Determination of effective rAAV-mediated gene transfer conditions to support chondrogenic differentiation processes in human primary bone marrow aspirates

Abstract

The genetic modification of freshly aspirated bone marrow may provide convenient tools to enhance the regenerative capacities of cartilage defects compared with the complex manipulation of isolated progenitor cells. In the present study, we examined the ability and safety of recombinant adeno-associated virus (rAAV) serotype 2 vectors to deliver various reporter gene sequences in primary human bone marrow aspirates over time without altering the chondrogenic processes in the samples. The results demonstrate that successful rAAV-mediated gene transfer and expression of the lacZ and red fluorescent protein marker genes were achieved in transduced aspirates at very high efficiencies (90-94%) and over extended periods of time (up to 125 days) upon treatment with hirudin, an alternative anticoagulant that does not prevent the adsorption of the rAAV-2 particles at the surface of their targets compared with heparin. Application of rAAV was safe, displaying neither cytotoxic nor detrimental effects on the cellular and proliferative activities or on the chondrogenic processes in the aspirates especially using an optimal dose of 0.5 mg ml(-1) hirudin, and application of the potent SOX9 transcription factor even enhanced these processes while counteracting hypertrophic differentiation. The current findings demonstrate the clinical value of this class of vector to durably and safely modify bone marrow aspirates as a means to further develop convenient therapeutic approaches to improve the healing of cartilage defects.

Chondrogenic Differentiation Processes in Human Bone Marrow Aspirates upon rAAV-Mediated Gene Transfer and Overexpression of the Insulin-Like Growth Factor I

“…The aspirates containing MSCs [34][35][36] were aliquoted in standard tissue culture plastic 96-well plates (100 mL of aspirate/well) and immediately transduced with the rAAV vectors by mixing 40 mL of vector solution with each aliquot (i.e., 8 · 10 5 functional recombinant viral particles, MOI = 10 -3) and then adding 50 mL of Dulbecco's modified Eagle's medium (DMEM) for an incubation time of 1.5 h at 37°C under 5% CO 2 . A volume of 60 mL of chondrogenic medium (4.5 g/L DMEM high glucose, 100 U/ mL penicillin and 100 mL/mL streptomycin, 6.25 mg/mL insulin, 6.25 mg/mL transferrin, 6.25 mg/mL selenious acid, 5.35 mg/mL linoleic acid, 1.25 mg/mL bovine serum albumin, 1 mM sodium pyruvate, 37.5 mg/mL ascorbate 2-phosphate, 10 -7 M dexamethasone, and 10 ng/mL TGF-b3) was then added per aspirate for an incubation at 37°C under 5% CO 2 for up to 21 days based on work adapted for cartilage repair 20,36 with medium change performed once per week for MSC chondrogenesis. [34][35][36] To avoid attachment on the bottom of the plates, the aspirates were carefully mixed after each medium change.…”

“…A volume of 60 mL of chondrogenic medium (4.5 g/L DMEM high glucose, 100 U/ mL penicillin and 100 mL/mL streptomycin, 6.25 mg/mL insulin, 6.25 mg/mL transferrin, 6.25 mg/mL selenious acid, 5.35 mg/mL linoleic acid, 1.25 mg/mL bovine serum albumin, 1 mM sodium pyruvate, 37.5 mg/mL ascorbate 2-phosphate, 10 -7 M dexamethasone, and 10 ng/mL TGF-b3) was then added per aspirate for an incubation at 37°C under 5% CO 2 for up to 21 days based on work adapted for cartilage repair 20,36 with medium change performed once per week for MSC chondrogenesis. [34][35][36] To avoid attachment on the bottom of the plates, the aspirates were carefully mixed after each medium change. For osteogenic and adipogenic differentiation, the aspirates were transduced using similar conditions as those described above and then induced either toward osteogenic differentiation using the StemPro Osteogenesis Differentiation Kit or adipogenic differentiation using the StemPro Adipogenesis Differentiation Kit (both from Life Technologies GmbH).…”

“…Transgene expression was also monitored by immunohistochemical analysis using a specific primary antibody. 20,36,39 Histological, immunocytochemical, and immunohistochemical analyses…”

“…Sections were stained with Hematoxylin and eosin (H&E) for cellularity, Toluidine blue for matrix proteoglycans, and Alizarin Red for matrix mineralization as previously described. 19,20,32,36 Immunohistochemistry was performed to monitor the expression of IGF-I, type-II, type-I, and type-X collagen, and SOX9 using specific primary antibodies, biotinylated secondary antibodies, and the ABC method with diaminobenzidine as the chromogen. 19,20,32,36 To control for secondary immunoglobulins, sections were processed with omission of the primary antibody.…”

“…Most notably, Pascher et al 34 and Ivkovic et al 35 employed adenoviral gene transfer to transduce marrow aspirates from rabbit and sheep, achieving relatively short-term levels of transgene expression in such samples in vitro (21 days). In marked contrast, we recently provided evidence that recombinant vectors derived from the human nonpathogenic, replication defective adenoassociated virus (AAV) are capable of effectively (*90% transduction efficiency) and persistently (up to 125 days) modify human marrow aspirates without deleterious effects in vitro, 36 in good agreement with the properties of the vectors (reduced toxicity and immunogenicity, maintenance of recombinant adeno-associated virus (rAAV) sequences as stable, safe episomes) that make them the currently preferred gene delivery system for clinical applications. 16 In the present study, in light of our recent work showing the benefits of applying an rAAV IGF-I vector to modulate the proliferative, biosynthetic, and chondrogenic activities in isolated human MSCs, 20 we examined the ability of this vector to deliver and mediate the expression of this mitogenic and proanabolic growth factor in primary human bone marrow aspirates as a potential approach to enhance the chondrogenic differentiation processes in the samples.…”