**1. Introduction**

The preeclamptic syndrome (PE), exclusive to human pregnancy, represents the main cause of fetal–maternal mortality and morbidity worldwide [1,2]. PE generally resolves at delivery with placenta removal, but it causes severe long-term complications for both the mother and the fetus, such as cardiovascular and neurological disorders, diabetes, and metabolic syndrome [3]. Despite almost three decades of intensive investigation, PE still remains an unsolved medical need.

Indeed, preeclampsia has a major social–economic impact due to the lack of effective therapies, except for a timely and often premature delivery. There were several unsuccessful attempts to find a resolutive cure for PE, ranging from new drug candidates to drug relocation. The main problem with preeclampsia is that, as a syndrome, it is multifactorial, a destructive mix of inflammation, endothelial damage, and immunological impairment [2,4]. Key features of PE are a maternal immune maladaptation towards the fetoplacental district with a shift towards Th1 immunity [5–7], increased placental release of proinflammatory cytokines (e.g., Tumor Necrosis Factor-α—TNF-α; Interleukin-6–IL-6), and anti-angiogenic factors (e.g., soluble FMS-like tyrosine kinase-1—sFlt-1) that promote aberrant placental angiogenesis and generalized endothelial cell activation and damage [8–11]. Therefore,

**Citation:** Nuzzo, A.M.; Moretti, L.; Mele, P.; Todros, T.; Eva, C.; Rolfo, A. Effect of Placenta-Derived Mesenchymal Stromal Cells Conditioned Media on an LPS-Induced Mouse Model of Preeclampsia. *Int. J. Mol. Sci.* **2022**, *23*, 1674. https://doi.org/10.3390/ ijms23031674

Academic Editor: Luisa Campagnolo

Received: 27 December 2021 Accepted: 28 January 2022 Published: 31 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

an ideal PE therapeutic approach must be able to contemporarily target all preeclamptic culprits and not to just mitigate a single clinical symptom as hypertension or inflammation.

The human placenta has been identified as a source of mesenchymal stromal cells (placenta-derived mesenchymal stromal cells—PDMSCs). PDMSCs could be isolated from the chorionic villi, the amnion, and the decidua and possess an increased self-renewal potential. Moreover, PDMSCs express stem cell markers (e.g., OCT-4, NANOG) and could differentiate into condrogenic, adipogenic, and osteogenic lineages [12–14]. Importantly, PDMSCs are characterized by unique immunologic and immune-regulatory properties, thus exerting a powerful immunosuppressive effect on T-cells [15–18]. Placental MSCs have been shown to promote angiogenic growth and to possess anti-inflammatory, anti-fibrotic, and cytoprotective abilities mediated by both direct cell-to-cell contact and/or specific trophic mediators more than cell differentiation [15,19].

Thus, PDMSCs may be an attractive therapeutic candidate for PE treatment. Recently, decidual MSCs were injected in vivo in a Th1 cell-induced PE-like mouse model demonstrating the ability to ameliorate PE-like symptoms as blood pressure and proteinuria [20]. In line with these results, an endotoxin-induced PE rat model infused with umbilical cord blood-derived MSCs showed decreased blood pressure, proteinuria, and inflammation relative to untreated controls [21]. Finally, commercially available placental mesenchymal cells were administered to hypertensive TLRs-induced pregnant mice, decreasing blood pressure, placental injury, and inflammation [22]. MSC-based therapy definitely sounds an intriguing potential multitarget therapeutic tool for preeclampsia.

Nevertheless, it could be hazardous to hypothesize a cell therapy for such a sensitive and delicate environment as human pregnancy for both ethical and biosafety reasons. No long-term studies on MSCs oncogenic potentials are available, and data about MSCs ability to invade maternal organs are contrasting [21,23–27].

Since mesenchymal stromal cells exerts their beneficial effects mainly through the release of trophic mediators, in the present study we tested the hypothesis that PDM-SCs' conditioned media (CM) could be used as an effective, ethical, and safe therapeutic approach for preeclampsia. Therefore, we evaluated the effects of PDMSCs-CM administration on maternal blood pressure, proteinuria, fetal outcome, and placental expression of sFlt-1, TNF-α, and IL-6 in an LPS-induced mouse model of preeclampsia.

## **2. Results**

### *2.1. PDMSCs Presented Proper Mesenchymal Stromal Cell Profile*

PDMSCs used for CM preparation presented proper mesenchymal stromal phenotype as assessed by flow cytometry. As previously published, cells were positive for CD105, CD166, CD90, and CD73, and negative for HLA-II, CD34 and CD45 (hematopoietic markers), and CD133 and CD31 (endothelial progenitor markers). PDMSCs were also negative for B cells, neutrophils, and macrophages markers CD20 and CD14 and for trophoblast and epithelial marker CD326, thus excluding any type of contamination [19,28]. RT-PCR detected the expression of typical stemness markers Oct-4 and Nanog in all PDMSCs cell lines [19,28].
