Generation and characterization of human hematopoietic cell lines expressing factor VIII.
Considering the plasticity of hematopoietic stem cells (HSC), they would be ideal targets for gene therapy of hemophilia A by virtue of their progeny providing immediate access to the bloodstream. However, several attempts to show expression of recombinant factor VIII (rFVIII) by primary hematopoietic cells and cell lines have failed; this failure was attributed to the inability of HSC to secrete rFVIII. Here we describe the generation of stable, FVIII-secreting hematopoietic cell lines representing different blood-cell types using a bicistronic lentiviral vector encoding for a B-domain-deleted FVIII (FVIII Delta B) and enhanced green fluorescence protein (EGFP). Transduced cell lines with erythroid and/or megakaryocytic background, (K562-F8 and TF-1-F8) secrete high levels of FVIII in the order of 76.4 and 41.6 ng FVIII:C/ml, whereas moderate and low levels are observed in B lymphoblastoid Raji-F8 cells and the T leukemia line Jurkat-F8 which secrete 6.73 and 1.83 ng FVIII:C/ml, respectively. The capacity to secrete rFVIII appeared to depend on factors related to the cell lineage rather than on the transduction efficacy. Stimulation of transduced cells with the protein kinase C (PKC)-activator phorbol myristate acetate (PMA) resulted in a marked augmentation of rFVIII secretion and enhanced green fluorescent protein (EGFP). Incubation with 0.1 and 1 ng/ml PMA resulted in up to 2.7-fold (K562-F8, Raji-F8) and 1.8-fold (293T-F8) increased rFVIII secretion. The established cell lines should be helpful in further elucidating mechanisms that are able to improve FVIII secretion in hematopoietic cells on a post-translational level and suggest reanalysis of hematopoietic cells as target for gene therapy of hemophilia.
Tonn
T
T
Institute for Biomedical Research, Georg-Speyer-Haus, Frankfurt/Main, Germany. ttonn@bsdhessen.de
Herder
C
C
Becker
S
S
Seifried
E
E
Grez
M
M
D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D005169:Factor VIII / Q000032:analysis / Q000235:genetics*; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000502:physiology*; D006801:Humans; D007938:Leukemia / Q000097:blood / Q000145:classification; D008223:Lymphoma / Q000097:blood / Q000145:classification; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D005169:Factor VIII