Isolation and characterization of CD73+CD39+CD146+ mesenchymal stem cell subset from bone marrow

Figure 1 Co-expression of CD73 and CD39 in bone marrow-derived mesenchymal stem cells. A: Representative flow cytometry analysis showed, as expected, 99.9% of isolated bone marrow mesenchymal stem cells were negative for CD45 (a1). Representative flow cytometry analysis showed 96.0% of CD45- population of bone marrow-derived mesenchymal stem cells were co-positive for CD73+ CD39+ (a2); B: Bar graph showing expression of CD73 and CD39 markers at passage 2 and passage 3 (n = 3). Results are mean ± SD.

Figure 2 Sorting strategy and purity post-sort for bone marrow-derived CD45-CD73+CD39+ subset. A: Sorting strategy for bone marrow (BM)-derived mesenchymal stem cells (MSCs) for first sorting (a1 and a2). Cell population was defined based on cell size, granularity, and single cells. Cells were gated on the viable population (a3). The cell fraction negative for CD45 was gated (a4) and cells which co-expressed CD73 and CD39 were gated and isolated based on single stained controls (a5). Controls not shown; B: Sorting strategy for BM-derived MSCs for second sort (b1 and b2). Cell population was defined based on cell size, granularity, and single cells. Cells were gated on the viable population (b3). The cell fraction negative for CD45 was gated (b4) and cells which co-expressed CD73 and CD39 were gated and isolated based on single stained controls (b5). Controls not shown; C: Bar graphs showing percentage of the CD73+CD39+ cell subset in samples obtained post-sorting.

Figure 3 CD45-CD73+CD39+ cell subset displays mesenchymal stem cell characteristics. A: Clonogenicity of CD45-CD73+CD39+ isolated mesenchymal stem cells (MSCs). Cells were plated at 100 cells/well in a 6 well plate. After 14 days cells were stained with crystal violet. Aggregates of ≥ 50 cells were defined as colonies (a1). The bar graph represents the average number of colonies. Results are mean ± SD (n = 2). Representative image of one colony at day 14 following fixation and crystal violet staining at 40 × and scale bar = 24 μm (a1-a3); B: Each bar graph represents treatment group mean ± SD (n = 3), P < 0.0001, MSC subset compared to negative control. CD45-CD73+CD39+ subset was cultured at 10000 cells/well and treated with osteogenic differentiation medium. On day 21, cells were fixed and stained with alizarin red. Untreated cells and osteoblasts were used as controls (b1). Representative images of untreated subset, osteoblast, treated subset at day 21 following fixative and stain at 40 × and scale bar = 24 μm (b2-b4). Following imaging, cells were measured for absorbance at 405 nm; C: Each bar graph represents treatment group mean ± SD (n = 3), P < 0.001, MSC subset compared to negative control. CD45-CD73+CD39+ subset was cultured at 100 cells/well and treated with adipogenic differentiation medium. On day 28, cells were fixed and stained with Oil Red O. Aggregates of ≥ 50 cells and vacuoles were defined as adipogenic colonies. Untreated cells and adipocytes were used as controls (c1). Representative images of untreated subset, adipocytes, treated subset at day 28 following fixative and stain at 40 × and scale bar = 24 μm (c2-c4); D: Each bar graph represents treatment group mean ± SD (n = 3), P < 0.001, MSC subset compared to negative control. Cells were plated in micromasses in a 6 well plate at density of 250000 cells/ well. After 7 days, cells were fixed and stained with Alcian blue (d1). Representative images of untreated subset, chondrocyte, treated subset at 7 days following fixative and stain at 40 × and scale bar = 24 μm (d2-d4). Cells were measured for absorbance at 600 nm.

Figure 4 In vitro and in vivo CD146 expression in CD45-CD73+CD39+ subset. A: Representative flow cytometry analysis showed the entire population of cells copositive for CD73 and CD39 were also positive for CD146 post-sorting and culture (a1-a3); B: Frequency of parent of bone marrow mesenchymal stem cells post sorting for cultured cells for passage 3 and passage 4 (n = 3) with phenotype CD45-CD73+CD39+ and CD45-CD73+CD39+CD146+ as bar graphs are mean ± SD. No significant difference was observed between passage 3 and passage 4; C: Representative immunofluorescence analysis showing colocalization of CD73, CD39, and CD146 in human bone marrow slides. Human bone marrow slide with isotype control was used to verify staining. Image was taken at 40 × (c1). Human tonsils were previously validated for expressing CD45, CD73, CD39, and CD146 and served as a positive control. Image was taken at 40 × (c2). Representative image of human bone marrow slide showing colocalization of subset at perivascular locations where the arrows show locations of CD73, CD39, and CD146 expression and represents the vessel. Image was taken at 40 × (c3).

Figure 5 CD45-CD73+CD39+CD146+ subset expressed in mobilized peripheral blood. A: Representative flow cytometry analysis showing target subset in mononuclear cells. Total CD45 negative fraction was gated (a1). t-Distribution Stochastic Neighbor Embedding (tSNE) plot in combination with flowSOM and cluster explorer was used. 1.8% of CD45- cells expressed CD73 and CD39 (a2). Only cells from the CD45- fraction presented in the t-SNE plot were gated[14], showing co-expression of CD73 and CD39 at 99.9% (a3). CD45-CD73+CD39+CD146+ population was not expressed in non-mobilized leukopaks (mononuclear cells) (a4); B: Representative flow cytometry analysis showing subset in single mobilized peripheral blood stem cells (PBSCs). CD45- fraction of total population was gated (b1). tSNE plot in combination with flowSOM and cluster explorer was used. 1.5% of CD45- cells expressed CD73 and CD39 (b2)[14]. Only cells from the CD45- fraction presented in the tSNE plot were gated, showing co-expression of CD73 and CD39 at 96.8% (b3)[14]. From CD45-CD73+CD39+ cells, approximately 80% are positive for CD146+ (b4); C: Representative flow cytometry analysis of target subset in single mobilized PBSCs. CD45 negative fraction of total population was gated (c1). tSNE plot in combination with flowSOM and cluster explorer was used. 5.7% of CD45- cells expressed CD73 and CD39 (c2)[14]. Only cells from cluster of CD45- fraction presented in the tSNE plot were gated and showed co-expression of CD73 and CD39 at 99.8% (c3)[14]. Among the CD45-CD73+CD39+ cells, 25.1% were positive for CD146+ (c4). All gates were established using Fluorescence MinusOne and unstained controls (A-C); D: Percent of CD45-CD73+CD39+CD146+ in single-mobilized PBSCs (< 1%). Bar graph is a result of mean ± SD (n = 6); E: Percent of CD45-CD73+CD39+CD146+ in dual-mobilized PBSCs (< 1%). Bar graph is a result of mean ± SD (n = 3).