Background Multipotent mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues and are considered as attractive candidates for the development of cell-based regenerative therapies. Currently, there are more than 200 clinical trials involving the use of MSCs for a wide variety of indications. However, variations in their isolation, expansion, and particularly characterization have made the interpretation of study outcomes or the rigorous assessment of therapeutic efficacy difficult. An unbiased characterization of MSCs is of major importance and essential to guaranty that only the most suitable cells will be used. The development of standardized and reproducible assays to predict MSC potency is therefore mandatory. The currently used quantification methodologies for the determination of the trilineage potential of MSCs are usually based on absorbance measurements which are imprecise and prone to errors. We therefore aimed at developing a methodology first offering a standardized way to objectively quantify the trilineage potential of MSC preparations and second allowing to discriminate functional differences between clonally expanded cell populations. Method MSCs originating from several patients were differentiated into osteoblasts, adipocytes, and chondroblasts for 14, 17, and 21 days. Differentiated cells were then stained with the classical dyes: Alizarin Red S for osteoblasts, Oil Red O for adipocytes, and Alcian Blue 8GX for chondroblasts. Quantification of differentiation was then performed with our newly developed digital image analysis (DIA) tool followed by the classical absorbance measurement. The results from the two techniques were then compared. Result Quantification based on DIA allowed highly standardized and objective dye quantification with superior sensitivity compared to absorbance measurements. Furthermore, small differences between MSC lines in the differentiation potential were highlighted using DIA whereas no difference was detected using absorbance quantification. Conclusion Our approach represents a novel method that simplifies the laboratory procedures not only for the quantification of histological dyes and the degree of differentiation of MSCs, but also due to its color independence, it can be easily adapted for the quantification of a wide range of staining procedures in histology. The method is easily applicable since it is based on open source software and standard light microscopy. Electronic supplementary material The online version of this article (10.1186/s13287-019-1170-8) contains supplementary material, which is available to authorized users.

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Background The impressive progress in the field of stem cell research in the past decades has provided the ground for the development of cell-based therapy. Mesenchymal stromal cells obtained from adipose tissue (AD-MSCs) represent a viable source for the development of cell-based therapies. However, the heterogeneity and variable differentiation ability of AD-MSCs depend on the cellular composition and represent a strong limitation for their use in therapeutic applications. In order to fully understand the cellular composition of MSC preparations, it would be essential to analyze AD-MSCs at single-cell level. Method Recent advances in single-cell technologies have opened the way for high-dimensional, high-throughput, and high-resolution measurements of biological systems. We made use of the cytometry by time-of-flight (CyTOF) technology to explore the cellular composition of 17 human AD-MSCs, interrogating 31 markers at single-cell level. Subcellular composition of the AD-MSCs was investigated in their naïve state as well as during osteogenic commitment, via unsupervised dimensionality reduction as well as supervised representation learning approaches. Result This study showed a high heterogeneity and variability in the subcellular composition of AD-MSCs upon isolation and prolonged culture. Algorithm-guided identification of emerging subpopulations during osteogenic differentiation of AD-MSCs allowed the identification of an ALP+/CD73+ subpopulation of cells with enhanced osteogenic differentiation potential. We could demonstrate in vitro that the sorted ALP+/CD73+ subpopulation exhibited enhanced osteogenic potential and is moreover fundamental for osteogenic lineage commitment. We finally showed that this subpopulation was present in freshly isolated human adipose-derived stromal vascular fractions (SVFs) and that could ultimately be used for cell therapies. Conclusion The data obtained reveal, at single-cell level, the heterogeneity of AD-MSCs from several donors and highlight how cellular composition impacts the osteogenic differentiation capacity. The marker combination (ALP/CD73) can not only be used to assess the differentiation potential of undifferentiated AD-MSC preparations, but also could be employed to prospectively enrich AD-MSCs from the stromal vascular fraction of human adipose tissue for therapeutic applications.