Biomimetic Hydrogels Promote Pseudoislet Formation to Improve Glycemic Control in Diabetic Mice.

Islet or ß-cell transplantation is currently considered to be the ideal treatment for diabetes, and three-dimensional (3D) bioprinting of a bionic pancreas with physiological stiffness is considered to be promising for the encapsulation and transplantation of ß-cells. In this study, a 5%GelMA/2%AlgMA hybrid hydrogel with pancreatic physiological stiffness was constructed and used for ß-cell encapsulation, 3D bioprinting, and in vivo transplantation to evaluate glycemic control in diabetic mice. The hybrid hydrogel had good cytocompatibility and could induce insulin-producing cells (IPCs) to form pseudoislet structures and improve insulin secretion. Furthermore, we validated the importance of betacellulin (BTC) in IPCs differentiation and confirmed that IPCs self-regulation was achieved by altering the nuclear and cytoplasmic distributions of BTC expression. In vivo transplantation of diabetic mice quickly restored blood glucose levels. In the future, 3D bioprinting of ß-cells using biomimetic hydrogels will provide a promising platform for clinical islet transplantation for the treatment of diabetes.

LINC MIR503HG Controls SC-ß Cell Differentiation and Insulin Production by Targeting CDH1 and HES1.

Stem cell-derived pancreatic progenitors (SC-PPs), as an unlimited source of SC-derived ß (SC-ß) cells, offers a robust tool for diabetes treatment in stem cell-based transplantation, disease modeling, and drug screening. Whereas, PDX1+/NKX6.1+ PPs enhances the subsequent endocrine lineage specification and gives rise to glucose-responsive SC-ß cells in vivo and in vitro. To identify the regulators that promote induction efficiency and cellular function maturation, single-cell RNA-sequencing is performed to decipher the transcriptional landscape during PPs differentiation. The comprehensive evaluation of functionality demonstrated that manipulating LINC MIR503HG using CRISPR in PP cell fate decision can improve insulin synthesis and secretion in mature SC-ß cells, without effects on liver lineage specification. Importantly, transplantation of MIR503HG-/- SC-ß cells in recipients significantly restored blood glucose homeostasis, accompanied by serum C-peptide release and an increase in body weight. Mechanistically, by releasing CtBP1 occupying the CDH1 and HES1 promoters, the decrease in MIR503HG expression levels provided an excellent extracellular niche and appropriate Notch signaling activation for PPs following differentiation. Furthermore, this exhibited higher crucial transcription factors and mature epithelial markers in CDH1High expressed clusters. Altogether, these findings highlighted MIR503HG as an essential and exclusive PP cell fate specification regulator with promising therapeutic potential for patients with diabetes.

Matrix stiffness triggers chemoresistance through elevated autophagy in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a signature of extremely high matrix stiffness caused by a special desmoplastic reaction, which dynamically stiffens along with the pathological process. The poor prognosis and low five-year survival rate of PDAC are partly owing to chemoresistance triggered by substrate stiffness. Understanding the potential mechanisms of matrix stiffness causing PDAC chemoresistance is of great significance. In this study, methacrylated gelatin hydrogel was used as platform for PANC-1 and MIA-PaCa2 cell culture. The results indicated that compared to soft substrate, stiff substrate distinctively reduced the gemcitabine sensitivity of pancreatic cancer. Intriguingly, transmission electron microscopy, immunofluorescence staining, western blot and qRT-PCR assay showcased that the number of autophagosomes and the expression of LC3 were elevated. The observations indicate that matrix stiffness may regulate the autophagy level, which plays a vital role during chemoresistance. In brief, soft substrate exhibited low autophagy level, while the counterpart displayed elevated autophagy level. In order to elucidate the underlying interaction between matrix stiffness-mediated cell autophagy and chemoresistance, rescue experiments with rapamycin and chloroquine were conducted. We found that inhibiting cell autophagy dramatically increased the sensitivity of pancreatic cancer cells to gemcitabine in the stiff group, while promoting autophagy-driven chemoresistance in the soft group, demonstrating that matrix stiffness modulated chemoresistance via autophagy. Furthermore, RNA-seq results showed that miR-1972 may regulate autophagy level in response to matrix stiffness. Overall, our research shed light on the synergistic therapy of PDAC combined with gemcitabine and chloroquine, which is conducive to promoting a therapeutic effect.

Diagnosis and prognosis of pancreatic cancer with immunoglobulin heavy constant delta blood marker.

BACKGROUND: Pancreatic cancer (PC) is highly malignant and difficult to detect, while few blood markers are currently available for diagnosing PC. METHODS: We obtained differential expression genes (DEGs) from GEO (gene expression omnibus) database and assessed by quantitative real-time polymerase chain reaction (qRT-PCR), receiver operating characteristic (ROC), univariate and multifactorial regression analysis, and survival analysis in our clinic center. Through the TCGA (the cancer genome atlas) database, we analyzed functional enrichment, different risk groups with survival analysis, immunological features, and the risk score established by the Cox regression model and constructed a nomogram. RESULT: Immunoglobulin heavy constant delta (IGHD) was remarkably upregulated in peripheral blood from PC patients, and IGHD was a potential independent biomarker for PC diagnosis (ROC sensitivity, 76.0%; specificity, 74.2%; area under the curve (AUC) = 0.817; univariate logistic regression analysis: odds ratio (OR) 1.488; 95% confidence interval (CI) 1.182-1.872; P < 0.001; multiple logistic: OR 2.097; 95% CI 1.276-3.389, P = 0.003). In addition, the IGHD expression was remarkably reduced after resectioning the primary tumor. High IGHD expression indicated higher lymphocyte infiltration and increased activities of immunological pathways in PC patients. KRAS and SMAD were observed with a prominent difference among top mutated genes between the two groups. The risk score predicted reliable clinical prognosis and drug responses. Furthermore, a nomogram with the risk score and clinical characteristics was constructed, showing a better predictive performance. CONCLUSION: IGHD is a valuable PC diagnosis, prognosis, and therapeutic response marker.

An artificial LAMA2-GelMA hydrogel microenvironment for the development of pancreatic endocrine progenitors.

The biomimetic pancreatic microenvironment improves the differentiation efficiency and function of human embryonic stem cell-derived ß-cells (SC-ß cells). Thus, a laminin subunit alpha 2-gelatin methacrylate (LAMA2-GelMA) hybrid hydrogel as a bionics carrier for the formation and maturation of endocrine lineage was developed in our research, based on pancreas proteomics analysis of postnatal mice. Pancreatic endocrine cells cultured on the hybrid hydrogel in vitro, which was composed of 0.5 µg/mL LAMA2 protein and 4% GelMA, the expression of transcription factors (TFs), including NKX6.1, NKX6.2, and NEUROD1 were upregulated. Single-cell transcriptomics was performed after LAMA2 knockdown during the early differentiation of pancreatic progenitor (PP) cells, a marked decrease in the forkhead box protein A2 (FOXA2+)/GATA-binding factor 6 (GATA6+) cluster was detected. Also, we clarified that as a receptor of LAMA2, integrin subunit alpha 7 (ITGA7) participated in Integrin-AKT signaling transduction and influenced the protein levels of FOXA2 and PDX1. In vivo experiments showed that, PP cells encapsulated in the LAMA2-GelMA hydrogel exhibited higher serum C-peptide levels compared to the GelMA and Matrigel groups in nude mice and reversed hyperglycemia more quickly in STZ-induced diabetic nude mice. Taken together, our findings highlighted the feasibility of constructing a pancreas-specific microenvironment based on proteomics and tissue engineering for the treatment of diabetes.

Glucose oxidase-amplified CO generation for synergistic anticancer therapy via manganese carbonyl-caged MOFs.

Carbon monoxide (CO) as one of the therapeutic gaseous molecules has been widely applied for treating various diseases, especially in cancer therapy. However, the in situ-triggered and efficient transport of CO to tumors are the primary obstacles that limit its clinical applicability. To address this obstacle, herein, a H2O2-triggered CO gas releasing nanoplatform has been designed by embedding manganese carbonyl (MnCO) into Zr (IV)-based metal-organic frameworks (MOFs). The porous structures of MOFs provide encapsulation capacity for glucose oxidase (GOx) loading, thereby catalyzing the endogenous glucose into gluconic acid and H2O2 to accelerate CO release and energy depletion. In the meantime, the Mn2+ produced by MnCO can react with intracellular H2O2 via the Fenton reaction to form cytotoxic •OH. Therefore, the synthesized gas nanogenerator demonstrated a synergistic efficacy of CO gas therapy, reactive oxygen species (ROS)-mediated therapy, and energy starvation to prevent tumor growth. Both in vitro and in vivo studies indicated that this multifunctional nanoplatform not only successfully inhibited tumors through a synergistic effect, but also provided a new technique for the creation of starvation/gas/chemodynamic combination therapy in a single material. STATEMENT OF SIGNIFICANCE: In this study, we developed a H2O2 responsive CO gas nanogenerator to augment the in-situ generation of CO gas for combined modality therapy of tumors. The nanogenerator was constructed by encapsulating glucose oxidase (GOx) and manganese carbonyl (MnCO) into UiO-67-bpy, which can catalyze the conversion of intracellular glucose to H2O2 for cutting off energy supply of cancer cells. Meanwhile, the cumulated H2O2 can trigger the release of CO for gas therapy and generation of •OH for chemodynamic therapy (CDT) via the Fenton-like reaction, thereby resulting in apoptosis of the cancer cells. Collectively, our designed nanotherapeutic agent not only displays the synergistic therapy efficacy of starvation-enhanced CO gas therapy and CDT, but also provides an efficient strategy for developing the intelligent nanocarrier for CO gas delivery and release.

Controllable self-assembly of thiophene-based π-conjugated molecule and further construction of pillar[5]arene-based host-guest white-light emission system.

Photoluminescence materials have been widely applied in biological imaging and sensing, anti-counterfeiting, light-emitting diodes, logic gates et al. The fabrication of luminescent materials with adjustable emission color by self-assembly of π-conjugated molecules has attracted particular attention. In this study, we designed and synthesized a thiophene-based α-cyanostyrene-derivative (TPPA), then investigate its self-assembly morphology and fluorescence emission under different organic solvents, different proportions of H2O/THF (DMSO) mixture and different pH conditions by UV, FL and SEM images. It was found that TPPA formed nanoparticles by self-assembly in organic solvent (THF or DMSO), accompanied by strong fluorescence emission. However, with the increase of water ratio, the fluorescence intensity decreased accompany with red shift, and the self-assembly morphology changed from nanoparticles to fibers. More interestingly, when pillar[5]arene (P5) was added to form host-guest complex with TPPA, white light emission could be successfully constructed when the ratio of TPPA to P5 was 1:20 and THF to water was 19:1.

Exosomes from ß-Cells Promote Differentiation of Induced Pluripotent Stem Cells into Insulin-Producing Cells Through microRNA-Dependent Mechanisms.

OBJECTIVE: Exosomes have emerged as potential tools for the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells (IPCs). Exosomal microRNAs are receiving increasing attention in this process. Here, we aimed at investigating the role of exosomes derived from a murine pancreatic ß-cell line and identifying signature exosomal miRNAs on iPSCs differentiation. METHODS: Exosomes were isolated from MIN6 cells and identified with TEM, NTA and Western blot. PKH67 tracer and transwell assay were used to confirm exosome delivery into iPSCs. qRT-PCR was applied to detect key pancreatic transcription gene expression and exosome-derived miRNA expression. Insulin secretion was determined using FCM and immunofluorescence. The specific exosomal miRNAs were determined via RNA-interference of Ago2. The therapeutic effect of 21 day-exosome-induced IPCs was validated in T1D mice induced by STZ. RESULTS: iPSCs cultured in medium containing exosomes showed sustained higher expression of MAFA, Insulin1, Insulin2, Isl1, Neuroud1, Nkx6.1 and NGN3 compared to control iPSCs. In FCM analysis, approximately 52.7% of the differentiated cells displayed insulin expression at the middle stage. Consistent with the gene expression data, immunofluorescence assays showed that Nkx6.1 and insulin expression in iPSCs were significantly upregulated. Intriguingly, the expression of pancreatic markers and insulin was significantly decreased in iPSCs cultured with siAgo2 exosomes. Transplantation of 21 day-induced IPCs intoT1D mice efficiently enhanced glucose tolerance and partially controlled hyperglycemia. The therapeutic effect was significantly attenuated in T1D mice that received iPSCs cultured with siAgo2 exosomes. Of the seven exosomal microRNAs selected for validation, miR-706, miR-709, miR-466c-5p, and miR-423-5p showed dynamic expression during 21 days in culture. CONCLUSION: These data indicate that differentiation of exosome-induced iPSCs into functional cells is crucially dependent on the specific miRNAs encased within exosomes, whose functional analysis is likely to provide insight into novel regulatory mechanisms governing iPSCs differentiation into IPCs.

Exosomal circ_0030167 derived from BM-MSCs inhibits the invasion, migration, proliferation and stemness of pancreatic cancer cells by sponging miR-338-5p and targeting the Wif1/Wnt8/ß-catenin axis.

Pancreatic cancer (PC) is one of the most lethal malignant tumors and has the lowest survival rate due to early metastasis and drug resistance. Exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) have emerged as crucial regulators of the progression of various tumors. These vesicles contain abundant circRNAs that have important biological functions. This study aimed to elucidate the role of exosomal circRNAs in PC progression. In this study, we successfully isolated BM-MSCs from human bone marrow based on their surface marker expression and osteogenic and adipogenic differentiation potential. We found that BM-MSC-derived exosomes significantly reduced the invasion, migration, and proliferation of PC cells, as well as tumor stemness. According to whole-transcriptome resequencing and clustering heat map analysis, we identified the key molecule circ_0030167 and miR-338-5p, its downstream target. We revealed that circ_0030167 mainly regulates miR-338-5p, enhances Wif1 expression, and inhibits the Wnt8/ß-catenin pathway, thereby inhibiting the stemness of PC cells and tumor progression. Overall, BM-MSC exosomal circ_0030167 contributes to the progression and stemness of PC cells via the miR-338-5p/wif1/Wnt 8/ß-catenin axis. Our study provides a new perspective for the treatment of PC.

Inhibition of Aß aggregates in Alzheimer's disease by epigallocatechin and epicatechin-3-gallate from green tea.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive accumulation of senile plaques, which are primarily composed of misfolded amyloid ß-peptide (Aß). Aß aggregates are believed to be a key factor in the pathogenesis of AD, affecting the nervous system in human body. The therapeutic potential of tea-derived polyphenolic compounds, (-)-epigallocatechin (EGC) and (-)-epicatechin-3-gallate (ECG), for AD was investigated by assessing their effects on the Cu2+/Zn2+-induced or self-assembled Aß40 aggregation using thioflavine T fluorescent spectrometry, inductively coupled plasma mass spectrometry, UV-Vis spectroscopy, transmission electron microscope, silver staining, immunohistochemistry, and immunofluorescence assays. EGC and ECG mildly bind to Cu2+ and Zn2+, and diminish the Cu2+- or Zn2+-induced or self-assembled Aß aggregates; they also modulate the Cu2+/Zn2+-Aß40 induced neurotoxicity on mouse neuroblastoma Neuro-2a cells by reducing the production of ROS. Metal chelating, hydrogen bonding or Van Der Waals force may drive the interaction between the polyphenolic compounds and Aß. The results demonstrate that green tea catechins EGC and ECG are able to alleviate the toxicity of Aß oligomers and fibrils. Particularly, ECG can cross the blood-brain barrier to reduce the Aß plaques in the brain of APP/PS1 mice, thereby protecting neurons from injuries. The results manifest the potential of green tea for preventing or ameliorating the symptoms of AD.

Autophagy-related gene expression classification defines three molecular subtypes with distinct clinical and microenvironment cell infiltration characteristics in colon cancer.

BACKGROUND: Multiple molecular subtypes with distinct clinical outcomes in colon cancer have been identified in recent years. Nonetheless, the autophagy-related molecular subtypes as well as its mediated tumor microenvironment (TME) cell infiltration characteristics have not been fully understood. METHODS: Based on the seven colon cancer cohorts with 1580 samples, we performed a comprehensive genomic analysis to explore the molecular subtypes mediated by autophagy-related genes. The single-sample gene-set enrichment analysis (ssGSEA) was used to quantify the relative abundance of each cell infiltration in the TME. Unsupervised methods were used to perform autophagy subtype clustering. Least absolute shrinkage and selection operator regression (LASSO) was used to construct autophagy characterization score (APCS) signature. RESULTS: We determined three distinct autophagy-related molecular subtypes in colon cancer. The three autophagy subtypes presented significant survival differences. Microenvironment analyses revealed the heterogeneous TME immune cell infiltration characterization between three subtypes. Cluster 1 autophagy subtype was characterized by abundant innate and adaptive immune cell infiltration. This subtype exhibited an enhanced stromal activity including activated pathways of epithelial-mesenchymal transition, TGF-ß and angiogenesis, and an increased infiltration of fibroblasts and endothelial cells. The expression of immune checkpoint molecules was also significantly up-regulated, which may mediate immune escape in Cluster 1 subtype. Cluster 2 subtype was characterized by relatively lower TME immune cell infiltration and enhanced DNA damage repair pathways. Cluster 3 subtype was characterized by the suppression of immunity. Patients with high APCS, with poorer survival, presented a significantly positive correlation with TME stromal activity. Low APCS, relevant to activated damage repair pathways, showed enhanced responses to anti-PD-1/PD-L1 immunotherapy. Two immunotherapy cohorts confirmed patients with low APCS exhibited prominently enhanced clinical response and treatment advantages. CONCLUSIONS: This study may help understand the molecular characterization of autophagy-related subtypes. We demonstrated the autophagy genes in colon cancer could drive the heterogeneity of TME immune cell infiltration. Our study represented a step toward personalized immunotherapy in colon cancer.

Brusatol Protects HepG2 Cells against Oxygen-Glucose Deprivation-Induced Injury via Inhibiting Mitochondrial Reactive Oxygen Species-Induced Oxidative Stress.

BACKGROUND: It has been reported that brusatol (BRU) reduces cellular reactive oxygen species (ROS) level under hypoxia; here the protective effect of BRU against oxygen-glucose deprivation/reoxygenation (OGD-R)-induced injury in HepG2 cells and against anoxia/reoxygenation (A/R)-induced injury in rat liver mitochondria was investigated. MATERIALS AND METHODS: OGD-R-induced HepG2 cell viability loss was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide and trypan blue staining. Mitochondrial ROS level in HepG2 cells was measured by MitoSOX staining. The cellular malondialdehyde and adenosine triphosphate level was measured by commercial kits. The mitochondrial membrane potential in HepG2 cells was measured by JC-1 staining. The protein level was detected by Western blotting. Rat liver mitochondria were separated by differential centrifugation. A/R-induced injury in isolated rat liver mitochondria was established by using a Clark oxygen electrode. The ROS generation in isolated mitochondria was evaluated using Amplex red/horseradish peroxidase. RESULTS: BRU reduced mitochondrial ROS level and alleviated oxidative injury in HepG2 cells, thereby significantly inhibited OGD-R-induced cell death. During OGD-R, BRU improved mitochondrial function and inhibited the release of cytochrome c. Furthermore, BRU showed a clear protective effect against A/R-induced injury in isolated rat liver mitochondria. When isolated rat liver mitochondria were pretreated with BRU, A/R-induced ROS generation was significantly decreased, and mitochondrial respiratory dysfunction was ameliorated. CONCLUSIONS: BRU pretreatment attenuated OGD-R-induced injury in HepG2 cells and A/R-induced injury in isolated rat liver mitochondria by inhibiting mitochondrial ROS-induced oxidative stress.

Reseeding endothelial cells with fibroblasts to improve the re-endothelialization of pancreatic acellular scaffolds.

Pancreatic transplantation remains the only cure for diabetes, but the shortage of donors limits its clinical application. Whole organ decellularized scaffolds offer a new opportunity for pancreatic organ regeneration; however inadequate endothelialization and vascularization can prevent sufficient transport of oxygen and nutrient supplies to the transplanted organ, as well as leading unwanted thrombotic events. In the present study, we explored the re-endothelialization of rat pancreatic acellular scaffolds via circulation perfusion using human skin fibroblasts (FBs) and human umbilical vein endothelial cells (HUVECs). Our results revealed that the cell adhesion rate when these cells were co-cultured was higher than under control conditions, and this increase was associated with increased release of growth factors including VEGF, FGFb, EGF, and IGF-1 as measured by ELISA. When these recellularized organs were implanted in vivo for 28 days in rat dorsal subcutaneous pockets, we found that de novo vasculature formation in the co-culture samples was superior to the control samples. Together these results suggest that endothelial cell and FB co-culture enhances the re-endothelialization and vascularization of pancreatic acellular scaffolds.

lncRNA Gm10451 regulates PTIP to facilitate iPSCs-derived ß-like cell differentiation by targeting miR-338-3p as a ceRNA.

iPSCs-derived insulin-producing cell transplantation is a promising strategy for diabetes therapy. Although there have been many protocols of mature, glucose-responsive ß cells induced in vitro over the past few years, many underlying problems remain to be resolved. As a crucial regulator, long noncoding RNAs (lncRNAs) participate in numerous biological processes, including the maintenance of pluripotency, and stem cell differentiation. In this study, we identified a novel lncRNA Gm10451 as a functional regulator for ß-like cell differentiation. Localized to the cytoplasm, Gm10451 regulates histone H3K4 methyltransferase complex PTIP to facilitate Insulin+/Nkx6.1+ ß-like cell differentiation by targeting miR-338-3p as a competing endogenous RNA (ceRNA). miR-338-3p has also been shown to suppress Nkx6.1+ early-stage ß-like cell differentiation by targeting PTIP. Following transplantation into streptozotocin (STZ)-mice, Gm10451 loss in ß-like cells prevented the expression of mature ß-cell makers, such as Insulin, Nkx6.1, and Mafa. Accordingly, hyperglycemia in the mice was not resolved. Taken together, this study provides an efficient epigenetic target for generating more mature and functional iPSCs-derived ß-like cells. We anticipate that pancreatic organoids, which are generated from human stem cells, biological materials, and epigenetic modifications, can be used in the future as a novel diabetes treatment option.

microRNA-690 regulates induced pluripotent stem cells (iPSCs) differentiation into insulin-producing cells by targeting Sox9.

BACKGROUND: The regulatory mechanism of insulin-producing cells (IPCs) differentiation from induced pluripotent stem cells (iPSCs) in vitro is very important in the phylogenetics of pancreatic islets, the molecular pathogenesis of diabetes, and the acquisition of high-quality pancreatic ß-cells derived from stem cells for cell therapy. METHODS: miPSCs were induced for IPCs differentiation. miRNA microarray assays were performed by using total RNA from our iPCs-derived IPCs containing undifferentiated iPSCs and iPSCs-derived IPCSs at day 4, day 14, and day 21 during step 3 to screen the differentially expressed miRNAs (DEmiRNAs) related to IPCs differentiation, and putative target genes of DEmiRNAs were predicted by bioinformatics analysis. miR-690 was selected for further research, and MPCs were transfected by miR-690-agomir to confirm whether it was involved in the regulation of IPCs differentiation in iPSCs. Quantitative Real-Time PCR (qRT-PCR), Western blotting, and immunostaining assays were performed to examine the pancreatic function of IPCs at mRNA and protein level respectively. Flow cytometry and ELISA were performed to detect differentiation efficiency and insulin content and secretion from iPSCs-derived IPCs in response to stimulation at different concentration of glucose. The targeting of the 3'-untranslated region of Sox9 by miR-690 was examined by luciferase assay. RESULTS: We found that miR-690 was expressed dynamically during IPCs differentiation according to the miRNA array results and that overexpression of miR-690 significantly impaired the maturation and insulinogenesis of IPCs derived from iPSCs both in vitro and in vivo. Bioinformatic prediction and mechanistic analysis revealed that miR-690 plays a pivotal role during the differentiation of IPCs by directly targeting the transcription factor sex-determining region Y (SRY)-box9. Furthermore, downstream experiments indicated that miR-690 is likely to act as an inactivated regulator of the Wnt signaling pathway in this process. CONCLUSIONS: We discovered a previously unknown interaction between miR-690 and sox9 but also revealed a new regulatory signaling pathway of the miR-690/Sox9 axis during iPSCs-induced IPCs differentiation.

Transcriptome Analysis of Induced Pluripotent Stem Cell (iPSC)-derived Pancreatic ß-like Cell Differentiation.

Diabetes affects millions of people worldwide, and ß-cell replacement is one of the promising new strategies for treatment. Induced pluripotent stem cells (iPSCs) can differentiate into any cell type, including pancreatic ß cells, providing a potential treatment for diabetes. However, the molecular mechanisms underlying the differentiation of iPSC-derived ß cells have not yet been fully elucidated. Here, we generated pancreatic ß-like cells from mouse iPSCs using a 3-step protocol and performed deep RNA sequencing to get a transcriptional landscape of iPSC-derived pancreatic ß-like cells during the selective differentiation period. We then focused on the differentially expressed genes (DEGs) during the time course of the differentiation period, and these genes underwent Gene Ontology annotation and Kyoto Encyclopedia of Genes and Genomes pathway analysis. In addition, gene-act networks were constructed for these DEGs, and the expression of pivotal genes detected by quantitative real-time polymerase chain reaction was well correlated with RNA sequence (RNA-seq). Overall, our study provides valuable information regarding the transcriptome changes in ß cells derived from iPSCs during differentiation, elucidates the biological process and pathways underlying ß-cell differentiation, and promotes the identification and functional analysis of potential genes that could be used for improving functional ß-cell generation from iPSCs.

Culture of iPSCs Derived Pancreatic ß-Like Cells In Vitro Using Decellularized Pancreatic Scaffolds: A Preliminary Trial.

Diabetes mellitus is a disease which has affected 415 million patients in 2015. In an effort to replace the significant demands on transplantation and morbidity associated with transplantation, the production of ß-like cells differentiated from induced pluripotent stem cells (iPSCs) was evaluated. This approach is associated with promising decellularized scaffolds with natural extracellular matrix (ECM) and ideal cubic environment that will promote cell growth in vivo. Our efforts focused on combining decellularized rat pancreatic scaffolds with mouse GFP+-iPSCs-derived pancreatic ß-like cells, to evaluate whether decellularized scaffolds could facilitate the growth and function of ß-like cells. ß-like cells were differentiated from GFP+-iPSCs and evaluated via cultivating in the dynamic circulation perfusion device. Our results demonstrated that decellularized pancreatic scaffolds display favorable biochemical properties. Furthermore, not only could the scaffolds support the survival of ß-like cells, but they also accelerated the expression of the insulin as compared to plate-based cell culture. In conclusion, these results suggest that decellularized pancreatic scaffolds could provide a suitable platform for cellular activities of ß-like cells including survival and insulin secretion. This study provides preliminary support for regenerating insulin-secreting organs from the decellularized scaffolds combined with iPSCs derived ß-like cells as a potential clinical application.

The dynamic three-dimensional culture of islet-like clusters in decellularized liver scaffolds.

Diabetes mellitus is a worldwide metabolic disease which constitutes a major threat to human health. Stem cells with the ability to differentiate into insulin-producing cells (IPCs) could provide unlimited sources of transplanted cells and solve allogeneic rejection problems. The decellularized scaffolds could provide IPCs with tissue microarchitecture and intact vascular systems. The goal of this study was to engineer intact whole rat liver scaffolds and repopulate the stem cell-derived IPCs into the scaffolds to discover whether the decellularized scaffolds could facilitate the growth and development of IPCs. Decellularized liver scaffolds were obtained using 1 % Triton X-100 with 0.1 % ammonium hydroxide. Architecture and composition of the original extracellular matrix were confirmed by morphologic, histological and immunolabeling examinations. Islet-like clusters were differentiated from Wharton's jelly mesenchymal stem cells (WJMSCs) by a three-step induction procedure. The differentiation was evaluated by morphology, RT-PCR, immunofluorescence and glucose stimulation experiments. The islet-like clusters were recellularized into the decellularized scaffolds by the portal-vein infusion method and cultured by the dynamic circulation perfusion device. After cultivation, hematoxylin-eosin staining, immunofluorescence and RT-PCR were conducted. Our results demonstrated that the decellularized rat liver scaffolds have favorable biochemical properties and could support the survival of WJMSC-derived IPCs. In addition, the three-dimensional decellularized scaffolds could enhance the expression of the insulin gene compared with two-dimensional plate culture. In conclusion, these findings suggested that the decellularized scaffolds could provide a suitable platform for cellular activities of IPCs such as survival, differentiation, proliferation and insulin secretion. This study provides fundamental support for regenerating insulin-secreting organs from the decellularized scaffolds combined with stem cell-derived IPCs as a potential clinical application.

Decellularization and Recellularization of Rat Livers With Hepatocytes and Endothelial Progenitor Cells.

Whole-organ decellularization has been identified as a promising choice for tissue engineering. The aim of the present study was to engineer intact whole rat liver scaffolds and repopulate them with hepatocytes and endothelial progenitor cells (EPCs) in a bioreactor. Decellularized liver scaffolds were obtained by perfusing Triton X-100 with ammonium hydroxide. The architecture and composition of the original extracellular matrix were preserved, as confirmed by morphologic, histological, and immunolabeling methods. To determine biocompatibility, the scaffold was embedded in the subcutaneous adipose layer of the back of a heterologous animal to observe the infiltration of inflammatory cells. Hepatocytes were reseeded using a parenchymal injection method and cultured by continuous perfusion. EPCs were reseeded using a portal vein infusion method. Morphologic and functional examination showed that the hepatocytes and EPCs grew well in the scaffold. The present study describes an effective method of decellularization and recellularization of rat livers, providing the foundation for liver engineering and the development of bioartificial livers.