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Expression of α-Smooth Muscle Actin Determines the Fate of Mesenchymal Stromal Cells.

Talele NP, Fradette J, Davies JE, Kapus A, Hinz B - Stem Cell Reports (2015)

Bottom Line: Pro-fibrotic microenvironments of scars and tumors characterized by increased stiffness stimulate mesenchymal stromal cells (MSCs) to express α-smooth muscle actin (α-SMA).Sorted α-SMA-positive MSCs exhibited high contractile activity, low clonogenicity, and differentiation potential limited to osteogenesis.We propose that α-SMA mediated contraction plays a critical role in mechanically regulating MSC fate by controlling YAP/TAZ activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada.

No MeSH data available.


Related in: MedlinePlus

Soft Substrate Culture Restores Lineage Differentiation of SMA(+) hMSCs(A–D) Sorted SMA(+) hMSCs were cultured on stiff and soft substrates and assessed for MF activation after 5 days, clonogenicity after 10 days, and differentiation potential after 14 days. MF activation was assessed by (B) western blotting, (C) immunofluorescence for α-SMA (red) and stress fibers (F-actin, green) (the scale bar represents 20 μm), and (D) qRT-PCR for fibrotic markers.(E) Self-renewal potential was quantified from CFU-F assays and qRT-PCR analysis of OCT4, SOX2, and DNMT1 transcripts.(F) Sorted SMA(+) were grown for 5 days on stiff and soft culture substrates and then transferred to conventional culture dishes for induction into adipogenic lineage (oil red O, PPARG) and osteogenesis (Alizarin Red S, RUNX2). The scale bar represents 50 μm.The graphs show averages ± SD from at least five independent experiments (∗p ≤ 0.05, ∗∗p ≤ 0.005, and ∗∗∗p ≤ 0.0005 using Student’s t test). See also Figure S3.
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fig3: Soft Substrate Culture Restores Lineage Differentiation of SMA(+) hMSCs(A–D) Sorted SMA(+) hMSCs were cultured on stiff and soft substrates and assessed for MF activation after 5 days, clonogenicity after 10 days, and differentiation potential after 14 days. MF activation was assessed by (B) western blotting, (C) immunofluorescence for α-SMA (red) and stress fibers (F-actin, green) (the scale bar represents 20 μm), and (D) qRT-PCR for fibrotic markers.(E) Self-renewal potential was quantified from CFU-F assays and qRT-PCR analysis of OCT4, SOX2, and DNMT1 transcripts.(F) Sorted SMA(+) were grown for 5 days on stiff and soft culture substrates and then transferred to conventional culture dishes for induction into adipogenic lineage (oil red O, PPARG) and osteogenesis (Alizarin Red S, RUNX2). The scale bar represents 50 μm.The graphs show averages ± SD from at least five independent experiments (∗p ≤ 0.05, ∗∗p ≤ 0.005, and ∗∗∗p ≤ 0.0005 using Student’s t test). See also Figure S3.

Mentions: We next addressed whether expression of α-SMA in hMSCs hallmarks reversible MF activation or terminal fibrogenic differentiation by culturing pure SMA(+) hMSCs for 5 days on soft culture substrates. α-SMA protein levels and sizes of cell-ECM focal adhesions decreased with decreasing substrate stiffness (Figure S3). Cell culture on 3 kPa soft substrates resulted in 2-fold reduced expression of α-SMA (Figures 3A and 3B), disappearance of α-SMA from stress fibers (Figure 3C), and generally reduced levels of fibrotic marker transcripts compared with hMSC(+) grown on stiff substrates (Figure 3D). The culture time required to deactivate MFs mechanically was 5 days (Figure S4). Reversibility of the fibrotic phenotype was not dependent on the degree of MF pre-activation. SMA(+) hMSCs lost the MF phenotype on soft substrate even when being sorted from TGF-β1-pre-treated heterogeneous populations and were not re-activated on soft substrate by adding TGF-β1 (Figure S4).


Expression of α-Smooth Muscle Actin Determines the Fate of Mesenchymal Stromal Cells.

Talele NP, Fradette J, Davies JE, Kapus A, Hinz B - Stem Cell Reports (2015)

Soft Substrate Culture Restores Lineage Differentiation of SMA(+) hMSCs(A–D) Sorted SMA(+) hMSCs were cultured on stiff and soft substrates and assessed for MF activation after 5 days, clonogenicity after 10 days, and differentiation potential after 14 days. MF activation was assessed by (B) western blotting, (C) immunofluorescence for α-SMA (red) and stress fibers (F-actin, green) (the scale bar represents 20 μm), and (D) qRT-PCR for fibrotic markers.(E) Self-renewal potential was quantified from CFU-F assays and qRT-PCR analysis of OCT4, SOX2, and DNMT1 transcripts.(F) Sorted SMA(+) were grown for 5 days on stiff and soft culture substrates and then transferred to conventional culture dishes for induction into adipogenic lineage (oil red O, PPARG) and osteogenesis (Alizarin Red S, RUNX2). The scale bar represents 50 μm.The graphs show averages ± SD from at least five independent experiments (∗p ≤ 0.05, ∗∗p ≤ 0.005, and ∗∗∗p ≤ 0.0005 using Student’s t test). See also Figure S3.
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Related In: Results  -  Collection

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fig3: Soft Substrate Culture Restores Lineage Differentiation of SMA(+) hMSCs(A–D) Sorted SMA(+) hMSCs were cultured on stiff and soft substrates and assessed for MF activation after 5 days, clonogenicity after 10 days, and differentiation potential after 14 days. MF activation was assessed by (B) western blotting, (C) immunofluorescence for α-SMA (red) and stress fibers (F-actin, green) (the scale bar represents 20 μm), and (D) qRT-PCR for fibrotic markers.(E) Self-renewal potential was quantified from CFU-F assays and qRT-PCR analysis of OCT4, SOX2, and DNMT1 transcripts.(F) Sorted SMA(+) were grown for 5 days on stiff and soft culture substrates and then transferred to conventional culture dishes for induction into adipogenic lineage (oil red O, PPARG) and osteogenesis (Alizarin Red S, RUNX2). The scale bar represents 50 μm.The graphs show averages ± SD from at least five independent experiments (∗p ≤ 0.05, ∗∗p ≤ 0.005, and ∗∗∗p ≤ 0.0005 using Student’s t test). See also Figure S3.
Mentions: We next addressed whether expression of α-SMA in hMSCs hallmarks reversible MF activation or terminal fibrogenic differentiation by culturing pure SMA(+) hMSCs for 5 days on soft culture substrates. α-SMA protein levels and sizes of cell-ECM focal adhesions decreased with decreasing substrate stiffness (Figure S3). Cell culture on 3 kPa soft substrates resulted in 2-fold reduced expression of α-SMA (Figures 3A and 3B), disappearance of α-SMA from stress fibers (Figure 3C), and generally reduced levels of fibrotic marker transcripts compared with hMSC(+) grown on stiff substrates (Figure 3D). The culture time required to deactivate MFs mechanically was 5 days (Figure S4). Reversibility of the fibrotic phenotype was not dependent on the degree of MF pre-activation. SMA(+) hMSCs lost the MF phenotype on soft substrate even when being sorted from TGF-β1-pre-treated heterogeneous populations and were not re-activated on soft substrate by adding TGF-β1 (Figure S4).

Bottom Line: Pro-fibrotic microenvironments of scars and tumors characterized by increased stiffness stimulate mesenchymal stromal cells (MSCs) to express α-smooth muscle actin (α-SMA).Sorted α-SMA-positive MSCs exhibited high contractile activity, low clonogenicity, and differentiation potential limited to osteogenesis.We propose that α-SMA mediated contraction plays a critical role in mechanically regulating MSC fate by controlling YAP/TAZ activation.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Tissue Repair and Regeneration, Matrix Dynamics Group, Faculty of Dentistry, University of Toronto, Toronto, ON M5S 3E2, Canada.

No MeSH data available.


Related in: MedlinePlus