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 agerelated 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
Figure 1. Left panel shows tubulogenesis assay from
formation, compared to young, healthy
counterparts 21. Our preliminary data in
freshly isolated and cultured SVF from young and old
freshly isolated SVF from young (2-4
mos)
rats. Brief culturing recovered the age-related decline in
and old (24 mos) Fischer-344 rats
the ability to form tubes in vitro. (Right) Calcein staining
supports this observation (Fig 1), as in
vitro
of cultured SVF made from young and old. (n=3/group)
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.
Figure 2. Left panels show confocal images of SVF constructs made from young (top) and old
(bottom) rats. Dextran infusion prior to explant labels perfused vessels, construct staining with
Gs-1 identifies total vascular elements. (Right) SVF constructs made from old rats exhibit less %
of perfused vessels compared to SVF from young rats. (n=3/group)
Fresh
Isolati
on
and
Cultur
e of
Strom
al
vascu
lar
fracti
on
(SVF).
For
this
partic
ular
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 adiposederived 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 resuspended 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 1315
_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 5azacytidine 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,
Figure 3. Microvascular repair potential of adipose-derived SVF. GFP+ SVF rat cells have the
or
ability to incorporate (arrows, A) and assemble into neovessels (B) after implantation to
GFP+),
contribute to an existing vasculature. GFP+ SVF cells associate with the coronary vasculature
it is
(arrows,toC)
of the repairing
ischemic
epicardium
4-weeks
post-SVF
construct
implant.assay,
WhiteSVF (from
possible
delineate
the different
activities
in this complex
setting.
For the
vasculogenesis
specific
either freshly isolated or cultured) are suspended (3 x 106 cells/mL) in ice-cold 3 mg/ml rat
bars = age
100 groups,
μm
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.
Neovascularization Analysis. After 4 weeks, dextran-rhodamine is injected and allowed to circulate for
15’ (200 MW/ml/g) before SVF constructs are explanted. We will extensively use confocal microscopy to
evaluate the newly formed microvasculatures in one of the explanted constructs. In our experience, it is critical
to have three-dimensional data for these experiments. In addition to accurately visualizing cell locations and
spatial distributions, accurate measurements of vascular length, diameter, branching and volume require 3-D
image sets. We have good experience evaluating vasculatures with both confocal and multiphoton microscopy
including advanced post-acquisition image analysis involving volume rendering, co-localization mapping, and
custom data extraction 33, 35-40.
Histology and Immuno-cytochemistry. The other construct is immediately placed into 4% PFA/PBS
overnight. The solution is changed to 30% sucrose every night for 3 days. Afterwards, SVF constructs are fixed
in Tissue-tek (Sakura) and frozen in liquid-nitrogen-cooled isopentane until they are sliced into 10 μm thick
cryosections using an ultramicrotome (Leica). The presence of grafted cells is detected by fluorescent in situ
hybridization (against the Y chromosome). Nuclei will be identified through DAPI staining (1:10000).
Perivascular cells are identified with a monoclonal anti-alpha SMC actin antibody (clone 1A4; Sigma
Immunochemicals) using a horseradish peroxidase (HRP) reporter system (Sigma Immunochemicals). General
histological structure is determined with hematoxylin and eosin or Masson’s Trichrome. Vessel density is
determined by counting discrete, dextran-rhodamine positive structures.
Potential problems and alternative strategies. We anticipate