Specific Aim 1: Evaluate whether advancing age influences the cell characteristics and neovascularization potential of SVF Hypothesis: SVF from old animals will exhibit differentiation capacity and neovascularization ability in an in vivo assay, but will be less effective compared to SVF from young animals.
a) Determine the autologous regenerative potential of freshly isolated and cultured SVF by examining age-related differences in cell type, concentration, function, and differentiation potential
b) Compare neovascularization potential and functional perfusion of freshly isolated and cultured SVF derived from young or old animals in an in vivo model.
Background and Preliminary Studies. Contained within adipose tissue is a heterogeneous cell population defined as the SVF. Consisting of endothelial cells, smooth muscle cells, blood cells and mesenchymal cells 7, SVF and/or subpopulations exhibit the potential to differentiate into various mesodermic lineages 8 and have the ability to induce vascular network formation through the reassembly of endothelial cells at the site of implantation 9 (Fig ?). In total, adipose-derived cells, which can include fresh isolates, lipoaspirates and cultured cells of various subpopulations, have exhibited the ability to differentiate into adipocytes 10, 11_ENREF_10, osteocytes 10, 11_ENREF_10, chondrocytes10 11, myocytes 11, smooth muscle cells 12, cardiomyocytes 13-15_ENREF_13, and endothelial cells 14, 16, amoung others17. Numerous studies have shown that adipose tissue from older animals and humans demonstrate weakened or deleterious signaling processes, such as lower proliferation rates and greater susceptibility to apoptosis 18. The general consensus remains that mesenchymal stem cells (MSCs) and progenitor cells lose proliferative and differentiation capacity with advancing age 1, 19_ENREF_1. A recent study in humans found that aging has less effect on proliferation and osteogenic differentiation potential in mesenchymal cells derived from adipose tissue compared to those from bone marrow 19. However, it remains relatively unknown how advancing age contributes to the differentiation potential of other cell lineages, as well as vascular-related functions of SVF, such as neovascularization._ENREF_14 Neovascularization entails an integrated series of vascular activities leading to the formation of a new mature microcirculation and includes angiogenesis, vascular inosculation, vessel maturation, and microvascular network stabilization, among other vascular activities 20. Recently, a subpopulation of SVF isolated from the adipose tissue of elderly individuals showed decreased angiogenic potential of progenitor cells, as measured by in vitro tube/network formation, compared to young, healthy counterparts 21. Our preliminary data in freshly isolated SVF from young (2-4 mos) and old (24 mos) Fischer-344 rats supports this observation (Fig 1), as in vitro tube formation was lower in SVF from old animals. In addition, more preliminary data shows total number and percent perfusion of microvessels formed in implanted constructs with freshly isolated SVF from old animals was diminished (Fig 2). Interestingly, a brief culture period (P0) for SVF from old rats recovered the number of angiogenic tubes formed (Fig 1), but we do not know if this would translate into improved in vivo neovascularization activity. We believe this difference between freshly isolated and cultured SVF warrants further investigation for two reasons: 1) ex vivo expansion of human cells for clinical trials is more than “minimal manipulation” and would require formal US Food and Drug Administration approval for use 22 and 2) there are many clinical trials currently utilizing autologous, freshly isolated SVF in ischemia/infarction diseases, of which older subjects may be negatively affected if their autologous cells are unable to exert angiogenic actions as expected 17.
Therefore, this specific aim has two main areas of focus. 1) Determine the autologous regenerative potential of freshly isolated and cultured SVF by examining age-related differences in cell type, concentration, function, and differentiation potential in vitro, and 2) Compare neovascularization potential and functional perfusion of freshly isolated and cultured SVF derived from young or old animals in an in vivo model.
Fresh Isolation and Culture of Stromal vascular fraction (SVF). For this particular aim, we will isolate SVF from ovarian fat pads of either adult (2-4 mos), middle-aged (12-14 mos) or aged (22-24 months) female Fisher-344 rats. The inclusion of the middle-age group for this aim allows us to evaluate a potential age-specific deterioration in regenerative capacity that may occur at the same time the rat begins to enter into a constant state of diestrus (which mimics menopause in humans) 23. While we realize that utilizing human adipose-derived SVF cells would be preferred, the liposuction sources from local plastic surgeons we receive at the CII tend to be females between 35-50 yrs old. Therefore, we chose to utilize a rat model to isolate SVF cells from in order to eliminate potential disease complications from hosts and to examine different but specific age groups. However, future studies intend to include human sources of adipose-derived cells. Harvested fat pads are washed in 0.1% BSA-PBS, finely minced and digested in 2 mg/ml Type I collagenase solution for 40 min at 37oC with vigorous shaking. Buoyant adipocytes are removed by centrifugation and the cell pellet re-suspended in 0.1% BSA-PBS as previously described 24. Cell count in fresh isolations from all groups is determined by using a Nucleocounter, while viability is assessed by live/dead assay (Invitrogen).
Because our prior study and preliminary data showed that SVF cultured on an epicardial patch can incorporate into the epicardial vasculature of hearts 24 (Fig 3C), we chose to again utilize this proven method of SVF cell delivery in the current study and evaluate the cell characteristics of the SVF on the Vicryl at the time of implant in addition to the fresh isolate SVF from the three age groups. Therefore, a subset of the SVF isolated as described above will be immediately plated (1x106 cells/cm2) with a piece of Vicryl 1x1.5 cm. The patches will be cultured for 14 days in DMEM with 10% FBS, changing the media every 4 days. After 14 days, three age-dependent SVF patches will be ready for characterization (ySVF, mSVF and oSVF patches). Viability is assessed through the use of live/dead assay at this 14-day time point (Invitrogen). To evaluate the cell characteristics of cultured SVF, SVF cells will then be trypsinized (1:10 dilution) directly from 14-day patches (ySVF, mSVF and oSVF patches).
Cell Characterization of SVF. In order to compare the regenerative cell characteristics of freshly isolated and cultured SVF from different age groups, we will evaluate the mesenchymal cell marker profile and differentiation potential in vitro. By evaluating a broad range of cell lineages, including adipogenic, chrondrogenic, osteogenic, cardiomyogenic, endothelial and smooth muscle cell, we will be able to compare and contrast the functional potential of our regenerative cell population to others and also describe how advancing age might alter these outcomes. A total of 3-5 rats from each age group will be used for each differentiation assay and will be performed in duplicate. Where possible, bone marrow-derived cells (BMC) will be isolated from the femur and tibia of the same rats as described previously 25 and used as positive controls.
Adipogenic, chondrogenic and osteogenic differentiation will be assessed for age-related alterations in differentiation potential as previously performed by Dr. Boyd 26. Briefly, SVF from either fresh isolation or trypsinized SVF patch are passaged onto six-well tissue culture plates at a concentration of 2.5x105 cells/well (35mm) for osteogenic and adipogenic induction. For chondrogenesis, a 10 mL cell suspension micromass (2x107 cells/mL) is allowed to adhere in a 35mm tissue culture dish for 1 h, and then media is added to prevent desiccation. After an overnight incubation at 37°C, 1mL of proliferation or differentiation media is added to the well. Osteogenic differentiation is qualitatively assessed via Von Kassa (silver nitrate) staining and subsequent phase and brightfield images, and quantitatively assessed via measurement of calcium using StanBio Calcium Assay. Adipogenesis differentiation is qualitatively assessed by staining for lipid deposits using Oil Red O, and quantitatively assessed by measurement of lipid content with Nile Red fluorescence. Chrondrogenesis differentiation is qualitatively assessed by Safranin O staining for glycosarninoglycans (GAG), and quantitatively assessed by measurement of GAG using 1,9-Dimethylmethylene Blue normalized to DNA using PicoGreen.
Cardiomyogenic differentiation SVF cells from fresh isolate or patches will be assessed for age-related alterations in cardiomyogenic differentiation potential as described for adipose-derived cells previously 13-15_ENREF_15. Briefly, cells will be seeded in six-well plates at 50,000 cells/well. Twenty-four hours after seeding, the cells are washed with phosphate buffered saline (PBS) twice. Then, the cells are treated with 5-azacytidine at 9 umol/L and incubated for 24 hours. Cells are washed and replaced with fresh RPMI medium and incubated in a CO2 incubator. The cells are observed daily, and the medium is changed once every 3 days until the experiment is terminated at 2 months. Primary monoclonal antibodies against the cardiac-specific myosin heavy chain (Chemicon Inc.), α-actinin (1:100 Chemicon Inc.) and Troponin I (dilution 1:200) (Chemicon Inc.) will be used to detect cardiomyocytes.
Endothelial Cell Differentiation. SVF cells from fresh isolate or patches will be assessed for age-related alterations in endothelial cell differentiation potential as described previously 16. To analyze in vitro endothelial differentiation, a 24-well cell culture plate is coated with Matrigel (8.8 mg/ml; BD Bioscience Biotech) in each well. SVF cells are suspended in endothelial differentiation medium at a concentration of 1x105 cells/ml and 0.5 ml of cell suspension is added to each well. Differentiation medium contains medium 199, 50 ng/ml VEGF, 10 ng/ml b-FGF, and 3% FBS. Cultures are incubated at 37°C in a 5% CO2 humidified atmosphere for 2–3 days observed with an inverted photomicroscope. FITC-coupled CD31, CD34 (BD Bioscience) are used at a 1:50 dilution. Cells are examined by fluorescence microscopy. For VE-cadherin (CD144, Santa Cruz Biotechnology), a goat polyclonal antibody against VEcadherin and streptavidin–biotin peroxidase detection system is used.
Smooth Muscle Differentiation. To assess the ability of SVF from fresh or cultured patches to differentiate into smooth muscle cells (SMC) as age progresses, we will perform SMC differentiation protocol as previously described 12. SVF reaching confluence will be induced into SMC by culturing in DMEM supplemented with 1% FBS, 5 ng/mL recombinant human TGF-b1 (R&D Systems, Minneapolis, MN), and 2.5 ng/mL recombinant human BMP4 (R&D Systems). Media is changed every 2 days, and 7 days after culture immunofluorescence was performed on methanol-fixed cells using the following SMC-specific primary antibodies: mouse monoclonal anti-α -SMA (C6198; Sigma-Aldrich), rabbit polyclonal anti-SM22α (ab14106; Abcam, Cambridge, United Kingdom), rabbit monoclonal anti-calponin (ab46794; Abcam, Cambridge, United Kingdom), and mouse monoclonal anti-SM-MHC (M7786; Sigma-Aldrich). FITC conjugated secondary antibody will be used to detect localization of anti- α-SMA and SM22α. Positive control cells will be a human aortic SMC (Invitrogen). Control samples consisted of cells without primary antibody and were used to assess background fluorescence.
Endothelial Cell Tube Formation Assay. Tubule formation of SVF is measured by In Vitro Angiogenesis Assay Kits (CHEMICON) as described 27 with slight modifications. Freshly isolated and P1 cultured SVF from all groups will be plated on matrigel coated flasks (10 mg/ml) at a concentration of 5x104 cells/flask. Cells are incubated for 6 hours at 37°C, 5% CO2. DMSO and Calcein AM solution (8ug/ml) are added to the flask after media was removed and incubated for 30-40 min. After 2x rinsing, flasks are imaged using Olympus IX51 fluorescent microscope. Three images/flask are processed using MetaMorph software to calculate tube formation numbers, n=5 SVF isolations/group. Additionally, tube length, tube areas and branch points will be assessed to complement in vitro angiographic characteristics.
Flow Cytometry. The recognized biologic properties of MSC’s have been defined by the International Society for Cellular Therapy (ISCT). According to their cell marker profile and definition for plastic-adherent cells isolated from tissue, the following CD marker profile is identified as MSCs: CD73+, CD90+, CD105+, CD34-, CD31-, CD11b/14-, and CD45- 28. Freshly isolated and cultured SVF from all age groups are harvested and cells (~2x106/age group) are stained with antibodies against the aforementioned CD markers. Phenotypic characterization will be calculated as percent positive of events above isotype control for that particular antibody.
Neovascularization Potential of SVF. The first in vivo model for this proposal is our microvascular construct model used to evaluate age-related neovascularization characteristics of SVF. This model is used to perform a vasculogenic assay, in which microvascular assembly and functional perfusion by the SVF cells are assessed. Based on the premise that the formation of a stable and functional microvasculature requires the presence of both endothelial cells and mural cells 29, 30, we developed this vasculogenesis model. When SVF is placed in a three-dimensional collagen gel and implanted, the SVF cells will form complete functional, hierarchical microvascular networks 31-34 (Fig 2). In addition to these in vivo uses of the microvascular construct, we can perform a variety of reconstitution experiments in order to assess SVF cell behavior. For example, by strategically using SVF cells derived from different ages of reporter rats (green fluorescent protein positive, or GFP+), it is possible to delineate the different activities in this complex setting. For the vasculogenesis assay, SVF (from specific age groups, either freshly isolated or cultured) are suspended (3 x 106 cells/mL) in ice-cold 3 mg/ml rat tail type I collagen (BD Biosciences) prepared with DMEM and pH neutralized with 1 M NaOH. SVF/collagen suspensions are plated into individual wells (0.25 ml/well) and placed in a 37°C incubator for 20 minutes to polymerize the collagen. Two newly formed constructs are implanted immediately in the subcutaneous position on the flanks of each anesthetized Rag 1 mouse. For implantation, a subcutaneous pocket is formed between the skin and the underlying muscle anterior to the pelvis using blunt dissection through a small skin incision. The incision is then closed with 6-0 suture and the animal is allowed to recover for 4 weeks. This vasculogenic assay allows us to directly compare age-related differences in the ability of SVF cells to assemble functional neovessels in vivo.