| Objective: In this study, we synthesized a biomimetic hydrogel(i.e. CS-IGF-1C) by immobilizing C domain of insulin-like growth factor(IGF-1) onto chitosan(CS). We determined the biocompatibility of the hydrogel as well as its impacts on mitogenic, anti-apoptotic, and pro-angiogenic characteristics of adipose-derived stem cells(ADSCs) in vitro. In a murine model of acute kidney injury(AKI), we transplanted ADSCs with CS-IGF-1C and investigated whether the hydrogel could improve ADSC survival and their transdifferentiation potential in vivo. Moreover, we attempted to elucidate the mechanism of increased beneficial effects of cell-based therapy for AKI. Our study provides scientific experimental basis for the use of tissue engineered approaches to promote kidney repair.Methods: 1. ADSCs were isolated from transgenic mice that express double-fusion protein green fluorescent protein(GFP)-firefly luciferase(Fluc). The expansive capacity of ADSCs was evaluated, with their transgenic properties assessed by fluorescent microscopy, flow cytometry(FCM), and bioluminescence imaging(BLI). In addition, FCM was employed to delineate the phenotypic profiling of ADSCs, and their multipotency was also appraised. 2. The C domain peptide of IGF-1(IGF-1C) was synthesized. Then IGF-1C was conjugated onto CS to obtain CS-IGF-1C. We used Fourier Transform infrared spectroscopy(FT-IR) to characterize the chemical structure, parallel plate rheometer to assess rheological properties, and scanning electron microscopy(SEM) to analyze the morphology of hydrogels. The gelation of CS and CS-IGF-1C was also examined. What’s more, culturing ADSCs on plates coated with hydrogels, cell morphology was observed and the cytocompatibility was assessed using live/dead staining. 3. Culturing ADSCs on plates coated with hydrogel, CCK-8 assay and BLI analysis were carried out to analyze cell expansion. Moreover, real-time RT-PCR was used to measure the expression of proliferation-related genes including HGF, IGF-1, and EGF. After ADSCs were challenged with H2O2, the BLI and Annexin V/PI staining were used to assess cell apoptosis, and the real-time RT-PCR was performed to measure the expression of apoptosis-related genes, such as Bax/Bad, Caspase-9/-3, and Fas/Fasl. In addition, real-time RT-PCR was also used to measure the expression of angiogenesis-related genes including Ang-1/-2, VEGF-A, HIF-1α, PLGF, PDGF-BB, b-FGF, and CCL5. 4. We induced acute renal ischemia/reperfusion injury in FVB mice. The animals were grouped into sham, PBS, CS, ADSCs, ADSCs/CS, and ADSCs/CS-IGF-1C. The BLI was used to evaluate survival of transplanted ADSCs. Periodic acid-Schiff(PAS) staining was conducted and the serum levels of creatinine and blood urea nitrogen(BUN) were measured to assess renal structural/functional recovery. Moreover, the effects of hydrogel on ADSC differentiation towards tubular epithelial cells and vascular endothelial cells were investigated 14 days after AKI. PCNA staining was used to evaluate renal cell proliferation at 3 days and the number of proliferative cells within hydrogel-injection regions and adjacent regions at 28 days. On the other hand, we performed TUNEL and Bax staining to measure renal cell apoptosis 3 days after injury. Additionally, CD31 and α-SMA staining were conducted to examine renal angiogenesis at different time points after AKI. CD31 staining was also used to appraise capillary ingrowth within hydrogel-injected regions at 14 days. Furthermore, VEGF-R2-luc transgenic mice in the C57BL/6J background were subjected to AKI, and the animals were grouped into sham, PBS, CS, ADSCs, ADSCs/CS, ADSCs/CS-IGF-1C. After injury, BLI was used to monitor VEGF-R2 gene expression in real time in order to elucidate the mechanism of stimulated neovascularization. In addition, we performed Masson’s trichrome, Picrosirius red, as well as collagen IV staining to examine collagen deposition, α-SMA staining to evaluate myofibroblast infiltration 2 months after AKI. MMP-2 immunostaining was also used to assess its expression in kidney. What’s more, real-time PCR was also performed to measure the expression of pro-fibrotic genes in renal tissue including TGF-β, Col1A1, and α-SMA.Results: 1. In in vitro culture, ADSCs possess robust expansive potential and GFP expression. BLI analysis revealed a strong correlation between cell number and Fluc activity(R2 = 0.99). ADSCs expressed surface markers of mesenchymal stem cells, including CD29, CD44, CD73, CD90, and CD105, while they were negative for the markers of hematopoietic cells, such as CD31, CD34, and CD45. In addition, these cells can be induced to differentiate into adipocytes, osteoblasts, and chondrocytes. 2. FT-IR analysis confirmed successful synthesis of CS-IGF-1C. Relological studies showed that the gelation temperature of CS and CS-IGF-1C hydrogels was between 35°C to 36°C. The SEM results demonstrated that the hydrogels possessed homogeneous and interconnected pores with averaged pore size of 50μm-100μm. In addition, both hydrogels exhibited excellent cytocompatibility. 3. When culturing ADSCs in hydrogel-coated plates, CS-IGF-1C increased cell expansive capacity and upregulated the expression of proliferation-related genes. Moreover, CS-IGF-1C hydrogel protected against H2O2-induced injury and downregulated the expression of apoptosis-related genes, in contrast, increased the expression of angiogenesis-related genes. 4. CS-IGF-1C hydrogel improved in vivo retention of ADSCs in injured kidney, and facilitated their transdifferentiation to tubular epithelial cells and vascular endothelial cells, thereby accelerating renal structural/functional recovery. In addition, ADSCs/CS-IGF-1C transplantation increased renal cell proliferation, decreased cell apoptosis, and promoted neovascularization at early period after AKI. BLI analysis revealed that the expression of VEGF-R2 in ADSCs/CS-IGF-1C-treated mice was significantly higher than that in other groups. Furthermore, the number of capillaries and proliferative cells within ADSCs/CS-IGF-1C-injected regions was markedly higher than other groups, indicating interaction between ADSCs and hydrogel. In addition, ADSCs/CS-IGF-1C treatment remarkably reduced collagen deposition, myofibroblast infiltration, and MMP-2 expression in damaged kidney at 2 months. Moreover, such treatment downregulated the expression of pro-fibrotic genes.Conclusions: 1. We developed an injectable thermosensitive biomimetic CS-IGF-1C hydrogel. 2. The CS-IGF-1C hydrogel is biocompatible and exerted pro-proliferative, anti-apoptotic, and pro-angiogenic effects on cultured ADSCs in vitro. 3. When co-transplanted into post-ischemic kidney, CS-IGF-1C hydrogel protected delivered ADSCs, enhanced their multi-differentiation potential, and promoted renal recovery. 4. These benefits can be attributed to the favorable niche produced by cell-hydrogel interplay which led to enhanced proliferation and reduced apoptosis of renal cells, stimulated angiogenesis as well as attenuated fibrosis. |
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