Cd105 pe

Expanded WJ-MSC (n = 3) supplemented with 10% hPL (n = 3, batch 50, 53, 54), hS (n = 3, batch 21, 54, 57) and FBS (n = 2, batch 1, 2) (Gibco, Life Technologies, Carlsbad, CA, USA) were characterized immunophenotypically using antibodies against the following human antigens—CD90-APC, CD73-PE/Cy7, CD105-PE, CD274-PE, CD45-APC/Cy7, CD34-PerCP-Cy5.5, CD31-PE and HLA-DR-Pacific Blue (Biolegend, San Diego, USA). Appropriate isotype controls for each of the antibodies were used. Cells cultures a 70–80% confluence were harvested using 0.25% trypsin-EDTA, (Gibco, Life Technologies, Carlsbad, CA, USA) and centrifuged at 1200 rpm × 6 min. Cell pellets were resuspended in 100 µL of 1× PBX with 5% Bovine Serum Albumin (Gibco, Life Technologies, Carlsbad, CA, USA) containing the corresponding antibody. Cell suspension was incubated for 30 min at 4 °C, washed with 1X PBS and resuspended in 200 µL 1× PBS. Flow cytometry analysis were carried out with a FACSCanto II™ instrument (BD, Franklin Lakes, NJ, USA) and data were analyzed with the FlowJo vX.7.0 software package (TreeStar, USA).

For staining of cells from bone marrow and blood, cells were first harvested and incubated with red blood cell lysis buffer (eBioscience, 00-4333) to lyse red blood cells, and passed through a 40-μm cell strainer to obtain single-cell suspensions. Cells were then stained with DAPI (Molecular Probe, D1306) to exclude dead cells from the analysis where indicated, and further analyzed for tdTomato expression. For staining of vascular c-Kit⁺ SPCs, cells were first stained with indicated conjugated-antibodies (1:50) for 30 min on ice. Conjugated-antibodies used in this study include c-Kit-PE (Biolegend, 105807), Sca-1-PE (BD biosciences, 553108), CD31-PE/Cy7 (Biolegend, 102523), CD34-AF647 (Biolegend, 103124), CD29-FITC (Abcam, ab21845), CD45-PerCP/Cy5.5 (Biolegend, 103131), CD105-PE (Biolegend, 120407), CD140a-PE (eBioscience, 12-1401-81), and α-SMA-AF488 (Abcam, ab184675). Corresponding control isotype antibodies were used as control. For analyses of glucose uptake, 2-NBDG (100 μM, Invitrogen, N13195) was added to cultured cells and incubated for 1 h at 37 °C. Cells were then washed with PBS, detached by trypsin, and suspended in PBS for further analysis. All prepared samples were analyzed by a BD LSR Fortessa II flow cytometer (Becton Dickinson) or BD ACCURI C6 flow cytometer (BD Biosciences). FlowJo v10 software (Tree Star) was used to analyze the flow cytometric data.

Similar to the aforementioned fibroblast extraction process, after removing vascular tissues, adipose tissues were minced and digested with 4 mg/mL type I collagenase for 20 min. Subsequently, the mixture was allowed to sit for 3–5 min before removing the supernatant. The supernatant was centrifuged to obtain primary ADSC, and proliferation was continued in the culture with Medium Essential Medium alpha (MEM-α; BI) containing 10% FBS. To identify surface markers of ADSC, the fluorescence-conjugated antibodies CD105-PE, CD73-PE, CD90-PE, CD34-PE, and CD45-PE (BioLegend, San Diego, CA, USA) were incubated with three sub-passages of ADSC several times and analyzed using flow cytometer (BD Pharmingen, San Diego, CA, USA). To identify the differentiation potential, ADSCs were cultured for several days, according to the instructions of the adipogenic (Cyagen, Santa Clara, CA, USA) and osteogenic (Cyagen) induction kits. We then visualized the differentiation results after Oil Red O (Solarbio) or Alizarin Red S (Solarbio) ing according to corresponding instructions under a microscope. We then used an alkaline phosphatase (ALP) kit (BI) to detect the stem cell properties of ADSCs. Finally, a light microscopic (Ningbo ShunYu Instruments Co., Ltd., Ningbo, China) analysis was performed.

Isolated cells were fixed with 2% formalin and stained with CD34-APC (Biolegend, 343607), CD90-PE (Biolegend, 328109), CD105-PE (Biolegend, 323205), CD146-APC (Biolegend, 361015), IgG2a-APC (Biolegend, 400221), or IgG1-PE (Biolegend, 400112) for 10  min at 4°C. Following the staining, the cells were washed with 1X PBS containing 0.5% BSA and 0.1% sodium azide and analysed by flow cytometry (FACS Canto II, BD Biosciences).

For phenotypic analysis BM-MSCs, UCT-MSCs and AT-MSCs were harvested and stained for 15 minutes at room temperature with the following monoclonal antibodies (mAbs): anti-human CD90 APC, CD73 PE, CD34 PE, CD200 PE, CD273 APC, CD274 PE, CD71 FITC, CD44 PE (BD Pharmingen, Heidelberg, Germany), anti-human HLA-DR PE, HLA-A,B,C FITC, CD144 (VE-cadherin) APC, CD31 FITC, CD105 PE (Biolegend, San Diego, CA, USA), CD45 FITC (Tonbo, Biosciences, San Diego, CA, USA) or with the appropriate fluorochrome-conjugated isotype-matched mAbs to establish background fluorescence. After incubation cells were washed with PBS, centrifuged at 1400 RPM for 5 minutes and suspended in PBS for flow-cytometry analysis. Samples were acquired using a FACSCalibur (Becton Dickinson, San Josè, CA, USA) and the data were analysed with CellQuest software (Becton Dickinson).

Surface markers of MSCs were assayed by flow cytometry analysis (Attune™ NxT Acoustic Focusing Cytometer, A24863, Thermo Fisher Scientific), and the same voltage parameters for FSC and SSC were used in all the assays; FSC was 90, and SSC was 255. Fluorescently labelled antibodies were CD147‐FITC (Biolegend, Cat# 306204), CD90‐PerCP/Cyanine5.5 (Biolegend, Cat# 328117), CD44‐PE/CY7 (Biolegend, Cat# 103029), CD34‐FITC (BD Biosciences, Cat# 560942), CD73‐APC (Biolegend, Cat# 344005), CD105‐PE (Biolegend, Cat# 800503), CD14‐FITC (Biolegend, Cat# 301803), CD45‐APC (BD Biosciences, Cat# 555485), CD29‐PE (Biolegend, Cat# 303003), PCNA‐PE (Biolegend, Cat# 307908) and Ki‐67‐APC (Biolegend, Cat# 350513).