*4.2. Preeclamptic Mouse Model Preparation and PDMSCs-CM Treatment*

The preeclamptic mouse model was prepared following a modified protocol from Wang et al. [57]. Briefly, C57BL/6NCrl virgin mice females (n = 30) and males (n = 10) at 4 weeks of age were purchased from Charles River Laboratories (Calco (LC), Italy). All mice were maintained on a 12 h/12 h dark and light cycle with relative humidity of 50–70% at 18–22 ◦C. Tap water and standard laboratory pelleted formula were provided.

Female mice were mated with males at 9 weeks of age and plug discovery was considered as day 0 of pregnancy. Female mice lacking copulation plugs (n = 20) were returned to the breeding colony. At day 11 of pregnancy, all pregnant females (n = 10) were removed from breeding cages and received intravenous tail injection of 1 µg/kg LPS solution in order to induce inflammation-mediated endothelial damage and hypertension. At day 12, after blood pressure measurements, mice were randomized into two groups as follows: (1) animals that received a single intravenous tail injection of 300 µL of plain unconditioned media (control group, n = 5); (2) animals that received a single intravenous tail injection of 300 µL PDMSCs-CM (CM group, n = 5) (Figure 1A). LPS was purchased from Sigma Aldrich (Milan, Italy). PDMSCs-CM was prepared as described above.

Maternal systolic blood pressure (SBP) was monitored by tail cuff plethysmography by using BP-2000 Series II Blood Pressure Analysis System, 2 channels mouse platform (Visitech Systems, Napa Pl, Apex, United States) from day 9 to 18 of pregnancy. Urine samples were collected at days 12, 13, and 17 of pregnancy and analyzed for protein content by Bradford assay (Sigma Aldrich, Milan, Italy). Mice were sacrificed by cervical dislocation at day 19 of pregnancy and uteri were removed. Maternal blood samples were taken from mice carotid and collected in heparin tubes to determine the following hematological parameters: RBC count, WBC count, Plt count, Htc, and Hb. All analyses were performed using a veterinary hematology analyzer. Serum was separated and used for measuring ALT, AST, urea, and creatinine. Placental weights, number of fetuses, and fetal weights were recorded. Placentae were collected and stored at −80 ◦C until the next molecular analysis.
