## 3 Results
### hAMSC Marker Expression is Modified by Age and Pathology  
At isolation and during passaging, hAMSCs were characterized by a spindle-shape, elongated morphology and adherence to plastic, and could be kept in culture until passage 5 without any change in morphology. Figure S1 shows that the morphology and size of hAMSCs belonging to the three Groups at p0 and p2 were very similar.  
An imaging-based flow cytometry apparatus was used to gate cells in best focus, select single cells, and determine the fluorescence intensity on the selected population of cell markers identifying mesenchymal stem cells (CD29, CD73, and CD105). Figure 2 shows representative panels of the analysis carried out on the Group 1 at p2. In Panel C, ten cells for each condition are shown as different channels: brightfield, FITC fluorescence, and side scatter. The same analysis was done with hAMSCs obtained from Groups 2 and 3 (not shown).  
Figure 2: Strategy of flow cytometric analysis for hAMSC phenotypic markers. Cells in best focus, i.e., those with higher Gradient root mean square (RMS) values, were identified (**A**) to draw brightfield scatter plots, (**B**) obtained by plotting Area vs. Aspect Ratio, to identify single cells events (identified by a square region). In (**C**), ten cells for condition are shown, five on the first four columns and five more on the last four columns, as different channels: Ch01 and Ch09 (brightfield); Ch02 (FITC fluorescence); Ch06 (side scatter). The intensity of Ch02 vs. normalized frequency (**D**) was used to calculate the statistics Count and Percent Gated on 10,000 single-cell events for sample. Ctrl = cells incubated in the absence of antibody. The analysis for p2 hAMSCs of Group 1 is depicted. FITC: fluorescein isothiocyanate.  
Using this strategy, we obtained that CD29 was expressed at lower levels of Group 3 in comparison with cells obtained from Group 1 (Figure 3A). No significant changes were found for CD105 (Figure 3B). On the other hand, CD73 expression was significantly decreased in Groups 2 and 3 both compared with Group 1 (Figure 3C).  
The same cytofluorimetric strategy was used for hematopoietic markers (not shown). From the isolation to the p2, the expression of CD14, CD34, and CD45 was negligible for all the hAMSCs from the three Groups, as expected for mesenchymal stem cells (Table 2). These results reproduce well those we have found in an earlier work on hAMSCs obtained from another cohort of women (n = 3), in which we showed at p0 and p2 negligible to null expression of hematopoietic markers, high expression of CD29 (p0 range: 66-86%; p2 range: 95-99%) and CD73 (p0 range: 88-91%; p2 range: 88-99%), and lower expression of CD105 (p0 range: 30-66%; p2 range: 40-76%) [25].  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Study Group** & **CD14** & **CD34** & **CD45** \\ \hline Group 1 (\textless{}40) & & & \\ p0 & \(3.4\pm 1.2\) & \(3.4\pm 1.9\) & \(3.9\pm 2.5\) \\ p2 & \(2.9\pm 1.3\) & \(3.6\pm 1.7\) & \(3.8\pm 2.0\) \\ \hline Group 2 (\textless{}40) & & & \\ p0 & \(0.8\pm 0.3\) & \(0.6\pm 0.7\) & \(0.9\pm 1.0\) \\ p2 & \(2.8\pm 1.6\) & \(0.9\pm 0.6\) & \(1.8\pm 1.8\) \\ \hline Group 3 (PE) & & & \\ p0 & \(0.8\pm 0.4\) & \(1.3\pm 0.2\) & \(2.1\pm 1.4\) \\ p2 & \(1.5\pm 1.2\) & \(1.0\pm 0.2\) & \(2.0\pm 1.5\) \\ \hline \hline \end{tabular} Values are shown as percentages of positive cells and expressed as mean \(\pm\) SD of triplicates for each isolate.  
\end{table}
Table 2: Hematopoietic markers of hAMSCs belonging to the three study groups.  
Figure 3: Mesenchymal marker expression in hAMSCs from all Groups. hAMSCs were evaluated for CD29 (**A**), CD105 (**B**), CD73 (**C**) markers at isolation (p0) and p2 by flow cytometry. Results are expressed as mean \(\pm\) SD of triplicates for each isolate (Group 1, n = 7; Group 2, n = 6; Group 3, n = 5). * \(P<0.05\); ** \(P<0.01\).