ORIGINAL ARTICLE BMP-6 is more efficient in bone formation than BMP-2 when overexpressed in mesenchymal stem cells O Mizrahi 1,4 , D Sheyn 2,4 , W Tawackoli 2 , I Kallai 1 , A Oh 2 , S Su 2 , X Da 2 , P Zarrini 2 , G Cook-Wiens 3 , D Gazit 1,2 and Z Gazit 1,2 Bone regeneration achieved using mesenchymal stem cells (MSCs) and nonviral gene therapy holds great promise for patients with fractures seemingly unable to heal. Previously, MSCs overexpressing bone morphogenetic proteins (BMPs) were shown to differentiate into the osteogenic lineage and induce bone formation. In the present study, we evaluated the potential of osteogenic differentiation in porcine adipose tissue- and bone marrow-derived MSCs (ASCs and BMSCs, respectively) in vitro and in vivo when induced by nucleofection with rhBMP-2 or rhBMP-6. Our assessment of the in vivo efficiency of this procedure was made using quantitative micro-computed tomography (micro-CT). Nucleofection efficiency and cell viability were similar in both cell types; however, the micro-CT analyses demonstrated that in both ASCs and BMSCs, nucleofection with rhBMP-6 generated bone tissue faster and of higher volumes than nucleofection with rhBMP-2. RhBMP-6 induced more efficient osteogenic differentiation in vitro in BMSCs, and in fact, greater osteogenic potential was identified in BMSCs both in vitro and in vivo than in ASCs. On the basis of our findings, we conclude that BMSCs nucleofected with rhBMP-6 are superior at inducing bone formation in vivo than all other groups studied. Gene Therapy (2013) 20, 370–377; doi:10.1038/gt.2012.45; published online 21 June 2012 Keywords: mesenchymal stem cells; bone morphogenetic proteins; bone formation; nucleofection INTRODUCTION Bone tissue is a specialized form of connective tissue that possesses a natural regenerative capacity. Nevertheless, 5–10% of all fractures and as many as 30% of patients with pre-existing conditions face impaired healing and significant morbidity, which in turn can also be economically burdensome. 1 To date, autologous bone grafting remains the ‘gold standard’ biological method used to promote nonunion fracture sites and spinal fusion in cases of intervertebral disc degeneration. 2,3 However, failure rates as high as 30% have been cited together with complications resulting from repeated interventions. 1 Mesenchymal stem cells (MSCs) have been isolated from various adult tissues, among which are bone marrow (BM) and adipose tissue. MSCs have been shown to differentiate successfully into osteogenic, chondrogenic and adipogenic lineages. 4–6 In addition, when genetically modified, these multipotent cells have displayed the ability to form and regenerate bone in vivo in multiple animal models, providing gene- and cell-mediated therapy for clinical orthopedic applications. 7–11 The bone morphogenetic protein (BMP) family and its 20 identified members are regarded today as key signaling proteins responsible for organization of tissue architecture. 12 It is widely known that BMPs have an important role in osteogenesis. 1,13 We previously showed that MSCs that have been genetically modified to overexpress BMP can produce regeneration in bone defects and form bone in vivo without the need for mass quantities of BMP proteins or harvested bone grafts. 7–10,14,15 To date, a number of studies have been conducted to determine the most suitable BMP for osteogenic differentiation in vitro and in vivo. Most of these studies have been conducted with the aid of viral gene delivery. 1,16–21 Experiments involving MSCs infected with adenoviruses carrying 14 different human isoforms of BMP revealed that BMP-2, -6, and -9 are the most potent inducers of osteoblast differentiation in MSCs. 17,21 BMP-2 and BMP-6 belong to two different subclasses, which were assigned based on similarities in amino acid sequences. 16 The use of BMP-2 in bone regeneration has been widely studied. As the FDA approved rhBMP-2 protein for clinical use in tibia and nonunion fractures, interest in this protein has become more widespread. 22 We previously showed that BMP-2 gene overexpression in MSCs induces bone formation and heals bone defects in vivo. 10,15,23–25 Although less popular, BMP-6 is also a prominent candidate for use in bone regeneration. Our studies have shown that BMP-6 is potent when used for osseous regeneration in bone defects and formation of bone in vivo. 8,9,26 Although viral gene delivery is more efficient than nonviral gene delivery, it poses several safety concerns. 27 In studies undertaken by us and others, nucleofection, a novel and efficient method for nonviral plasmid DNA delivery into MSCs, 28 has been used to overexpress rhBMP-2 and -6 in order to induce ectopic bone formation, 23 form spinal fusion, 9 and repair vertebral fractures. 8 Despite the use of nucleofection in numerous studies involving BMPs, the in vitro and in vivo efficiency of this process when used with various members of the BMP family in different types of adult stem cells has not yet been tested. Thus, our goal in the present study was to evaluate and compare the in vivo and 1 Skeletal Biotech Laboratory, Hebrew University-Hadassah, Faculty of Dental Medicine, Jerusalem, Israel; 2 Cedars-Sinai Medical Center, Department of Surgery, Regenerative Medicine Institute, Los Angeles, CA, USA and 3 Cedars-Sinai Medical Center, Samuel Oschin Comprehensive Cancer Institute, Los Angeles, CA, USA. Correspondence: Dr Z Gazit, Department of Surgery, Regenerative Medicine Institute, Stem Cell Therapeutics Research Lab, Cedars-Sinai Medical Center, 8700 Beverly Blvd. SSB3, Rm 357, Los Angeles, CA 90048, USA. E-mail: zulma.gazit@csmc.edu 4 These authors contributed equally to this work. Received 23 February 2012; revised 2 May 2012; accepted 8 May 2012; published online 21 June 2012 Gene Therapy (2013) 20, 370–377 & 2013 Macmillan Publishers Limited All rights reserved 0969-7128/13 www.nature.com/gt