Figure 1

Generation of GDNF-secreting MSCs. (A) Schematic diagram illustrates the key elements of the lentiviral plasmid containing a viral 2A bicistronic transgene cassette (2A peptide depicted below with an arrowhead denoting the cleavage site) to co-express EGFP and GDNF driven by a short EF1α (sEF1α) promoter. Cis-acting sequences contain cPPT (central polypurine tract) and WPRE (woodchuck posttranscriptional regulatory element). (B) Immunoblots of HEK293T lysates for GDNF (top panel) or EGFP (middle panel) transfected with the lentiviral plasmids carrying the following transgenes: HEK293 (lane 2), EGFP (lane 3), GDNF (lane 4), GDNF-EGFP fusion (lane 5), and two GDNF-2A-EGFP constructs (lane 6,7). Various protein bands are denoted along the right side of the panels. Native GDNF appears as two bands due to the variable retention or cleavage of the signal peptide portion. The bands for GDNF-2A are approximately 5KDa heavier than GDNF due to retention of the 2A peptide up to the cleavage site. Alpha-tubulin (TUBA) was used as the protein loading control (bottom panel). (C) PC12 cells were transduced using lentivectors (LV) expressing EGFP, GDNF, or GDNF-2A-EGFP, respectively. ELISA analysis of media demonstrates an efficient GDNF secretion from the cells transduced with LV-GDNF-2A-EGFP, similar to LV-GDNF transduction, whereas secretion is minimal in controls (PC12 or PC12 transduced with LV-EGFP). n = 4 for each group, ***p < 0.001. (D) Immunocytofluorescence shows minimal GDNF expression in MSCs transduced by LV-EGFP, while MSCs transduced by LV-GDNF-2A-EGFP develop substantial GDNF expression 3 days after transduction (scale bar = 100 μm). (E) Immunoblots of cell lysates of MSCs transduced with LV-GDNF-2A-EGFP show EGFP and GDNF expression, which is absent in naïve MSCs. (F) ELISA of media shows that naïve MSCs secreted low levels of GDNF, which increased five-fold in the MSCs after LV-GDNF-2A-EGFP transduction, n = 4 for each group, ***p < 0.001.