Bone marrow-derived mesenchymal stem cells were isolated from a 33-year old, healthy male donor. The cells were immortalized by the human telomerase reverse transcriptase (hTERT) gene and transfected with the GLP-1 fusion protein and thymidine kinase of Herpes simplex virus (HSV) Type 1, a suicide gene, to enable controlled elimination of the cells in vivo.
Mesenchymal stem cells are isolated from bone marrow aspirates by their adherence to plastic and then immortalized with the catalytic subunit of the human telomerase reverse transcriptase gene. The hMSC-TERT cell line is transfected with an expression vector encoding both GLP-1 fusion protein (see below) and thymidine kinase of Herpes simplex virus (HSV) Type 1. The dimeric GLP-1 construct corresponds to human preproglucagon (89–145), followed by a second GLP-1 (7–37) sequence. To target GLP-1 to the secretory pathway, the stromelysin signal and leader sequence (acc. no. P24347 aa 1–97) are used. After repeated single-cell cloning, the production cell line 79TM217/18K5 is used to produce a master cell bank according to good manufacturing practice (GMP).
Cells are grown in EMEM, FCS (10% ) and L-glutamine (2 mM).