title: Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors: assessment of immunophenotype and differentiation capacity in vitro.
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.
authors.affiliation: Department of Immunology, Faculty of Health Sciences, Institute for Cellular and Molecular Medicine and MRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria, P.O. Box 2034, Pretoria, 0001, South Africa.
authors.forename: Fiona A
authors.initials: FA
authors.lastname: van Vollenstee
authors.affiliation: Department of Immunology, Faculty of Health Sciences, Institute for Cellular and Molecular Medicine and MRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria, P.O. Box 2034, Pretoria, 0001, South Africa.
authors.forename: Carlo
authors.initials: C
authors.lastname: Jackson
authors.affiliation: Plastic and Reconstructive Surgeon, Private Practice, Pretoria, South Africa.
authors.forename: Danie
authors.initials: D
authors.lastname: Hoffmann
authors.affiliation: Department of Immunology, Faculty of Health Sciences, Institute for Cellular and Molecular Medicine and MRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria, P.O. Box 2034, Pretoria, 0001, South Africa.
authors.forename: Marnie
authors.initials: M
authors.lastname: Potgieter
authors.affiliation: Department of Immunology, Faculty of Health Sciences, Institute for Cellular and Molecular Medicine and MRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria, P.O. Box 2034, Pretoria, 0001, South Africa.
authors.forename: Chrisna
authors.initials: C
authors.lastname: Durandt
authors.affiliation: Department of Immunology, Faculty of Health Sciences, Institute for Cellular and Molecular Medicine and MRC Extramural Unit for Stem Cell Research and Therapy, University of Pretoria, P.O. Box 2034, Pretoria, 0001, South Africa. michael.pepper@up.ac.za.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Pepper
keywords: ASC labeling; Adipose derived stromal/stem cells (ASCs); Green fluorescent protein (GFP); Lentiviral vector; Viral transduction
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