Cell culture
The BM-MSCs were obtained from the Catholic Institute of Cell Therapy (CIC; Catholic MASTER Cells, CIC, Korea). Human bone marrow aspirates were obtained from the iliac crest of healthy donors aged 20 to 55 years with approval from the Institutional Review Board of Seoul St. Mary's Hospital (approval numbers KIRB-00344-009 and KIRB-00362-006). Bone marrow aspirates from each donor who consented were collected and sent to the good manufacturing practice (GMP)-compliant facility of the CIC (Seoul, Korea; www.cic.re.kr) for isolation, expansion, and quality control of human BM-MSCs. The MSC growth medium was Dulbecco's modified Eagle's medium (DMEM)-low glucose (Gibco), supplemented with 7% fetal bovine serum (FBS; Gibco), 15 ng/µL IGF-1 and 125 pg/µL bFGF. The BM-MSCs were added to 100-mm tissue culture dishes (TPP) and incubated in the growth medium at 37°C with 5% CO2. The medium was replaced every other day. The cells were detached when they reached 70 to 90% confluence and were replated at a density of 5 × 103/cm2. The cells were expanded through two to four passages at the GMP-compliant facility. Human umbilical vein endothelial cells (HUVECs) were obtained from Promocell and cultured in EGM-2 complete medium (Lonza). The HUVECs were plated into 100-mm tissue culture dishes (TPP) at a density of 2.5 × 103/cm2 and incubated at 37°C with 5% CO2. The media were replaced every other day. The C2C12 cells were obtained from ATCC and cultured in 100-mm tissue culture dishes (TPP) at a density of 5 × 103/cm2. The C2C12 growth medium consisted of Dulbecco's modified Eagle's medium (DMEM)-high glucose (Gibco) supplemented with 10% fetal bovine serum (FBS; Gibco), and the cells were incubated at 37°C with 5% CO2.
Generation of engineered SDF1α-eMSCs
The BM-MSCs (Catholic MASTER Cells) were obtained from Catholic Institute of Cell Therapy (CIC, Seoul, Korea). To immortalize the MSCs, replication-incompetent lentiviral vectors containing hTERT and c-Myc were prepared and transfected into the cells. To produce immortalized MSCs expressing SDF-1α, replication-incompetent lentiviral vectors expressing the human SDF-1α were prepared and then transfected into the immortalized MSCs. Then, SDF1α-eMSCs were isolated as a monoclonal cell population using the limiting dilution method. The final monoclonal cells were selected based on SDF-1α protein secretion, proliferation rate, and other MSC phenotypes. The SDF1α-eMSCs were cultured in low-glucose Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 10 ng/mL basic fibroblast growth factor (bFGF, Peprotech) and 2 µg/mL doxycycline (Clonetech) at 37°C and 5% CO2.
Production of conditioned medium (CM)
The BM-MSCs and SDF1α-eMSCs were cultured at 37°C with 5% CO2, and when the cultures became confluent, the cells were rinsed with DPBS and serum-free medium was replaced with 1% Gibco™ Antibiotic-Antimycotic. The culture was maintained for 72 h. After 72 h, the medium was collected and centrifuged at 1500 rpm for 10 min to remove cell debris. The supernatant was harvested and stored at − 70°C.
Protection assay (FACS)
When HUVECs and C2C12s reached 100% confluency, the medium was replaced with EBM or serum-free DMEM to mimic starvation conditions. The CM of each cell was combined with 50% in EBM or DMEM. Cells cultured in EGM were the positive control (Normal group), and those cultured in EBM or serum-free DMEM were the negative control (Control group). After 24 h in starvation culture, flow cytometric analyses were performed using a Membrane Permeability/Dead Cell Apoptosis Kit with YO-PRO™-1 and propidium iodide (PI; both Invitrogen, V13243). The cells (1 x 106 cells/mL) were stained with 0.1 µM YO-PRO™-1 and 1.5 µM PI. After incubation on ice for 30 min, the cell fluorescence was analyzed using flow cytometry (BD FACS).
Viability/cytotoxicity assay
Viability and cytotoxicity were assessed using a LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen, L3224) according to the manufacturer's instructions. The medium was exchanged with phenol-red-free DMEM with a calcein and ethidium homodimer-1 dye mixture and incubated for 30 min; then, the labeled cells were analyzed under a fluorescence microscope (Nikon).
Migration assay
The HUVECs (5 × 104) were seeded onto the upper layer of a Transwell™ insert (8-µm pore) with EBM. The inserts were placed in a 24-well plate containing the test media (EGM2, EBM, BM-MSCS CM, or SDF1α-eMSCs CM). The C2C12 cells (5 × 104) were seeded onto the upper layer of Transwell™ insert (8-µm pore) with serum-free DMEM-high glucose and placed in a 24-well plate containing the test media (10% FBS + DMEM, serum-free DMEM, BM-MSCS CM, or SDF1α-eMSCs CM). After 16 h of culture, the cells were fixed with 4% PFA for 10 min, and staining was performed using 0.1% crystal violet (Sigma-Aldrich) for 10 min. After rinsing the Transwell™ membrane with distilled water, the upper side of the membrane was gently wiped with a cotton swab to remove non-migrated cells. The migrated cells were counted using a light microscope, and the stained area of the membrane was calculated using ImageJ software.
Tube formation assay
Matrigel growth factor reduced base membrane matrix (Corning) was added to each well of µ-slide (ibidi). HUVECs were stained with 1uM cell tracker (Invitrogen) and then cells (3 × 105/ml) were added onto Matrigel-coated well containing media (EGM2, EBM, BM-MSCS CM, SDF1α-eMSCs CM) and then incubated at 37°C with 5% CO2 for 8 hours. After 8hrs, the cells were visualized using a light microscope, and formation of tube structures was calculated using ImageJ software.
Western blotting
Cell lysates were prepared in RIPA buffer () with 0.5M EDTA () and Phosphatase Inhibitor Cocktail (). Total cell protein was quantified using Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23225). Equal amounts of total protein sample were taken and Fluorescent-Compatible Sample Buffer (Invitrogen, LC2570) with 10X Bolt™ Sample Reducing Agent (Invitrogen, B0009) was added. Next, each sample was heated to 80°C for 15 min and then centrifuged at 16,000 × g for 3 min at 4°C. The supernatant was collected for western blotting. Equal amounts of loaded protein were resolved by Nupage™ 4 to 12% Bis-Tris Protein Gel (Invitrogen, NP0321BOX) in each experiment. Next, proteins were transferred to iBlot™ NC Transfer Stacks (Invitrogen, IB301002) and membranes were incubated with primary antibody overnight at 4 ◦C, and the secondary antibody for 1 h at room temperature. For quantification of target signal expression, all samples to be compared were run on the same gel and imaged. Total protein normalization was carried out using No-Stain™ Protein Labeling Reagent (Invitrogen, A44717) according to the manufacturer's instructions. Bands were quantified using iBright™ FL1500 Imaging System (Invitrogen, A44115). The antibodies used in western blotting were anti-Erk1/2 (cell signaling, #4696), anti-phospho Erk1/2 (cell signaling, #4376), anti-Akt (cell signaling, #4693), anti-Akt (cell signaling, #4051), anti-HSP 90α/β (santa cruz, sc-13119), donkey anti-mouse IgG Alexa Fluor Plus 680 (abcam, ab175774) and donkey anti-rabbit IgG Alexa Fluor Plus 800 (abcam, ab216773).
Hindlimb ischemia model
All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of The Catholic University of Korea (Approval number: CUMC-2021-0056-02). IACUC and the Department of Laboratory Animals (DOLA) at The Catholic University of Korea, Songeui Campus accredited the Korea Excellence Animal Laboratory Facility at the Korea Food and Drug Administration in 2017, and the facility acquired full AAALAC International accreditation in 2018. All animal procedures conformed to the guidelines from Directive 2010/63/EU of the European Parliament addressing the protection of animals used for scientific purposes or the NIH guidelines. The Balb/c nude mice (8 weeks old, male, 20–25 g) were procured from Orientbio, Korea, and were anesthetized with 2% isoflurane and body temperature was maintained on a 37°C heating pad to prevent cooling during procedure. Ischemia was induced by ligating the proximal of superficial epigastric artery its bifurcation into common femoral artery, occluding the distal and proximal ends of the femoral artery with double-knotted suture (7 − 0 silk) and the intervening 2–3 mm of the artery excised. Subsequently, cells (1 x 106) were injected into two intramuscular sites of the medial hindlimb at a volume of 20 uL per shot. The experimental groups were (1) Control (PBS), (2) BM-MSC, and (3) SDF1α-eMSCs. Additionally, mice received ketoprofen (2 mg/kg) intraperitoneally for three days following the surgery.
Blood flow and limb salvage measurements
To evaluate regenerative efficacy, animals were tracked by serial monitoring of hind limb blood perfusion using a laser Doppler perfusion imaging system (Omegawave, Japan) at Days 0, 7, 14, 21 and 28 post surgery. The blood flow from the knee joint to the toe region was quantified by analyzing color-coded digital images, and the perfusion rate was calculated. In addition, at Day 28 post implantation, the percentages of five statuses (limb loss, foot necrosis, tip necrosis, toe necrosis or limb salvage) were quantified. Limb loss score was graded as whole limb loss (5), limb loss (4), foot necrosis (3), tip necrosis (2), toe necrosis (1) or limb salvage (0).
Determination of fibrosis
Masson's trichrome (MT) staining (Sigma, St. Louis, MO, USA) was performed to determine the area of fibrotic tissue in the ischemic hind limb. Briefly, three paraffin slides were pre-incubated in a 37°C dry oven before deparaffinization and rehydration. The paraffin sections were then re-fixed for one hour in 56°C Bouin's solution. These sections were stained using Weigert's iron hematoxylin solution for 15 min at room temperature and further stained with Biebrich scarlet-acid fuchsin solution for 20 min at room temperature. Finally, the sections were counterstained with aniline blue for 15 min, followed by incubation in 1% acetic acid for 1 min at room temperature. Extensive washes were performed between each step. The collagen fibers appeared blue and viable skeletal muscle appeared red. Imaging of the tissue sections was performed with a slide scanner (Pannoramic MIDI). All other items, including the fibrotic area, were quantified using the ImageJ software.
Immunohistochemistry analyses
At the time of sacrifice, the limb tissues were fixed in 4% paraformaldehyde overnight and then paraffin blocks were made. The tissue was sectioned into 5 µm cross sections starting at the top of the apex using a microtome (Leica, RM2255, Germany). The sections were stored at − 20°C before use. After deparaffinization and rehydration, antigen retrieval with target retrieval solution (DAKO) was performed in a humid chamber. The sections were blocked and incubated with diluted primary antibody (Dako) at 4°C overnight. The primary antibodies used in this study were mouse anti-CD31 (Novus, AF3628; 1:200), rabbit anti-αSMA (Abcam, ab5617; 1:200), rabbit anti-laminin (Sigma, L9393; 1:200), mouse anti-CD68 (Abcam, ab31630; 1:200), rabbit anti-CD206 (Abcam, ab64693; 1:200) and rabbit anti-iNOS (Abcam, ab15323; 1:200). After washing three times with 1% Tween® 20 in PBS, the samples were incubated with secondary antibody for 90 min at room temperature in the dark. The secondary antibody used in this study was anti-goat Alexa Fluor 488 (Invitrogen; 1:400) and anti-mouse Alexa Fluor 647 (Invitrogen; 1:400). After washing three times with PBS, the sections were stained with DAPI solution (VectaShield) for nuclear staining and then mounted on slides. Quantification was performed for five random microscopic fields using a fluorescence microscope (Nikon) and was calculated using imageJ. Each image was used for statistical analysis.
Statistical analyses
All data are presented as the mean ± standard error of the mean (SEM). The statistical significance (P < 0.05) was determined by a two-tailed t test using the GraphPad Prism Software.