Studies focusing on the role of stem cells in bladder regeneration have mostly used bone marrow-derived stem cells. Furthermore, none of the earlier reports have discussed the transplantation of adipose-derived stem cell sheets generated in temperature-responsive culture dishes into rats, the evaluation of physiological functional recovery by cystometry, or confirmation of their long-term viability and differentiation. In this study, we investigated the effects of adipose-derived stem cell (ASC) sheets in a rat model of detrusor underactivity.

Mature male syngeneic Lewis rats were obtained for ASC sheet production. To produce GFP-expressing ASC sheets, male Lewis rats [LEW-Tg(CAG-EGFP)1Ys] were used, which express GFP in tissue cells throughout the body under the control of the CAG promoter. ASC sheet transplantation was performed on 10-week-old female syngeneic Lewis rats. The experiments involving animals and genetic recombination were approved by the Research Promotion Organization of Tottori University (32-027, 20-Y-23).
Based on the intervention methods, the experimental animals were subdivided into four groups containing eight animals each: the first group underwent induction of bladder cryo-injury followed by application of ASC sheets to the injury site (cryo-injury + ASC sheet group); the second group underwent bladder cryo-injury induction but did not receive ASC sheets (cryo-injury group); the third group underwent the surgical procedure without cryo-injury induction or application of ASC sheets (sham operation group); and the fourth group did not undergo any treatment (control group).
The engineering of the ASC sheet involved washing of the adipose tissue obtained from inguinal subcutaneous fat tissue of male Lewis rats, followed by dissection into small pieces. ASCs were obtained via centrifugation and filtered through a mesh filter. The cell pellets were seeded into culture dishes. Subsequently, temperature-responsive culture dishes were used to prepare the cell sheets.
In this study, we used a rat bladder cryoinjury model. A cryo-injury procedure was performed on the bladder three days prior to ASC sheet transplantation. A small incision was made in the midline of the lower abdomen in the supine position and an aluminum rod, cooled with dry ice, was placed in contact with the anterior wall of the bladder to induce cryoinjury. This was followed by placing the bladder in the abdominal cavity, and closure of the wound. In the sham operation group, the bladder was similarly exposed, only saline was injected, and no cryo-injury was administered.
Three days after cryo-injury induction, the rats were anesthetized with isoflurane, as described above, and a midline incision was made in the lower abdomen to expose the damaged part of the bladder. In the cryo-injury + ASC sheet group, the ASC sheet was applied to the cryo-injured area. For the cryo-injury and sham operation groups, the bladder was re-exposed, and saline was injected into the bladder; however, the ASC sheet was not applied. Finally, the wound was closed and the operation was completed. Seven days after these surgeries, the rats were euthanized after cystometry, and their bladders were removed for histological and reverse transcription polymerase chain reaction (RT-PCR) analyses.
To confirm the viability and differentiation of ASC sheets, GFP-expressing ASC sheets were prepared from inguinal adipose tissue of male LEW-Tg (CAG-EGFP)1Ys rats using the same method. In accordance with the procedure described above, cryo-injury was induced in 25 female Lewis rats, and three days later, GFP-expressing ASC sheets were transplanted into the bladder. Five rats were euthanized at 3, 7, 14, 21, and 28 days after implantation. The abdomen of euthanized Lewis rats was cut open and observed under a fluorescent stereomicroscope to confirm ASC sheet viability under excitation light. The bladder was removed and fixed in 10% formalin. Double immunofluorescence staining for GFP and vWF was performed.

・Cystometry
The results of cystometry evaluation are shown in Fig. . No significant differences were found in the maximum intravesical pressure, injection volume, and voided volume between the four groups; however, significant differences were evident in the pressure threshold (P=0.016), residual volume (P=0.011), voiding efficiency (P=0.020), and percentage of urine overflow (P=0.001) (Fig. 1).

・Histopathology
Acute inflammatory findings were observed in the cryo-injury group based on histopathological analysis. In contrast, in the cryo-injury + ASC sheet group, chronic inflammation caused by infiltration of inflammatory cells was observed; however, the degree of tissue necrosis was low, and granuloma formation, which occurs during the wound healing process, was also confirmed. Furthermore, the bladder tissue from each group was stained with the anti-vWF antibody, and a comparison of the mean number of capillaries in the 4× field of view was performed between the groups. The number of capillaries was significantly higher in the cryo-injury + ASC sheet group than that in the other groups.

・RT-PCR analysis
The mRNA levels of VEGF and HGF were significantly higher in the cryo-injury + ASC sheet group in comparison to the cryo-injury group (P=0.045 and P=0.037 respectively). The four groups showed no significant differences in bFGF levels, although the mRNA expression was comparatively higher in the cryo-injury + ASC sheet group.

・Immunofluorescence based analysis of GFP
Following application of GFP-expressing ASC sheets, green fluorescence of the sheets was observed under excitation light. In the anterior bladder wall, green fluorescence was observed in all rats up to day 14 (100%) and in four out of five rats on day 21 (80%). At 28 days post-implantation, fluorescence was observed in one of five rats (20%). At every time point, green fluorescence was confined only to the bladder implantation site and was not visible in the other organs. Immunostaining of the removed bladder showed many GFP-positive cells at the ASC sheet application site. On day 3, double immunofluorescence staining showed that GFP-positive cells did not merge with the vWF-positive cells. However, 7 days after sheet transplantation, the GFP-positive cells merged with vWF-positive cells, indicating that the cells had differentiated into blood vessels (Fig. 2).

Transplantation of ASC sheets after cryo-injury resulted in recovery of bladder contraction at a relatively early stage. Several mechanisms have been proposed for stem cell wound healing, including differentiation, homing, and immunoregulation.[1] A large body of evidence suggests that neovascularization induction is strongly involved in tissue repair and wound healing in ASCs. Recently, it has been reported that ASCs secrete factors such as VEGF, PDGF, IGF, HGF, b-FGF, SDF-1, TGF-β, and GDF11, which promote the differentiation of ASCs and fibroblasts into endothelial cells.[2, 3]  Furthermore, this study showed that ASCs marked with GFP differentiated into cells that provided structural elements of the capillary system and survived for up to 28 days after transplantation. As a result, stable blood perfusion to the injured bladder wall is maintained, and acute inflammation is suppressed, suggesting that these combined effects may lead to a relatively early recovery of injured bladder function.

Adipose-derived stem cell (ASC) sheets transplanted into the bladder of cryo-injured rats differentiated into blood vessels and restored bladder contractile function seven days after transplantation.

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Zhou X, et al. Multiple Injections of Autologous Adipose-Derived Stem Cells Accelerate the Burn Wound Healing Process and Promote Blood Vessel Regeneration in a Rat Model. Stem Cells Dev, 2019, 28(21): 1463-72
Liang J, et al. Safety analysis in patients with autoimmune disease receiving allogeneic mesenchymal stem cells infusion: a long-term retrospective study. Stem Cell Res Ther, 2018, 9(1): 312

Continence 2S2 (2022) 100216
DOI: 10.1016/j.cont.2022.100216