Human amniotic fluid stem cell differentiation along smooth muscle lineage

Abstract

Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-β1. Molecular assays revealed higher level of α smooth muscle actin (α-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the α-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.

Keywords: fetal cells; multipotent; myogenic; regenerative medicine; tissue engineering.

Figure 1.

A) Growth rate of hAFSCs and SMhAFSCs cultured for 7 d. *P < 0.05, **P < 0.01, ***P < 0.001. B) Phase-contrast images of hAFSCs and SMhAFSCs (21 d of differentiation; view ×20) C) mRNA expression of smooth muscle markers (αSMA, desmin, calponin, and myosin heavy chain) analyzed after 7, 11, 14, and 21 d by real-time PCR in hAFSCs, SMhAFSCs, and hBSMCs. Results for each condition are from triplicate experiments, and values are expressed in relative units as the mean. *P< 0.05, **P< 0.01, ***P< 0.001.

Figure 2.

Left panels: transmission electron microscopy in hAFSCs (A, B), SMhAFSCs (C, D), and SMCs (E, F). N, nucleus. Asterisks (*) indicate swollen mitochondria (A); hashtags (#) indicate glycogen (C); arrows indicate normal mitochondria (A) or thin filaments (D); arrowheads indicate dense bodies (D, F). Scale bars = 2 μm (A, C, E); 1 μm (B, D, F). Right panels: immunofluorescence analysis for α-SMA, desmin, and smoothelin. SMhAFSCs were positive for all 3 smooth muscle markers (H, K, N) compared to SMCs (I, J, O), while hAFSCs were negative (G, J, M). SMhAFSCs contained an increased presence of α-SMA positively stained microfilaments (dotted white lines), respectively, after 7 d (P) and 14 d (Q) of selective culture conditions.

Figure 3.

A) Uptake and metabolism of [U-¹³C]-glucose in hAFSCs and SMhAFSCs. B, C) ¹³CO₂ production present in culture medium (B) and released into the air contained in a sealed culture flask (C). SMhAFSCs demonstrated a higher production of ¹³CO₂ compared to hAFSCs. *P< 0.05. D) Proliferation assay (a.u., arbitrary units) after FBS starvation in hAFSCs, SMhAFSCs, and hBSMCs. All cell populations were seeded on differentiation medium (see Materials and Methods). Proliferation was assessed as total DNA/cells. Similar results were obtained by evaluating cell proliferation by means of total cells/well. Proliferation index is the ratio between the proliferative effect obtained in response to any stimulus and the proliferation of unstimulated control cells (cells grown in medium+1% FBS; proliferation index of 1.0). *P< 0.05, **P< 0.01, ***P< 0.001 vs. control unstimulated cells (ANOVA followed by Dunnet test).

Figure 4.

Patch-clamp analysis of SMhAFSCs. A, B) Voltage step stimulation protocol (A) employed to elicit outward currents in SMhAFSCs and the response to K⁺ channel blockers, 5 mM 4-AP and 2 mM TEA (B). C) Current-voltage relationships of peak (IPEAK) and steady-state (ISS) currents before and after application of 4-AP and TEA. *P< 0.05. D) hAFSC and SMhAFSC dimensions, estimated by measurements of cell membrane capacitance. **P< 0.01. E) Outward current traces recorded in SMhAFSCs before and after application of 1 μM carbachol. F) Current-voltage relationships of peak (IPEAK) and steady-state (ISS) currents before and after application of CCH. *P< 0.05, **P< 0.01.

Figure 5.

A, B) Time-lapse analysis under phase-contrast microscopy of a single hAFSC (A) and SMhAFSC (B) before and after 10⁻⁴ M carbachol stimulation. C) Cells were compared before and after contractile stimulation in both hAFSCs and SMhAFSCs, as reported in a surface/time chart with surface expressed as percentage of the initial cell surface. D) In a collagen lattice gel assay, macroscopic contractile activity was evaluated in both hAFSCs and SMhAFSCs after KCl (60 mM) stimulation. Gel with SMhAFSCs (gray line) had a significant reduction of gel area compared to hAFSCs (black line). Assay was performed in triplicate.