Article
Construction of a XYLT1-overexpressing mesenchymal stem cell line for cartilage tissue engineering
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Authors
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Michel Remling
- Universitätsmedizin Göttingen, Klinik für Unfallchirurgie, Orthopädie und plast. Chirurgie, Göttingen, Germany
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Kai O. Böker
- Universitätsmedizin Göttingen, Klinik für Unfallchirurgie, Orthopädie und plast. Chirurgie, Göttingen, Germany
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Isabel Faust-Hinse
- Herz- und Diabeteszentrum NRW, Universitätsklinik der Ruhr-Universität Bochum, Institut für Laboratoriums- und Transfusionsmedizin, Bad Oeynhausen, Germany
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Wolfgang Lehmann
- Universitätsmedizin Göttingen, Klinik für Unfallchirurgie, Orthopädie und plast. Chirurgie, Göttingen, Germany
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Arndt F. Schilling
- Universitätsmedizin Göttingen, Klinik für Unfallchirurgie, Orthopädie und plast. Chirurgie, Göttingen, Germany
| Published: | October 25, 2022 |
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Outline
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Objectives: The extracellular matrix (ECM) plays a vital role in the homeostasis and differentiation of mesenchymal stem cells (MSCs). In the process of ECM synthesis, Xylosyltransferase I (gene: XYLT1, protein: XT-I) catalyses the rate-limiting step in the glycosylation of proteoglycans which is crucial for a physiological ECM composition. It has been shown that hypofunctional XT-I can affect the chondrogenic maturation process, leading to a premature chondrocyte phenotype. However, little is known about the direct effect of XYLT1-overexpression on the chondrogenic differentiation of MSCs. Our approach is to establish a stable XYLT1-overexpressing mesenchymal stem cell line which will be used subsequently to investigate its chondrogenic differentiation potential. We want to shed light onto the role of XT-I-activity in cartilage differentiation.
Methods: Human bone marrow (hBM) MSCs were isolated from femoral necks and characterized using fluorescence-activated cell sorting (FACS) and in-vitro differentiation into adipogenic, osteogenic and chondrogenic lineage. Different gene transfer methods (i.e., nucleofection, lipofection, lentiviral transduction) with a XYLT1-coding plasmid were performed in primary hBM MSCs and immortalized SCP1 MSCs. Transfection efficacy was analyzed via quantitative PCR and fluorescence microscopy. Successful differentiation of MSCs was detected through qPCR and histological stainings.
Results and conclusion: FACS-analysis and in-vitro differentiation of the isolated hBM MSCs revealed multipotency which was confirmed via qPCR and histological staining. Lentiviral transduction of hBM MSCs showed nearly 100 % GFP-positive cells but the efficacy could not be confirmed through qPCR due to slow division rate. A stable 2.5-fold overexpression of XYLT1 could be obtained in immortalized SCP1 through lentiviral transduction. Chondrogenic differentiation of XYLT1-overexpressing SCP1 showed slightly delayed expression of cartilage hypertrophy marker Collagen X.
Our results indicate that XT-I-activity seems to influence cartilage hypertrophy within chondrogenic differentiation of MSCs. However, XYLT1-expression seems to be highly regulated since stable overexpression never exceeded 2.5-fold. Further research will be necessary to understand the mechanisms behind XT-I-activity and the differentiation of MSCs which might reveal new therapeutical or scientific targets for cartilage tissue engineering.
