Single-Cell Sorting of Immunophenotyped Mesenchymal Stem Cells from Human Exfoliated Deciduous Teeth.

The mesenchymal stem cells (MSCs) of an organism possess an extraordinary capacity to differentiate into multiple lineages of adult cells in the body and are known for their immunomodulatory and anti-inflammatory properties. The use of these stem cells is a boon to the field of regenerative biology, but at the same time, a bane to regenerative medicine and therapeutics owing to the multiple cellular ambiguities associated with them. These ambiguities may arise from the diversity in the source of these stem cells and from their in vitro growth conditions, both of which reflect upon their functional heterogeneity. This warrants methodologies to provide purified, homogeneous populations of MSCs for therapeutic applications. Advances in the field of flow cytometry have enabled the detection of single-cell populations using a multiparametric approach. This protocol outlines a way to identify and purify stem cells from human exfoliated deciduous teeth (SHEDs) through fluorescence-assisted single-cell sorting. Simultaneous expression of surface markers, namely, CD90-fluorescein isothiocyanate (FITC), CD73-peridinin-chlorophyll-protein (PerCP-Cy5.5), CD105-allophycocyanin (APC), and CD44-V450, identified the "bright," positive-expressors of MSCs using multiparametric flow cytometry. However, a significant drop was observed in percentages of quadruple expressors of these positive markers from passage 7 onwards to the later passages. The immunophenotyped subpopulations were sorted using the single-cell sort mode where only two positive and one negative marker constituted the inclusion criteria. This methodology ensured the cell viability of the sorted populations and maintained cell proliferation post sorting. The downstream application for such sorting can be used to evaluate lineage-specific differentiation for the gated subpopulations. This approach can be applied to other single-cell systems to improve isolation conditions and for acquiring multiple cell surface marker information.

MSCProfiler: a single cell image processing workflow to investigate mesenchymal stem cell heterogeneity.

Single cell cytometry has demonstrated plausible immuno-heterogeneity of mesenchymal stem cells (MSCs) owing to their multivariate stromal origin. To contribute successfully to next-generation stem cell therapeutics, a deeper understanding of their cellular morphology and immunophenotype is important. In this study, the authors describe MSCProfiler, an image analysis pipeline developed using CellProfiler software. This workflow can extract geometrical and texture features such as shape, size, eccentricity and entropy, along with intensity values of the surface markers from multiple single cell images obtained using imaging flow cytometry. This screening pipeline can be used to analyze geometrical and texture features of all types of MSCs across different passages hallmarked by enhanced feature extraction potential from brightfield and fluorescent images of the cells.