TRANSLATIONAL AND CLINICAL RESEARCH Nonvirally Engineered Porcine Adipose Tissue-Derived Stem Cells: Use in Posterior Spinal Fusion DIMA SHEYN,a GADI PELLED,a YORAM ZILBERMAN,a FARAHNAZ TALASAZAN,b JONATHAN M. FRANK,b DAN GAZIT,a,b ZULMA GAZITa,b a

Skeletal Biotechnology Laboratory, Hebrew University-Hadassah Medical Center, Jerusalem, Israel; bDepartment of Surgery, Cedars Sinai Medical Center, Los Angeles, California, USA

Key Words. Adipose tissue-derived stem cells • Nonviral gene delivery • Genetic engineering • Bone morphogenetic protein • Posterior spinal fusion

ABSTRACT Multiple factors alter intervertebral disc volume, structure, shape, composition, and biomechanical properties, often leading to low back pain. Spinal fusion is frequently performed to treat this problem. We recently published results of our investigation of a novel system of in vivo bone formation, in which we used nonvirally nucleofected human mesenchymal stem cells that overexpress a bone morphogenetic protein gene. We hypothesized that primary porcine adipose tissue-derived stem cells (ASCs) nucleofected with plasmid containing recombinant human bone morphogenetic protein-6 (rhBMP-6) could induce bone formation and achieve spinal fusion in vivo. Primary ASCs were isolated from freshly harvested porcine adipose tissue. Overexpression of rhBMP-6 was achieved ex vivo by using a nucleofection technique. Transfection efficiency was monitored by assessing a parallel transfection involving an enhanced

green fluorescent protein reporter gene and flow cytometry analysis. rhBMP-6 protein secreted by the cells was measured by performing an enzyme-linked immunosorbent assay. Genetically engineered cells were injected into the lumbar paravertebral muscle in immunodeficient mice. In vivo bone formation was monitored by a quantitative microcomputed tomography (␮CT). The animals were euthanized 5 weeks postinjection, and spinal fusion was evaluated using in vitro ␮CT and histological analysis. We found formation of a large bone mass adjacent to the lumbar area, which produced posterior spinal fusion of two to four vertebrae. Our data demonstrate that efficient bone formation and spinal fusion can be achieved using ex vivo, nonvirally transfected primary ASCs. These results could pave the way to a novel biological solution for spine treatment. STEM CELLS 2008;26:1056 –1064

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Low back pain is a common complaint voiced by the adult population [1–3]. Multiple elements, including aging and genetic and environmental factors, contribute to disc degeneration. As people age, the intervertebral disc undergoes alterations in volume, structure, shape, composition, and biomechanical properties [2]. Although the etiology and pathophysiology of disc degeneration remain unknown [2, 3], disc degeneration can be described clinically as a loss of two major disc functions: stability and mobility [4, 5]. Pathological conditions, such as spondylosis, scoliosis, spondylolisthesis, tumor, infection, posttraumatic fracture, and instability, can produce an abnormal relationship between adjacent vertebral structures. Fusion models have been designed to stabilize the spinal column by removing intervertebral articulations and repositioning vertebral segments in appropriate, mechanically advantageous alignment. Spinal fusion ranks as the second most common lumbar spine procedure, with approximately 46,500 lumbar spinal arthrodeses performed each year in the United States. Autologous bone graft derived from the iliac crest is the standard procedure used for spinal fusion; nevertheless, the number of complications involv-

ing surgical harvest of this graft material is high, and the incidence of morbidity is estimated at 7%–25% [6]. Adipose tissue-derived stem cells (ASCs) [7] are excellent candidates for stem cell-based therapy for bone regeneration because they have the potential to differentiate into various mesenchymal lineages, including bone-forming cells [8, 9]. Our preliminary studies showed the ability of ASCs to differentiate in vitro into osteogenic and chondrogenic lineages (data not shown). Both human bone marrow-derived mesenchymal stem cells (BM-MSCs) and ASCs have proved effective in regenerating bone defects in animals. Genetically engineered BMMSCs were shown to regenerate nonunion fractures in nude mice [10, 11]. Quarto et al. [11] used autologous BM-MSCs delivered on a macroporous hydroxyapatite carrier to heal large bone defects (⬎4.0 cm) and reported preliminary success in achieving callus formation after 2 months. Dragoo et al. [12, 13] achieved bone induction in NOD/SCID mice by using human ASCs expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). The ability of rhBMP-2-expressing human ASCs to heal critically sized bone defects in the nude rat femur has also been demonstrated [14]. Recently, autologous human ASCs were shown to induce bone formation and regeneration in a 7-year-old girl with multiple calvarial fractures [15]. The

Correspondence: Zulma Gazit, Ph.D., Skeletal Biotech Laboratory, Hebrew University of Jerusalem–Hadassah Medical Campus, Jerusalem 91120, Israel. Telephone: 972-2-6757625; Fax: 972-2-6757628; e-mail: [email protected] Received November 1, 2007; accepted for publication January 10, 2008; first published online in STEM CELLS EXPRESS January 24, 2008. ©AlphaMed Press 1066-5099/2008/$30.00/0 doi: 10.1634/stemcells.2007-0858

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simplicity of the surgical procedure, the easy and repeatable access to subcutaneous adipose tissue, and the uncomplicated enzyme-based isolation procedures make this source of autologous adult MSCs most attractive to researchers and clinicians [16]. Only local anesthesia is necessary to harvest ASCs, the patient is subjected only to minimal discomfort, and large quantities of these cells can be obtained repeatedly. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-␤ superfamily and are known for their potential to induce bone formation and osteogenic differentiation of MSCs [10, 17–20]. Various BMPs exert different effects on bone formation in vivo following adenovirus-mediated direct gene transfer [21–23]. Recently, it was shown that rhBMP-2, -6, and -9 are the most potent osteogenic inducers of MSCs among the BMP family [24]. Previous studies have indicated that human BM-MSCs nonvirally transfected with the rhBMP-9 gene were able to induce prominent bone formation in vivo [6]. Nonviral gene delivery approaches hold great promise for the development of efficient and safe cell-mediated gene therapy for clinical orthopedic applications. Viral gene delivery is efficient but less safe than nonviral gene delivery. There is thus an enormous need to develop efficient novel nonviral vectors so that we may advance gene and cell therapy to the clinical setting. Furthermore, nonviral vectors have greater potential to provide nucleic acid-based drugs that closely resemble traditional pharmaceutical agents. Nucleofection, a novel form of electropermeabilization, is an efficient physical method of delivery of plasmid DNA into primary cells and established cell lines [25, 26]. Recently, it was shown that nucleofection, developed by Amaxa Biosystems (Cologne, Germany, http://www. amaxa.com), efficiently transfects human BM-MSCs with a reporter gene [26] and a therapeutic gene from the BMP family and that the transfected cells can be used to create ectopic bone formation in vivo [6]. On the basis of our earlier findings, we hypothesized that primary porcine adipose tissue-derived stem cells that have been nucleofected with a plasmid encoding the rhBMP-6 gene will express and secrete biologically effective levels of rhBMP-6 and that these cells can induce posterior spinal fusion after their direct injection into the vicinity of the intervertebral disc. This injectable treatment is minimally invasive and more effective than bone marrow transplantation.

MATERIALS

AND

METHODS

Isolation of Porcine ASCs Porcine ASCs were isolated from adipose tissues obtained from the subcutaneous fat pads of euthanized adult female Yorkshire pigs 19 –20 weeks old. The adipose tissues were cut into small pieces, placed on a shaker, and treated with 0.075% collagenase (SigmaAldrich, St. Louis, http://www.sigmaaldrich.com) in phosphatebuffered saline (PBS) at 37°C for 40 – 60 minutes. The collagenase was inactivated by addition of complete growth medium (Dulbecco’s modified Eagle’s medium [DMEM] containing 10% fetal calf serum [FCS]; Biological Industries, Beth Haemek, Israel, http:// www.bioind.com). The mononuclear cells were plated in tissueculture dishes at a density of 10 ⫻ 106 to 15 ⫻ 106 cells per 100-mm culture plate, in 5% CO2/95% air at 37°C. The medium was changed after 72 hours and again every 3– 4 days. The ASCs were expanded in culture up to the fifth passage.

Nucleofection and the Establishment of a Nonviral Gene Delivery Protocol With the aid of nucleofection (Amaxa Biosystems) technology, including a Nucleofector device and an MSC-specific nucleofection

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buffer, 106 or 2 ⫻ 106 porcine ASCs were transfected with 5, 10, or 15 ␮g of pCMV-EGFP-N1 (hereafter referred to as pEGFP), which encodes the enhanced green fluorescent protein (EGFP) gene, according to the manufacturer’s protocol for human MSCs. The nucleofection procedure was performed using program G22, which we found to be optimal in our previously reported preliminary studies [6]. Immediately after nucleofection, the cells were transferred to 100-mm plates, which contained 10 ml of complete growth medium (including 20% FCS), and were maintained in culture for 24 hours. Viable cells were counted after trypan blue staining by using light microscopy 24 hours post-transfection. Cell viability was calculated as the ratio of surviving cells to cells initially nucleofected, as previously reported [6]. Nucleofected cells were analyzed 24 hours after transfection by performing a flow cytometry analysis for the expression of green fluorescent protein (GFP) and by determining the percentage of nucleofected cells among the total viable cells. After the nucleofection conditions were calibrated, the ASCs were nucleofected with pCMV-cDNA3-rhBMP-6 (hereafter referred to as prhBMP-6).

Real-Time Reverse Transcriptase-Polymerase Chain Reaction To estimate the rhBMP-6 gene expression profile, ASCs from three different donors were nucleofected by following the same method described earlier and grown in confluent conditions in culture for 21 days. Total RNA was extracted from nucleofected porcine ASCs by using Trizol reagent on days 1, 2, 4, 7, 10, 14, and 21 after nucleofection. Total RNA was first treated with DNase I (Promega, Madison, WI, http://www.promega.com) and then retrotranscribed using random hexamers and reverse transcriptase (RT) (Promega); rhBMP-6 gene expression was quantitatively analyzed using realtime RT-polymerase chain reaction (PCR). Real-time quantitative PCR was performed with the aid of an ABI 7300 Prism system (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems. com) by using an absolute quantification method and Assay-onDemand gene expression assays (Applied Biosystems). The number of transcript copies was determined using a standard curve.

In Vitro Protein Secretion Assay To estimate the amount of rhBMP-6 that was secreted, ASCs from three different donors were nucleofected using the method described above and grown in confluent conditions in culture for 21 days. Media from the flasks were collected on days 1, 2, 4, 7, 10, 14, and 21 after nucleofection. To quantify the rhBMP-6 secreted during 24 hours, the media were changed 24 hours before sampling at every time point tested. An enzyme-linked immunosorbent assay (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) was performed to measure the amount of rhBMP-6 protein secreted into the culture media by the nucleofected porcine ASCs. After sampling the media, we lifted and counted cells so that we could normalize the secreted protein to 106 cells.

Alkaline Phosphatase Activity In Vitro To induce osteogenic differentiation in vitro, adipose tissue-derived ASCs were nucleofected with prhBMP-6 and plated at a density of 7 ⫻ 104 cells per cm2 in maintenance DMEM containing 10% FCS, 0.05 mM ascorbic acid-2-phosphate, and 10 mM ␤-glycerophosphate (Sigma-Aldrich). To provide a positive control of osteogenic induction, non-nucleofected cells were cultured in an induction medium consisting of maintenance medium plus 0.1 ␮M dexamethasone as an induction agent. To provide negative controls, ASCs were nucleofected with the pEGFP reporter, and non-nucleofected cells were cultured in maintenance medium only. Six days after nucleofection or addition of osteogenic supplements, an alkaline phosphatase (ALP) colorimetric assay was performed as previously described [27]. Briefly, the cells were lysed with alkaline buffer solution (Sigma-Aldrich) containing 0.5% Triton X-100 and 10 mM MgCl2. For the ALP assay, the cell lysates were incubated with assay buffer containing 0.75 M 2-amino-2-methyl-1-propranolol (pH 10.3) for 10 minutes at 37°C with p-nitrophenylphosphate as a substrate.

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Animal Studies Our institutional animal care and use committee approved all the procedures used in this study and agreed that care of the animals was consistent with that outlined in the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals. For the spinal fusion procedure, immunodeficient (NOD/ SCID) mice were anesthetized by administration of an intraperitoneal injection of a xylazine-ketamine mixture [28] (100 mg/kg ketamine and 3.3 mg/kg xylazine). Nucleofected porcine ASCs (5 ⫻ 106) were resuspended in 50 ␮l of fibrin gel (Tisseel kit; Baxter, Vienna, Austria, http://www.baxter.com) and injected into the lumbar paravertebral muscle of each mouse as previously described [20]. As a control group, nontreated ASCs were injected in the same manner. The mice were housed in a specific pathogen-free animal facility for 5 weeks. In the donor cell contribution experiment, the samples were harvested after 2 and 5 weeks. At the end of this period, the mice were euthanized, their fused spines were harvested, and bone formation and spinal fusion in these animals were analyzed.

Microcomputed Tomography To obtain a detailed qualitative and quantitative three-dimensional (3D) analysis, formation of the fusion mass was evaluated using microcomputed tomography (␮CT). Five weeks after cells injection, the mice were euthanized, and their lumbar vertebrae, intact muscles, and soft tissue were dissected and measured using a Desktop Cone-Beam ␮CT Scanner (␮CT 40; Scanco Medical AG, Bassersdorf, Switzerland, http://www.scanco.ch). Microtomographic slices were acquired at 1,000 projections and reconstructed at a nominal spatial resolution of 16 ␮m. A constrained 3D gaussian filter (␴ ⫽ 0.8; support ⫽ 1) was used to suppress some noise found in the volumes. Bone tissue was cut away from the marrow and soft tissue by using a global thresholding procedure [29]. Newly formed bone tissue was separated from intact spines by applying a manual contouring method. For reference spinal bone tissue, we used posterior portions of lumbar vertebrae that were contoured in the same manner. In addition to the visual assessment of structural images, morphometric indices were determined from the microtomographic data sets by using direct 3D morphometry [30]. Structural metrics measured using ␮CT are closely correlated with those measured using standard histomorphometry [31]. The following morphometric indices were determined for the newly formed bone and vertebral bone control tissue: (a) total volume of bone (TV; in mm3), including new bone and soft tissue cavities; (b) volume of mineralized bone tissue (BV; in mm3); (c) bone volume density based on the BV/TV ratio; (d) average bone thickness (in mm); (e) degree of anisotropy determined from the ratio of maximal to minimal radii of the mean intercept length ellipsoid [30]; and (f) connectivity density (1/mm3). The connectivity (C) of a two-component system— bone and marrow—is derived directly from the Euler number (E) by following the formula C ⫽ 1 ⫺ E, if all trabeculae and bone marrow cavities are connected without isolated marrow cavities inside the bone [32]. Connectivity was normalized by examining tissue volume and is reported as connectivity density.

Histological Analysis The harvested lumbar spines, intact muscle, and soft tissue were fixed in 4% formalin for at least 24 hours, decalcified by 0.5 M EDTA solution (pH 7.4) for 7 days, passed through an increasing grade series of ethanol baths, and embedded in paraffin. Five-micrometer-thick sections were cut from each paraffin block with the aid of a motorized microtome (Leica Microsystems, Nussloch, Germany, http://www. leica.com). The slides were heated at 65°C for 45 minutes; this process was followed by deparaffinization. Hematoxylin and eosin (H&E) staining and bone matrix-specific Masson’s trichrome staining were performed as previously reported [10].

Contribution of Injected ASCs to Spinal Fusion Genetically engineered ASCs were labeled with Vybrant CM-DiI (Invitrogen, Eugene, OR, http://www.invitrogen.com) cell-labeling solution 24 hours after nucleofection. Labeling was performed by resuspending the cells in serum-free DMEM at a concentration of

Engineered Porcine ASCs Form Spinal Fusion 106 cells per milliliter, mixing the cell suspension with Vybrant CM-DiI solution (15 ␮l per milliliter of the 106-cell suspension), and incubating the cells in the dark on a shaker for 30 minutes in an atmosphere of 95% air/5% CO2 at 37°C. The CM-DiI-labeled genetically engineered ASCs were injected into the mouse paravertebral muscle in the manner described earlier. The implants were harvested 2 or 5 weeks after the injections, fixed, decalcified using 0.5 M EDTA solution, and embedded in paraffin. Five-micrometerthick sections were deparaffinized and rehydrated by passing them through a descending grade series of ethanol baths, after which they were counterstained with 4⬘,6-diamidino-2-phenylindole (DAPI; catalog no. D9542; Sigma-Aldrich) for 5 minutes and mounted with glycerol-polyvinyl-alcohol (GVA; Zymed Laboratories, San Francisco, http://www.invitrogen.com). The CM-DiI and DAPI stains were visualized using fluorescent microscopy.

Vimentin Immunohistochemical Analysis An immunohistochemical assay was performed to detect the expression of porcine vimentin on paraffin sections of fused spines by using a HistoMouse-SP kit (catalog no. 95-9541; Zymed Laboratories). Sections of tissue were deparaffinized with xylene, rehydrated in a descending grade series of ethanol baths, and rinsed in PBS. The antigens were retrieved enzymatically by using an antigen retrieval kit (catalog no. ab8212; Abcam, Cambridge, U.K., http://www.abcam.com) Endogenous peroxidase activity was removed by treatment with 0.1% H2O2 for 10 minutes. A primary antibody that reacts with human, porcine, and equine vimentin but not with mouse vimentin (catalog no. ab8069; Abcam) was diluted 1:10 in PBS and applied to the slides for 1 hour at room temperature. After incubation with the primary antibody, the slides were rinsed in PBS, and a secondary goat anti-mouse IgG antibody (biotin-conjugated; Zymed Laboratories) was applied to the slides at room temperature for 10 minutes. After they had been washed with PBS, the slides were incubated with horseradish peroxidase conjugated to streptavidin and then stained with 3-amino-9-ethylcarbazole dye. The slides were counterstained with hematoxylin, washed, attached with GVA (Zymed Laboratories), and visualized with the aid of light microscopy.

Statistical Analysis Assays were performed using ASCs obtained from three different donors. For each donor, the samples within each assay were assayed in three repeated experiments. All mean values in Results and figures are displayed with their SEs. Statistical tests for significance were performed using Student’s t test, and the minimal criterion for significance was determined to be a probability level less than 0.05.

RESULTS Porcine MSC Transfection and Evaluations of Transfection Efficiency and Cell Viability Porcine ASCs were transfected with pEGFP by applying the nucleofection technique under different nucleofection conditions to quantitatively evaluate the transfection efficiency and viability of the cells. The conditions tested included different cell numbers per sample and different amounts of plasmid DNA (Fig. 1A). Transfection efficiency was evaluated by performing a flow cytometric analysis. The results are presented as the percentage of GFP-expressing cells among total live cells. The cells were considered transfected if the fluorescent signal intensity was greater than the autofluorescent signal of nontransfected cells. These results do not reflect the expression intensity of the transfected cells but rather the ratio of transfected cells to total viable cells. The optimal nucleofection conditions were found to comprise 2 ⫻ 106 cells per sample and 10 ␮g of plasmid DNA, which resulted in approximately 50% cell viability and more than 60% transfection efficiency (Fig. 1B). All additional experiments were performed using these conditions.

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Figure 1. Nucleofection condition optimization. (A): The optimal conditions of nucleofection of ASCs were calibrated using nucleofection of plasmid enhanced GFP. The viability of the cells was calibrated by counting the surviving cells 24 hours postnucleofection. The efficiency of the nucleofection was evaluated by flow cytometry. (B): Flow cytometric analysis of the chosen conditions. Bars indicate SE (n ⫽ 3). Abbreviations: FSC-H, forward scattering; GFP, green fluorescent protein.

Figure 2. ASCs nucleofected with rhBMP-6 expressed and secreted rhBMP-6. The gene expression was evaluated using quantitative reverse transcription-polymerase chain reaction (A), and the protein secretion was quantified using an enzyme-linked immunosorbent assay during the 3 weeks after nucleofection (B). The gene expression and protein secretion by ASCs nucleofected with EGFP were set as day 0. The osteogenic differentiation of genetically modified ASCs in vitro was evaluated using colorimetric alkaline phosphatase assay 7 days postnucleofection (C). Bars indicate SE (n ⫽ 3). Abbreviations: ALP, alkaline phosphatase; ASC, adipose tissue-derived stem cell; GFP, green fluorescent protein; h, hours; rhBMP, recombinant human bone morphogenetic protein.

Nucleofection of ASCs with rhBMP-6 —In Vitro Evaluations ASCs were transfected with rhBMP-6 using the nucleofection system, and the optimal conditions were found. The transfected rhBMP-6 gene expression was evaluated using real-time quantitative RT-PCR. Gene expression was assessed at several time points during the first 3 weeks after transfection and was found to reach a maximal expression of 5.2 ⫻ 107 ⫾ 1.6 ⫻ 107 copies of mRNA per 1 ␮g of RNA on day 2 after nucleofection (n ⫽ 3). The gene expression of rhBMP-6 gradually declined, and on day 21, it reached a level of 1.4 ⫻ 105 ⫾ 2.2 ⫻ 104 copies of mRNA per 1 ␮g of RNA (n ⫽ 3), which was close to the basal level of rhBMP-6 detected in pEGFP-nucleofected ASCs (Fig. 2A). This level of gene expression is very similar to previously reported gene expression in human MSCs nucleofected with plasmids encoding rhBMP-2 and rhBMP-9 [6]. To estimate the efficiency of protein synthesis after rhBMP-6 gene transfer, we performed an immunoassay of conditioned media collected from cultures of porcine ASCs nucleowww.StemCells.com

fected with prhBMP-6. These porcine ASCs secreted rhBMP-6 on day 1 postnucleofection at a level of 4.78 ⫾ 0.97 ng/106 cells per 24 hours (n ⫽ 3), whereas ASCs transfected with pEGFP secreted rhBMP-6 at levels of only 0.56 ⫾ 0.07 ng/106 cells per 24 hours. These results correlate with findings in our previous studies, in which prhBMP-2 was transfected using the same method in human MSCs [6]. The profile of protein secretion followed the profile of gene expression, and protein secretion peaked on day 2 at a level of 45.4 ⫾ 2.89 ng/106 cells per 24 hours (n ⫽ 3). After 3 weeks in culture, rhBMP-6 protein secretion declined to 0.2 ⫾ 0.02 ng/106 cells per 24 hours, which was similar to the level, detected in the control pEGFPtransfected ASCs (Fig. 2B).

Osteogenic Potential of rhBMP-6 In Vitro Following the evaluation of gene expression and protein secretion of rhBMP-6, we tested the osteogenic potential of the nucleofected ASCs. The genetically modified ASCs were grown in vitro in maintenance conditions (without differentiation sup-

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Figure 3. Spinal fusion bone mass formation imaged by microcomputed tomography (␮CT). The spines were harvested 5 weeks after the injection of genetically engineered adipose tissue-derived stem cells and analyzed by ␮CT. The new bone formation was contoured manually and is depicted in orange color on the three-dimensional (3D) reconstructed images. Shown are a representative lateral view of 3D image of fused spine on the left side and coronal sections in two-dimensional and 3D views on the right.

plements), and the osteogenic differentiation was evaluated using a quantitative colorimetric alkaline phosphatase assay. The ASCs nucleofected with prhBMP-6 showed the highest level of osteogenic activity, 1.53 ⫾ 0.22 nmol of ALP/minute per ␮g of protein (n ⫽ 3), compared with the control group transfected with pEGFP and the control group of non-nucleofected cells, which had 0.57 ⫾ 0.09 nmol of ALP/minute per ␮g of protein (n ⫽ 3) and 0.11 ⫾ 0.04 nmol of ALP/minute per ␮g of protein (n ⫽ 3), respectively (Fig. 2C).

Spinal Fusion Formation Evaluated Using ␮CT and Histological Analysis Genetically engineered porcine ASCs overexpressing the rhBMP-6 gene were injected into the paraspinal muscles of immunodeficient mice. Bone formation was analyzed using ␮CT (Figs. 3, 4) and histological analysis (Fig. 5). As a control, nonengineered ASCs were injected in the same manner; however, no bone formation was observed (data not shown). We were able to demonstrate that the bone formation bridged two or three spine segments. Extensive bone formation on and adjacent to posterior elements of the spine actually constituted a bridging mass of bone fusion above and covering several spinal segments. The structural parameters measured using ␮CT showed that the bone fusion mass that formed de novo in the mice did not differ from intact vertebral bone tissue (Fig. 3). The TV, including cavities within the tissue, was found to be 13.3 ⫾ 3.3 mm3 (n ⫽ 5). The BV, not including cavities, reached 8.5 ⫾ 2.4 mm3 (n ⫽ 5). To evaluate the morphological and structural properties of the ectopically formed bone tissue and compare them with those of native bone, we compared ectopically formed bone tissue to the posterior part of intact vertebrae that had not fused (Fig. 4). The bone volume density, which is the BV/TV ratio, was found to be 0.64 ⫾ 0.04 (n ⫽ 5) in the fusion bone mass and 0.8 ⫾ 0.04 (n ⫽ 3) in the posterior part of the vertebrae. Average bone thickness indicates the solidness of bone tissue compartments [30]. The average bone thickness of the fusion mass was very similar to that of control tissue: 0.16 ⫾ 0.01 mm (n ⫽ 5)

and 0.19 ⫾ 0.01 mm (n ⫽ 3), respectively. The parameter of connectivity density is used to describe the porosity of the bone sample and to show how branched the bone tissue structure is [30]. The fusion mass displayed a connectivity density of 24.19 ⫾ 3.19 1/mm3 (n ⫽ 5), whereas the control tissue exhibited a connectivity density of 16.97 ⫾ 3.87 1/mm3 (n ⫽ 3). The degree of anisotropy was also found to be similar in both tissues: 1.78 ⫾ 0.11 (n ⫽ 5) in the fusion mass and 1.52 ⫾ 0.08 (n ⫽ 3) in the control bone tissue. Overall, none of the structural parameters differed significantly between the two groups according to Student’s t test (p ⬎ 05). Standard H&E and bone matrix-specific Masson’s trichrome staining revealed a well-organized fusion mass on the posterior aspect of the mouse spines (Fig. 5A, 5C). Light microscopy of the bone architecture revealed an organized fusion mass with a lamellar microstructure, which contained large compartments of bone marrow within it and highly resembled the intact vertebral bone tissue morphologically (Fig. 5A–5C). To illustrate the histological staining, we have included a schematic diagram in Figure 5B, which depicts the three main components: intact vertebral bone tissue, newly formed bone tissue, and bone marrow. Conversely, the control nonnucleofected cells that were injected in the same manner were found in rather small amounts, mostly undifferentiated (Fig. 6E). Several cells had an adipocyte-like morphology, indicating that some cells could have undergone adipogenic differentiation.

Contribution of Injected ASCs to the Newly Fused Bone Mass The genetically modified cell therapeutic approach described in this report raises the question, what was the contribution of injected genetically engineered cells to the newly fused bone mass? We chose two methods to pursue this question. First, we stained the genetically engineered porcine ASCs with the fluorescent, biologically inactive tracing dye CM-DiI. Second, we performed immunohistochemical staining for the intracellular mesenchymal marker protein vimentin by using an antibody that “recognizes” the porcine molecule but not the mouse one. CM-

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Figure 4. Quantitative analysis of structural properties of new bone formation using microcomputed tomography. (A): Representative threedimensional image of the new bone formed in the spinal fusion (in orange) on the left side was compared with posterior part of intact vertebra, a representative image of which is depicted on the right side. (B): The total tissue volume and actual bone volume. Bone volume density is calculated as the ratio of the bone volume and total volume (C), average bone thickness (D), connectivity density (E), and degree of anisotropy (F) are structural parameters of bone. Bars indicate SE (spinal fusion mass, n ⫽ 5; control vertebra, n ⫽ 3).

Figure 5. Spinal fusion bone mass formation imaged by histology. Histological analysis revealed that bone fusion mass adjusted to the intact vertebrae 5 weeks after injecting the nucleofected cells. The bone formation included morphologically normal bone structures and bone marrow. The sections are presented in H&E standard staining (A); a schematic map of the histological sections in which green indicates new bone, red indicates bone marrow, and blue indicates the intact vertebrae (B); and Masson’s trichrome staining (C).

DiI-stained porcine ASCs were shown to form cartilage-like islands in the paraspinal muscle after 2 weeks in vivo and in bone tissue after 5 weeks in vivo (Fig. 6). Most of the cells forming the cartilage-like islands were stained with CM-DiI, indicating that these islands were composed of the genetically modified ASCs that had been injected. However, the bone structures formed after 5 weeks were not composed of CM-DiIlabeled cells alone (Fig. 6), indicating that the bone tissue is a chimera of donor (CM-DiI-labeled) cells and host (nonlabeled) cells. These findings were confirmed by examining paraffinembedded tissue sections that had been subjected to an immunohistochemical stain designed to detect porcine vimentin (Fig. 6G– 6J). The immunohistochemical staining technique produced an intracellular (red) stain in the fusion bone mass (Fig. 6G, 6H) www.StemCells.com

but no stain in the osteocytes that formed intact mouse vertebrae (Fig. 6J). These results show that most of the fusion bone mass was formed by the injected cells, although there are indications that some bone was formed of donor-host chimerical tissue.

DISCUSSION Our data demonstrate, for the first time, the following: (a) primary ASCs that have been nonvirally transfected with plasmid rhBMP-6 ex vivo can secrete biologically active rhBMP-6; and (b) after these cells have been injected into the lumbar paravertebral muscle of immunodeficient mice, the cells can induce functional bone tissue formation and efficient posterior

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Figure 6. Contribution of the donor adipose tissue-derived stem cells (ASCs) to the spinal fusion bone mass formation. Porcine ASCs nucleofected with bone morphogenetic protein 6 were stained with CM-DiI, and 5 ⫻ 106 cells were injected into paraspinal muscle of NOD/SCID mice. The muscles were harvested 2 weeks after the injection (A, B) and 5 weeks after the injection (C–J). The sections were counterstained with 4⬘,6diamidino-2-phenylindole (DAPI) (A–D, F). The control non-nucleofected cells were imaged using H&E stain (E) and fluorescent CM-DiI and DAPI stains (F). The sections of fused spines were stained with immunohistochemical staining for the porcine mesenchymal marker gene vimentin (G–J). New bone tissue (G), new cartilage-like tissue (H), and mouse intact vertebrae are depicted at magnifications of ⫻10, ⫻20, and ⫻40. (I): Schematic map indicating the regions of the spine. In (A–F), white arrows indicate intact bone, and green arrows indicate new bone formation.

spinal fusion. We optimized the nucleofection conditions of the porcine ASCs so that we could gain maximal viability and efficiency. The outcome of this optimized protocol was approximately 50% cell viability and more than 60% transfection efficiency, as detected using flow cytometry 24 hours after nucleofection. These results are close to what we obtained using human bone marrow-derived MSCs [6] and human ASCs [25]. The gene expression and protein secretion profiles demonstrated high peaks on day 2 postnucleofection; however, the levels of transgene expression and protein secretion declined until they reached basal levels. These profiles were observed in ASCs that were also nucleofected with pEGFP. The profiles were previously observed in human ASCs nucleofected with the EGFP reporter gene [25]. In this stem cell-mediated therapeutic approach, the transient expression of the transgene is advantageous because induction of osteogenic differentiation is needed only for a short period. The genetically modified ASCs that expressed and secreted rhBMP-6 were deemed competent to induce massive bone formation and vast spinal fusion in a minimally invasive injection approach. The quality and structural properties of the newly formed bone, as tested by performing quantitative ␮CT, were comparable to those of native bone; nevertheless, in a comparison with intact vertebrae, the new bone formation appeared much younger and less mature.

The bone fusion mass contained donor porcine ASCs; this was verified by CM-DiI and immunohistochemical staining. The expression of porcine vimentin by most cells forming the new bone formation indicated the vast contribution of these cells to de novo bone tissue formation. Genetic engineering of ASCs to produce rhBMP-6 had a major effect on tissue formation; the nonengineered ASCs did not induce bone formation. In an attempt to produce spine fusion in rodents, direct nonviral delivery of osteogenic genes efficiently induced ectopic bone formation in vivo [33]. However, the bone tissue that developed was insufficient to bridge the vertebrae and induce spinal fusion. That was probably due to the low number of progenitor cells in the region that were able to respond to the secreted BMP and induce major bone formation. In other studies in which MSCs were used, we and others relied on adenoviral vectors to genetically modify the cells [22, 34]. The use of adenoviral vectors for clinical applications remains doubtful, however, given the relatively high safety of ex vivo nonviral gene delivery [21]. To date, autograft bone implantation in conjunction with added rhBMP-2 protein is the only way to induce ectopic bone formation and spinal fusion in the clinical setting [21, 22]. In this study, we created conditions that come as close as possible to those required for clinical applications. First, in

Sheyn, Pelled, Zilberman et al. adults, the reservoir of available adipose tissue-derived stem cells is greater than that of bone marrow-derived MSCs, and these cells can be obtained with less risk of morbidity [8]. Second, the nonviral gene delivery method we used to genetically engineer the ASCs ex vivo is safe and transient, limiting overexpression of the osteogenic gene to a short period of a few weeks. There is a potential advantage in the transient expression of the osteogenic transgene: short-term expression may be preferred for skeletal regeneration applications. This advantage was previously exhibited only by adenoviral vectors, which induced transient overexpression of BMPs [34 –36]. Third, the therapeutic transgene of BMP is gradually overexpressed, translated, and secreted by the injected mammalian cells, rather than being produced by prokaryotes, as is recombinant BMP that is locally delivered in high nonphysiological doses with a high immunogenicity. We used porcine adipose tissue-derived ASCs to explore the osteogenic potential of these cells within the context of spinal fusion. The data generated in this study will enable us to take the next essential step to preclinical studies involving large animals, because the porcine model for spine fusion is clinically relevant [37] and porcine ASCs have been extensively investigated [38]. An injection-based therapeutic system carries great advantages. First, it saves the patient from the invasive and traumatic surgical treatments currently practiced for degenerative disc

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diseases. Second, the injection system enables the clinician to control bone formation in vivo and offer subsequent treatments to shape the newly formed bone and produce incremental fusion. Genetically engineered ASCs have great clinical potential compared with autologous bone grafts, as does endogenously produced BMP compared with recombinant BMP with respect to the immunogenic response.

CONCLUSION The proposed therapeutic model involving the use of genetically modified adipose tissue-derived adult stem cells has great potential in various applications of bone tissue engineering. In the present study, we identified engineered bone tissue that morphologically and structurally resembled native tissue. In future studies, we intend to validate the biomechanical properties of the engineered tissue, because the fused section of spine should bear the load of the degenerated spine.

DISCLOSURE

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CONFLICTS

The authors indicate no potential conflicts of interest.

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