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Regulating Gene Expression to Promote Osteoblastic Differentiation of Stem Cells
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - Ångström, Polymerkemi. (Translational Chemical Biology Laboratory)
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
Abstract [en]

Bone is a tissue that heals by itself, unless the defect is too large (critical size). Today, novel regenerative medicine approaches have emerged as an alternative to treat such defects. This thesis explores alternative therapeutic strategies for bone tissue engineering which are biocompatible and clinically translatable. Many types of scaffolds that can act as reservoirs for growth factors such as rh-BMP-2 have been developed for bone tissue engineering in the past. However, the role of cross-linking chemistries that are employed to make hydrogels on the integrity and function of the loaded growth factors is not well understood. In this thesis, we have explored the influence of cross-linking chemistry on rh-BMP-2 integrity and bioactivity both in-vitro and in-vivo. These studies have demonstrated that thiol-Michael addition cross-linking chemistry greatly affects the integrity and bio-functionality of the loaded protein BMP-2 and leads to poor bone formation in an in-vivo rat model. On the other hand, hydrogels employing hydrazone chemistry did not significantly affect the integrity and bioactivity of BMP-2, which lead to a superior bone formation in-vivo. Since the high dose of rh-BMP-2 is known to confer many side effects, alternative ex-vivo strategies involving transient transfection of BMP-2 expressing plasmid DNA and silencing of anti-osteogenic genes using siRNA are developed. Our optimized method involves rapid transfection of hMSCs in suspension (5 minutes) with plasmid DNA followed by centrifugation and encapsulation in a hydrogel not only reduced cytotoxicity but also lead to efficient osteoblast differentiation of stem cells. Furthermore, this thesis presents the role of ECM-derived polymer HA in interacting with siRNA and trafficking across the plasma membrane, presumably through CD44 receptors and successfully silencing the target gene in-vitro. We explored the potential of such a non-cationic transfection method to deliver functional siRNA (anti-Pleckho-1 siRNA) in MSCs and compared it with commercially available cationic lipid LipofectamineTMRNAiMAX, using our optimized suspension transfection method. Our novel ex-vivo strategy employing HA hydrogels enabled efficient silencing of BMP-2 signaling pathway antagonist Pleckho-1 while avoiding the cytotoxicity issues in 3D, which further qualifies them for potential clinical application for cell-based therapies. 

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. , s. 106
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1758
HSV kategori
Forskningsprogram
Kemi med inriktning mot polymerkemi
Identifikatorer
URN: urn:nbn:se:uu:diva-369660ISBN: 978-91-513-0540-0 (tryckt)OAI: oai:DiVA.org:uu-369660DiVA, id: diva2:1271021
Disputas
2019-02-13, Häggsalen, Ångströmlaboratoriet, Lägerhyddsvägen 1, Uppsala, 13:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-01-23 Laget: 2018-12-15 Sist oppdatert: 2019-02-18
Delarbeid
1. Rational Design of Biomaterials for Growth Factor Delivery: Impact of Hydrogel Crosslinking Chemistry on the In Vitroand In VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2
Åpne denne publikasjonen i ny fane eller vindu >>Rational Design of Biomaterials for Growth Factor Delivery: Impact of Hydrogel Crosslinking Chemistry on the In Vitroand In VivoBioactivity of Recombinant Human Bone Morphogenetic Protein-2
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-369656 (URN)
Tilgjengelig fra: 2018-12-14 Laget: 2018-12-14 Sist oppdatert: 2018-12-15
2. Innovative strategy for 3D transfection of primary human stem cells with BMP-2 expressing plasmid DNA: A clinically translatable strategy for ex vivogene therapy
Åpne denne publikasjonen i ny fane eller vindu >>Innovative strategy for 3D transfection of primary human stem cells with BMP-2 expressing plasmid DNA: A clinically translatable strategy for ex vivogene therapy
2019 (engelsk)Inngår i: International Journal of Molecular Sciences, ISSN 1422-0067, E-ISSN 1422-0067, Vol. 20, nr 1, artikkel-id 56Artikkel i tidsskrift (Fagfellevurdert) Published
Abstract [en]

Ex vivo gene therapy offers enormous potential for cell-based therapies, however, cumbersome in vitro cell culture conditions have limited its use in clinical practice. We have optimized an innovative strategy for the transient transfection of bone morphogenetic protein-2 (BMP-2) expressing plasmids in suspended human stem cells within 5-min that enables efficient loading of the transfected cells into a 3D hydrogel system. Such a short incubation time for lipid-based DNA nanoparticles (lipoplexes) reduces cytotoxicity and at the same time reduces the processing time for cells to be transplanted. The encapsulated human mesenchymal stromal/stem cells (hMSCs) transfected with BMP-2 plasmid demonstrated high expression of an osteogenic transcription factor, namely RUNX2, but not the chondrogenic factor (SOX9), within the first three days. This activation was also reflected in the 7-day and 21-day experiment, which clearly indicated the induction of osteogenesis but not chondrogenesis. We believe our transient transfection method demonstrated in primary MSCs can be adapted for other therapeutic genes for different cell-based therapeutic applications.

sted, utgiver, år, opplag, sider
Basel, Switzerland: MDPI, 2019
Emneord
hydrogel; DNA; transfection; ex vivo; hyaluronic acid
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-369655 (URN)10.3390/ijms20010056 (DOI)000459747700056 ()30583610 (PubMedID)
Forskningsfinansiär
EU, FP7, Seventh Framework Programme, FP7/2007-2013/607868Swedish Foundation for Strategic Research , SBE13-0028
Tilgjengelig fra: 2018-12-14 Laget: 2018-12-14 Sist oppdatert: 2019-03-18bibliografisk kontrollert
3. Hyaluronic acid facilitates non-cationic siRNA delivery and gene silencing in CD44 positive cells
Åpne denne publikasjonen i ny fane eller vindu >>Hyaluronic acid facilitates non-cationic siRNA delivery and gene silencing in CD44 positive cells
Vise andre…
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-369657 (URN)
Tilgjengelig fra: 2018-12-14 Laget: 2018-12-14 Sist oppdatert: 2018-12-15
4. New insight on siRNA transfection in three dimensions: Improved gene silencing in human mesenchymal stem cells encapsulated in hyaluronicacid hydrogel
Åpne denne publikasjonen i ny fane eller vindu >>New insight on siRNA transfection in three dimensions: Improved gene silencing in human mesenchymal stem cells encapsulated in hyaluronicacid hydrogel
(engelsk)Manuskript (preprint) (Annet vitenskapelig)
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-369658 (URN)
Tilgjengelig fra: 2018-12-14 Laget: 2018-12-14 Sist oppdatert: 2018-12-15

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