GRP94 is an IGF-1R chaperone and regulates beta cell death in diabetes.

High workload-induced cellular stress can cause pancreatic islet ß cell death and dysfunction, or ß cell failure, a hallmark of type 2 diabetes mellitus. Thus, activation of molecular chaperones and other stress-response genes prevents ß cell failure. To this end, we have shown that deletion of the glucose-regulated protein 94 (GRP94) in Pdx1+ pancreatic progenitor cells led to pancreas hypoplasia and reduced ß cell mass during pancreas development in mice. Here, we show that GRP94 was involved in ß cell adaption and compensation (or failure) in islets from leptin receptor-deficient (db/db) mice in an age-dependent manner. GRP94-deficient cells were more susceptible to cell death induced by various diabetogenic stress conditions. We also identified a new client of GRP94, insulin-like growth factor-1 receptor (IGF-1R), a critical factor for ß cell survival and function that may mediate the effect of GRP94 in the pathogenesis of diabetes. This study has identified essential functions of GRP94 in ß cell failure related to diabetes.

Alpha-1 Antitrypsin Augmentation Therapy in Chronic Pancreatitis Patients Undergoing Total Pancreatectomy and Islet Autotransplantation: A Randomized, Controlled Study.

Stress-induced islet graft loss during the peri-transplantation period reduces the efficacy of islet transplantation. In this prospective, randomized, double-blind clinical trial, we evaluated the safety and efficacy of 60 mg/kg human alpha-1 antitrypsin (AAT) or placebo infusion weekly for four doses beginning before surgery in chronic pancreatitis (CP) patients undergoing total pancreatectomy and islet autotransplantation (TP-IAT). Subjects were followed for 12 months post-TP-IAT. The dose of AAT was safe, as there was no difference in the types and severity of adverse events in participants from both groups. There were some biochemical signals of treatment effect with a higher oxygen consumption rate in AAT islets before transplantation and a lower serum C-peptide (an indicator of islet death) in the AAT group at 15 min after islet infusion. Findings per the statistical analysis plan using a modified intention to treat analysis showed no difference in the C-peptide area under the curve (AUC) following a mixed meal tolerance test at 12 months post-TP-IAT. There was no difference in the secondary and exploratory outcomes. Although AAT therapy did not show improvement in C-peptide AUC in this study, AAT therapy is safe in CP patients and there are experiences gained on optimal clinical trial design in this challenging disease.

Immortalized Mesenchymal Stromal Cells Overexpressing Alpha-1 Antitrypsin Protect Acinar Cells from Apoptotic and Ferroptotic Cell Death.

Chronic pancreatitis (CP) is a progressive inflammatory disorder that impairs endocrine and exocrine function. Our previous work suggests that mesenchymal stem/stromal cells (MSCs) and MSCs overexpressing alpha-1 antitrypsin (AAT-MSCs) could be therapeutic tools for CP treatment in mouse models. However, primary MSCs have a predisposition to undergo senescence during culture expansion which limits their therapeutic applications. Here we generated and characterized immortalized human MSCs (iMSCs) and AAT-MSCs (iAAT-MSCs) and tested their protective effect on 2,4,6-Trinitrobenzenesulfonic acid (TNBS) -induced acinar cell death in an in vitro cell culture system. Primary MSCs were immortalized by transduction with simian virus 40 large T antigen (SV40LT), and the resulting iMSC and iAAT-MSC lines were analyzed for proliferation, senescence, phenotype, and multi-differentiation potential. Subsequently, the impact of these cells on TNBS-induced cell death was measured and compared. Both apoptosis and ferroptosis pathways were investigated by assessing changes of critical factors before and after cell treatment. Coculture of iMSCs and iAAT-MSCs with acinar cell lines inhibited early apoptosis induced by TNBS, reduced ER stress, and reversed TNBS-induced protein reduction at tight junctions. Additionally, iMSCs and iAAT-MSCs exerted such protection by regulating mitochondrial respiration, ATP content, and ROS production in TNBS-induced acinar cells. Furthermore, iMSCs and iAAT-MSCs ameliorated ferroptosis by regulating the ferritin heavy chain 1 (FTH1)/protein disulfide isomerase (PDI)/glutathione peroxide 4 (GPX4) signaling pathways and by modulating ROS function and iron generation in acinar cells. These findings identified ferroptosis as one of the mechanisms that leads to TNBS-induced cell death and offer mechanistic insights relevant to using stem cell therapy for the treatment of CP.

Deletion of Spinophilin Promotes White Adipocyte Browning.

Browning of white adipose tissue (WAT) is suggested as a promising therapeutic approach to induce energy expenditure and counteract obesity and its associated complications. Systemic depletion of spinophilin (SPL) increases metabolism and improves energy balance in mice. In this study, we explored the mechanistic insight of SPL action in WAT browning. Gene expression and mitochondria tracker staining showed that visceral white adipose tissue (vWAT) harvested from SPL KO mice had a higher expression of classic browning-related genes, including uncoupling protein 1 (UCP1), Cell death inducing DFFA like effector A (CIDEA) and PR domain containing 16 (PRDM16), as well as a higher mtDNA level compared to vWAT from wild type (WT) control mice. When adipogenesis was induced in pre-adipocytes harvested from KO and WT mice ex vivo using the PPAR-γ agonist rosiglitazone (Rosi), SPL KO cells showed increased browning marker gene expression and mitochondria function compared to cells from WT mice. Increased PPAR-γ protein expression and nucleus retention in vWAT from SPL KO mice after Rosi treatment were also observed. The effect of SPL on vWAT browning was further confirmed in vivo when WT and KO mice were treated with Rosi. As a result, SPL KO mice lost body weight, which was associated with increased expression of browning maker genes in vWAT. In summary, our data demonstrate the critical role of SPL in the regulation of WAT browning.

Stem Cell Therapy Improves Human Islet Graft Survival in Mice via Regulation of Macrophages.

Islet/ß-cell transplantation offers great hope for patients with type 1 diabetes. We assessed the mechanisms of how intrahepatic coinfusion of human α-1 antitrypsin (hAAT)-engineered mesenchymal stromal cells (hAAT-MSCs) improves survival of human islet grafts posttransplantation (PT). Longitudinal in vivo bioluminescence imaging studies identified significantly more islets in the livers bearing islets cotransplanted with hAAT-MSCs compared with islets transplanted alone. In vitro mechanistic studies revealed that hAAT-MSCs inhibit macrophage migration and suppress IFN-γ-induced M1-like macrophages while promoting IL-4-induced M2-like macrophages. In vivo this translated to significantly reduced CD11c+ and F4/80+ cells and increased CD206+ cells around islets cotransplanted with hAAT-MSCs as identified by multiplex immunofluorescence staining. Recipient-derived F4/80+and CD11b+ macrophages were mainly present in the periphery of an islet, while CD11c+ and CD206+ cells appeared inside an islet. hAAT-MSCs inhibited macrophage migration and skewed the M1-like phenotype toward an M2 phenotype both in vitro and in vivo, which may have favored islet survival. These data provide evidence that hAAT-MSCs cotransplanted with islets remain in the liver and shift macrophages to a protective state that favors islet survival. This novel strategy may be used to enhance ß-cell survival during islet/ß-cell transplantation for the treatment of type 1 diabetes or other diseases.

A Novel Cellular Therapy to Treat Pancreatic Pain in Experimental Chronic Pancreatitis Using Human Alpha-1 Antitrypsin Overexpressing Mesenchymal Stromal Cells.

Chronic pancreatitis (CP) is characterized by pancreatic inflammation, fibrosis, and abdominal pain that is challenging to treat. Mesenchymal stromal cells (MSCs) overexpressing human alpha-1 antitrypsin (hAAT-MSCs) showed improved mobility and protective functions over native MSCs in nonobese diabetic mice. We investigated whether hAAT-MSCs could mitigate CP and its associated pain using trinitrobenzene sulfonic acid (TNBS)-induced CP mouse models. CP mice were given native human MSCs or hAAT-MSCs (0.5 × 106 cells/mouse, i.v., n = 6-8/group). The index of visceral pain was measured by graduated von Frey filaments. Pancreatic morphology and pancreatic mast cell count were analyzed by morphological stains. Nociceptor transient receptor potential vanilloid 1 (TRPV1) expression in dorsal root ganglia (DRG) was determined by immunohistochemistry. hAAT-MSC-treated CP mice best preserved pancreatic morphology and histology. MSC or hAAT-MSC infusion reduced abdominal pain sensitivities. hAAT-MSC therapy also suppressed TRPV1 expression in DRG and reduced pancreatic mast cell density induced by TNBS. Overall, hAAT-MSCs reduced pain and mitigated pancreatic inflammation in CP equal to MSCs with a trend toward a higher pancreatic weight and better pain relief in the hAAT-MSC group compared to the MSC group. Both MSCs and hAAT-MSCs might be used as a novel therapeutic tool for CP-related pain.

Inhibition of NFAM1 suppresses phospho-SAPK/JNK signaling during osteoclast differentiation and bone resorption.

We have recently demonstrated NFAT activating protein with ITAM motif 1 (NFAM1) signaling increases osteoclast (OCL) formation/bone resorption associated with the Paget's disease of bone, however, the underlying molecular mechanisms of the NFAM1 regulation of OCL differentiation and bone resorption remains unclear. Here, we showed that RANK ligand stimulation enhances NFAM1 expression in preosteoclast cells. Conditioned media collected from RANKL stimulated RAW264.7 NFAM1 knockdown (KD) stable cells showed inhibition of interleukin-6 (2.5-fold), tumour necrosis factor-α (2.2-fold) and CXCL-5 (3-fold) levels compared to wild-type (WT) cells. Further, RANKL stimulation significantly increased p-STAT6 expression (5.5-fold) in WT cells, but no significant effect was observed in NFAM1-KD cells. However, no changes were detected in signal transducer and activator of transcription 3 levels in either of cell groups. Interestingly, NFAM1-KD suppressed the RANKL stimulated c-fos, p-c-Jun and c-Jun N-terminal kinase (JNK) activity in preosteoclasts. We further showed that the suppression of JNK activity is through inhibition of p-SAPK/JNK in these cells. In addition, NFATc1 expression, a critical transcription factor associated with osteoclastogenesis is significantly inhibited in NFAM1-KD preosteoclast cells. Interestingly, NFAM1 inhibition suppressed the OCL differentiation and bone resorption capacity in mouse bone marrow cell cultures. We also demonstrated inhibition of tartrate-resistant acid phosphatase expression in RANKL stimulated NFAM1-KD preosteoclast cells. Thus, our results suggest that NFAM1 control SAPK/JNK signaling to modulate osteoclast differentiation and bone resorption.

Minimizing Post-Infusion Portal Vein Bleeding during Intrahepatic Islet Transplantation in Mice.

Although the liver is currently accepted as the primary transplantation site for human islets in clinical settings, islets are transplanted under the kidney capsule in most rodent preclinical islet transplantation studies. This model is commonly used because murine intrahepatic islet transplantation is technically challenging, and a high percentage of mice could die from surgical complications, especially bleeding from the injection site post-transplantation. In this study, two procedures that can minimize the incidence of post-infusion portal vein bleeding are demonstrated. The first method applies an absorbable hemostatic gelatin sponge to the injection site, and the second method involves penetrating the islet injection needle through the fat tissue first and then into the portal vein by using the fat tissue as a physical barrier to stop bleeding. Both methods could effectively prevent bleeding-induced mouse death. The whole liver section showing islet distribution and evidence of islet thrombosis post-transplantation, a typical feature for intrahepatic islet transplantation, were presented. These improved protocols refine the intrahepatic islet transplantation procedures and may help laboratories set up the procedure to study islet survival and function in pre-clinical settings.

A Mouse Model for Chronic Pancreatitis via Bile Duct TNBS Infusion.

Chronic pancreatitis (CP) is a complex disease involving pancreatic inflammation and fibrosis, glandular atrophy, abdominal pain and other symptoms. Several rodent models have been developed to study CP, of which the bile duct 2,4,6 -trinitrobenzene sulfonic acid (TNBS) infusion model replicates the features of neuropathic pain seen in CP. However, bile duct drug infusion in mice is technically challenging. This protocol demonstrates the procedure of bile duct TNBS infusion for generation of a CP mouse model. TNBS was infused into the pancreas through the ampulla of Vater in the duodenum. This protocol optimized drug volume, surgical techniques, and drug handling during the procedure. TNBS-treated mice showed features of CP as reflected by bodyweight and pancreas weight reductions, changes in pain-associated behaviors, and abnormal pancreatic morphology. With these improvements, mortality associated with TNBS injection was minimal. This procedure is not only critical in generating pancreatic disease models but is also useful in local pancreatic drug delivery.

Overexpression of alpha-1 antitrypsin in mesenchymal stromal cells improves their intrinsic biological properties and therapeutic effects in nonobese diabetic mice.

Islet/ß cell dysfunction and death caused by autoimmune-mediated injuries are major features of type 1 diabetes (T1D). Mesenchymal stromal cells (MSCs) have been used for the treatment of T1D in animal models and clinical trials. Based on the anti-inflammatory effects of alpha-1 antitrypsin (AAT), we generated human AAT engineered MSCs (hAAT-MSCs) by infecting human bone marrow-derived MSCs with the pHAGE CMV-a1aT-UBC-GFP-W lentiviral vector. We compared the colony forming, differentiation, and migration capacity of empty virus-treated MSCs (hMSC) and hAAT-MSCs and tested their protective effects in the prevention of onset of T1D in nonobese diabetic (NOD) mice. hAAT-MSCs showed increased self-renewal, better migration and multilineage differentiation abilities compared to hMSCs. In addition, polymerase chain reaction array for 84 MSC-related genes showed that 23 genes were upregulated, and 3 genes were downregulated in hAAT-MSCs compared to hMSCs. Upregulated genes include those critical for the stemness (ie, Wnt family member 3A [WNT3A], kinase insert domain receptor [KDR]), migration (intercellular adhesion molecule 1 [ICAM-1], vascular cell adhesion protein 1 [VICAM-1], matrix metalloproteinase-2 [MMP2]), and survival (insulin-like growth factor 1 [IGF-1]) of MSCs. Pathway analysis showed that changed genes were related to growth factor activity, positive regulation of cell migration, and positive regulation of transcription. In vivo, a single intravenous infusion of hAAT-MSCs significantly limited inflammatory infiltration into islets and delayed diabetes onset in the NOD mice compared with those receiving vehicle or hMSCs. Taken together, overexpression of hAAT in MSCs improved intrinsic biological properties of MSCs needed for cellular therapy for the treatment of T1D.

Alpha-1 antitrypsin suppresses macrophage activation and promotes islet graft survival after intrahepatic islet transplantation.

Alpha-1 antitrypsin (AAT) has protective functions in animal islet transplantation models. While the therapeutic effect of AAT therapy is currently being tested in clinical trials, we investigated the mechanism of AAT protection in a clinically relevant marginal intrahepatic human islet transplantation model. In recipients receiving islets and AAT, 68.9% (20/29) reached normoglycemia, compared to 35.7% (10/28) in those receiving islets only, at 60 days posttransplant (PT). AAT-treated mice had lower serum levels of inflammatory cytokines immediately PT. Reduced M1 macrophages were observed in livers of AAT-treated recipients compared to controls as evidenced by flow cytometry and RNA-seq transcriptional profiling analysis. In vitro AAT suppressed IFN-γ-induced M1 macrophage activation/polarization via suppression of STAT1 phosphorylation and iNOS production. AAT inhibits macrophage activation induced by cytokines or dying islets, and consequently leads to islet cell survival. In a macrophage depletion mouse model, the presence of M1 macrophages in the liver contributed to graft death. AAT, through suppressing macrophage activation, protected transplanted islets from death and dysfunction in the human islet and NOD-SCID mouse model. The protective effect of AAT was confirmed in a major mismatch allogeneic islet transplantation model. Taken together, AAT suppresses liver macrophage activation that contributes to graft survival after transplantation.

GRP94 regulates M1 macrophage polarization and insulin resistance.

Macrophage polarization contributes to obesity-induced insulin resistance. Glucose-regulated protein 94 (GRP94) is an endoplasmic reticulum (ER) chaperone specialized for folding and quality control of secreted and membrane proteins. To determine the role of GRP94 in macrophage polarization and insulin resistance, macrophage-specific GRP94 conditional knockout (KO) mice were challenged with a high-fat diet (HFD). Glucose tolerance, insulin sensitivity, and macrophage composition were compared with control mice. KO mice showed better glucose tolerance and increased insulin sensitivity. Adipose tissues from HFD-KO mice contained lower numbers of M1 macrophages, with lower expression of M1 macrophage markers, than wild-type (WT) mice. In vitro, WT adipocytes cocultured with KO macrophages retained insulin sensitivity, whereas those cultured with WT macrophages did not. In addition, compared with WT bone marrow-derived macrophages (BMDMs), BMDMs from GRP94 KO mice exhibited lower expression of M1 macrophage marker genes following stimulation with LPS or IFN-γ, and exhibited partially increased expression of M2 macrophage marker genes following stimulation with interleukin-4. These findings identify GRP94 as a novel regulator of M1 macrophage polarization and insulin resistance and inflammation.

Islet Harvest in Carbon Monoxide-Saturated Medium for Chronic Pancreatitis Patients Undergoing Islet Autotransplantation.

Stresses encountered during human islet isolation lead to unavoidable ß-cell death after transplantation. This reduces the chance of insulin independence in chronic pancreatitis patients undergoing total pancreatectomy and islet autotransplantation. We tested whether harvesting islets in carbon monoxide-saturated solutions is safe and can enhance islet survival and insulin independence after total pancreatectomy and islet autotransplantation. Chronic pancreatitis patients who consented to the study were randomized into carbon monoxide (islets harvested in a carbon monoxide-saturated medium) or control (islets harvested in a normal medium) groups. Islet yield, viability, oxygen consumption rate, ß-cell death (measured by unmethylated insulin DNA), and serum cytokine levels were measured during the peri-transplantation period. Adverse events, metabolic phenotypes, and islet function were measured prior and at 6 months post-transplantation. No adverse events directly related to the infusion of carbon monoxide islets were observed. Carbon monoxide islets showed significantly higher viability before transplantation. Subjects receiving carbon monoxide islets had less ß-cell death, decreased CCL23, and increased CXCL12 levels at 1 or 3 days post transplantation compared with controls. Three in 10 (30%) of the carbon monoxide subjects and none of the control subjects were insulin independent. This pilot trial showed for the first time that harvesting human islets in carbon monoxide-saturated solutions is safe for total pancreatectomy and islet autotransplantation patients.

Clathrin-mediated Endocytosis of Alpha-1 Antitrypsin is Essential for its Protective Function in Islet Cell Survival.

Cytokine-induced pancreatic ß cell death plays a pivotal role in both type 1 and type 2 diabetes. Our previous study showed that alpha-1 antitrypsin (AAT) inhibits ß cell death through the suppression of cytokine-induced c-Jun N-terminal kinase (JNK) activation in an islet transplantation model. The aim of this study was to further understand how AAT impacts ß cells by studying AAT endocytosis in human islets and a ßTC3 murine insulinoma cell line. Methods: In vitro, human islets and ßTC3 cells were stimulated with cytokines in the presence or absence of chlorpromazine (CPZ), a drug that disrupts clathrin-mediated endocytosis. Western blot, real-time PCR and cell death ELISA were performed to investigate ß cell death. The oxygen consumption rate (OCR) was measured on human islets. In vivo, islets were harvested from C57BL/6 donor mice treated with saline or human AAT and transplanted into the livers of syngeneic mice that had been rendered diabetic by streptozotocin (STZ). Islet graft survival and function were analyzed. Results: AAT was internalized by ß cells in a time- and dose-dependent manner. AAT internalization was mediated by clathrin as treatment with CPZ, profoundly decreased AAT internalization, cytokine-induced JNK activation and the downstream upregulation of c-Jun mRNA expression. Similarly, addition of CPZ attenuated cytokine-induced caspase 9 cleavage (c-casp 9) and DNA fragmentation, which was suppressed by AAT. Treatment of donor mice with AAT produced AAT internalization in islets, and resulted in a higher percentage of recipients reaching normoglycemia after syngeneic intraportal islet transplantation. Conclusion: Our results suggest that AAT is internalized by ß cells through clathrin-mediated endocytosis that leads to the suppression of caspase 9 activation. This process is required for the protective function of AAT in islets when challenged with proinflammatory cytokines or after islet transplantation.

Insights into hypoxic adaptation in Tibetan chicken embryos from comparative proteomics.

Tibetan chicken, an indigenous breed, is highly adapted to the extreme environment of the Qinghai-Tibet Plateau. It serves as a model organism to identify genetic differences between hypoxia-adapted and lowland breeds. However, the mechanisms underlying hypoxia adaptation are yet unclear. This study aimed to identify differently abundant proteins (DAPs) and elucidate the mechanisms involved in hypoxic adaptation in the Tibetan chicken. In this study, we obtained proteome data for the embryonic heart tissues of Tibetan and Chahua chickens incubated under hypoxia (TCH and CHH) and normoxia (TCN and CHN) using isobaric tags for relative and absolute quantitation (iTRAQ) technology. We identified 4210 proteins from 53,352 unique peptides in the heart tissue of chicken embryos. Pairwise TCH vs. CHH, TCH vs. TCN, CHH vs. CHN, and TCN vs. CHN comparisons revealed 118, 176, 103, and 162 differently abundant proteins, respectively. Several key proteins (EGLN1, MAP2K2, MYLK, QARS, NOTCH2, and MYH7) and pathways (glutathione metabolism, PPAR signaling pathway, and vascular smooth muscle contraction) were identified and considered important candidates for high-altitude adaptation in Tibetan chicken. This study provides novel insights into the chicken embryonic heart tissue and furthers the current understanding of the mechanisms of survival among animals in high-altitude environments.

Carbon Monoxide Inhibits Islet Apoptosis via Induction of Autophagy.

AIM: Carbon monoxide (CO) functions as a therapeutic molecule in various disease models because of its anti-inflammatory and antiapoptotic properties. We investigated the capacity of CO to reduce hypoxia-induced islet cell death and dysfunction in human and mouse models. RESULTS: Culturing islets in CO-saturated medium protected them from hypoxia-induced apoptosis and preserved ß cell function by suppressing expression of proapoptotic (Bim, PARP, Cas-3), proinflammatory (TNF-α), and endoplasmic reticulum (ER) stress (glucose-regulated protein 94, grp94, CHOP) proteins. The prosurvival effects of CO on islets were attenuated when autophagy was blocked by specific inhibitors or when either ATG7 or ATG16L1, two essential factors for autophagy, was downregulated by siRNA. In vivo, CO exposure reduced both inflammation and cell death in grafts immediately after transplantation, and enhanced long-term graft survival of CO-treated human and mouse islet grafts in streptozotocin-induced diabetic non-obese diabetic severe combined immunodeficiency (NOD-SCID) or C57BL/6 recipients. INNOVATION: These findings underline that pretreatment with CO protects islets from hypoxia and stress-induced cell death via upregulation of ATG16L1-mediated autophagy. CONCLUSION: Our results suggested that CO exposure may provide an effective means to enhance survival of grafts in clinical islet cell transplantation, and may be beneficial in other diseases in which inflammation and cell death pose impediments to achieving optimal therapeutic effects. Antioxid. Redox Signal. 28, 1309-1322.

Genome methylation and regulatory functions for hypoxic adaptation in Tibetan chicken embryos.

Tibetan chickens have unique adaptations to the extreme high-altitude environment that they inhabit. Epigenetic DNA methylation affects many biological processes, including hypoxic adaptation; however, the regulatory genes for DNA methylation in hypoxic adaptation remain unknown. In this study, methylated DNA immunoprecipitation with high-throughput sequencing (MeDIP-seq) was used to provide an atlas of the DNA methylomes of the heart tissue of hypoxic highland Tibetan and lowland Chahua chicken embryos. A total of 31.2 gigabases of sequence data were generated from six MeDIP-seq libraries. We identified 1,049 differentially methylated regions (DMRs) and 695 related differentially methylated genes (DMGs) between the two chicken breeds. The DMGs are involved in vascular smooth muscle contraction, VEGF signaling pathway, calcium signaling pathway, and other hypoxia-related pathways. Five candidate genes that had low methylation (EDNRA, EDNRB2, BMPR1B, BMPRII, and ITGA2) might play key regulatory roles in the adaptation to hypoxia in Tibetan chicken embryos. Our study provides significant explanations for the functions of genes and their epigenetic regulation for hypoxic adaptation in Tibetan chickens.

Adipose stem cells from chronic pancreatitis patients improve mouse and human islet survival and function.

BACKGROUND: Chronic pancreatitis has surgical options including total pancreatectomy to control pain. To avoid surgical diabetes, the explanted pancreas can have islets harvested and transplanted. Immediately following total pancreatectomy with islet autotransplantation (TP-IAT), many islet cells die due to isolation and transplantation stresses. The percentage of patients remaining insulin free after TP-IAT is therefore low. We determined whether cotransplantation of adipose-derived mesenchymal stem cells (ASCs) from chronic pancreatitis patients (CP-ASCs) would protect islets after transplantation. METHODS: In a marginal mass islet transplantation model, islets from C57BL/6 mice were cotransplanted with CP-ASCs into syngeneic streptozotocin-treated diabetic mice. Treatment response was defined by the percentage of recipients reaching normoglycemia, and by the area under the curve for glucose and c-peptide in a glucose tolerance test. Macrophage infiltration, ß-cell apoptosis, and islet graft vasculature were measured in transplanted islet grafts by immunohistochemistry. mRNA expression profiling of 84 apoptosis-related genes in islet grafts transplanted alone or with CP-ASCs was measured by the RT2 Profiler™ Apoptosis PCR Array. The impact of insulin-like growth factor-1 (IGF-1) on islet apoptosis was determined in islets stimulated with cytokines (IL-1ß and IFN-γ) in the presence and absence of CP-ASC conditioned medium. RESULTS: CP-ASC-treated mice were more often normoglycemic compared to mice receiving islets alone. ASC cotransplantation reduced macrophage infiltration, ß-cell death, suppressed expression of TNF-α and Bcl-2 modifying factor (BMF), and upregulated expressions of IGF-1 and TNF Receptor Superfamily Member 11b (TNFRSF11B) in islet grafts. Islets cultured in conditioned medium from CP-ASCs showed reduced cell death. This protective effect was diminished when IGF-1 was blocked in the conditioned medium by the anti-IGF-1 antibody. CONCLUSION: Cotransplantation of islets with ASCs from the adipose of chronic pancreatitis patients improved islet survival and islet function after transplantation. The effects are in part mediated by paracrine secretion of IGF-1, suppression of inflammation, and promotion of angiogenesis. ASCs from chronic pancreatitis patients have the potential to be used as a synergistic therapy to enhance the efficacy of islet transplantation following pancreatectomy.

Adipose Stem Cell Therapy Mitigates Chronic Pancreatitis via Differentiation into Acinar-like Cells in Mice.

The objective of this study was to assess the capacity of adipose-derived mesenchymal stem cells (ASCs) to mitigate disease progression in an experimental chronic pancreatitis mouse model. Chronic pancreatitis (CP) was induced in C57BL/6 mice by repeated ethanol and cerulein injection, and mice were then infused with 4 × 105 or 1 × 106 GFP+ ASCs. Pancreas morphology, fibrosis, inflammation, and presence of GFP+ ASCs in pancreases were assessed 2 weeks after treatment. We found that ASC infusion attenuated pancreatic damage, preserved pancreas morphology, and reduced pancreatic fibrosis and cell death. GFP+ ASCs migrated to pancreas and differentiated into amylase+ cells. In further confirmation of the plasticity of ASCs, ASCs co-cultured with acinar cells in a Transwell system differentiated into amylase+ cells with increased expression of acinar cell-specific genes including amylase and chymoB1. Furthermore, culture of acinar or pancreatic stellate cell lines in ASC-conditioned medium attenuated ethanol and cerulein-induced pro-inflammatory cytokine production in vitro. Our data show that a single intravenous injection of ASCs ameliorated CP progression, likely by directly differentiating into acinar-like cells and by suppressing inflammation, fibrosis, and pancreatic tissue damage. These results suggest that ASC cell therapy has the potential to be a valuable treatment for patients with pancreatitis.

Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide an unlimited cell source to treat cardiovascular diseases, the leading cause of death worldwide. However, current hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. Here, we examined two important factors that can affect functions of the nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. To examine the first factor, we prepared hiPSC cardiac spheroids with four different sizes by varying cell number per spheroid (∼0.5k, ∼1k, ∼3k, ∼7k cells/spheroid). Spheroids with ∼3k cells/spheroid was found to maximize the beneficial effects of the 3D spheroid microenvironment. This result was explained with a semi-quantitative theory that considers two competing factors: 1) the improved 3D cell-cell adhesion, and 2) the reduced oxygen supply to the center of spheroids with the increase of cell number. Also, the critical role of electrical conductivity of silicon nanowires has been confirmed in improving tissue function of hiPSC cardiac spheroids. These results lay down a solid foundation to develop suitable nanowired hiPSC cardiac spheroids as an innovative cell delivery system to treat cardiovascular diseases. STATEMENT OF SIGNIFICANCE: Cardiovascular disease is the leading cause of death and disability worldwide. Due to the limited regenerative capacity of adult human hearts, human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have received significant attention because they provide a patient specific cell source to regenerate damaged hearts. Despite the progress, current human hiPSC-CMs retain an immature phenotype that leads to difficulties for integration with adult myocardium after transplantation. To address this, we recently utilized electrically conductive silicon nanowires (e-SiNWs) to facilitate self-assembly of hiPSC-CMs to form nanowired hiPSC cardiac spheroids. Our previous results showed addition of e-SiNWs effectively enhanced the functions of the cardiac spheroids and improved the cellular maturation of hiPSC-CMs. In this manuscript, we examined the effects of two important factors on the functions of nanowired hiPSC cardiac spheroids: (1) cell number per spheroid (i.e., size of the spheroids), and (2) the electrical conductivity of the e-SiNWs. The results from these studies will allow for the development of suitable nanowired hiPSC cardiac spheroids to effectively deliver hiPSC-CMs for heart repair.