Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors : assessment of immunophenotype and differentiation capacity in vitro
dc.contributor.author | Van Vollenstee, Fiona A. | |
dc.contributor.author | Jackson, Carlo Stephan | |
dc.contributor.author | Hoffmann, Danie | |
dc.contributor.author | Potgieter, Marnie | |
dc.contributor.author | Durandt, Chrisna | |
dc.contributor.author | Pepper, Michael Sean | |
dc.contributor.email | michael.pepper@up.ac.za | en_ZA |
dc.date.accessioned | 2016-09-12T13:52:26Z | |
dc.date.issued | 2016-10 | |
dc.description | Supplementary Figure 1 Gaussian distribution curve of GFP positive transduced cells. Adherent ASCs (48 000 cells) were transduced with different dilutions of lentiviral vector stock solutions and GFP expression was measured over 10 post-transduction passages using flow cytometry. The following amounts of lentivirus vector stock solution - 0-, 5-, 10-, 25-, 50-, 100-, 150-, 200-, 250- and 300 µl – were utilized to determine the optimal titer for ASC transduction. Data at all post transduction passages (T), with the exception of T0 and T3 fits with the Gaussian distribution curve. The interpolated value for 100 % transduction was found to be 363.18 µl lentiviral vector stock solution. (TIFF 130 kb) | |
dc.description | Supplementary Figure 2 Overlaying plots comparing the unstained control, non-transduced and transduced GFP positive cells for individual markers. The plots are from one biological replicate at a specific post-transduction passage. Each plot displays the expression of an individual antibody marker or GFP expression in the cell cytoplasm. (A) CD73 BV510; (B) CD90 PC5; (C) CD105 PE; (D) CD34 PC; (E) CD45 KO; and (F) GFP. (TIFF 388 kb) | |
dc.description.abstract | Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studies | en_ZA |
dc.description.department | Immunology | en_ZA |
dc.description.librarian | hb2016 | en_ZA |
dc.description.uri | http://link.springer.com/journal/10616 | en_ZA |
dc.identifier.citation | Van Vollenstee, F.A., Jackson, C., Hoffmann, D., Potgieter, M., Durandt, C. & Pepper, M.S. Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors : assessment of immunophenotype and differentiation capacity in vitro. Cytotechnology (2016) 68: 2049-2060. doi:10.1007/s10616-016-9945-6. | en_ZA |
dc.identifier.issn | 0920-9069 (print) | |
dc.identifier.issn | 1573-0778 (online) | |
dc.identifier.other | 10.1007/s10616-016-9945-6 | |
dc.identifier.uri | http://hdl.handle.net/2263/56721 | |
dc.language.iso | en | en_ZA |
dc.publisher | SpringerLink | en_ZA |
dc.rights | © The Author(s) 2016. This article is published with open access at Springerlink.com. | en_ZA |
dc.subject | ASC labeling | en_ZA |
dc.subject | Adipose derived stromal/stem cells (ASCs) | en_ZA |
dc.subject | Green fluorescent protein (GFP) | en_ZA |
dc.subject | Lentiviral vector | en_ZA |
dc.subject | Virl transduction | en_ZA |
dc.title | Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors : assessment of immunophenotype and differentiation capacity in vitro | en_ZA |
dc.type | Article | en_ZA |
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