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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

Clonal and Differentiation Potentials Are Higher in Enriched α-SMA-Negative Than in α-SMA-Positive hMSC Populations(A) hMSCs spontaneously acquire the MF phenotype, resulting in a heterogeneous population of α-SMA-positive (red) and -negative cells (nuclei only, blue). The scale bar represents 50 μm. The cell spreading area was measured as a function of α-SMA expression.(B) Heterogeneous hMSC populations were sorted into small (lower 25%) and large hMSCs (upper 25%) using forward scatter in FACS and then immunostained for α-SMA in suspension or analyzed by western blotting.(C) Small size-sorted hMSC, now defined as “SMA(−)” and large-size sorted “SMA(+)” hMSCs, were cultured for 1 day on stiff substrates to assess α-SMA (red) and stress fibers (F-actin, green). Transcript levels were quantified for pro-fibrotic markers ACTA2 (α-SMA), TGFB2 (TGF-β2), COL1A1 (collagen type I), COL3A1 (collagen type III), and CTGF (CCN2) using quantitative (q)RT-PCR.(D and E) Sorted SMA(−) and SMA(+) hMSCs were assessed for clonogenicity in functional CFU-F and single cell cloning assays and assessed for (E) levels of self-renewal markers OCT4, SOX2, and DNMT1.(F) Sorted SMA(−) and SMA(+) hMSCs were lineage-induced in adipogenic and osteogenic induction medium and assessed for adipogenesis using oil red O staining and qRT-PCR analysis for PPARG and osteogenesis using Alizarin Red S staining and qRT-PCR analysis for RUNX2.The scale bars represent (A) 50 μm and (C and F) 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 S2.
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fig2: Clonal and Differentiation Potentials Are Higher in Enriched α-SMA-Negative Than in α-SMA-Positive hMSC Populations(A) hMSCs spontaneously acquire the MF phenotype, resulting in a heterogeneous population of α-SMA-positive (red) and -negative cells (nuclei only, blue). The scale bar represents 50 μm. The cell spreading area was measured as a function of α-SMA expression.(B) Heterogeneous hMSC populations were sorted into small (lower 25%) and large hMSCs (upper 25%) using forward scatter in FACS and then immunostained for α-SMA in suspension or analyzed by western blotting.(C) Small size-sorted hMSC, now defined as “SMA(−)” and large-size sorted “SMA(+)” hMSCs, were cultured for 1 day on stiff substrates to assess α-SMA (red) and stress fibers (F-actin, green). Transcript levels were quantified for pro-fibrotic markers ACTA2 (α-SMA), TGFB2 (TGF-β2), COL1A1 (collagen type I), COL3A1 (collagen type III), and CTGF (CCN2) using quantitative (q)RT-PCR.(D and E) Sorted SMA(−) and SMA(+) hMSCs were assessed for clonogenicity in functional CFU-F and single cell cloning assays and assessed for (E) levels of self-renewal markers OCT4, SOX2, and DNMT1.(F) Sorted SMA(−) and SMA(+) hMSCs were lineage-induced in adipogenic and osteogenic induction medium and assessed for adipogenesis using oil red O staining and qRT-PCR analysis for PPARG and osteogenesis using Alizarin Red S staining and qRT-PCR analysis for RUNX2.The scale bars represent (A) 50 μm and (C and F) 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 S2.

Mentions: To answer whether loss of hMSC stem cell features on stiff substrates is a consequence of MF activation and α-SMA expression, we sorted α-SMA-positive and α-SMA-negative hMSC from heterogeneous hMSC using flow cytometry (Figure 2A). Because no cell surface marker reliably identifies MFs, we sorted based on the observation that α-SMA-expressing hMSCs exhibited about six times larger spreading area than α-SMA-negative cells (Figure 2A). After live-sorting the lower and higher quartile of hMSCs by size, the large-size-sorted fraction contained no α-SMA-negative cells, whereas only 2.3% of the small-sorted population were α-SMA-positive (Figure 2B). For the remainder of this study, size-sorted hMSCs were thus termed SMA(−) (small) and SMA(+) (large). The pro-fibrotic character of SMA(+) hMSCs extended beyond expression of α-SMA as shown by higher mRNA levels of pro-fibrotic markers ACTA2, COL1A1, COL3A1, CTGF, and TGFB2 as compared to SMA(−) hMSCs (Figure 2C).


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)

Clonal and Differentiation Potentials Are Higher in Enriched α-SMA-Negative Than in α-SMA-Positive hMSC Populations(A) hMSCs spontaneously acquire the MF phenotype, resulting in a heterogeneous population of α-SMA-positive (red) and -negative cells (nuclei only, blue). The scale bar represents 50 μm. The cell spreading area was measured as a function of α-SMA expression.(B) Heterogeneous hMSC populations were sorted into small (lower 25%) and large hMSCs (upper 25%) using forward scatter in FACS and then immunostained for α-SMA in suspension or analyzed by western blotting.(C) Small size-sorted hMSC, now defined as “SMA(−)” and large-size sorted “SMA(+)” hMSCs, were cultured for 1 day on stiff substrates to assess α-SMA (red) and stress fibers (F-actin, green). Transcript levels were quantified for pro-fibrotic markers ACTA2 (α-SMA), TGFB2 (TGF-β2), COL1A1 (collagen type I), COL3A1 (collagen type III), and CTGF (CCN2) using quantitative (q)RT-PCR.(D and E) Sorted SMA(−) and SMA(+) hMSCs were assessed for clonogenicity in functional CFU-F and single cell cloning assays and assessed for (E) levels of self-renewal markers OCT4, SOX2, and DNMT1.(F) Sorted SMA(−) and SMA(+) hMSCs were lineage-induced in adipogenic and osteogenic induction medium and assessed for adipogenesis using oil red O staining and qRT-PCR analysis for PPARG and osteogenesis using Alizarin Red S staining and qRT-PCR analysis for RUNX2.The scale bars represent (A) 50 μm and (C and F) 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 S2.
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fig2: Clonal and Differentiation Potentials Are Higher in Enriched α-SMA-Negative Than in α-SMA-Positive hMSC Populations(A) hMSCs spontaneously acquire the MF phenotype, resulting in a heterogeneous population of α-SMA-positive (red) and -negative cells (nuclei only, blue). The scale bar represents 50 μm. The cell spreading area was measured as a function of α-SMA expression.(B) Heterogeneous hMSC populations were sorted into small (lower 25%) and large hMSCs (upper 25%) using forward scatter in FACS and then immunostained for α-SMA in suspension or analyzed by western blotting.(C) Small size-sorted hMSC, now defined as “SMA(−)” and large-size sorted “SMA(+)” hMSCs, were cultured for 1 day on stiff substrates to assess α-SMA (red) and stress fibers (F-actin, green). Transcript levels were quantified for pro-fibrotic markers ACTA2 (α-SMA), TGFB2 (TGF-β2), COL1A1 (collagen type I), COL3A1 (collagen type III), and CTGF (CCN2) using quantitative (q)RT-PCR.(D and E) Sorted SMA(−) and SMA(+) hMSCs were assessed for clonogenicity in functional CFU-F and single cell cloning assays and assessed for (E) levels of self-renewal markers OCT4, SOX2, and DNMT1.(F) Sorted SMA(−) and SMA(+) hMSCs were lineage-induced in adipogenic and osteogenic induction medium and assessed for adipogenesis using oil red O staining and qRT-PCR analysis for PPARG and osteogenesis using Alizarin Red S staining and qRT-PCR analysis for RUNX2.The scale bars represent (A) 50 μm and (C and F) 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 S2.
Mentions: To answer whether loss of hMSC stem cell features on stiff substrates is a consequence of MF activation and α-SMA expression, we sorted α-SMA-positive and α-SMA-negative hMSC from heterogeneous hMSC using flow cytometry (Figure 2A). Because no cell surface marker reliably identifies MFs, we sorted based on the observation that α-SMA-expressing hMSCs exhibited about six times larger spreading area than α-SMA-negative cells (Figure 2A). After live-sorting the lower and higher quartile of hMSCs by size, the large-size-sorted fraction contained no α-SMA-negative cells, whereas only 2.3% of the small-sorted population were α-SMA-positive (Figure 2B). For the remainder of this study, size-sorted hMSCs were thus termed SMA(−) (small) and SMA(+) (large). The pro-fibrotic character of SMA(+) hMSCs extended beyond expression of α-SMA as shown by higher mRNA levels of pro-fibrotic markers ACTA2, COL1A1, COL3A1, CTGF, and TGFB2 as compared to SMA(−) hMSCs (Figure 2C).

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