A Sample of the Culture-Expanded Ascs Described Above Were Frozen and Preserved in Liquid

Flow Cytometry

Methods

A sample of the culture-expanded ASCs described above were frozen and preserved in liquid nitrogen, thawed, and expanded for one additional passage (total of 5 passages) prior to flow cytometric analysis. Cells from 7 of the 10 pigs were harvested by either trypsinization (0.05%) or EDTA (5mM) treatment, washed, and incubated with appropriate antibodies. Antibodies were selected based upon reported affinities to porcine antigens: anti-CD29 (0.5mg/ml, Becton Dickinson Biosciences (BD) #552369), anti-CD31 (0.2mg/ml, BD #555027), anti-CD34 (0.2mg/ml, R&D Systems #AF3890), anti-CD45RA (0.5mg/ml, BD #522546), anti-CD90 (0.5mg/ml, BD #555595), and anti-CD105 (20ml, Abcam #53318). Appropriate secondary antibodies and controls were employed: BD #550617, #554001, #555748, #557273, and Santa Cruz #2348. After antibody incubation, the cells were washed and resuspended in a propidium iodide solution (BD #556463) to identify dead cells, which comprised less than 5% of the total cell population. Samples were analyzed using a FACSCaliburTM flow cytometer and CellQuest software (BD Biosciences). Ten to twenty thousand events were acquired per antibody set. Positive staining cells were identified as events with fluorescence intensity greater than the 99th percentile of the corresponding control sample.

Results

Flow cytometric analysis was performed to determine whether ASC-associated stromal (CD29, CD90, CD105) and mesenchymal stem cell (CD34) markers were expressed within the collected ASCs. Representative histograms for the different CD antigens examined are shown in Supplemental Figure 1a. The average percent of cells expressing a particular antigen among ASCs from different pigs is shown in Supplemental Figure 1b. The cells did not express CD45, a hematopoietic cell marker, however nearly all expressed CD29, CD34 and CD90. CD105 (Endoglin) was scored as positive in only 54.1 ± 16.0% of the cells. The small shift of the entire population of CD105+ cells from controls (S.F. 1a, lower right) likely indicates a higher percentage of cells actually express CD105, but do so only at low levels. A vascular endothelial marker, CD31 was present at low levels in 5 of the 8 samples examined (17.9 ± 8.7%), and nearly absent in the remaining 3 samples (1.9 ± 0.9%), resulting in significant overall variability (11.9 ± 7.7% CD31+). No correlation could be found between CD31-positivity in these cells and either culture doubling times or bone formation in vivo (not shown).