Therapeutic effect of adipose stromal vascular fraction spheroids for partial bladder outlet obstruction induced underactive bladder

Abstract

Background: Underactive bladder (UAB) is a common clinical problem but related research is rarely explored. As there are currently no effective therapies, the administration of adipose stromal vascular fraction (ad-SVF) provides a new potential method to treat underactive bladder.

Methods: Male Sprague-Dawley rats were induced by partial bladder outlet obstruction (PBOO) for four weeks and randomly divided into three groups: rats treated with PBS (Sham group); rats administrated with ad-SVF (ad-SVF group) and rats performed with ad-SVF spheroids (ad-SVFsp group). After four weeks, urodynamic studies were performed to evaluate bladder functions and all rats were sacrificed for further studies.

Results: We observed that the bladder functions and symptoms of UAB were significantly improved in the ad-SVFsp group than that in the Sham group and ad-SVF group. Meanwhile, our data showed that ad-SVF spheroids could remarkably promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation in bladder tissue than that in the other two groups. Moreover, ad-SVF spheroids increased the expression levels of bFGF, HGF and VEGF-A than ad-SVF. IVIS Spectrum small-animal in vivo imaging system revealed that ad-SVF spheroids could increase the retention rate of transplanted cells in bladder tissue.

Conclusions: Ad-SVF spheroids improved functions and symptoms of bladder induced by PBOO, which contributes to promote angiogenesis, suppress cell apoptosis and stimulate cell proliferation. Ad-SVF spheroids provide a potential treatment for the future patients with UAB.

Keywords: Bladder outlet obstruction; Spheroid; Stromal vascular fraction; Underactive bladder.

Fig. 1

Characterization of ad-SVF and ad-SVF spheroids. A Representative flow cytometry histograms of ad-SVF. B Representative images of CM-DiI (red) labelled ad-SVF spheroids and nuclear marked DAPI (blue). Ad-SVF spheroids culture at day 3 (brightfield). C Representative images of CM-DiI labelled ad-SVF spheroids and nuclear marked DAPI (cross section). Scale bar = 100 μm

Fig. 2

Enhanced expression of growth factors in ad-SVF spheroids. A–C Profiles for the cumulative release of bFGF, HGF, and VEGF-A from ad-SVF and ad-SVF spheroids as determined by ELISA at 24, 48, 72 h after seeding. D, E Relative abundance of bFGF/GAPDH, HGF/GAPDH, and VEGF-A/GAPDH were quantified according to western blots at day 3. F Representative images of migration effect on HUVEC. G Quantitative analysis of the mean number of migrated cells per field was performed. H Live/Dead fluorescence imaging of ad-SVF and ad-SVF spheroids. I Quantitative analysis of cell viability between ad-SVF and ad-SVF spheroids. Scale bar = 100 μm. *p < 0.05 (ad-SVF vs ad-SVFsp)

Fig. 3

Ad-SVF spheroids promoted angiogenesis. A Representative images of tube formation assay under different culture medium. B–E Quantification of total length, total segment length, number of branches, and number of nodes were analyzed by ImageJ’s angiogenesis plugin. F Representative images of CM-DiI labelled cells (red), the expression of CD31 by immunofluorescence (green), and nuclear marked DAPI (blue) in Matrigel of each group (Arrows represent vessel). G, H Semi-quantitative analysis of the mean percentage of CD31 positive cells and the mean fluorescent intensity of CD31. Scale bar = 100 μm. *p < 0.05 (ad-SVF, ad-SVFsp vs Sham); ^#p < 0.05 (ad-SVFsp vs ad-SVF)

Fig. 4

Ad-SVF spheroids improved bladder functions. A Representative images of urodynamics recording in each group (the peaks mean maximum pressure during bladder contraction). B Statistical chart of MBC in each group. C Statistical chart of PVR in each group. D Statistical chart of Pmax in each group. E Representative images of isolated bladder. F Statistical chart of bladder wet weight in each group. *p < 0.05 (ad-SVF, ad-SVFsp vs Sham); ^#p < 0.05 (ad-SVFsp vs ad-SVF)

Fig. 5

Ad-SVF spheroids attenuated the damage of underactive bladder. A–C Representative images of HE, Masson, and CD31 staining in each group (black arrows indicate small blood vessels). D, E Semi-quantitative analysis of the percentage of collagen fibers and MVD CD31 was performed by using Image J software. Scale bar = 100 μm. *p < 0.05 (ad-SVF, ad-SVFsp vs Sham); ^#p < 0.05 (ad-SVFsp vs ad-SVF)

Fig. 6

Ad-SVF spheroids promoted cell proliferation in underactive bladder. A, B Representative images of CM-DiI labelled cells (red), the expression of Ki67 by immunofluorescence (green), and nuclear marked DAPI (blue) in each group at 48 h and 72 h after cell transplantation. C, D Semi-quantitative analysis of the mean percentage of Ki67 positive cells at 48 h and 72 h after cell transplantation. Scale bar = 100 μm. *p < 0.05 (ad-SVF, ad-SVFsp vs Sham); ^#p < 0.05 (ad-SVFsp vs ad-SVF)

Fig. 7

Cell tracking in rats treated with CM-Dil and Dil-C18(5)-DS labelled ad-SVF or ad-SVF spheroids at 4 weeks after cell transplantation. A Representative images of CM-DiI labelled cells (red), the expression of CD31 by immunofluorescence (green), and nuclear marked DAPI (blue) in each group. (Arrows represent vessel). B, C Semi-quantitative analysis of the mean percentage of CD31 positive cells and the mean fluorescent intensity of CD31. D Representative fluorescence images of bladders in vivo imaging system. E Semi-quantitative of CM-Dil and Dil-C18(5)-DS labelled cells in bladder of each group. *p < 0.05 (ad-SVF, ad-SVFsp vs Sham); ^#p < 0.05 (ad-SVFsp vs ad-SVF)

Fig. 8

Effects of ad-SVF spheroids on cell apoptosis and proliferation. A Representative images of TUNEL staining in each group. B Representative images of PCNA staining in each group. C, D Semi-quantitative analysis of TUNEL-positive cells and PCNA-positive cells was performed by using Image J software. E Representative western blot images of AKT, pAKT, GSK-3β, pGSK-3β, and GAPDH. F Relative abundance of AKT/GAPDH, pAKT/GAPDH, GSK-3β/GAPDH and pGSK-3β/GAPDH were quantified according to western blots. Scale bar = 100 μm. *p < 0.05 (ad-SVF, ad-SVFsp vs Sham); ^#p < 0.05 (ad-SVFsp vs ad-SVF)