Wild-type (WT) (C57BL/6) and type 2 diabetic (DM2) mice (BKS.Cg-m +/+Lepr
) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) to study the effect of diabetes on ASC physiology. Selected assays were also performed on ASCs isolated from WT mice following induction of type 1 diabetes (DM1) via streptozotocin injection (STZ) (Sigma-Aldrich, St. Louis, MO, USA) as previously described
. Only mice with blood glucose levels of greater than 350 mg/dL were considered diabetic and used for further analysis. All protocols were approved by the Stanford Administrative Panel on Laboratory Animal Care.
ASC niche analysis
WT and DM2 murine inguinal fat pads were harvested and manually disrupted for real-time quantitative polymerase chain reaction as described below.
ASC harvest and culture
ASCs were isolated from WT, DM1, and DM2 murine inguinal fat pads, minced, and digested for 1 hour at 37°C using collagenase I (Roche Applied Science, Indianapolis, IN, USA). The reaction was stopped with the addition of supplemented media, pelleted via centrifugation, and subjected to erythrocyte lysis in accordance with the instructions of the manufacturer (Sigma-Aldrich). The remaining cells were pelleted again, forming the stromal vascular fraction (SVF). The SVF was either cultured under standard conditions (37°C in 5% CO2) in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Life Technologies, Grand Island, NY, USA) containing 1 g/L of glucose to purify for ASCs or used immediately for flow cytometric/single-cell analyses. Cultured cells were used at or before passage 2, and all analyses were run in triplicate unless otherwise stated.
In vitroMatrigel tubulization assays
PKH26-labeled WT and DM2 ASCs alone or mixed with calcein-labeled human umbilical vein endothelial cells (HUVECs) (Life Technologies) were cultured for 12 hours under hypoxic conditions on a 24-well plate (4 × 104 cells per well) coated with growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). ASC and HUVEC tubule counts were determined in five random high-power fields per well, respectively, by using an inverted Leica DMIL microscope (Leica Microsystems, Wetzlar, Germany).
In vivoMatrigel plug assay and CD31 immunohistochemistry
WT or DM2 ASCs (8 × 105) (cultured not more than two passages) were suspended in 250 μL of growth factor-reduced Matrigel (BD Biosciences) and injected in a subcutaneous fashion on the dorsum of WT mice (n = 4). Plugs were harvested at day 10, and 7-μm-thick frozen sections were immunohistochemically stained for the commonly used vascular marker platelet/endothelial cell adhesion molecule 1 (PECAM1/CD31, a transmembrane glycoprotein expressed on the surface of platelets, endothelial cells, and subsets of hematopoietic cells but particularly concentrated at the intercellular junctions of endothelial cells), followed by ImageJ (National Institutes of Health, Bethesda, MD, USA) quantification
In vitroadipogenic differentiation
WT and DM2 ASCs were seeded in standard six-well tissue culture plates (1.5 × 105 cells per well), and adipogenic differentiation medium—consisting of DMEM (1 g/L glucose), 10% fetal bovine serum, 1% penicillin/streptomycin, 10 μg/mL insulin, 1 μM dexamethasone, 0.5 mM methylxanthine, and 200 μM indomethacin—was added after cell attachment. Oil red O staining was performed after 7 days of incubation.
In vitroosteogenic differentiation
WT and DM2 ASCs were seeded in standard six-well tissue culture plates (1.0 × 105 cells per well) and grown to at least 80% confluence before being cultured in osteogenic differentiation medium, which consisted of DMEM (1 g/L glucose) supplemented with 10% FBS, 1% penicillin/streptomycin, 100 μg/mL ascorbic acid, and 10 mM β-glycerophosphate. Photometric quantification of Alizarin red stain was performed after 14 days to assay extracellular mineralization as previously described
In vitrohydrogel bioscaffold seeding
WT and DM2 ASCs (1 × 105) were suspended in 15 μL of growth media and seeded within a previously described 5% collagen-pullulan hydrogel bioscaffold
[7, 8]. Seeded scaffolds were placed in growth media and incubated at 37°C in 5% CO2 prior to proliferation and survival analyses and RNA/protein harvest.
In vitroproliferation and survival
After hydrogel bioscaffold seeding, a live-dead assay (Live/Dead Cell Viability Assay) was performed at multiple time points to assess WT and DM2 ASC viability in accordance with the instructions of the manufacturer (Life Technologies). ASC proliferation was compared between hydrogel-seeded WT and DM2 cells at multiple time points by using an MTT assay (Vybrant MTT Cell Proliferation Assay Kit; Invitrogen, Grand Island, NY, USA).
Real-time quantitative polymerase chain reaction
Total RNA was isolated from ground WT and DM2 fat pads or hydrogel-seeded ASCs by using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA) and transcribed to cDNA (Superscript First-Strand Synthesis Kit; Invitrogen). Real-time quantitative polymerase chain reactions (qPCRs) were performed by using TaqMan gene expression assays (Applied Biosystems, Foster City, CA, USA) for murine Mmp-9 (matrix metalloproteinase 9, Mm00442991_m1), Cxcl-12 (stromal cell-derived factor-1/Sdf-1, Mm00445552_m1), Vegf-a (vascular endothelial growth factor-A, Mm01281447_m1), Eng (endoglin, Mm00468256_m1), Hgf (hepatocyte growth factor, Mm01135193_m1), Mmp-3 (matrix metalloproteinase 3, Mm00440295_m1), Cxcr-4 (chemokine receptor 4, Mm01292123_m1), Fgf-2 (fibroblast growth factor 2, Mm00433287_m1), Fgfr-2 (fibroblast growth factor receptor 2, Mm01269930_m1), Pdgf-a (platelet-derived growth factor-A, Mm01205760_m1), Pdfgr-a (platelet-derived growth factor receptor-A, Mm01205760_m1), and Angpt-1 (angiopoietin 1, Mm00456503_m1) by using a Prism 7900HT Sequence Detection System (Applied Biosystems). Expression levels of the target genes were normalized to Actb (beta actin, Mm01205647_g1) or B2m (beta-2-microglobulin, Mm00437764_m1).
Angiogenic cytokine protein production from hydrogel-seeded WT and DM2 ASCs was quantified by using a Mouse Angiogenesis Array Kit (R&D Systems, Minneapolis, MN, USA). Pixel density of each spot in the array was quantified and normalized to controls by using ImageJ (National Institutes of Health).
In vivomurine ischemia model
A model of graded soft tissue ischemia was created on the dorsum of WT mice, as described previously
. Briefly, a full-thickness three-sided peninsular flap was elevated, and a thin silicone sheet was inserted to separate the skin from the underlying fascia. A size-matched hydrogel bioscaffold was inlaid between the silicon sheet and skin flap either alone or following the seeding of 2.5 × 106 WT or diabetic (type 2 or 1) ASCs (cultured not more than two passages) (n = 5). Unseeded scaffolds were used as controls. The skin flap was then sutured into place, and the demarcating necrotic area was quantified 10 days post-op. The proximal surviving flap was processed for neovascular growth via CD31 immunohistochemistry.
Microfluidic single-cell gene expression analysis
ASCs isolated from freshly harvested WT, DM2, and DM1 SVF (obtained as described above) by using the surface marker profile CD45-/CD31-/CD34+ (to exclude contaminating CD45+ hematopoietic and CD31+ endothelial cells found within the SVF and to select for a putatively stem-like subset of CD34+ cells
[22, 24]) were analyzed and sorted as single cells by using a Becton Dickinson FACSAria flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) into 6 μL of lysis buffer. Propodium iodide exclusion was used to ensure that only live cells were sorted. Reverse transcription and low cycle pre-amplification were performed by using Cells Direct (Invitrogen) with TaqMan assay primer sets (Applied Biosystems) in accordance with the specifications of the manufacturers. cDNA was loaded onto 96.96 Dynamic Arrays (Fluidigm, South San Francisco, CA, USA) for qPCR amplification by using Universal PCR Master Mix (Applied Biosystems) with a uniquely compiled TaqMan assay primer set as previously described
Results are presented as mean ± standard error of the mean. Data analysis was performed by using a Student t test. Results were considered significant for P values of not more than 0.05. For single-cell transcriptional data, a Kolmogorov-Smirnov (K-S) test was used to compare empirical distributions, followed by an adaptive fuzzy c-means clustering algorithm as previously described