Cell sheet formation enhances the therapeutic effects of adipose-derived stromal vascular fraction on urethral stricture

Abstract

Urethral stricture (US) is a common disease in urology, lacking effective treatment options. Although injecting a stem cells suspension into the affected area has shown therapeutic benefits, challenges such as low retention rate and limited efficacy hinder the clinical application of stem cells. This study evaluates the therapeutic impact and the mechanism of adipose-derived vascular fraction (SVF) combined with cell sheet engineering technique on urethral fibrosis in a rat model of US. The results showed that SVF-cell sheets exhibit positive expression of α-SMA, CD31, CD34, Stro-1, and eNOS. In vivo study showed less collagen deposition, low urethral fibrosis, and minimal tissue alteration in the group receiving cell sheet transplantation. Furthermore, the formation of a three-dimensional (3D) tissue-like structure by the cell sheets enhances the paracrine effect of SVF, facilitates the infiltration of M2 macrophages, and suppresses the TGF-β/Smad2 pathway through HGF secretion, thereby exerting antifibrotic effects. Small animal in vivo imaging demonstrates improved retention of SVF cells at the damaged urethra site with cell sheet application. Our results suggest that SVF combined with cell sheet technology more efficiently inhibits the early stages of urethral fibrosis.

Keywords: Cell sheet; Fibrosis; Stem cell therapy; Stromal vascular fraction; Urethral stricture.

Schematic diagram of the anti-fibrotic and therapeutic effects of SVF-cell sheet on urethral stricture remodeling (By Figdraw).

Fig. 1

SVFs identification (A–B) Representative flow cytometry histograms of primary (A, P0) and first-generation (B, P1) adipose-derived stromal vascular fraction (SVF). Results indicated that they express hematopoietic (CD45 and CD34), mesenchymal (CD90 and CD106), endothelial (CD31 and VEGFR2), and T regulatory cell (CD4) markers. The blue lines represent isotype controls. (C) Determination and statistics of colony-forming units (CFU) of first-generation (P1) and second-generation (P2) SVF. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Cell sheet formation and identification. (A) A SVF-cell sheet was observed under an inverted microscope (scale bar: 100 μm). (B) SVF-cell sheet formation after 14 days of continuous culture. (C) H-E staining results of the SVF-cell sheet. The cell sheet consists of 3- or 4-layers of cells (scale bar: 50 μm). (D) SEM image of the SVF-cell sheet (scale bar: 10 μm). (E) The result of immunofluorescence showed positive staining of α-SMA, CD31, CD34, Stro-1, and eNOS in the SVF-cell sheet (scale bar: 50 μm).

Fig. 3

In vivo tracing and cellular localization of transplanted SVF cells. (A) Cell tracking of CM-Dil-labeled cells in penile urethral tissues from SVF group and Cell sheet group at 7, 14, and 28 days after US (scale bar: 50 μm). (B) Representative images of luciferase signals in the rats were indicated after transplantation of SVF suspension cells or SVF-cell sheets for 1, 4, and 17 days.

Fig. 4

Transplanted SVF cell sheets alleviate the progression of urethral fibrosis and promote structural repair. (A) Representative micro-ultrasound images of penile urethras in sham, US, SVF, and Cell sheet groups at 28 days after surgery. The arrows indicate the location of hyperechoic tissue and the narrowing of the urethral lumen. CS corpus cavernosum, U urethral lumen. (B–C) Representative microscopic results of hematoxylin and eosin (H&E) and Masson's trichrome staining of urethral tissue in sham, US, SVF, and Cell sheet groups at 28 days postoperatively (scale bar: 100 μm). US group urethral tissues showed densely arranged collagen bundles and sparse smooth muscle. The SVF and Cell sheet groups tissues showed mild submucosal fibrosis and less tissue alteration. Arrows indicate sites of collagen deposition. (D) Immunofluorescence analysis of AE1/AE3 in the repair site urethra (scale bar: 50 μm).

Fig. 5

Transplanted SVF-cell sheet reduces collagen expression and promotes angiogenesis in urethral stricture rats. (A–B) At 28 days after surgery, the expression of Collagen III, Collagen I, Smad2, Phospho-Smad2 (p-Smad2), VEGFA, and bFGF proteins in urethral tissues of Sham, US, SVF, and Cell sheet group was measured by western blot. Quantification was performed using the relative abundance data of the target proteins to GAPDH. (C) Gene expression of FN1, COL3A1, bFGF, CTGF, TNF-α, Gata3, and Txb21 in urethral tissues was measured by real-time PCR in Sham, US, SVF, and Cell sheet groups at 28 days after surgery. (D) Immunofluorescence staining of CD31 and CD34 expression in urethral tissues of Sham, US, SVF, and Cell sheet groups 28 days postoperatively. (scale bar: 50 μm). (E) Quantitative expression levels of CD31 and CD34 in the urethral after different treatments. Data are shown as mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6

SVF-cell sheet alleviates urethral fibrosis, which may be related to changes in the phenotypic distribution of periurethral macrophages. (A–B) Immunofluorescence staining of F4/80, CD86, and CD163 in urethral sections from Sham, US, SVF, and Cell sheet groups at 28 days after surgery (scale bar: 50 μm). (C) Semi-quantitative statistics of CD86⁻and CD206-positive cells. (D) Gene expression of Arg1 and Nos2 in urethral tissues was measured by real-time PCR in Sham, US, SVF, and Cell sheet groups 28 days after surgery. (E) SVF cells or SVF-cell sheets were co-cultured with LPS/IFN-γ-treated M1 macrophages, and RT-qPCR was performed to measure M1 macrophage markers and M2 macrophage markers at the mRNA level. Data are shown as mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7

TGF-β induced in vitro fibrosis model and three-dimensional cell sheet tissue-like structures promote enhanced expression of pro-regenerative cytokine genes, especially HGF. (A–C) The SV-HUC-1 human urinary tract epithelial cell line was treated with TGF-β1 at 5 ng/mL and constructed the co-culture system with SVF. The expression levels of Fibronectin, Collagen I and Collagen III were measured by western blot and qRT-PCR. (D) qRT-PCR results showed that gene expression of interaction-related proteins, including β-catenin (cell-cell interaction) and Connexin 43 (gap junction), was increased in the 3D-cell sheet group compared with the 2D-SVF group. (E) Quantitative gene expression of the pro-regenerative cytokines VEGF, HGF, and IL-10 was also increased, and bFGF gene expression was decreased in the 3D-cell sheet group. (F) The supernatants of 3D-SVF cell sheets and 2D-cultured SVF cells were taken and subjected to ELISA, which showed that 3D-SVF cell sheets secreted higher levels of HGF. (G) 28 days after transplantation of SVF-cell sheets, the supernatant of tissue homogenate from the transplanted area was taken for ELISA, and the results showed that the SVF-cell sheet group continuously secreted HGF. (H) Gene expression of HGF in urethral tissues was measured by real-time PCR in Sham, US, SVF, and Cell sheet groups 28 days after surgery. Data are shown as mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 8

SVF reverses TGF-β-induced fibrosis by HGF released through paracrine effects. (A–B) Recombinant HGF at different concentrations and treatment times was added to the in vitro fibrosis model. Protein expression levels of Fibronectin, Collagen III, p-Smad2, Smad2, and TGF-β1/2 were measured and quantified by Western blot. (C) FN, COL1A1, COL3A1, and ACTA2 gene expression levels were determined by qRT-PCR. (D) SVF cells treated with 1 μg/mL HGF-neutralizing antibody were co-cultured with TGF-β-stimulated SV-HUC-1. Protein expression levels of Fibronectin, Collagen III, p-Smad2, Smad2, and TGF-β1/2 were measured and quantified by Western blot. (E) FN, COL1A1, COL3A1, and ACTA2 gene expression levels were determined by qRT-PCR. Data are shown as mean ± SD.*P < 0.05, **P < 0.01, ***P < 0.001.