Cd105

Six months after the surgical procedures, the filling tissue of cerebral cortex lesions were cut into small pieces and fixed with glutaraldehyde (2.5%) at 4 ℃ overnight. They were then fixed with 1% osmium acid for 1.5 h. Dehydration with gradient ethanol and overnight polymerization in epoxy encapsulated medium (Sigma, St. Louis, MO, USA) was done. The tissues were settled for 10 h at 60℃, ultra-thin slices prepared by Ultramicrotome (Leica EM UC7, EMS, USA) and stained with uranium acetate and lead citrate (EMS, USA). Myelin sheath and newborn synapses were observed under a transmission electron microscope (TEM, 80 KV, Zeiss, Germany). The fate of hUCMSCs was also determined. The paraffin slices were dewaxed and the EDTA buffer solution used to repair the antigen. The slices were then treated with BSA, incubated for 30 min, and CD90 (mouse anti-human, 1:200, Bioss, Beijing, China) and CD105 (Rabbit anti-human, 1:200, Bioss, Beijing, China) mixtures added. Secondary antibodies and DAPI were successively used, the slices were sealed, CD90 and CD105 co-expressed cells were observed under a fluorescence microscope. In order to track the cell fate of the hUCMSCs in SC and CB group, we arranged immunohistochemistry of Doublecortin (DCX, Rabbit anti-human, 1:1000, Abcam, Cambridge, UK) and NeuN (Rabbit anti-human, 1:1000, Abcam, Cambridge, UK) staining.

Cells of the fourth generation in logarithm growth phase were digested and cell density was adjusted to approximately 1×10⁶/mL. The cell suspension was analyzed by flow cytometry to detect ADSC-specific antigens. Antibodies used in the experiment included anti-CD31, CD49, CD90, CD106 (BD biosciences, New Jersey, USA), CD34 (Santa Cruz, Texas, USA), CD45 (AbD Serotec, North Carolina, USA), CD73 and CD105 (Bioss, Beijing, China). The data was analyzed using CellQuest software (BD Biosciences, New Jersey, USA).

Passage 2 to 3 MSCs were lifted by means of trypsin, counted and put in sterile Falcon tubes at aliquots of 1 × 10⁶ cells/tube at least. The following antibody panel was used for characterization of porcine cells: CD14 (AbD Serotec), CD29 (BD Pharmingen), CD31 (BD Biosciences), CD34 (Abcam + Goat anti-rabbit IgG (PE-cy5.5), Life Technologies), CD44 (Biolegend), CD45 (Genway Biotech), CD73 (Biolegend), CD90 (BD Biosciences), CD105 (Bioss Antibodies), CD146 (Genetex). For human cells, following antibodies were used CD45, CD90, CD105, CD73, CD235a, CD34 (BD Biosciences), CD31 (BioLegend), CD33, CD14 (Beckman Coulter), and CD146 (Miltenyi Biotec) (antibodies summarized in Tables 2, 3). The cells were stained with the antibodies singularly and according to manufacturer's protocol and were assessed using a BD LSRII flow cytometer, analyzing at least 20,000 events (Becton Dickinson, Franklin Lakes, NJ, USA). The obtained data was analyzed with FlowJo software (TreeStar Inc., Ashland, OR, USA).