Downregulation of Melanoma Cell Adhesion Molecule (MCAM/CD146) Accelerates Cellular Senescence in Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells

Abstract

Therapeutic applications of mesenchymal stem cells (MSCs) for treating various diseases have increased in recent years. To ensure that treatment is effective, an adequate MSC dosage should be determined before these cells are used for therapeutic purposes. To obtain a sufficient number of cells for therapeutic applications, MSCs must be expanded in long-term cell culture, which inevitably triggers cellular senescence. In this study, we investigated the surface markers of human umbilical cord blood-derived MSCs (hUCB-MSCs) associated with cellular senescence using fluorescence-activated cell sorting analysis and 242 cell surface-marker antibodies. Among these surface proteins, we selected the melanoma cell adhesion molecule (MCAM/CD146) for further study with the aim of validating observed expression differences and investigating the associated implications in hUCB-MSCs during cellular senescence. We observed that CD146 expression markedly decreased in hUCB-MSCs following prolonged in vitro expansion. Using preparative sorting, we found that hUCB-MSCs with high CD146 expression displayed high growth rates, multilineage differentiation, expression of stemness markers, and telomerase activity, as well as significantly lower expression of the senescence markers p16, p21, p53, and senescence-associated β-galactosidase, compared with that observed in hUCB-MSCs with low-level CD146 expression. In contrast, CD146 downregulation with small interfering RNAs enhanced the senescence phenotype. In addition, CD146 suppression in hUCB-MSCs caused downregulation of other cellular senescence regulators, including Bmi-1, Id1, and Twist1. Collectively, our results suggest that CD146 regulates cellular senescence; thus, it could be used as a therapeutic marker to identify senescent hUCB-MSCs.

Significance: One of the fundamental requirements for mesenchymal stem cell (MSC)-based therapies is the expansion of MSCs during long-term culture because a sufficient number of functional cells is required. However, long-term growth inevitably induces cellular senescence, which potentially causes poor clinical outcomes by inducing growth arrest and the loss of stem cell properties. Thus, the identification of markers for evaluating the status of MSC senescence during long-term culture may enhance the success of MSC-based therapy. This study provides strong evidence that CD146 is a novel and useful marker for predicting senescence in human umbilical cord blood-derived MSCs (hUCB-MSCs), and CD146 can potentially be applied in quality-control assessments of hUCB-MSC-based therapy.

Keywords: Bmi-1; Cell surface marker; Cellular senescence; Human umbilical cord blood-derived mesenchymal stem cells; Id1; Melanoma cell adhesion molecule (CD146); Mesenchymal stem cell-based therapy; Twist1.

Figure 1.

Senescence phenotype of cells following expansion. (A): Cumulative PD values from three different donors were analyzed. (B): Cell proliferation was checked by measuring fold-increases, with results normalized to the growth observed at passage (P) 5 (set as one-fold; mean ± SD; n = 2). (C): Stemness markers were quantified by quantitative real-time polymerase chain reaction (q-PCR; mean ± SD; n = 3; ∗, p < .05; ∗∗p, < .01). (D): Telomerase activity was analyzed in telomeric repeat amplification protocol assays (mean ± SD; n = 4; ∗, p < .05; ∗∗, p < .01). (E, F): Senescence-related proteins were measured by immunoblotting (E) or qPCR (F) (mean ± SD; n = 3; ∗p, < .05; ∗∗, p < .01). (C, E–F): Expression levels were normalized to β-actin, with the expression levels at P5 defined as 1. (G): The cells were stained to measure SA β-gal) expression, and quantitation was achieved by determining the percentage of SA β-gal-positive cells (upper panel; mean ± SD; n = 4; ∗, p < .05; ∗∗, p < .01). Cell areas at three passages were compared. The black lines indicate the cell margins that were drawn on the T75 flask, with the results normalized to the mean area at P5, which was defined as 1 (lower panel; mean ± SD; n = 20; ∗∗, p < .01). (H): Osteogenic and adipogenic lineages were measured by staining for alkaline phosphatase (ALP) or Oil Red O, respectively. (G, H): Scale bar = 50 μm. Abbreviations: MSC, mesenchymal stem cell; OD, optical density; PD, population doubling; pho-p53, phospho-p53; pho-Rb, phospho-retinoblastoma; SA β-gal, senescence-associated β-galactosidase; TRAP, telomeric repeat amplification protocol.

Figure 2.

Screening for cell surface proteins in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) with altered expression during senescence. (A): Flow cytometric analysis of both early-stage (passage [P] 4) and late-stage (P10) hUCB-MSCs based on the cell surface expression of putative MSCs markers. (B): Heat map analysis showing downregulated cell surface proteins at a late passage (P10) compared with those observed at an early passage (P4). (C): To confirm the downregulation of cell surface proteins shown in panel B, the protein expression levels of CD47, CD71, CD106, CD146, CD165, CD274, and EGFR were measured by flow cytometry at early (P4) and late (P10) stages in hUCB-MSCs from 25 different donors (mean ± SD; n = 25; ∗, p < .05; ∗∗, p < .01). CD146 expression showed the most significant decline in late-stage cells (black box). (D): CD146 expression was quantified by using flow cytometry at the indicated passages (mean ± SD; n = 3; ∗∗, p < .01). Abbreviation: EGFR, epidermal growth factor receptor.

Figure 3.

Immunophenotyping results of sorted in human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), based on CD146 expression. (A): The percentage of CD146+ hUCB-MSCs was analyzed, and CD146+ and CD146− populations were sorted by flow cytometry. The plots shown indicate the CD146 staining profile (filled graph) versus isotype-control staining profile (unfilled graph; mean ± SD; n = 3). (B): The immunophenotypic characteristics of CD146+ and CD146− cells were examined by flow cytometry. Both CD146+ and CD146− cells were strongly positive for the MSC-specific surface markers CD29, CD73, CD90, CD105, and CD166, and they were negative for CD14 and CD45 (mean ± SD; n = 3).

Figure 4.

Senescence phenotypes of cells, based on CD146 expression. (A): Cell growth was measured by determining the cumulative PD. (B): Stemness genes were assessed by quantitative real-time polymerase chain reaction (qPCR) at passage (P) 9 (mean ± SD; n = 3; ∗∗, p < .01). (C): Telomerase activities was measured at P9 by using the TRAP assay (mean ± SD; n = 4; ∗∗, p < .01). (D, E): Expression of cell cycle inhibitors was measured by immunoblotting (D) and qPCR at P9 (E) (mean ± SD; n = 3; ∗∗, p < .01). (B, D, E): Expression levels were normalized to β-actin, with the expression levels in CD146+ cells defined as 1. (F): CD146− cells showed strong SA β-gal staining at P9 and P12 (right panel; mean ± SD; n = 4; ∗, p < .05; ∗∗, p < .01). (G): Cell areas were normalized to the mean area in CD146+ cells, which was defined as 1 at P9 (right panel; mean ± SD; n = 30; ∗∗, p < .01). The black lines indicate the cell margins. (H): In each population, multilineage differentiation was examined by ALP staining, von Kossa staining, and Oil Red O staining. Quantitative results was significantly reduced in CD146− cells at P9 (lower panel; mean ± SD; n = 3; ∗, p < .05; ∗∗, p < .01). (F–H): Scale bar = 50 μm. Abbreviations: ALP, alkaline phosphatase; OD, optical density; PD, population doubling; pho-p53, phospho-p53; SA β-gal, senescence-associated β-galactosidase; TRAP, telomeric repeat amplification protocol.

Figure 5.

CD146 knockdown in human umbilical cord blood-derived mesenchymal stem cells accelerates the senescence process. (A): Cell growth was monitored by measuring cumulative PD. (B): Cells were assessed for their expression of stemness genes by quantitative real-time polymerase chain reaction (qPCR) at passage (P) 9 (mean ± SD; n = 3; ∗∗, p < .01). (C): Telomerase activities was measured at P9 using the telomerase PCR enzyme-linked immunosorbent assay kit (mean ± SD; n = 4; ∗∗, p < .01). (D): Expression of the cell cycle inhibitors was measured by immunoblotting at P9, with β-actin serving as a loading control fold (right panel; mean ± SD; n = 4; ∗, p < .05; ∗∗, p < .01). (E): qPCR data showing the mRNA expression levels (p16 and Bmi-1) at P9 (mean ± SD; n = 3; ∗∗, p < .01). (B, D, E): Expression levels were normalized to β-actin, with the expression levels in naïve defined as 1. (F): SA β-gal-positive cells were measured at P9 and P12. Results are shown as mean ± SD (n = 4; ∗∗, p < .01). (G): Cell areas compared at P9, which was normalized to the mean area in naïve cells, defined as 1 (mean ± SD; n = 25; ∗∗, p < .01). The black lines indicate the cell margins. (H): Multilineage differentiation was assessed by quantifying the percentage of positively stained cells at P9 (mean ± SD; n = 3; ∗, p < .05). (F–H): Scale bar = 50 μm. Abbreviations: OD, optical density; PD, population doubling; pho-p53, phospho-p53; SA β-gal, senescence-associated β-galactosidase; siCD146, CD146 small interfering RNA; siCon, small interfering scrambled RNA; siRNA, small interfering RNA; TRAP, telomeric repeat amplification protocol.

Figure 6.

Involvement of CD146 in Id and Twist-family gene expression. (A): The expression of Id- and Twist-family genes was analyzed after human umbilical cord blood-derived mesenchymal stem cell (hUCB-MSC) expansion (both at passage [P] 5 and P12, mean ± SD; n = 3; ∗∗, p < .01). The expression levels of all genes were normalized to that of β-actin in hUCB-MSCs at P5, which was defined as 1-fold expression. (B): CD146+ and CD146− hUCB-MSCs were sorted by flow cytometry and examined for Id- or Twist-family gene expression at P9 (mean ± SD; n = 3; ∗∗, p < .01). The expression levels of all genes were normalized to that of β-actin in CD146+ cells, which was defined as 1-fold expression. (C): The gene-expression levels of Id1 and Twist1 significantly decreased siCD146 compared with the expression levels in naïve and siCon-treated cells (mean ± SD; n = 3; ∗∗, p < .01). The expression levels of all genes were normalized to that of β-actin in naïve cells, which was defined as 1-fold expression. Abbreviations: siCD146, CD146-specific siRNA; siCon, control siRNA; siRNA, small-interfering RNA.