biostudies-literatureUnknown12Smolinska ABackground: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due to different degrees of differentiation of cell subpopulations poses a considerable challenge in preclinical studies. The cells at a pluripotent-like stage represent a stem cell population of interest for many researchers worldwide, which is worthy of identification, isolation, and functional characterization. In the current study, we asked whether Wharton's jelly-derived MSCs (WJ-MSCs) which express stage-specific embryonic antigen-4 (SSEA-4) can be considered as a pluripotent-like stem cell population. Methods: SSEA-4 expression in different culture conditions was compared and the efficiency of two cell separation methods were assessed: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After isolation, SSEA-4+ cells were analyzed for the following parameters: the maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-related gene expression, proliferation potential, clonogenicity, secretome profiling, and the ability to form spheres under 3D culture conditions. Results: FACS allowed for the enrichment of SSEA-4+ cell content in the population that lasted for six passages after sorting. Despite the elevated expression of stemness-related genes, SSEA-4+ cells neither differed in their proliferation and clonogenicity potential from initial and negative populations nor exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to form smaller spheroids and exhibited increased survival under 3D conditions. Conclusion: Despite the transient expression of stemness-related genes, our findings could not fully confirm the undifferentiated pluripotent-like nature of the SSEA-4+ WJ-MSC population cultured in vitro.Frontiers in cell and developmental biology1227034https://www.ebi.ac.uk/biostudies/studies/S-EPMC10917976biostudies-literatureStemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton's jelly mesenchymal stem/stromal cells.PMC10917976Piwocka KSulejczak DKominek AJaniec JSarnowska ASmolinska AChodkowska MfalseStemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton's jelly mesenchymal stem/stromal cells.Background: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due to different degrees of differentiation of cell subpopulations poses a considerable challenge in preclinical studies. The cells at a pluripotent-like stage represent a stem cell population of interest for many researchers worldwide, which is worthy of identification, isolation, and functional characterization. In the current study, we asked whether Wharton's jelly-derived MSCs (WJ-MSCs) which express stage-specific embryonic antigen-4 (SSEA-4) can be considered as a pluripotent-like stem cell population. Methods: SSEA-4 expression in different culture conditions was compared and the efficiency of two cell separation methods were assessed: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After isolation, SSEA-4+ cells were analyzed for the following parameters: the maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-related gene expression, proliferation potential, clonogenicity, secretome profiling, and the ability to form spheres under 3D culture conditions. Results: FACS allowed for the enrichment of SSEA-4+ cell content in the population that lasted for six passages after sorting. Despite the elevated expression of stemness-related genes, SSEA-4+ cells neither differed in their proliferation and clonogenicity potential from initial and negative populations nor exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to form smaller spheroids and exhibited increased survival under 3D conditions. Conclusion: Despite the transient expression of stemness-related genes, our findings could not fully confirm the undifferentiated pluripotent-like nature of the SSEA-4+ WJ-MSC population cultured in vitro.2024-01-01T00:00:00Z20242026-06-12T10:05:56.39Z2025-04-04T12:34:59.906ZS-EPMC109179763845507310.3389/fcell.2024.1227034