*4.1. PDMSCs Conditioned Media Preparation*

PDMSCs-CM was prepared as previously described [19,31]. Briefly, placentae from healthy women with a singleton physiological pregnancy were collected immediately after delivery. Physiological pregnancy was defined as term normotensive pregnancy and no signs of preeclampsia or FGR. Exclusion criteria were congenital malformations, chromosomal abnormalities (in number and/or structure), maternal and/or intrauterine infections, cardiovascular diseases, metabolic syndrome, diabetes, and immunological disorders.

PDMSCs were isolated by enzymatic digestion and gradient as previously described [19,28,31]. PDMSCs were next resuspended in Dulbecco's Modified Minimum Essential Medium (DMEM, Gibco, Life Technologies, Monza, Italy) supplemented with 10% fetal bovine serum (FBS Australian origin, Life Technologies, Monza, Italy) and

maintained at 37 ◦C and 5% CO2. At every passage, physiological PDMSCs were characterized by flow cytometry for the expression of the following antigens: HLA-I, HLA-DR, CD105, C166, CD90, CD34, CD73, CD133, CD20, CD326, CD31, CD45, and CD14 (Miltenyi Biotech, Bologna, Italy). PDMSCs were analyzed by semiquantitative PCR to assess gene expression levels of stem cell markers Oct-4 and Nanog. Primers were designed as previously described [19].

At passage three of culture, after obtaining a pure PDMSCs population, cells were plated and expanded in 1264 cm<sup>2</sup> EasyFill cell factories (Carlo Erba, Cornaredo (MI), Italy) at a concentration of 3 × 106 cells. When cells reached confluency, media was removed and replaced by 400 mL of DMEM LG without FBS. After 48 h of culture, CM was collected, filtered, and stored at −20 ◦C until use.
