Mesenchymal stem cell (MSC)-derived exosomes have been recognized as new candidates for the treatment of degenerative diseases or injury and may provide an alternative to cell-based therapy. However, the compositions in MSC-derived exosomes are highly influenced by the microenvironment in which their original cells reside. Here, we hypothesized that a nitric oxide (NO)-releasing polymer can boost the proangiogenic compositions of exosomes and enhance their proangiogenic capacity. Our results demonstrated that exosomes, released from human placenta-derived MSCs (hP-MSCs) by NO stimulation, augment the angiogenic effects of human umbilical vein endothelial cells (HUVECs) in vitro. Moreover, exosomes released from hP-MSCs by NO stimulation revealed superior angiogenic effects and ameliorated limb function in a murine model of hind limb ischemia. Further analysis demonstrated that increased VEGF and miR-126 levels in exosomes released from hP-MSCs by NO stimulation were identified as a novel mechanism contributing to the increased capacity of these exosomes to promote angiogenic processes. In conclusion, designing specific microenvironments for in vitro stem cell culture, such as those containing bioactive material, will facilitate the development of customized exosomes encapsulating a beneficial composition of stem cells for cell-free therapeutic applications.

Islet transplantation is considered the most promising therapeutic option with the potential to cure diabetes. However, efficacy of current clinical islet transplantation is limited by long-term graft dysfunction and attrition. We have investigated the therapeutic potential of a silk fibroin macroporous (SF) scaffold for syngeneic islet transplantation in diabetic mice. The SF scaffold was prepared via lyophilisation, which enables incorporation of active compounds including cytokines, peptide and growth factors without compromising their biological activity. For the present study, a heparin-releasing SF scaffold (H-SF) in order to evaluate the versatility of the SF scaffold for biological functionalisation. Islets were then co-transplanted with H-SF or SF scaffolds in the epididymal fat pad of diabetic mice. Mice from both H-SF and SF groups achieved 100% euglycaemia, which was maintained for 1 year. More importantly, the H-SF-islets co-transplantation led to more rapid reversal of hyperglycaemia, complete normalisation of glucose responsiveness and lower long-term blood glucose levels. This superior transplantation outcome is attributable to H-SF-facilitated islet revascularisation and cell proliferation since significant increase of islet endocrine and endothelial cells proliferation was shown in grafts retrieved from H-SF-islets co-transplanted mice. Better intra-islet vascular reformation was also evident, accompanied by VEGF upregulation. In addition, when H-SF was co-transplanted with islets extracted from vegfr2-luc transgenic mice in vivo, sustained elevation of bioluminescent signal that corresponds to vegfr2 expression was collected, implicating a role of heparin-dependent activation of endogenous VEGF/VEGFR2 pathway in promoting islet revascularisation and proliferation. In summary, the SF scaffolds provide an open platform as scaffold development for islet transplantation. Furthermore, given the pro-angiogenic, pro-survival and minimal post-transplantation inflammatory reactions of H-SF, our data also support the feasibility of clinical implementation of H-SF to improve islet transplantation outcome.Statement of Significance1) The silk fibroin scaffold presented in the present study provides an open platform for scaffold development in islet transplantation, with heparinisation as an example.2) Both heparin and silk fibroin have been used clinically. The excellent in vivo therapeutic outcome reported here may therefore be clinically relevant and provide valuable insights for bench to bed translation.3) Compared to conventional clinical islet transplantation, during which islets are injected via the hepatic portal vein, the physical/mechanical properties of silk fibroin scaffolds create a more accessible transplantation site (i.e., within fat pad), which significantly reduces discomfort.4) Islet implantation into the fat pad also avoids an instant blood mediated inflammatory response, which occurs upon contact of islet with recipient’s blood during intraportal injection, and prolongs survival and function of implanted islets.Download high-res image (96KB)Download full-size image

Transplantation of endothelial cells (ECs) holds great promise for treating various kinds of ischemic diseases. However, the major challenge in ECs-based therapy in clinical applications is to provide high quality and enough amounts of cells. In this study, we developed a simple and efficient system to direct endothelial differentiation of mouse embryonic stem cells (ESCs) using a controllable chitosan nitric oxide (NO)-releasing hydrogel (CS-NO). ESCs were plated onto the hydrogel culture system, and the expressions of differentiation markers were measured. We found that the expression of Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) increased obviously under the controlled NO releasing environment. Moreover, the Flk-1 upregulation was accompanied by the activation of the phospho-inositide-3 kinase (PI3K)/Akt signaling. We also found that in the presence of the PI3K inhibitor (LY294002), the endothelial commitment of ESCs was abolished, indicating the importance of Akt phosphorylation in the endothelial differentiation of ESCs. Interestingly, in the absence of NO, the activation of Akt phosphorylation alone by using AKT activator (SC-79) did not profoundly promote the endothelial differentiation of ESCs, suggesting an interdependent relationship between NO and the Akt phosphorylation in driving endothelial fate specification of ESCs. Taken together, we demonstrated that NO releasing in a continuous and controlled manner is a simple and efficient method for directing the endothelial differentiation of ESCs without adding growth factors.Statement of SignificanceFascinating data continues to show that artificial stem cell niche not only serve as a physical supporting scaffold for stem cells proliferation, but also as a novel platform for directing stem cell differentiation. Because of the lack of proper microenvironment for generating therapeutic endothelial cells (ECs) in vitro, the source of ECs for transplantation is the major limitation in ECs-based therapy to clinical applications. The current study established a feeder cell-free, 2-dimensional culture system for promoting the differentiation processes of embryonic stem cells (ESCs) committed to the endothelial lineage via using a nitric oxide (NO) controlled releasing hydrogel (CS-NO). Notably, the NO releasing from the hydrogel could selectively up-regulate Flk-1 (early ECs marker) and VE-cadherin (mature ECs marker) in the absence of growth factors, which was of crucial importance in the endothelial differentiation of ESCs. In summary, the current study proposes a simple and efficient method for directing the endothelial differentiation of ESCs without extra growth factors.Download high-res image (184KB)Download full-size image

The differentiated cell lineages from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) have shown their potential in regenerative medicine. However, the functional and transcriptional microRNA (miRNA) expression pattern during endothelial differentiation has yet to be characterized.In this study, hESCs and hiPSCs were differentiated into endothelial cells (ECs). Then the endothelial-related gene profiling and miRNA profiling of hiPSCs, hESCs, hiPSCs derived endothelial cells (hiPSC-ECs), hESC derived endothelial cells (hESC-ECs) and human umbilical vein endothelial cells (HUVECs) were compared using RT-PCR Array. The data was analyzed using the data analysis system on QIAGEN’s website.Our analysis demonstrated that the endothelial differentiation was triggered after EB formation and the EC-associated genes were up-regulated swiftly in both hESC-EBs and hiPSC-EBs; hiPSC-ECs and hESC-ECs had the similar EC-associated gene expression patterns. Moreover, we report here the first miRNA profiling study of hiPSC-ECs. Analyzing 376 unique miRNAs, we have identified several interesting miRNAs, including miR-20a, miR-20b, miR-222, miR-210, which have been previously reported to be involved in endothelial differentiation and show surprising expression patterns across our samples. We also identified novel miRNAs, such as miR-125a-5p, miR-149, miR-296-5p, miR-100, miR-27b, miR-181a and miR-137, which were up-regulated in both hiPSC-ECs and hESC-ECs during endothelial differentiation.hiPSC-ECs and hESC-ECs exhibited a high degree of similarity with HUVECs in EC-associated genes expression. And the miRNA profiling analysis revealed significant differences between hiPSCs and hESCs, but a high degree of similarity between hiPSC-ECs and hESC-ECs.

Endothelial cell therapy has been implicated to enhance tissue regeneration and vascularization in ischemic kidney. However, no published study has yet examined direct effects of endothelial cell treatment in kidney recovery. This study investigated the therapeutic efficacy of endothelial cells in a mouse model with acute kidney injury (AKI). Thus, human embryonic stem cells-derived endothelial cells (hESC-ECs) labeled with a reporter system encoding a double fusion reporter gene for firefly luciferase (Fluc) and green fluorescent protein (GFP) were characterized by Fluc imaging and immunofluoresence staining. Cultured hESC-ECs (1×106) were injected into ischemic kidney shortly after AKI. Survival of the transplanted hESC-ECs was monitored in vivo from day 1 to 14 after endothelial cell transplantation and potential impact of hESC-EC treatment on renal regeneration was assessed by histological analyses. We report that a substantial level of bioluminescence activity was detected 24 h after hESC-EC injection followed by a gradual decline from 1 to 14 d. Human ESC-ECs markedly accelerated kidney cell proliferation in response to ischaemia-induced damage, indicated by an elevated number of BrdU+ cells. Co-expression of Sca-1, a kidney stem cell proliferation marker, and BrdU further suggested that the observed stimulation in renal cell regeneration was, at least in part, due to increased proliferation of renal resident stem cells especially within the medullary cords and arteriole. Differentiation of hESC-ECs to smooth muscle cells was also observed at an early stage of kidney recovery. In summary, our results suggest that endothelial cell therapy facilitates kidney recovery by promoting vascularization, trans-differentiation and endogenous renal stem cell proliferation in AKI.

Stem cell therapy has been proved to be an effective approach to ameliorate the heart remodeling post myocardial infarction (MI). However, poor cell engraftment and survival in ischemic myocardium limits the successful use of cellular therapy for treating MI. Here, we sought to transplant adipose derived-mesenchymal stem cells (AD-MSCs) with a hydrogel (NapFF-NO), naphthalene covalently conjugated a short peptide, FFGGG, and β-galactose caged nitric oxide (NO) donor, which can release NO molecule in response to β-galactosidase. AD-MSCs, either from transgenic mice that constitutively express GFP and firefly luciferase (Fluc), or express Fluc under the control of VEGFR2 promoter, were co-transplanted with NapFF-NO hydrogel into murine MI models. Improved cell survival and enhanced cardiac function were confirmed by bioluminescence imaging (BLI) and echocardiogram respectively. Moreover, increasing VEGFR2-luc expression was also tracked in real-time in vivo, indicating NapFF-NO hydrogel stimulated VEGF secretion of AD-MSCs. To investigate the therapeutic mechanism of NapFF-NO hydrogel, cell migration assay, paracrine action of AD-MSCs, and histology analysis were carried out. Our results revealed that condition medium from AD-MSCs cultured with NapFF-NO hydrogel could promote endothelial cell migration. Additionally, AD-MSCs showed significant improvement secretion of angiogenic factors VEGF and SDF-1α in the presence of NapFF-NO hydrogel. Finally, postmortem analysis confirmed that transplanted AD-MSCs with NapFF-NO hydrogel could ameliorate heart function by promoting angiogenesis and attenuating ventricular remodeling. In conclusion, NapFF-NO hydrogel can obviously improve therapeutic efficacy of AD-MSCs for MI by increasing cell engraftment and angiogenic paracrine action.