Introduction
It is well known that resident adipose stem/stromal cells (ASCs) are a heterogeneous
population of multipotent cells 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 cells of mesodermal origin including osteocytes,
chondrocytes and adipocytes. Adipose derived stromal cells offer great therapeutic potential
in multiple medical fields, including, orthopedics, cardiology, oncology and degenerative
diseases, to name a few. Combining different disciplines of medicine and engineering, organ
and tissue repair can be achieved through tissue engineering and regenerative medicine.
Adipose derived stromal cells (ASCs) can be utilized as biological vehicles for vector-based
gene delivery systems, since they home to sites of inflammation and infection in vivo. In order
to reach the long-term aim of clinical translation of cell-based therapy, preclinical safety and
efficacy need to be shown in animal models. This has motivated the development of
standardized isolation, characterization and differentiation operating procedures as well as an in vivo tracking system for ASCs and lentiviral vector transduction for a vector-based gene delivery system.
Methodology
Human ASCs were isolated from lipoaspirate, expanded in culture, immunophenotyped using flow cytometery and induced to differentiate into adipogenic, osteogenic and chondrogenic lineages. Tri-lineage differentiation was confirmed by microscopy. The ASCs were then transduced with green fluorescent protein (GFP)-expressing lentiviral vectors in vitro. The effect of the GFP lentiviral vector on ASCs was investigated by studying ASC immunophenotypic expression of surface markers as well as their capacity to differentiate into osteocytes, chondrocytes and adipocytes.
Results
The isolated and expanded cell population, from harvested lipoaspirate adhered to recommended ASC identity criteria. The heterogeneity of ASCs was confirmed by the presence of sub-populations. Transduction efficiency in ASC cultures of approximately 80% was observed after introducing a total of 300 μl of concentrated lentiviral vector suspension per 4.8 x 104 cells. No immunophenotypic differences were observed between GFP positive and GFP negative cultures. Flow cytometric analysis revealed a progressive increase in GFP expression following in vitro expansion of transduced ASCs. Both non-transduced and transduced cultures successfully differentiated into osteocytes, chondrocytes and adipocytes.
Conclusion
The isolated and expanded cell population conformed to the recommended characterization criteria. Heterogeneity was demonstrated with the identification of immunophenotypic sub-populations and semi-quantification of adipogenesis was performed. ASCs were efficiently transduced using the GFP lentiviral vectors produced in our facility. In addition, transduced ASCs maintained adherence to plastic, ASC immunophenotype and were able to differentiate successfully into cells of the three lineages of mesodermal origin. This optimized GFP-ASC transduction technique offers a feasible tracking system as well as a vector-based gene delivery system for future preclinical studies. / Dissertation (MSc)--University of Pretoria, 2015. / tm2015 / Immunology / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/45939 |
| Date | January 2015 |
| Creators | Van Vollenstee, Fiona A. |
| Contributors | Pepper, Michael Sean, Potgieter, Marnie |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2015 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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