Construction of vascular tissues with macro-porous nano-fibrous scaffolds and smooth muscle cells enriched from differentiated embryonic stem cells

Abstract

Vascular smooth muscle cells (SMCs) have been broadly used for constructing tissue-engineered blood vessels. However, the availability of mature SMCs from donors or patients is very limited. Derivation of SMCs by differentiating embryonic stem cells (ESCs) has been reported, but not widely utilized in vascular tissue engineering due to low induction efficiency and, hence, low SMC purity. To address these problems, SMCs were enriched from retinoic acid induced mouse ESCs with LacZ genetic labeling under the control of SM22α promoter as the positive sorting marker in the present study. The sorted SMCs were characterized and then cultured on three-dimensional macro-porous nano-fibrous scaffolds in vitro or implanted subcutaneously into nude mice after being seeded on the scaffolds. Our data showed that the LacZ staining, which reflected the corresponding SMC marker SM22α expression level, was efficient as a positive selection marker to dramatically enrich SMCs and eliminate other cell types. After the sorted cells were seeded into the three-dimensional nano-fibrous scaffolds, continuous retinoic acid treatment further enhanced the SMC marker gene expression level while inhibited pluripotent maker gene expression level during the in vitro culture. Meanwhile, after being implanted subcutaneously into nude mice, the implanted cells maintained the positive LacZ staining within the constructs and no teratoma formation was observed. In conclusion, our results demonstrated the potential of SMCs derived from ESCs as a promising cell source for therapeutic vascular tissue engineering and disease model applications.

Figure 1. Sorting of SMCs from RA-induced SM22α^−/−

^LacZ ESCs. (A) Morphology change of SM22α^−/−LacZ ESCs under the treatment of DMSO (upper panel) or 10⁻⁵ M RA (lower panel) respectively as indicated time. (B) Comparison of β-gal staining of cells treated with either DMSO (left panel) or 10⁻⁵ M RA (right panel). The upper panel showed that β-gal staining positive cells accumulated with RA treatment time, while only sporadic β-gal staining positive cells existed in DMSO-treated cell population. The lower panel showed representative magnified images by day 8. Scale bar = 100 µm. (C–D) Using 5-chloromethylfluorescein di-β-D-galactopyranoside (FDG) to react with intracellular glutathione. In LacZ-positive cells derived from RA-induced SM22α^−/−LacZ ESCs, the FDG–glutathione adduct was converted to a bright green fluorescent product and LacZ-positive cells were subsequently sorted through a GFP channel (C) and cultured (D). Left panel: bright field; middle panel: GFP channel; right panel: merge. Scale bar = 100 µm. (E) Immunofluorescence staining of SMCs derived from sorted LacZ-positive cells with SMC-specific marker α-SMA antibody. The nuclei were co-stained with DAPI. Scale bar = 100 µm. (F) Gene expression of sorted cells analyzed by quantitative real-time PCR, compared to undifferentiated ESCs (control, Ctrl) or cells before sorting. Before: before sorting; After: after sorting. *p<0.05.

Figure 2. Representative SEM micrographs of NF scaffold and sorted SMCs seeded on the scaffold.

(A–B) Low and high magnification images of the NF scaffold respectively. The cells were seeded and cultured for 24 hours and observed at low (C) or high (D) magnifications. Arrows indicate the cell aggregates inside the pores of scaffolds.

Figure 3. In vitro culture of sorted SMCs on 3D NF scaffolds.

(A) Gene expression profiles of sorted SMCs before (Ctrl) and after seeded on NF scaffolds and cultured under the treatment of RA or DMSO for 2 weeks, respectively. (B) Histology of constructs cultured for 2 weeks with H–E staining. Left panel: DMSO treatment; right panel: RA treatment. (C) Immunofluorescence staining of α-SMA for constructs cultured for 2 weeks. Upper panel: DMSO treatment; Lower panel: RA treatment. Red: positive α-SMA expression; Blue: nuclei. Scale bar = 50 µm. *p<0.05.

Figure 4. Histology of constructs implanted subcutaneously for 2 weeks.

(A) β-gal staining. (Left) blank scaffold implants; (Right) cell-scaffold construct implants. Blue: positive LacZ staining; Red: nuclei. (B) H&E staining of cell-scaffold construct implants. Scale bar = 200 µm.