Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro

Abstract

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24^pos cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24^pos and CD24^neg BMSCs identified a limited number of genes with increased expression in CD24^pos cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24^pos cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.

Keywords: bone; bone marrow mesenchymal stem cells; differentiation; osteoblast.

FIG. 1.

CD24 is induced immediately upon osteoblast differentiation. (A) Heatmap representing the expression changes of 192 differential expressed cell surface proteins (represented by 251 different probes) during osteoblast differentiation compared to adipocyte differentiating BMSC. Red is higher expressed in osteoblasts differentiating BMSC, Green is higher expressed in adipocyte differentiation BMSC. (1 donor, n = 3 per time point). (B) Next generation RNA sequencing data of CD24 mRNA expression in osteoblast differentiating human mesenchymal stromal cells from two different donors (n = 1 per donor). (C) qPCR analyses of CD24 mRNA expression in osteoblast differentiating BMSCs in two different donors illustrate that CD24 is quickly induced upon osteogenic differentiation and reduce to basal levels just prior extracellular matrix mineralization (n = 2 per donor). BMSC, bone marrow-derived mesenchymal stem/stromal cell; qPCR, quantitative PCR. Color images are available online.

FIG. 2.

CD24 is expressed in a subset of BMSC and induced upon osteogenic differentiation. (A) Flow cytometry gating of CD24 positive cells. After gating cells, the CD24 positive cells (αCD24-PE, Fl-2) are gated against autofluorescent signals (Fl-1). Example of (1) Isotype (Upper right), (2) undifferentiated BMSC (lower left), and (3) 7 days osteogenic differentiated BMSCs (lower right). (B) Percentage of CD24^pos cells determined by flow cytometry. Analyses of cell surface expression of CD24 before and after 7 days of osteoblast differentiation in three different BMSC donors and different passages. (C) Fold change increase in the number of CD24^pos cells after 7 days of osteogenic differentiating BMSCs compared to undifferentiated BMSCs. Fold changes are derived from the FACS experiments in Fig. 2B. (D) CD24 cell surface expression during osteogenic and adipogenic differentiating BMSC (two different BMSC donors, n = 2 per time point). Color images are available online.

FIG. 3.

CD24 marks a subset of hMSC with reduced osteogenic and adipogenic differentiation capacity. (A) To investigate the functional differences of the CD24 cell populations in BMSCs, we sorted the cells using flow cytometry. Three populations were sorted: CD24^pos, CD24^neg, and unsorted cells (NS), and differentiated into adipocytes and osteoblasts. (B) CD24 expression determined 2 days after sorting for CD24 cell surface expression. Gene expression levels are relative to GAPDH. (n = 2). (C) Differentiation of the sorted population resulted in a reduced mineralization. In the CD24 positive sorted cells. (D) CD24^pos sorted cells have a reduced adipogenic differentiation potential. Cells were fixed after 18 days of differentiation and stained with DAPI (nuclei, total cell number) and ORO (Adipocytes). For each condition, five independent images were analyzed (from two different wells). Quantification shows that only 12% of the CD24^pos cells were differentiated into ORO positive adipocytes whereas the 25%–30% of the unsorted and CD24^neg cells were differentiated into ORO positive adipocytes. (E) Quantification (n = 10) of the data represented in Fig. 3D. Left panel total nr cells (counted by the DAPI positive nuclei). Right panel: percentage of ORO positive adipocytes. DAPI, 4′,6-diamidino-2-phenylindole; NS, non-sorted; ORO, Oil Red O. Color images are available online.

FIG. 4.

CD24^pos population derived from proliferating and osteogenic differentiated cells have lower differentiation capacity. (A) Double sorting strategy to obtain and investigate CD24^pos cells that are present in undifferentiated human BMSCs and that are formed from CD24^neg cells upon osteoblast differentiation. Proliferating human BMSCs were sorted in three populations: CD24^pos, CD24^neg, and unsorted cells (NS). All populations were osteogenic differentiated for 7 days, trypsinized, and a second round of cell sorting was applied from the CD24^neg and unsorted population (NS) to obtain another CD24^neg, CD24^pos and unsorted population NS. The cells were seeded and cultured in osteogenic differentiation media for another 14 days. Cells were fixed and the extracellular matrix mineralization was stained with Alizarin Red (B) Calcium concentration in the media after differentiation of the different populations of Fig. 4A. (C) Percentage of CD24^pos cells after sorting of proliferating cells (left panel) or after the second sort of osteogenic differentiated populations (right panel). Color images are available online.

FIG. 5.

Differential expressed genes in CD24^pos cells have reduced expression of genes involved in mitotic cell cycle. (A) FACSorting strategy of proliferating BMSCs that express CD24 on surface for the RNAseq. (B) Gene expression analyzes comparing the CD24^pos and CD24^neg BMSCs. In red the genes that are significantly (FDR 1%) differentially expressed between the two populations. In brackets the number of genes significantly different (1%FDR) with a fold change = 2 (n = 3 donors). (C) Validation of genes higher (red) or lower (green) expressed in CD24^pos cells compared to CD24^neg cells. Left panel gene expression data from the RNAseq analyses and right panel analyses of the same genes by qPCR in independently sorted cells (n = 3 donors). (D) Venn diagram of the genes associated with extracellular matrix, cell adhesion and cell motility and enriched in the CD24^pos population. qPCR, quantitative PCR. Color images are available online.

FIG. 6.

CD24 positive cells have lower proliferative capacity. (A) Ki67 analyses in proliferating BMSCs. Proliferating BMSC double stained with Ki67-Alexa488 and CD24-PE and analyzed on a BD Accuri. The CD24 positive and negative cells were gated (panel 2) and analyzed for the percentage Ki67^pos cells (n = 4, 2 donors). (B) EDU analyses of proliferating BMSCs and after 7 days osteogenic differentiation. Proliferating and osteogenic differentiating (7 days) BMSCs were pulse labeled with EDU and stained with CD24-PE and analyzed on a BD Accuri. The CD24 positive and negative cells were gated (panel 2) and analyzed for the percentage EDU^pos cells (n = 6, 1 donor). Color images are available online.