Nonviral gene transfer to human meniscal cells. Part I ...

International Orthopaedics (SICOT) DOI 10.1007/s00264-014-2410-2

ORIGINAL PAPER

Nonviral gene transfer to human meniscal cells. Part I: transfection analyses and cell transplantation to meniscus explants Hsiao-Ping Lee & Gunter Kaul & Magali Cucchiarini & Henning Madry

Received: 30 May 2014 / Accepted: 2 June 2014 # SICOT aisbl 2014

Abstract Purpose Our aim was to evaluate whether nonviral vectors can genetically modify primary human juvenile and adult meniscal fibrochondrocytes at low toxicity in vitro and to test the hypothesis that transfected human meniscal fibrochondrocytes transplanted into longitudinal defects and onto human medial meniscus explant cultures are capable of expressing transgene products in vitro. Methods Eighteen nonviral gene transfer systems were examined to identify the best suited method for an efficient transfection of primary cultures of juvenile and adult human meniscal fibrochondrocytes using luciferase and lacZ reporter gene constructs and then transplanted to meniscus explant cultures. Results Gene transfer systems FuGENE 6, GeneJammer, TurboFectin 8, calcium phosphate co-precipitates and GeneJuice led to minimal toxicity in both cell types. Nanofectin 2 and JetPEI resulted in maximal luciferase activity in both cell types. Maximal transfection efficiency based on X-gal staining following lacZ gene transfer was achieved using Lipofectamine 2000, revealing a mean transfection efficiency of 8.6 % in human juvenile and of 8.4 % in adult meniscal fibrochondrocytes. Transfected, transplanted meniscal fibrochondrocytes adhered to the meniscal tissue and continued to express the transgene for at least five days following transfection. Conclusions Nonviral gene transfer systems are safe and capable of transfecting both juvenile and adult human meniscal Presented in part at the 55th Annual Meeting of the Orthopaedic Research Society, 2009, Las Vegas, NV, USA H.0.05). Differences in luciferase activity for each transfection system between juvenile and adult human meniscal fibrochondrocytes were not significant (P>0.05).

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Fig. 4 Time course of transgene expression following transplantation of luc-transfected human adult meniscal fibrochondrocytes onto human meniscus explants. Luciferase activity was not significantly different from the negative controls on days 7, 10 and 12

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Fig. 3 Transfection efficiency in human juvenile (a) and adult meniscal fibrochondrocytes (b). Efficiencies were determined by X-gal staining following transfection with a lacZ expression plasmid vector. Lf Lipofectamine 2000, JPI JetPEI, Nf2 Nanofectin 2, ET EcoTransfect, TL TransITLT1, DC DMRIE-C

Transplantation of lacZ-transfected human adult meniscal fibrochondrocytes Gene transfer into human adult meniscal fibrochondrocytes was mediated with Lipofectamine 2000, because it led to the highest transfection efficiency. Transfected meniscal fibrochondrocytes transplanted into a longitudinal (5 mm) defect and onto the femoral aspect of the avascular part of human medial meniscus explants surrounding the lesion in organ culture were visualised by X-gal staining. Stereoscopic examination of cultured meniscal explants showed a pattern of multiple X-gal-positive foci, distributed over the seeded meniscal explant’s surface (Fig. 4). In contrast, transplanted human adult meniscal fibrochondrocyte cultures exposed to Lipofectamine 2000 alone without plasmid vector did not result in positive X-gal staining. Histological analysis of transverse sections of the meniscal organ cultures showed transplanted X-gal-positive and X-gal-negative meniscal fibrochondrocytes attached to the margins of the longitudinal defect. Analysis of the surface of the human meniscus explants similarly revealed transplanted X-gal-positive and X-

gal-negative meniscal fibrochondrocytes which formed a new cell layer attached to the surface of the explant. There was no detectable activity based on X-gal staining in the untransfected controls. Time course of transgene expression following transplantation of luc-transfected human adult meniscal fibrochondrocytes onto human meniscus explants Finally, a study on the time course of transgene expression following transplantation of luc-transfected human adult meniscal fibrochondrocytes applying Lipofectamine 2000 (the best compound identified here) onto human meniscus explants demonstrated a peak at day one after transfection (15,388±398 RLU/explant), followed by a decrease in expression on days two (6,926±215 RLU/explant) and five (1,731±591 RLU/explant) (Figs. 4 and 5). Luciferase activity was not significantly different from the negative controls (untransfected meniscal fibrochondrocytes) on day 12, the longest time point evaluated (P>0.05).

Discussion To the best of our knowledge, the efficacy of a large number of nonviral gene transfer systems to safely transfect human meniscal fibrochondrocytes has not been systematically evaluated to date. The results of this study demonstrate that among the 18 nonviral systems tested, FuGENE 6, GeneJammer, TurboFectin 8, calcium phosphate co-precipitates and

International Orthopaedics (SICOT) Fig. 5 Human medial meniscus explant culture and meniscal fibrochondrocyte transplantation. A longitudinal defect was established in the avascular central area of the femoral aspect of human adult meniscal fragments. Isolated meniscal fibrochondrocytes transfected with Lipofectamine 2000 were then seeded into the longitudinal defect (arrowheads) and onto the surface of meniscal explant cultures. a, c, e Transplantation of untransfected fibrochondrocytes. b, d, f Transplantation of transfected fibrochondrocytes. a, b Macroscopic views of human medial meniscus explant cultures seeded with untransfected (a) or lacZ-transfected meniscal fibrochondrocytes (b). Note the presence of X-gal-positive transplanted cell clones (blue colour) at the edge of the lesion and surrounding the lesion (b). Representative histological sections of human medial meniscus explant cultures 2 days following transplantation of untransfected (c, e) or lacZtransfected meniscal fibrochondrocytes (d, f) into longitudinal defects (number sign, c, d) or onto the surface surrounding the lesion (e, f) stained with haematoxylin and eosin (c–f). Note the presence of transplanted cells (arrowheads) at the edges of the lesion (c, d) and the transplanted cells adhering to the surface of the repair tissue in several cell layers (asterisks) (e, f). Magnification ×4 (c–f)

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E GeneJuice led to minimal cytotoxicity in both juvenile and adult human meniscal fibrochondrocytes. Maximal luciferase activity in both cell types resulted from transfection with Nanofectin 2 and JetPEI. Maximal transfection efficiency was achieved using Lipofectamine 2000, corresponding to a mean transfection efficiency of 8.6 % in human juvenile and of 8.4 % in human adult meniscal fibrochondrocytes. Transfected meniscal fibrochondrocytes transplanted into meniscal defects and onto human medial meniscus explants adhered to the tissue and continued to express a transgene for at least five days after transfection in vitro. The nonviral gene transfer system Lipofectamine 2000 was the most effective of all 18 systems tested to mediate gene transfer based on X-gal staining of lacZ-transfected meniscal

F fibrochondrocytes. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids (DNA and RNA) and has been extensively applied in both continuous and primary cell lines in the field of orthopaedic research [16, 28, 35]. Although the reported efficiencies of 8–9 % in primary human meniscal fibrochondrocytes are lower than viral gene transfer systems such as recombinant adeno-associated viral vector systems (80–90 %) [17], such efficiencies are sufficient and adequate for transfection-based approaches in addition to the advantages of providing no virus-derived material in the target cell 7, 19, [28]. Of note, transfection efficiency (the percentage of transfected cells in a cell population) does not necessarily correlate with the magnitude of a biological response or with therapeutic efficacy [19]. Previous

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studies have shown that transfection of only 1 % of cells was sufficient to achieve biological effects in vivo [25]. Moreover, in the context of intra-articular approaches and transgene expression such as following gene transfer to meniscal fibrochondrocytes in vivo, the effect of the produced protein has to be restricted to the meniscal lesion, as a high intraarticular level of therapeutic factors, for example transforming growth factor beta (TGF-β), leads to undesirable side effects [33]. Therefore, future studies need to show whether this efficiency is enough to elicit physiological responses both in vitro and in vivo. Interestingly, while meniscus fibrochondrocytes share similarities with articular chondrocytes, transgene expression based on luciferase activity was nevertheless not efficient with FuGENE 6, an established gene transfer system suitable for articular chondrocytes, including osteoarthritic and juvenile chondrocytes [22]. Nakata and co-workers reported a transfection efficiency of 31 % using FuGENE 6 based on X-gal staining [26]. Fibrochondrocytes embed themselves in matrix mainly composed of type I collagen, while chondrocytes are surrounded by a collagen matrix chiefly composed of type II collagen. This highlights the important fact that nonviral gene transfer systems have to be optimised for each target cell, showing that a transfection system effective in one cell type may not work in another [22, 28]. Moreover, we could not report differences in cytotoxicity and luciferase activity between juvenile and adult human meniscal fibrochondrocytes. Transfection of juvenile human meniscal fibrochondrocytes was performed because meniscal lesions, particularly based on a discoid lateral meniscus, may occur already in paediatric patients [30, 32]. A possible application of transfected primary human meniscal fibrochondrocytes is the repopulation of meniscal lesions with transfected cells overexpressing a therapeutic gene [6, 31]. Here, transfected meniscal fibrochondrocytes would need to attach to the meniscal tissue surrounding the lesion, remain viable and continue to express their transgene for a therapeutically relevant period of time. The data presented here show that transfected meniscal fibrochondrocytes adhere to both the meniscal surface and to the tissue surrounding the lesion and continue to express the transgene product at least five days after transfection. Goto and co-workers were the first to show that meniscal cells can be transduced with adenoviral and retroviral vectors and that in vitro gene transfer to intact human and lapine menisci is both possible by direct adenoviral delivery and indirect transplantation of meniscal cells modified with a retroviral vector [10]. In these studies, transgene expression persisted for several weeks in vitro. When myoblasts transduced with adenoviral vectors were injected intraarticularly in the knee of the adult mice, the engineered muscle cells adhered to ligaments, capsule and synovium [6]. A major strength of our study is the detailed evaluation of a large number of nonviral gene transfer systems to safely

transfect human meniscal fibrochondrocytes. Limitations of this study include the use of a monolayer culture compared to three-dimensional culture systems and the lack of a therapeutic gene. In conclusion, we have identified an optimal nonviral gene transfer system that is safe and capable of transfecting both juvenile and adult human meniscal fibrochondrocytes, which, when transplanted to meniscal tissue in vitro, continue to express the transgene. These data may be of value for combined cell transplantation and gene therapy approaches [29, 15] using growth factors (e.g. fibroblast growth factor 2) that promote meniscal repair [27]. Acknowledgments We thank the members of the Department of Orthopaedic Surgery for providing human meniscal tissue. Conflict of interest The authors declare that they have no conflict of interest. The authors alone are responsible for the content and writing of the paper. Authors’ contributions HPL participated in the design of the study, carried out the transfection and cell viability studies and performed the statistical analysis. GK participated in its design, carried out the transplantation studies and performed the statistical analysis. MC participated in the study design and draft. HM conceived and designed the study, coordinated it and drafted the manuscript. All authors read and approved the final manuscript.

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