Aims/Introduction: Adipose-derived mesenchymal stem cell (ASC) transplantation is a promising therapy for diabetic nephropathy (DN). However, intravascular administration of ASCs is associated with low engraftment in target organs. Therefore, we considered applying the cell sheet technology to ASCs. In this study, ASC sheets were directly transplanted into the kidneys of a DN rat model, and therapeutic consequences were analyzed. Materials and Methods: Adipose-derived mesenchymal stem cells were isolated from adipose tissues of 7-week-old enhanced green fluorescent protein rats, and ASC sheets were prepared using a temperature-responsive culture dish. A DN rat model was established from 5-week-old Spontaneously Diabetic Torii fatty rats. Seven-week-old DN rats (n = 21) were assigned to one of the following groups: sham-operated (n = 6); ASC suspension (6.0 9 10 6 cells/mL) administered intravenously (n = 7); six ASC sheets transplanted directly into the kidney (n = 8). The therapeutic effect of the cell sheets was determined based on urinary biomarker expression and histological analyses. Results: The ASC sheets survived under the kidney capsule of the DN rat model for 14 days after transplantation. Furthermore, albuminuria and urinary tumor necrosis factor-a levels were significantly lower in the ASC sheets transplanted directly into the kidney group than in the sham-operated and ASC suspension administered intravenously groups (P < 0.05). Histologically, the ASC sheets transplanted directly into the kidney group presented mild atrophy of the proximal tubule and maintained the renal tubular structure. Conclusions: Transplantation of ASC sheets directly into the kidney improved transplantation efficiency and suppressed renal injury progression. Therefore, the ASC sheet technology might be a promising novel treatment for DN.Although strict glycemic control is reported to suppress the progression of DN 6,7 , there is no treatment to stop DN progression. Recently, mesenchymal stem cell (MSC) transplantation has received substantial attention as a therapy for DN. MSCs can differentiate into adipogenic and osteogenic cells 8 , and possess clinically useful properties, such as low antigenicity 9,10 and paracrine effects through cytokines 11,12 . In particular, adipose-derived mesenchymal stem cells (ASCs) represent an attractive cell source owing to their abundance in the body 13,14 .In animal experiments on DN, intravascular administration of ASC suspensions was shown to suppress the progression of

Renal fibrosis is one of the largest global health care problems, and microvascular (MV) injury is important in the development of progressive fibrosis. Although conventional cell therapy suppresses kidney injury via the role of vasoprotective cytokines, the effects are limited due to low retention of administered cells. We recently described that transplantation of hepatocyte growth factor (HGF)transgenic mesothelial cell sheets showed a remarkable cell survival and strong therapeutic effects in a rat renal fibrosis model. Due to the translational hurdles of transgenic cells, we here applied this technique for allogeneic transplantation using rat bone marrow mesenchymal stromal cells (MSCs). MSC sheets were transplanted onto the kidney surface of a rat renal ischemia-reperfusion-injury model and the effects were compared between those in untreated rats and those receiving intravenous (IV) administration of the cells. We found that donor-cell survival was superior in the cell sheet group relative to the IV group, and that the cell sheets secreted HGF and vascular endothelial growth factor (VEGF) up to day 14. Transplantation of cell sheets increased the expression of activated HGF/VEGF receptors in the kidney. There was no evidence of migration of transplanted cells into the kidney parenchyma. Additionally, the cell sheets significantly suppressed renal dysfunction, MV injury, and fibrosis as compared with that observed in the untreated and IV groups. Furthermore, we demonstrated that the MSC sheet protected MV density in the whole kidney according to threedimensional microcomputed tomography. In conclusion, MSC sheets strongly prevented renal fibrosis via MV protection, suggesting that this strategy represents a potential novel therapy for various kidney diseases. STEM CELLS TRANSLATIONAL MEDICINE 2019;8:1330-1341 SIGNIFICANCE STATEMENTCell therapy has been applied to various diseases that are resistant to conventional therapy. Renal fibrosis is one of the most difficult issues, and many researchers have tried intravascular administration of stem cells. The present study revealed that transplantation of mesenchymal stromal cell (MSC) sheets onto the kidney showed dramatic effects, including long-term cell survival, amelioration of kidney dysfunction, vasoprotection, and antifibrosis, compared with intravenous administration of MSCs. These results suggest that cell sheet technology improves the effects of cell therapy in various aspects. Therefore, cell sheet therapy could be a potential novel therapeutic approach for various kidney diseases in the future.

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Background Although hepatocyte growth factor (HGF) has antifibrotic effects and is involved in angiogenesis and vasodilation, systemic administration of HGF to prevent kidney fibrosis is not a feasible strategy for suppressing interstitial fibrosis in patients with CKD. Methods We investigated a novel therapy involving HGF transgenic cell sheets grown in culture from human mesothelial cells and administered to rats with unilateral ureteral obstruction (UUO). We compared progression of fibrosis in rats with UUO that received one of five interventions: HGF-transgenic mesothelial cell sheets transplanted to the kidney surface, HGF-transgenic mesothelial cell sheets transplanted to thigh, mesotherial cell sheets transplanted to kidney, no sheets, or HGF injections. Results HGF transgenic cell sheets transplanted to the kidney strongly suppressed the induction of myofibroblasts and collagen in the kidney for 28 days; other interventions did not. Additionally, the HGF-secreting cell sheets ameliorated loss of peritubular capillaries and maintained renal blood flow. Conclusions These findings suggest that cell sheet therapy is a novel and promising strategy for inhibiting progressive fibrosis in CKD.

Treatment using the cell sheet technology has been applied to various organs, including the cornea, heart, esophagus, periodontium, cartilage, middle ear, and lungs. It has been shown that the therapeutic efficacy of cell sheet transplantation involves 2 aspects, supplementation of cells and provision of cytokines to the affected organ. In addition, cell sheet transplantation also promotes repair of damage through the paracrine effects of cytokines derived from the transplanted cells. It is known that in cases of cell transplantation by injection, the transplanted cells are less likely to differentiate into renal tissue to supply cells, but repair is promoted by the actions of the transplanted cell-derived renotropic factors. Renal function requires functional conjugation of various tissues, including blood vessels, glomeruli, renal tubules, and collecting ducts. It is difficult to supply the necessary cells directly to the affected site of the renal tissue composed of complex structures. On the contrary, the 2-dimensional cell sheet can produce proteins such as erythropoietin, and is thus suitable for transplantation into the living body. It would be desirable to develop cell sheet therapy for the suppression of kidney damage in the future, taking advantage of the beneficial characteristics of cell sheets.