Cd34 pe

After taking the parents’ informed consent, fresh human umbilical cord samples were obtained during caesarean deliveries of healthy newborn infants for healthy mothers. Wharton jelly was retrieved at the moment of birth from term deliveries. Collagenase II enzyme (IgG, C. histoliticum, Biological Life Science, USA) was used to isolate UC-MSCs, which were then digested and maintained in 2% foetal bovine serum and 1x Pen/Strep (Invitrogen, CA, USA). Cells were then incubated at 37° C, 5% CO2 till cells will reach 70–80% confluence, rinsed with phosphate buffer saline (PBS), and trypsinized at 37° C with 0.25% trypsin-EDTA (Invitrogen) for 5 min. After ultracentrifugation, cell pellets precipitate were resuspended with medium and maintained as first-passage cultures [23 (link)]. Every tube contained 1 × 10⁵ cells were mixed with 10 µL of monoclonal antibodies against the surface markers: mesenchymal surface markers CD 29 PE, CD34 PE, and CD 90 PE (Beckman coulter, USA) in the dark at 4° C. The same species isotypes were used as a negative control. After incubation, 2 ml of PBS containing 2% fetal calf serum (FCS) solution were supplied, centrifuged and cells were recovered with fresh medium. Cell evaluation was done using CYTOMICS FC 500 Flow Cytometer (Beckman coulter, FL, USA), and CXP Software version 2.2 was used for analysis.

Phenotypic analysis of MSC was performed during monolayer expansion (prior to encapsulation in the hydrogel) and throughout scaffold culture. Briefly, to perform phenotypic analysis MSC were incubated with fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated mouse anti-human antibodies CD34-PE, CD45-FITC, HLA-DR-FITC, CD44-FITC, CD73-PE, CD90-FITC, CD105-PE and CD166-PE (Beckman Coulter, Brea, CA, USA) for 30 minutes at room temperature. Negative and isotype (FITC and PE) controls were performed. After immunofluorescence staining, for each sample 10,000 events were counted by Gallios flow cytometer (Beckman Coulter, Brea, CA, USA).

hBM-MSCs and hWJ-MSCs were detached and counted; 1 × 10⁵ cells were incubated at RT for 20 min with the following directly conjugated mouse-anti human antibodies: CD90 FITC (Beckman Coulter, Fullerton, CA, USA), CD73 APC (Miltenyi Biotec, Gladbach, Germany), CD105 PE (Beckman Coulter, Fullerton, CA, USA), CD45 PC7 (Beckman Coulter, Fullerton, CA, USA), HLA class-II FITC (Beckman Coulter, Fullerton, CA, USA), CD14 PC7 (Beckman Coulter, Fullerton, CA, USA), and CD34 PE (Beckman Coulter, Fullerton, CA, USA). The isotype-matched immunoglobulins IgG1 FITC (Beckman Coulter, Fullerton, CA, USA), IgG1 PE (Beckman Coulter, Fullerton, CA, USA), IgG1 APC (Beckman Coulter, Fullerton, CA, USA), and IgG1 PC7 (Beckman Coulter, Fullerton, CA, USA) were used as negative controls under the same conditions. At least 15,000 total events were acquired with a BD FACSVerse flow cytometer (Becton Dickinson, BD, NJ, USA). Further analysis and plots were generated using the BD FACSuite analysis software. Statistics are summarized in Table S1 (Supplementary Materials).