Additional file 1 of Identification of ALP+/CD73+ defining markers for enhanced osteogenic potential in human adipose-derived mesenchymal stromal cells by mass cytometry
Creators: name: Canepa, Daisy D.
name: Casanova, Elisa A.
name: Arvaniti, Eirini
name: Tosevski, Vinko
name: Märsmann, Sonja
name: Eggerschwiler, Benjamin
name: Halvachizadeh, Sascha
name: Buschmann, Johanna
name: Barth, André A.
name: Plock, Jan A.
name: Claassen, Manfred
name: Hans-Christoph Pape
name: Cinelli, Paolo
Publisher: name: figshare
Year: 2021
Subjects: subject: Space Science
subject: Cell Biology
subject: Biotechnology
subject: Immunology
subject: FOS: Clinical medicine
subject: FOS: Clinical medicine
subject: Developmental Biology
subject: Hematology

description: Additional file 1: Figure S1. In vitro chondrogenic and adipogenic categorization of 17 AD-MSCs A) Sum of the pixels acquired at the three time points (day 14, 17, 21) for chondrogenic (left) and adipogenic (right) differentiation of all 17 AD-MSC lines and interquartile categorization into «good», «intermediate», and «bad» AD-MSCs. C) Summary of the categorization of all 17 AD-MSCs for the three differentiation lineages (osteogenic, chondrogenic, and adipogenic). interm. = intermediate. Figure S2. UMAP analyses in the 17 human AD-MSC lines. A) UMAP projections of all 31 markers in 17 AD-MSC lines. Each dot represents one cell. Blue denotes minimal, green intermediate, and red high expression. Figure S3. Analyses of the osteogenic subpopulation. A) Means of the percentage of alkaline phosphatase (ALP) positive cells and CD73 positive cells in the three AD-MSC categories during the five analyzed days of osteogenic differentiation (d0, d1, d2, d3, d4). Error bars represent the mean ± s.d. of the percentage of positive cells present in «good» (n = 6), «intermediate» (n = 4),and «bad» (n = 7) AD-MSCs. B) Pearson correlations of the ALP frequency measured by CyTOF at day 0, 1, 2, 3, 4 with the staining intensities measured at day 14, 17, and 21 for osteogenic differentiation. Red dots represent «good», green «intermediate» (interm.), and black «bad» differentiating lines. Error bars indicate the triplicates of the staining and are presented as mean ± s.d. For statistical analyses, the one-way ANOVA Dunnett’s multiple comparisons test was used to compare each day of the “good” AD-MSCs with the same day of “intermediate” and “bad” categories: * p≤ 0.05, ** p≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001. ns=not significant. Figure S4. ALP+/CD73+ Sorting analysis and prediction of osteogenic differentiation potential. A) Gating strategy for FACS sorting for the following subpopulations: ALP+/CD73+, ALP-/CD73low, and ALP-/CD73high. B) Alizarin Red staining and quantification of the sorted subpopulations in four AD- MSC lines (F04, F14, F22, F28) after 14, 17, and 21 days. Control sorted are unstained cells, which were run through the FACS sorting machine. Depicted are triplicates of undifferentiated cells (control) and cells cultured with the differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. C) Categorization of the new AD-MSC lines (depicted in green) together with all the 17 already analyzed lines, based on Alizarin Red quantification after 14, 17, and 21 days of osteogenic differentiation and interquartile distribution of the five new AD-MSCs (depicted in violet). D) Alizarin Red staining and quantification of five new AD-MSCs: two «good» (F08, F26), one «intermediate» (F23), and two «bad» (F20, F24). Depicted are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation conditions (differentiation). Error bars indicate triplicates of the staining and are presented as mean ± s.d. E) Histograms of median intensities of expression of selected markers (CD73 and ALP) in F05, F14, F22 and F28 AD-MSC lines from passage 3 (p3) till passage 20 (p20). Black is the lowest intensity and white represents the highest intensity. F) Alizarin Red staining and quantification of F22 at passage p5, p9, and p20 after 14, 17, and 21 days of osteogenic differentiation. Depicted are triplicates of undifferentiated cells (control) and cells cultured under osteogenic differentiation medium (differentiation). Error bars indicates the triplicates of the staining and are presented as mean ± s.d. Figure S5. ALP+/CD73+ cells are present in the human fat tissue and stromal vascular fraction A) Hematoxylin/Eosin (H&amp;E) and immunohistochemistry staining of human fat tissue for ALP, CD73, and CD31. Scale 100 μm. B) Gating strategy for sorting the selected subpopulations (CD45- /ALP+/CD73+, CD45-/ALP-/CD73low, CD45-/ALP-/CD73high) in the SVFs. C) Alizarin Red staining and pixel quantification of sorted SVF fractions (CD45-/ALP+/CD73+, CD45-/ALP-/CD73low, CD45-/ALP- /CD73high) after 21 days of osteogenic differentiation in vitro. Control sorted are unstained SVFs, which were run through the FACS sorting machine. Depicted are triplicates of undifferentiated cells (control) and cells cultured with osteogenic differentiation medium (differentiation). Error bars indicate the triplicates of the staining and are presented as mean ± s.d. Table S1. Mass cytometry antibody panel. Table S2. Osteogenic differentiation barcoding schema. Table S3. Barcoding plan prediction experiment. Table S4: Barcoding plan for the passage experiment.