Characterization and response to inflammatory stimulation of endometrial-derived mesenchymal stem/stromal cells

Background aims: The human endometrium has emerged as an attractive source of endometrial-derived mesenchymal stem/stromal cells (eMSCs) that can be easily isolated by non-invasive procedures. The prominent capacity of the endometrium for efficient and scarless regeneration each menstrual cycle indicates the increased eMSC immunomodulatory and pro-angiogenic properties. Herein the authors investigated the molecular responses of eMSCs to an inflammatory environment and whether those intrinsic responses affected their functional attributes. Methods: Human eMSCs immunophenotypic, transcriptional and secretory profiles were evaluated at passage three (P3) and passage eight (P8) to determine culture effects. Functionally, P3 and P8 non-induced and TNF-alpha/IFN-gamma-induced eMSCs were interrogated for their capacity to suppress stimulated peripheral blood mononuclear cell (PBMC) proliferation, whereas non-induced eMSCs were assessed for their support to vascular network formation in co-cultures with human umbilical vein endothelial cells in vitro. Results: Non-induced P3 and P8 eMSCs exhibited similar spindle-shaped morphology and clonogenic capacity. Nevertheless, P8 eMSCs showed reduced growth rate capacity and telomere length. The eMSCs displayed the typical MSC-related immunophenotypic profile, with P3 and P8 eMSCs expressing high levels (>98%) of CD140/3, intermediate levels (35-60%) of CD146 and SUSD2 and low levels (-8%) of NG2 pericytic markers. Non-induced P3 and P8 showed similar transcriptional and secretory profiles, though the expression of immunomodulatory HLA-G and IL-8 genes was significantly downregulated in P8 compared with P3 eMSCs. Upon TNF-alpha/IFN-gamma induction, eMSCs showed an immunophenotypic profile similar to that of non-induced eMSCs, except for significant upregulation of HLA-DR protein expression in both induced P3 and P8 eMSCs. However, induced P3 and P8 eMSCs showed significant upregulation of CD10, HLA-G, IDO, IL-6, IL-8, LIF and TSG gene expression compared with non-induced cultures. TNF-alpha/IFN-gamma induction strongly increased the secretion of inflammatory-/angiogenesis-related molecules, whereas growth factor secretion was similar to the non-induced eMSCs. Functionally, P3 and P8 eMSCs showed a strong inhibitory effect on stimulated PBMC proliferation and the capacity to support neovascularization in vitro. Conclusions: The authors' study suggests that serial expansion does not affect eMSC immunophenotypic, transcriptional and secretory profiles. This is directly reflected by the functional immunomodulatory and pro-angiogenic properties of eMSCs, which remain unaltered until P8 in vitro. However, exposure of eMSCs to inflammatory environments enhances their immunomodulatory transcriptional and inflammatory-/angiogenesisrelated secretory profiles. Therefore, the resulting evidence of eMSCs serial expansion and exposure to inflammation could serve as a foundation for improved eMSCs manufacturing and potential clinical translation efforts. Published by Elsevier Inc. on behalf of International Society for Cell & Gene Therapy.