Stemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton’s jelly mesenchymal stem/stromal cells

Abstract

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.

The Role of Mesenchymal Stem/Stromal Cells in Head and Neck Cancer – Regulatory Mechanisms of Tumorigenic and Immune Activity, Chemotherapy Resistance, and Therapeutic Benefits of the Use of Stromal Cell-Based Pharmacological Strategies

“…Further in vitro research of MSCs has allowed their identification based on other features, like the presence of a subset of other characteristic surface markers and the ability to form colonies [40,48,[52][53][54]. One set of markers proposed for the identification of MSCs includes the superficial proteins, for instance smooth muscle actin (SMA), Gremlin-1 (GREM1), Meflin (ISLR), PDPN (Podoplanin), STRO-1, and stage-specific embryonic antigen 4 (SSEA-4) [55][56][57][58][59][60][61]. Interestingly, the presence of a key SMA marker allows MSCs to be differentiated from activated fibroblasts/myofibroblasts in the ECM.…”

“…Even so, fibroblasts have been differentiated from MSCs by co-expression of fibroblast activation protein alpha (FAP-α) and fibroblast specific protein 1 (FSP, also known as S100A4) [69,70]. Gremlin-1 (GREM1) is also indicated to be an important candidate marker of MSCs [56,57]. Unfortunately, markers such as ISLR, PDPN, STRO-1, and SSEA-4 are also expressed in other cell lineages and hence have limited value.…”