hAMSCs Cultured as Spheroids Increased Both the Expression of Pluripotent Markers and the Mesenchymal Stromal Cell Differentiation Potential In order to examine whether spheroid culture maintained or increased the multipotency and the differentiation capacity of hAMSCs, cell spheroids were dissociated in a single-cell suspension, plated onto culture flasks, grown as a monolayer, and used for further analysis

hAMSCs Cultured as Spheroids Increased Both the Expression of Pluripotent Markers and the Mesenchymal Stromal Cell Differentiation Potential In order to examine whether spheroid culture maintained or increased the multipotency and the differentiation capacity of hAMSCs, cell spheroids were dissociated in a single-cell suspension, plated onto culture flasks, grown as a monolayer, and used for further analysis. potential of both 3D culture-derived conditioned medium (3D CM) and their exosomes (EXO) was assessed against 2D culture products. In particular, tubulogenesis assays revealed increased capillary maturation in the presence of 3D CM compared with both 2D CM and 2D EXO. Furthermore, 3D CM had a greater effect on inhibition of PBMC proliferation than both 2D CM and 2D EXO. To support this data, hAMSC spheroids kept in our 3D culture system remained viable and multipotent and secreted considerable amounts of both angiogenic and immunosuppressive factors, which were detected at lower levels in 2D cultures. This work reveals the placenta as an important source of MSCs that can be used for eventual clinical applications as cell-free therapies. 1. Introduction Adult stem cells are extensively used for regenerative medicine because of their multilineage potential and regenerative properties. These cells exist in different tissues, including fat [1], bone marrow [2], the umbilical cord [3], and placenta tissue [4], where they participate in the maintenance of stem cell niches and tissue homoeostasis [5]. Though the pathophysiologic functions of mesenchymal stem cells (MSCs) are under investigation, the multipotency of these cells suggests a role in tissue regeneration, wound healing, and/or tissue repair after transplantation [6]. Indeed, MSCs are capable of self-renewal and differentiation into several mesenchymal lineages both and [10]. Despite the availability of various cell sources for the use of MSCs in the field of regenerative medicine, the ethical issues regarding the source have become an important clinical concern. Indeed, most of the data on this topic have been thus far generated using bone marrow-derived MSCs (BM-MSCs) [11], while increasing evidence supports the use of neonatal tissues, such as umbilical cord tissue and placenta tissue (e.g., amniotic membrane) [12, 13], as better sources of MSCs. Placenta-derived MSCs (PD-MSCs) have several advantages, such as being abundant, easy to KRIBB11 obtain without invasiveness, and readily cultured to a sufficient number for transplantation, thus precluding ethical issues concerning allografting [14]. Furthermore, placenta tissue derives from pregastrulation embryonic cells, conferring its plasticity to the derived cells [14]. Recently, the therapeutic effect of PD-MSCs in the field of regenerative medicine has been shown [15]. Indeed, different types of placenta cells have been described [4], and among these, human amnion-derived mesenchymal stem cells (hAMSCs) have been shown to have immunosuppressive properties both and [16, 17]. Tuca et al. found that hAMSCs participated in both angiogenesis and reepithelialization [18] and the beneficial effect of hAMSCs in inhibition of inflammation and induction of neuronal repair in autoimmune encephalomyelitis mice has been shown [17]. Notably, it has been demonstrated that the main mechanism for MSCs’ beneficial effects on injured tissue is represented by their capacity to migrate into damaged areas and exert a trophic effect because of secretion of bioactive factors acting on the injured microenvironment to facilitate tissue repair. On the other hand, another hypothetical mechanism refers to KRIBB11 the differentiation of MSCs into functional cells that replace damaged tissue. However, there is evidence concerning poor KRIBB11 grafting of transplanted MSCs in spite of substantial therapeutic effects in lung and kidney cartilage injuries, diabetes, myocardial infarction, and other diseases. Tissue repair mechanisms through transplantation of MSCs are most likely due to the production of cytokines and paracrine factors, though this is currently a subject of some debate [19, 20]. An study showed that the conditioned medium produced by umbilical cord MSCs promotes cutaneous wound healing [3], and various studies indicate that amnion-derived cells secrete soluble factors with immunomodulatory capacity [13]. It has also been shown that the administration of conditioned medium derived from hAMSCs favored the repair process after acute myocardial infarction in mouse models [21] and was able to reduce lung fibrosis in a bleomycin Ctsl mouse model [22]. Moreover, prostaglandin-mediated immunosuppressive effects were shown for conditioned medium derived from hAMSCs [23]. In recent years, microvesicles extracted from supernatant of MSC cells have been used to induce angiogenesis.