Ex vivo expansion of haematopoietic stem cells and gene therapy development.

The results of a research project on ex vivo expansion of human haematopoietic stem cells (HSC) and development of gene therapy, funded by the Swiss National Research Program 46, are summarised and discussed in the context of current progresses and difficulties in these fields. A routine method for ex vivo expansion of human HSC is not yet available. However, stem cell biology has progressed importantly in recent years; ex vivo expansion of human HSC should become possible in the near future.

Ex vivo expansion of hematopoietic stem cells and gene therapy development.

The results of a research project on ex vivo expansion of human haematopoietic stem cells (HSC) and development of gene therapy, funded by the Swiss National Research Program 46, are summarised and discussed in the context of current progresses and difficulties in these fields. A routine method for ex vivo expansion of human HSC is not yet available. However, stem cell biology has progressed importantly in recent years; ex vivo expansion of human HSC should become possible in the near future.

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system.

TNF-alpha induces the generation of Langerin/(CD207)+ immature Langerhans-type dendritic cells from both CD14-CD1a and CD14+CD1a- precursors derived from CD34+ cord blood cells.

CD34+ cell-derived hematopoietic precursors amplified with FLT3-ligand, thrombopoietin and stem cell factor became, after a 6-day induction with GM-CSF, IL-4 and TGF-beta1, HLA-DR+, CD1a+, CD83-, CD86-, CD80- cells. A fraction of them expressed Langerin, Lag, and E-cadherin, resembling epidermal Langerhans cells (LC). TNF-alpha added for the last 3 days only marginally induced CD83 expression, but strikingly increased the proportion of immature Langerin+CD83- LC.

Efficient transduction of primary human B lymphocytes and nondividing myeloma B cells with HIV-1-derived lentiviral vectors.

We studied the transduction of primary human B lymphocytes and myeloma cells with lentiviral vectors. In peripheral blood B cells that had been activated with helper T cells (murine thymoma EL-4 B5) and cytokines, multiply attenuated HIV-1-derived vectors pseudotyped with vesicular stomatitis virus (VSV) G-envelope protein achieved the expression of green fluorescence protein (GFP) in 27% +/- 12% (mean +/- 1 SD; median, 27%) of B cells in different experiments. When compared in parallel cultures, the transducibility of B cells from different donors exhibited little variation.