Figure 1
In vitro characterisation of genetically modified hMSC cell transplants. (A) Plasmid DNA constructs used for genetic modification of human bone marrow-derived stromal cells (hMSC) in order to obtain hMSC-EGFP, hMSC-NT3 and hMSC-NT3-EGFP cell populations. CMV: Cytomegalovirus immediate early promotor + enhancer. EGFP: enhanced green fluorescent protein. pA: SV40 early mRNA polyadenylation signal. NT3: neurothrophin-3. IRES: internal ribosome entry site. (B) Representative standard PCR and RT-PCR analysis on DNA and mRNA isolated from the different genetically modified hMSC populations used in this study (see numbers below pictures) indicating the presence of transgenic EGFP and/or NT3 DNA and mRNA sequences. M: length marker. GAPDH: glyceraldehyde-3-phosphate dehydrogenase. (C) Representative real-time RT-PCR analysis on mRNA isolated from the different genetically modified hMSC populations used in this study (see numbers below pictures) showing quantitative differences in the level of transgenic EGFP and/or NT3 mRNA transcripts/1000 copies GAPDH; nd: no data available. (D) Representative ELISA measurement on supernatant samples from the different genetically modified hMSC populations used in this study (see numbers below pictures) showing quantitative differences in the level of NT3 secretion in picogram/105 cells/24 hours. (E) Representative flow cytometric analysis of EGFP expression by hMSC-EGFP and hMSC-NT3-EGFP populations showing quantitative differences in the level of transgenic EGFP protein expression. SSC: side scatter.