Differentiation of WT and ATF6-deficient hMSCs from hESCs was carried out as previously described27 (link), 29 (link), 32 (link), 34 (link). Briefly, embryoid bodies were plated on Matrigel in differentiation medium (αMEM (Invitrogen) medium supplemented with 10% FBS (Gemcell), 5 ng/ml TGFβ (Human Zyme), 10 ng/ml bFGF (JPC), and 1% penicillin/streptomycin (Gibco)). About 10 days later, differentiated cells were passaged once. To further purify hMSCs, the differentiated cells were then subjected to fluorescence activated cell sorting (FACS) by evaluating the hMSC-specific surface markers (CD73, CD90, and CD105). The antibodies of hMSC-specific markers used in FACS were as follows: anti-CD90-FITC (BD Bioscience, 555595), anti-CD73-PE (BD Bioscience, 550257), and anti-CD105-APC (BD Bioscience, 17-1057-42). Anti-IgG-FITC (BD Biosciences, 555748), anti-IgG-PE (BD Biosciences, 555749), and anti-IgG-APC (BD Biosciences, 555751) antibodies were also used as isotype controls. The differentiation potentials of hMSC towards osteoblasts, chondrocytes and adipocytes were verified by histochemical staining with von Kossa (osteogenesis), Alcian blue (chondrogenesis), and Oil red O (adiopogenesis) Kit (IHC World), respectively.
Anti cd73 pe
Manufactured by BD
Sourced in United States
Anti-CD73-PE is a fluorescently-labeled monoclonal antibody that binds to the CD73 cell surface protein. CD73 is an ectonucleotidase that catalyzes the conversion of extracellular adenosine monophosphate (AMP) to adenosine. The PE (phycoerythrin) fluorescent label allows for the detection and analysis of CD73-expressing cells using flow cytometry.
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Lab products found in correlation
Anti cd90 fitc, by BD (10 mentions)
Anti cd105 pe, by BD (8 mentions)
Anti cd45 pe, by BD (7 mentions)
Anti cd34 pe, by BD (7 mentions)
Anti cd34 fitc, by BD (7 mentions)
Anti cd45 fitc, by BD (6 mentions)
Anti cd105 apc, by BD (5 mentions)
Anti cd44 fitc, by BD (5 mentions)
Anti cd90 pe, by BD (4 mentions)
Anti cd31 pe, by BD (4 mentions)
Anti cd146 pe, by BD (3 mentions)
Anti cd45 apc, by BD (3 mentions)
Anti hla dr fitc, by BD (3 mentions)
Anti cd90 apc, by BD (3 mentions)
Anti hla dr pe, by BD (3 mentions)
Cellquest software, by BD (3 mentions)
Anti cd105 fitc, by BD (3 mentions)
Facscalibur, by BD (3 mentions)
Anti cd31 fitc, by BD (3 mentions)
Anti igg apc, by BD (2 mentions)
36 protocols using anti cd73 pe
1
Differentiation of hMSCs from hESCs
2
Stem Cell Marker Expression Analysis
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The primary antibodies used were as follows (company, catalogue number): anti-ERCC6 (Abcam, ab96098), anti-NANOG (Abcam, ab21624), anti-SOX2 (Santa Cruz, sc-17320), anti-OCT4 (Santa Cruz, sc-5279), anti-SMA (Sigma, A5228), anti-TUJ1 (Sigma, T2200), anti-FOXA2 (Cell Signaling Technology, 8186S), anti-CD90-FITC (BD Bioscience, 555595), anti-CD73-PE (BD Bioscience, 550257), anti-CD105-APC (BD Bioscience, 17-1057-42), anti-IgG-FITC (BD Biosciences, 555748), anti-IgG-PE (BD Biosciences, 555749), anti-IgG-APC (BD Biosciences, 555751), anti-Lamin B (Santa Cruz, sc-6217), anti-LAP2 (BD Bioscience, 611000), anti-Ki67 (ZSGB-BIO, ZM0166), anti-P16 (BD Bioscience, 550834), anti-γ-H2AX (Millipore, 05-636), anti-Nestin (Millipore, MAB5326), anti-PAX6 (Covance, PRB-278P), anti-CPD (Cosmo Bio, TMD-2), anti-cleaved PARP (Cell Signaling Technology, 9541), anti-β-Actin (Santa Cruz, sc69879), anti-GAPDH (Santa Cruz, sc-25778), and anti-hCD31 (BD Bioscience, 555445).
3
Flow Cytometry Analysis of Immune Cells
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The following anti-human monoclonal antibodies (mAbs) were used for flow cytometry: anti-CD3-ECD, -CD4-ECD, -CD4-PC5, -CD25-PE, -GITR-FITC, -FOXP3-FITC, -CD39-FITC, -CD39-PE, -CD73-PE, -CD122-FITC, -CD132-FITC, -TGFβ-PE, -IL-10-PE, and -CTLA4-PE. All mAbs and their respective isotypes, which served as negative controls for surface and intracellular staining, were purchased from Beckman Coulter, except for antibodies to FOXP3, and CD39, which were purchased from eBioscience. Anti-CD73-PE and anti-IL-10 were purchased from BD Pharmingen and antibodies to GITR, TGF-β, CD132, and CTLA4 from R&D Systems. Before use, all antibodies were titrated using resting as well as activated PBMC.
4
Immunophenotyping of Aged ASCs and DFAT Cells
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For identification of immunophenotypes, aging ASCs and DFAT cells were harvested at P4. Cells were trypsinized, and viability was assessed using 0.4% Trypan Blue (Gibco) and found to be greater than 95%. Then, cells were separated into tubes containing 5 × 105 cells each. The following mouse monoclonal anti-human antibodies conjugated with fluorescein isothiocyanate-conjugated (FITC) and phycoerythrin (PE) were used: anti-CD29-PE, anti-CD31-PE, anti-CD73-PE, anti-CD90-PE, anti-CD105-PE, anti-CD106-PE, anti-CD146-PE (all from BD Biosciences, CA, USA): anti-CD34-FITC, and anti-CD44-FITC (Beckman coulter Inc.); and immunoglobulin G1 isotype control (BD Biosciences). Each aliquot was incubated in the dark at 4 °C for 20 min. Then, cell pellets were washed with PBS and resuspended in 0.1% bovine serum albumin (BSA)/PBS. Flow cytometric data were analyzed with Guava Express Plus version 5.3 software (Guava Technology).
5
Characterization of dNK Cell Phenotypes
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The dNK cells were cultured in 6-well plates (5 × 105 cells/well) for 24 hours. The mononuclear cells were resuspended in staining buffer, and then immediately stained with a range of monoclonal antibodies, namely anti-CD56 TULY56 FITC (Catalog#: 11-0566-42, eBioscience, USA), anti-CD16 PE (Catalog#: 12-0168-42, eBioscience, USA), anti-CD39 APC (Catalog#: 17-0399-41, eBioscience, USA), anti-CD107 APC H4A3 (Catalog#: 560664, BD Pharmingen, USA), anti-CD314 PE (Catalog#: 557940, BD Pharmingen, USA), anti-CD336 PE (Catalog#: 558563, BD Pharmingen, USA), anti-CD337 PE (Catalog#: 558407, BD Pharmingen, USA), anti-CD73 PE (Catalog#: 550257, BD Pharmingen, USA), anti-Annexin V FITC (Catalog#: 556419, BD Pharmingen, USA), anti-DAPI (Catalog#: 564907, BD Pharmingen, USA) and anti-CD45 APCCY7 (Catalog#: 348805, BD Pharmingen, USA). After incubation at room temperature for 30 min, the cells were then washed and resuspended in PBS for flow cytometry analysis (CytoFLEX, eBioscience, USA). The strategy for multidimensional flow cytometry analysis is shown in the Supplementary Material, Figure S1
6
Isolation and Characterization of Adipose-Derived Stem Cells
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According to methods previously reported,25 , 26 ADSCs were isolated from human adipose tissue obtained from patients who were undergoing liposuction at the Department of Plastic Surgery, the First Hospital of China Medical University. Adherent cells were cultured in a growth medium [DME F12 (HyClone, USA), 10% FBS, 1% Penicillin‐Streptomycin Solution (Gibco, USA)] at 37°C/5% CO2 and saturated humidity. ADSCs were passaged after reaching 90% confluence.
Multi‐lineage potential assay and flow cytometry analysis were performed to identify characteristics of ADSCs, as we have done in the past.4 The antibodies including anti‐CD34‐FITC, anti‐CD45‐PE, anti‐CD44‐FITC, anti‐CD73‐PE and anti‐CD105‐PE were purchased from BD biosciences (USA).
Multi‐lineage potential assay and flow cytometry analysis were performed to identify characteristics of ADSCs, as we have done in the past.
7
Immunophenotyping of Myogenic Cells
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The myogenic nature of purified cells was evaluated by flow cytometry analysis performed on CytoFLEX (Beckman Coulter) as previously described (Awaya et al., 2012 (link); Lapan and Gussoni, 2012 (link)); the following panel of antibodies was used to determine immunophenotype: anti-CD56 PC7 (Beckman Coulter, USA, A21692), anti-CD146 PE (Beckman Coulter, USA, A07483), anti-CD166 PE (Beckman Coulter, USA, A22361), anti-CD73 PE (BD Pharmingen, USA, 550257), anti-CD105 APC (R&D Systems, USA, FAB1097A-100), and anti-CD45 PC5 (Beckman Coulter, USA, A07785). Data were analyzed using the CytExpert 2.0 (Beckman Coulter). The phenotypic characteristics of the obtained cells are illustrated in the Supplementary Material, Figure S2A
8
Immunophenotyping of Mesenchymal Cell Types
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The immunophenotypes of the BM-DFATs, SC-DFATs, BM-MSCs, ASCs at passage 2 were identified using flow cytometry as previously described [13 (link)]. The cells grown to 60% confluence were suspended at a density of 5 × 105 cells per tube and incubated with various anti-human antibodies conjugated with phycoerythrin (PE) or allophycocyanin (APC). The following antibodies were used: anti-CD73-PE, anti-CD90-APC, anti-CD105-PE, anti-CD31-PE, anti-CD45-APC, anti-HLA-DR-PE, anti-CD106-PE, anti-CD54-APC, and anti-CD36-PE (all from BD Biosciences, San Jose, CA). Mouse IgG1-PE, mouse IgG1-APC, mouse IgG2a-PE, mouse IgG2b-APC, and mouse IgM-PE (all from BD Biosciences) were used as negative controls. The fluorescence intensity of the cells was evaluated by a FACSAria flow cytometer (Becton Dickinson, Bedford, NJ), and data were analyzed using FlowJo software (version 10.6.1, FlowJo, Ashland, OR). Positive cells were counted and compared with the signal of corresponding immunoglobulin isotypes. A minimum of 1 × 104 events were recorded for each sample, and analysis was performed at least three separate times for each condition tested.
9
Characterization of Porcine cATMSC-Derived Extracellular Vesicles
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Porcine cATMSC-EV were identified by bead-based flow cytometry, screening for EV and MSC markers. EV were covalently coupled to 4-μm aldehyde/sulphate-latex beads (Invitrogen-ThermoFisher Scientific) with a 15 min incubation, and then blocked for 2 h with BCB buffer (PBS, 0.1% BSA, and 0.01% sodium azide (NaN3); Sigma Aldrich). EV-coupled beads were centrifuged at 2000×g for 10 min and re-suspended in BCB buffer. Next, beads were incubated for 30 min at RT with the fluorochrome-conjugated antibodies anti-CD73-PE and anti-CD90-PE-Cy7 (1:50; both from BD); or the primary Ab anti-CD9 (Clone VJ1/20; 1:10), anti-CD63 (Clone TEA3/18; 1:10), anti-CD81 (Clone 5A6; 1:10), anti-CD29 (1:10; BD), anti-CD44 (1:10; AbD Serotec) or IgG isotype control (1:10; Abcam) followed by incubation with the FITC-goat F(ab')2 anti-mouse IgG (1:10; Bionova) or A488-rabbit anti-rat IgG (1:100; AbD Serotec). EV-coupled beads were washed with BCB buffer and spun down at 2000×g for 10 min after each step. Data was acquired in a FACSVerse flow cytometer (BD), and analysed with FlowJo® v10 (BD).
10
hESC-derived Mesenchymal Stem Cell Protocol
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The hMSCs were generated from hESCs. Briefly, embryoid bodies derived from hESCs were seeded on 6-well plates coated with Matrigel (BD Biosciences, 354230), the cells were cultured for about 14 days in hMSCs differentiation medium, which was performed as previously reported (Liang et al., 2021; (link)Chu et al., 2022 (link)). The differentiation medium was changed every other day until the fibroblast-like cells appeared and reached confluent. Subsequently, the cells were then transferred into hMSCs culture medium for a continuous culture. Finally, cell sorting was conducted utilizing the fluorescence activating cell sorter (FACS) system (BD FACS Influx). CD73, CD90, and CD105 triple-positive cells were collected and further characterized by surface antigen markers, including positive marker CD44, and negative markers CD34 and CD45. The following antibodies were used for FACS: anti-CD73-PE (BD Biosciences, 550257), anti-CD90-FITC (BD Biosciences, 555595), and anti-CD105-APC (BD Biosciences, 17-1057-42), anti-CD44-FITC (BD Biosciences, 550989), anti-CD34-FITC (BD Biosciences, 555821), and anti-CD45-FITC (BD Biosciences, 555482).
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