Endothelial Cells Promote Pseudo-islet Function Through BTC-EGFR-JAK/STAT Signaling Pathways.

Interactions between cells are of fundamental importance in affecting cell function. In vivo, endothelial cells and islet cells are close to each other, which makes endothelial cells essential for islet cell development and maintenance of islet cell function. We used endothelial cells to construct 3D pseudo-islets, which demonstrated better glucose regulation and greater insulin secretion compared to conventional pseudo-islets in both in vivo and in vitro trials. However, the underlying mechanism of how endothelial cells promote beta cell function localized within islets is still unknown. We performed transcriptomic sequencing, differential gene analysis, and enrichment analysis on two types of pseudo-islets to show that endothelial cells can promote the function of internal beta cells in pseudo-islets through the BTC-EGFR-JAK/STAT signaling pathway. Min6 cells secreted additional BTC after co-culture of endothelial cells with MIN6 cells outside the body. After BTC knockout in vitro, we found that beta cells functioned differently: insulin secretion levels decreased significantly, while the expression of key proteins in the EGFR-mediated JAK/STAT signaling pathway simultaneously decreased, further confirming our results. Through our experiments, we elucidate the molecular mechanisms by which endothelial cells maintain islet function in vitro, which provides a theoretical basis for the construction of pseudo-islets and islet cell transplants for the treatment of diabetes mellitus.

Long non-coding RNA LINC00930 targeting miR-6792-3p/ZBTB16 regulates the proliferation and EMT of pancreatic cancer.

Emerging evidence suggests the dysregulation of long non-coding RNAs (lncRNAs) involved in pancreatic cancer (PC). However, the function of LINC00930 in PC has not been elaborated. In this study, we found that LINC00930 was significantly down-regulated in PC cell lines and tissues, and associated with tumor size, lymphatic metastasis, TNM stage and poor prognosis. According to the bioinformatics database, the downregulation of LINC00930 was a common event in PC associated with prognosis and EMT. Overexpression of LINC00930 inhibited the aggressive cancer phenotypes including proliferation, metastasis and epithelial-mesenchymal transition (EMT) of PC in vitro and in vivo. Bioinformatics and dual-luciferase reporter assay indicated that miR-6792-3p could directly bind to LINC00930. Additionally, the Zinc finger and BTB domain containing 16 (ZBTB16) was significantly declined in PC, which was predicted to be the downstream gene of miR-6792-3p. MiR-6792-3p mimic rescued the decreased proliferation, metastasis and EMT caused by ZBTB16 in PC cells. The LINC00930/miR-6792-3p/ZBTB16 axis was associated with the malignant progression and process of PC. The relative expression of LINC00930 was negatively correlated with the expression of miR-6792-3p and was closely linked with ZBTB16 levels in PC. LINC00930 might serve as a potential prognostic biomarker and therapeutic target for PC.

Dual-crosslinking gelatin-hyaluronic acid methacrylate based biomimetic PDAC desmoplastic niche enhances tumor-associated macrophages recruitment and M2-like polarization.

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by deposition of desmoplastic matrix (including collagen and hyaluronic acid). And the interactions between tumor-associated macrophages (TAMs) and tumor cells play a crucial role in progression of PDAC. Hence, the appropriate model of tumor cell-macrophage interaction within the unique PDAC TME is of significantly important. To this end, a 3D tumor niche based on dual-crosslinking gelatin methacrylate and hyaluronic acid methacrylate hydrogels was constructed to simulate the desmoplastic tumor matrix with matching compressive modulus and composition. The bionic 3D tumor niche creates an immunosuppressive microenvironment characterized by the downregulation of M1 markers and upregulation of M2 markers in TAMs. Mechanistically, RNA-seq analysis revealed that the PI3K-AKT signaling pathway might modulate the phenotypic balance and recruitment of macrophages through regulating SELE and VCAM-1. Furthermore, GO and GSEA revealed the biological process of leukocyte migration and the activation of cytokine-associated signaling were involved. Finally, the 3D tumor-macrophage niches with three different ratios were fabricated which displayed increased M2-like polarization and stemness. The utilization of the 3D tumor niche has the potential to provide a more accurate investigation of the interplay between PDAC tumor cells and macrophages within an in vivo setting.

Synthesis and biological evaluation of novel quaternary ammonium antibody drug conjugates based on camptothecin derivatives.

Antibody drug conjugates (ADCs) have emerged as a highly promising class of cancer therapeutics, comprising antibodies, effector molecules, and linkers. Among them, DS-8201a with DXd as the effector molecule, has shown remarkable anti-tumor efficacy against solid tumors, sparking a surge of interest in ADCs with camptothecin derivatives as ADC effector molecules. In this study, we introduced and successfully constructed quaternary ammonium ADCs utilizing camptothecin derivatives WL-14 and CPTS-1 for the first time. All four ADCs displayed excellent stability under physiological conditions and in plasma, facilitating their prolonged circulation in vivo. Moreover, the four ADCs, employing Val-Cit or Val-Ala dipeptide linkers effectively achieved complete release of the effector molecules via cathepsin B. Although, the in vitro antitumor activity of these ADCs was comparatively limited, the development of quaternary ammonium ADCs based on novel camptothecin derivatives as effector molecules is still a viable and promising strategy. Significantly, our study provides valuable insights into the crucial role of linker optimization in ADCs design.

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.

Silver-catalysed three-component reactions of alkynyl aryl ketones, element selenium, and boronic acids leading to 3-organoselenylchromones.

An Ag-catalysed three-component reaction of alkynyl aryl ketones bearing an ortho-methoxy group, element selenium, and arylboronic acid, providing a facile route to selenofunctionalized chromone products has been developed. This protocol features high efficiency and high regioselectivity, and the use of selenium powder as the selenium source. Mechanistic experiments indicated that the combined oxidative effect of (bis(trifluoroacetoxy)iodo)benzene and oxygen in the air pushes the catalytic redox cycle of the Ag catalyst and the phenylselenium trifluoroacetate formed in situ is the key intermediate of the PIFA-mediated 6-endo-electrophilic cyclization and selenofunctionalization reaction of alkynyl aryl ketones.

Down-regulation of miR-18b-5p protects against splenic hemorrhagic shock by directly targeting HIF-1α/iNOS pathway.

Splenic hemorrhagic shock is a typical emergency in surgery, seriously threatening human beings' life. Emerging evidence shows that microRNAs (miRNAs) are closely related to inflammation and immunity in the body. However, the detailed effects and underlying mechanisms of miRNAs on the immune function of splenic hemorrhagic shock have not been revealed yet. In the present study, we construct the rat hemorrhagic shock model, and the rats are further cured with splenic blood transport clipping recanalization (SBTCR). MiR-18b-5p was highly expressed in the spleen of hemorrhagic shock rats detected by the qRT-PCR assay. Functionally, down-regulation of miR-18b-5p notably inhibited the levels of SOD1, iNOS and IL-6 in macrophages isolated from splenic tissues detected by qRT-PCR and ELISA assays. In addition, inhibition of miR-18b-5p significantly decreased the M1/M2 ratio of macrophages. Besides, knockdown of miR-18b-5p obviously reduced the Th1/Th2 ratio of CD4+ T cells. Moreover, HIF-1α was predicted as a target gene of miR-18b-5p, which was further confirmed by dual-luciferase reporter assay, and HIF-1α was negatively associated with miR-18b-5p. Furthermore, overexpression of HIF-1α partially restored the effects of miR-18b-3p on inflammation and immunity in macrophages. Taken together, miR-18b-5p may be a novel therapeutic candidate target in splenic hemorrhagic shock treatment.

A "Double-Locked" and Enzyme/pH-Activated Theranostic Agent for Accurate Tumor Imaging and Therapy.

Theranostic agents for concurrent cancer therapy and diagnosis have begun attracting attention as a promising modality. However, accurate imaging and identification remains a great challenge for theranostic agents. Here, we designed and synthesized a novel theranostic agent H6M based on the "double-locked" strategy by introducing an electron-withdrawing nitro group into 1-position of a pH-responsive 3-amino-ß-carboline and further covalently linking the hydroxamic acid group, a zinc-binding group (ZBG), to the 3-position of ß-carboline to obtain histone deacetylase (HDAC) inhibitory effect for combined HDAC-targeted therapy. We found that H6M can be specifically reduced under overexpressed nitroreductase (NTR) to produce H6AQ, which emits bright fluorescence at low pH. Notably, H6M demonstrated a selective fluorescence imaging via successive reactions with NTR (first "key") and pH (second "key"), and precisely identified tumor margins with a high S/N ratio to guide tumor resection. Finally, H6M exerted robust HDAC1/cancer cell inhibitory activities compared with a known HDAC inhibitor SAHA. Therefore, the NTR/pH-activated theranostic agent provided a novel tool for precise diagnosis and efficient tumor therapy.

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.

Enhanced vascularization and biocompatibility of rat pancreatic decellularized scaffolds loaded with platelet-rich plasma.

The ultimate goal of pancreatic tissue engineering is to create a long-lived substitute organ to treat diabetes. However, the lack of neovascularization and the occurrence of immune response limit the efficacy of tissue-engineered pancreas after in vivo transplantation. Platelet-rich plasma (PRP) is an autologous platelet concentrate containing a large number of growth factors and immunoregulatory factors. The aim of this study was to evaluate rat pancreatic decellularized scaffold (PDS) loaded with PRP for vascularization, host inflammatory response and macrophage polarization in an animal model. The study results indicated that compared to PDS, PRP-loading PDS exhibited the enhanced mechanical properties and released growth factors in a slow and sustained manner to supplement the loss of growth factors during decellularization. In vitro, human umbilical vein endothelial cells (HUVECs) were seeded in PDS and PRP-loading PDS, and cultured in the circular perfusion system. When compared with PDS, PRP-loading PDS significantly promoted the colonization, proliferation and pro-angiogenic genes expression of cells on scaffolds. In vivo, PDS loaded with PRP then re-endothelialized with HUVECs were implanted subcutaneously in rats, which enhanced the angiogenesis of scaffolds, inhibited the host inflammatory response, and induced the polarization dominated by pro-regenerative M2 macrophages that also facilitated tissue vascular regeneration. Thus, the re-endothelialized PRP-loading PDS may represent a promising bioengineered pancreas with sustained vascularization and excellent biocompatibility.

Novel ß-carboline-based indole-4,7-quinone derivatives as NAD(P)H: Quinone-oxidoreductase-1 inhibitor with potent antitumor activities by inducing reactive oxygen species, apoptosis, and DNA damage.

Eighteen new ß-carboline-based indole-4,7-quinone derivatives (12a-i and 13a-i) were designed and synthesized, and their in vitro and in vivo antiproliferative activities were studied. Most of target compounds showed strong inhibition on three human tumor cells' proliferation. In particular, the most active compound 13g not only displayed more prominent antiproliferative activities than ß-lapachone, a clinical antitumor candidate, but also exerted significant NAD(P)H: quinone-oxidoreductase-1 (NQO1) inhibitory activity and NQO1-dependent cytotoxicity in HT29 cells. Furthermore, 13g dose-dependently induced high ROS levels in HT29 cells, and selectively inhibited cancer cell but not non-tumor colon cell proliferation in vitro. Importantly, 13g promoted HT29 cell apoptosis and DNA damage by regulating relative apoptotic proteins and H2AX expression. Finally, 13g displayed significant growth inhibition of HT29 human colorectal adenocarcinoma xenograft in mice without overt toxicity.

Ophiopogonin A Alleviates Hemorrhagic Shock-Induced Renal Injury via Induction of Nrf2 Expression.

Ophiopogonin, including Ophiopogonin A, B, C, D, is an effective active component of traditional Chinese medicine Ophiopogon japonicus which has a wide range of pharmacological effects such as protecting myocardial ischemia, resisting myocardial infarction, immune regulation, lowering blood glucose, and anti-tumor. However, the functions of ophiopogonin A on hemorrhagic shock (HS)-induced renal injury remain unclear. First, this study constructed an HS rat model and hypoxia HK-2 cell model to assess the effects of ophiopogonin A in vivo and in vitro. In vivo, HE and TUNEL staining show that ophiopogonin A dose-dependently inhibits HS-induced tissue damage and apoptosis. Moreover, ophiopogonin A dose-dependently downregulates the levels of blood urea nitrogen (BUN), creatinine (Cr), KIM-1, NGAL, iNOS, TNF-α, IL-1ß, and IL-6 in HS rats kidney tissues, and decreases the number of MPO-positive cells. In vitro, we get similar results that ophiopogonin A dose-dependently improves hypoxia-induced HK-2 cell apoptosis and damage. In addition, ophiopogonin A dose-dependently increases the expression of NF E2-related factor 2 (Nrf2), while knockdown of Nrf2 reverses the functions of ophiopogonin A in vivo and in vitro. Furthermore, ophiopogonin A dose-dependently promotes the phosphorylation of ERK in HS kidney tissues and hypoxia-treated HK-2 cells, suggesting that ophiopogonin A functions via the p-ERK/ERK signaling pathway.

Conjugation of DM1 to anti-CD30 antibody has potential antitumor activity in CD30-positive hematological malignancies with lower systemic toxicity.

An anti-CD30 antibody-drug conjugate incorporating the antimitotic agent DM1 and a stable SMCC linker, anti-CD30-MCC-DM1, was generated as a new antitumor drug candidate for CD30-positive hematological malignancies. Here, the in vitro and in vivo pharmacologic activities of anti-CD30-MCC-DM1 (also known as F0002-ADC) were evaluated and compared with ADCETRIS (brentuximab vedotin). Pharmacokinetics (PK) and the safety profiles in cynomolgus monkeys were assessed. Anti-CD30-MCC-DM1 was effective in in vitro cell death assays using CD30-positive lymphoma cell lines. We studied the properties of anti-CD30-MCC-DM1, including binding, internalization, drug release and actions. Unlike ADCETRIS, anti-CD30-MCC-DM1 did not cause a bystander effect in this study. In vivo, anti-CD30-MCC-DM1 was found to be capable of inducing tumor regression in subcutaneous inoculation of Karpas 299 (anaplastic large cell lymphoma), HH (cutaneous T-cell lymphoma) and L428 (Hodgkin's disease) cell models. The half-lives of 4 mg/kg and 12 mg/kg anti-CD30-MCC-DM1 were about 5 days in cynomolgus monkeys, and the tolerated dose was 30 mg/kg in non-human primates, supporting the tolerance of anti-CD30-MCC-DM1 in humans. These results suggest that anti-CD30-MCC-DM1 presents efficacy, safety and PK profiles that support its use as a valuable treatment for CD30-positive hematological malignancies.

Experimental research on the effect of microRNA-21 inhibitor on a rat model of intervertebral disc degeneration.

Intervertebral disc degeneration is associated with angiogenesis and is the primary cause of disc-associated disease. Several studies have indicated the importance of microRNA (miR)-21 in angiogenesis. Thus, the present study aimed to validate the role and underlying mechanisms of miR-21 in a rat model of intervertebral disc degeneration. A total of 60 specific-pathogen-free Sprague-Dawley rats were used for in vivo experiments. A rat model of intervertebral disc degeneration was established and miR-21 inhibitor (antagomiR-21) was administered. The vertebral pulp and annulus fibrosus were isolated for immunohistochemical analysis of hypoxia inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) expression. Lumbar spine proteoglycan content was detected with the phloroglucinol method. Disc cell apoptosis was detected with terminal deoxynucleotidyl-transferase-mediated dUTP nick end labeling staining. It was revealed that antagomiR-21 treatment decreased the expression of HIF-1α and VEGF in the vertebral pulp and annulus fibrosus. Furthermore, antagomiR-21 treatment increased proteoglycan content and inhibited cell apoptosis in lumbar spines from model rats with intervertebral disc degeneration. In conclusion, antagomiR-21 treatment exerted a protective role in a rat model of intervertebral disc degeneration, which may provide the basis for a potential therapeutic approach in the treatment of disc-associated diseases.

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.

Increased half-life and enhanced potency of Fc-modified human PCSK9 monoclonal antibodies in primates.

Blocking proprotein convertase subtilisin kexin type 9 (PCSK9) binding to low-density lipoprotein receptor (LDLR) can profoundly lower plasma LDL levels. Two anti-PCKS9 monoclonal antibodies (mAbs), alirocumab and evolocumab, were approved by the FDA in 2015. The recommended dose is 75 mg to 150 mg every two weeks for alirocumab and 140mg every two weeks or 420 mg once a month for evolocumab. This study attempted to improve the pharmacokinetic properties of F0016A, an IgG1 anti-PCKS9 mAb, to generate biologically superior molecules. We engineered several variants with two or three amino acid substitutions in the Fc fragment based on prior knowledge. The Fc-modified mAbs exhibited increased binding to FcRn, resulting in prolonged serum half-life and enhanced efficacy in vivo. These results demonstrate that Fc-modified anti-PCKS9 antibodies may enable less frequent or lower dosing of antibodies by improved recycling into the blood.

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.

Controlling the blood glucose of type 1 diabetes mice by co-culturing MIN-6 ß cells on 3D scaffold.

T1D is an autoimmune disease, which may be caused by lack of insulin-secreting ß cells due to damage of autoimmune system. Living with T1D is a challenge for the child and the family; cell transplantation is a treatment option for diabetes in children. To establish a microenvironment suitable for cell growth and proliferation as well as for sustained cellular function, we used MIN-6 ß cells as seed cells and SF-IV collagen as a 3D composite scaffold to construct artificial pancreas in this experiment. The cell viabilities were determined by MTT assay, and the response of cells to different glucose concentrations was observed by glucose stimulation test. Artificial pancreas was transplanted into the abdominal cavity of T1D mice, and the changes of blood glucose were monitored. After 10 days, insulin expression was detected by immunohistochemical method, and the claybank stained area showed effectiveness of insulin secretion. A series of experiments showed that implantation of 3D cell scaffold into the abdominal cavity can effectively control the blood glucose level of T1D mice. It also had longer-lasting hypoglycemic effects than simple cell transplantation, which was expected to become a new method for the treatment of T1D.

Differentiation of iPSCs into insulin-producing cells via adenoviral transfection of PDX-1, NeuroD1 and MafA.

AIMS: The aim of this study was to evaluate the effect of PDX-1 (pancreatic and duodenal homeobox-1), NeuroD1 (neurogenic differentiation-1) and MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homolog A) in the differentiation of induced pluripotent stem cells (iPSCs) into insulin-producing cells and to explore this new approach of cell transplantation therapy for type 1 diabetes in mice. METHODS: iPSCs were infected with adenovirus (Ad-Mouse PDX-1-IRES-GFP, Ad-Mouse NeuroD1-IRES-GFP and Ad-Mouse Mafa-IRES-GFP) and then differentiated into insulin-producing cells in vitro. RT-PCR was applied to detect insulin gene expression, immunofluorescence to identify insulin protein, and mouse insulin enzyme-linked immunosorbent assay (ELISA) was used to evaluate the amount of insulin at different concentration of glucose. Insulin-producing cells were transplanted into the liver parenchyma of diabetic mice. Immunohistochemistry, intraperitoneal glucose tolerance test (IPGTT) and fasting blood glucose (FBG) were performed to assess the function of insulin-producing cells. RESULTS: Insulin biosynthesis and secretion were induced in iPSCs and insulin-producing cells were responsive to glucose in a dose-dependent manner. Gene expression of the three-gene-modified embryoid bodies (EBs) was similar to the mouse pancreatic ß cell line MIN6. Transplantation of insulin-producing cells into type I diabetic mice resulted in hyperglycemia reversal. CONCLUSIONS: The insulin-producing cells we obtained from three-gene-modified EBs may be used as seed cells for tissue engineering and may represent a cell replacement strategy for the production of ß cells for the treatment of type 1 diabetes.