral blood cells, effciency of MDR1 gene transfer was assessed by: (1) Drug selection and culture in presence of 50 ng/ml doxorubicin, 10 ng/ml colchicine and 0.85 μg/ml taxol. In uninfected control, 1–2% of CFU‐GM and CFU‐GEMM were found to be drug‐resistant, while 14–31% of original clonogenic activity was found after 2 weeks of culture of transduced cells. Efficiency of MDR1 transfer was significantly enhanced by prestimulation with cytokines, and found to be significantly superior in CB‐derived compared to BM‐derived progenitors. (2) Analysis of MDR1 gene expression by evaluating MDR1 mRNA through polymerase chain reaction. MDR1 expression was very low in cultures of uninfected controls, whereas, after drug selection, MDR1 mRNA levels in transduced cells was as high as in the MDR1 retroviral producer line (positive controls). (3) Flow cytometiric analysis of the expression of CD34 and P‐glycoprotein, the product of the MDR1 gene. After MDR1 transduction and 2 weeks of culture, membrane expression of P‐glycoprotien, was found on 17–25% of viable CD34+ cells. (4) Cytochemical localization by APAAP staining of P‐glycoprotein. No specific localization was found in untransduced controls, whereas transduced and cultured CB‐cells expressed P‐glycoprotein on plasma and nuclei membrane. In conclusion, MDR1 gene transfer into CB‐ and BM‐derived progenitor cells seems a feasible and attractive approach to generate a drug‐resistant haemopoiesis.

Functional and Morphological Characterization of Immunomagnetically Selected CD34+ Hematopoietic Progenitor Cells

We evaluated the potential of immunomagnetically selected (miniMACS) progenitor cells to give rise to colony‐forming cells and their precursors, detected as long‐term culture‐initiating cells (LTC‐IC), as well as their capacity to expand in liquid cultures. A 90% mean purity, a 43.2% yield and a 55.8‐fold enrichment were achieved from normal bone marrow. When corrected for enrichment, the mean number of committed progenitor cells and the frequency of LTC‐IC (evaluated by means of limiting dilution assay [LDA]) were not statistically different in low density mononuclear cells or in the CD34‐enriched fractions. In five cases CD34+ selected cells grown in a stroma‐free long‐term bone marrow culture system with the addition of stem cell factor, interleukin 3, interleukin 6 and GM‐CSF every 48 h, showed a 15 (±15) and 31 (±21) mean colony forming unit‐granulocyte/macrophage fold increase on cultures at days 7 and 14. However, when corrected for enrichment, the expansion capability of these cells was significantly lower than that of the unseparated fraction, particularly after the first week. Immediately after separation, electron microscopy revealed that the CD34+ selected fraction contained more than 45% of well‐differentiated myeloid cells (MPO+ ), with iron beads preferentially clustered at one pole of the cell surface and sometimes already endocytosed in pinocytic vesicles. After 24 h and 48 h incubation at 37°C, the majority of the cells showed no iron particles, but about 30% of the cells were iron‐labeled phagocytic cells. The percentage of apoptotic cells with internalized iron was negligible. These data show that immunomagnetically separated CD34+ cells may have a slightly impaired short‐term expansion capability, but give rise to both committed and more primitive progenitor cells. During the separation, the iron beads are internalized, rapidly processed in the cytoplasm and do not seem to interfere with in vitro growth.

The effect of interleukin‐12 in ex‐vivo expansion of human haemopoietic progenitors

Summary. We evaluated progenitor cell proliferation in cultures supplemented by different cytokine combinations in the presence or absence of IL‐12. In cultures of low density cells, cytokine combinations including IL‐12 were associated to a greater proliferation (up to 6.7 ± 2.5 CFU‐GM fold expansion). However, in cultures of purified CD34+ cells the more efficient cytokine combination (147 ± 49 CFU‐GM fold expansion) was SCF, IL‐3, IL‐11 and MlP‐la, and the addition of IL‐12 did not further enhance expansion of progenitors.

Platelet quality and reduction of HLA expression in acid-treated platelet concentrates

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Gene transfer-mediated generation of drug-resistant hemopoiesis.

Autologous- or allogeneic-bone marrow transplantation are increasingly used to overcome the myelosuppressive effects of high dose chemotherapy administered to cancer patients. Transfer of the multidrug resistance (MDR) gene in hemopoietic progenitors has been proposed as a tool to administer higher and possibly more curative doses of chemotherapy. Murine models have demonstrated that retrovirus-mediated MDR transfer in bone marrow cells can render animals resistant to myeloablative doses of Taxol, and in vitro studies have shown that MDR-transduced human CD34+ cells can generate drug-resistant multipotential hemopoietic progenitors such as long term culture-initiating cells. Given these results, phase I clinical trials are currently under way to evaluate feasibility and treatment-related toxicity of MDR gene transfer in cancer patients by means of safe retroviral vectors. Finally, Taxol treatment of MDR transduced mice and human CD34+ cells have indicated that MDR is a dominant selectable marker in vitro and in vivo, and vectors carrying both MDR and non selectable genes such as beta-globin or glucocerebrosidase could be used in the next future for gene therapy of inherited disorders like thalassemia or Gaucher disease.