Activin A expression regulates multipotency of mesenchymal progenitor cells

Abstract

Introduction: Bone marrow (BM) stroma currently represents the most common and investigated source of mesenchymal progenitor cells (MPCs); however, comparable adult progenitor or stem cells have also been isolated from a wide variety of tissues. This study aims to assess the functional similarities of MPCs from different tissues and to identify specific factor(s) related to their multipotency.

Methods: For this purpose, we directly compared MPCs isolated from different adult tissues, including bone marrow, tonsil, muscle, and dental pulp. We first examined and compared proliferation rates, immunomodulatory properties, and multidifferentiation potential of these MPCs in vitro. Next, we specifically evaluated activin A expression profile and activin A:follistatin ratio in MPCs from the four sources.

Results: The multidifferentiation potential of the MPCs is correlated with activin A level and/or the activin A:follistatin ratio. Interestingly, by siRNA-mediated activin A knockdown, activin A was shown to be required for the chondrogenic and osteogenic differentiation of MPCs. These findings strongly suggest that activin A has a pivotal differentiation-related role in the early stages of chondrogenesis and osteogenesis while inhibiting adipogenesis of MPCs.

Conclusions: This comparative analysis of MPCs from different tissue sources also identifies bone marrow-derived MPCs as the most potent MPCs in terms of multilineage differentiation and immunosuppression, two key requirements in cell-based regenerative medicine. In addition, this study implicates the significance of activin A as a functional marker of MPC identity.

Figure 1

Activin A expression profile, proliferative rate, and immunosuppressive characteristics of MPCs from different tissue sources. (a) ELISA quantification of secreted activin A. Results are expressed as the mean of secretion in 48-hour supernatants of three cell cultures (mean ± S.D.). (b) Activin A:follistatin ratio by real-time RT-PCR. (c) Cell-proliferation rate determined by using CCK-8. BM-MPCs, T-MPCs, M-MPCs, and DP-MPCs were plated in 96-well plates at 1 × 10⁴ cells per well and cultured in growth medium for 48, 96, 144, and 240 hours. (d) Proliferative activity of T cells stimulated with PHA. Responding PBMCs were stimulated with PHA at 5 μg/ml in the presence or absence of 5 × 10⁴ MPCs. The proliferative response of T cells is represented as a percentage of PHA control (mean ± SD of triplicates). *P ≤ 0.05, versus BM-MPCs; **P ≤ 0.05 versus PHA.

Figure 2

In vitro-induced differentiation of MPCs from different tissue sources. (a-d) Chondrogenic differentiation evaluated after 21 days in micropellet culture. (a) Immunohistochemical analysis of cartilage markers (COL2 and AGN) and alcian blue (AB) and picrosirius (PS) staining in pellet cultures of MPCs. (d) Expression of COL2, a specific marker for chondrogenesis, measured in differentiated MPC cultures compared with day 0 (D0) cultures, determined by quantitative RT-PCR with GAPDH as an internal control. (b-e) Osteogenic differentiation of MPCs assessed after 21-day culture in osteogenic medium. (b) All MPCs tested exhibited ALP activity and produced a mineralized matrix stained with alizarin red. T-MPCs displayed less alizarin red staining compared with BM-MPCs, M-MPCs, and DP-MPCs. (e) Significant upregulation of the expression of osteocalcin (OC), an osteoblast-specific gene, compared with undifferentiated MPCs was observed after osteogenic induction of BM-MPCs, T-MPCs, M-MPCs, and DP-MPCs compared with D0 cultures. (c-f) Adipogenic differentiation of MPCs cultured in adipogenic medium for 21 days. (c) All MPCs tested exhibited lipid-containing intracellular vacuoles that stained with oil red O at the end of the differentiation period. (f) MPCs cultured in adipogenic medium upregulated the expression of adipocyte-specific genes (PPAR-γ and LPL) compared with MPCs at D0. Quantitative RT-PCR data represent the mean ± SD in three independent experiments. *P ≤ 0.05, versus D0; **P ≤ 0.05, versus BM-MPCs.

Figure 3

Role of activin A in MPC multipotency. BM-MPCs transiently transfected either with control siRNA or with activin A siRNA were analyzed. (a) Activin A secretion level in the supernatant of transfected BM-MPCs quantified by ELISA 3 days (D3) after transfection showed the efficiency of knockdown by using activin A siRNA. (b, c) Effect of activin A knockdown on BM-MPC and T-MPC chondrogenic potential. Chondrogenic differentiation was evaluated after 21 days in micropellet culture. (b) Immunohistochemical analysis of cartilage markers (COL2 and AGN) in pellet cultures of BM-MPCs and T-MPCs as well as alcian blue (AB) and picrosirius red (PS) staining revealed that, compared with the untransfected and Control siRNA, MPCs transfected with activin A siRNA exhibited less chondrogenic potential. (c) Quantitative RT-PCR also showed decreased expression of COL2 in MPCs transfected with activin A siRNA, compared with the cells transfected with control siRNA (c). (b, c) Effect of activin A knockdown on BM-MPC osteogenic potential. Both the expression levels of the osteogenic markers (OC and ALP), determined by quantitative RT-PCR, and alizarin red staining, revealed a decrease in the osteogenic activity of BM-MPCs with activin A knockdown. (b, c) Effect of activin A knockdown on BM-MPC adipogenic potential. Both expression levels of the adipogenic markers (PPAR-γ and LPL), determined by quantitative RT-PCR and oil red O staining, revealed an increase in the adipogenic potential of BM-MPCs with activin A knockdown, compared with BM-MPCs transfected with the irrelevant Control siRNA. RT-PCR data represent the mean ± SD of three independent experiments. *P ≤ 0.05, versus Control siRNA.

Figure 4

Relation between Sox2 expression levels and activin A:follistatin expression ratio in MPCs from different tissue sources. (a) Western blot analysis of Sox2 expression in BM-MPCs, T-MPCs, M-MPCs, and DP-MPCs before and after a 48-hour treatment with 10 ng/ml of activin A. (b) Activin A:follistatin expression ratio determined by quantitative RT-PCR in BM-MPCs before (day 0 (0)) and after their differentiation along chondrogenic, osteogenic, and adipogenic lineages. (c) mRNA expression levels of Sox2 in undifferentiated (day 0 (0)) and chondro-, osteo- and adipo-differentiated BM-MPCs. The values at day 0 (0) corresponding to the mRNA ratio of activin A:follistatin (b) to the mRNA level of Sox2 (c) relative to GAPDH were assigned the values of 100% ± SD. *P ≤ 0.05, versus Day 0.