Detection of PD-L1-Expressing Myeloid Cell Clusters in the Hyaluronan-Enriched Stroma in Tumor Tissue and Tumor-Draining Lymph Nodes.

Expression of the transmembrane protein PD-L1 is frequently upregulated in cancer. Because PD-L1-expressing cells can induce apoptosis or anergy of T lymphocytes through binding to the PD1 receptor, the PD-L1-mediated inhibition of activated PD1+ T cells is considered a major pathway for tumor immune escape. However, the mechanisms that regulate the expression of PD-L1 in the tumor microenvironment are not fully understood. Analysis of organotypic tumor tissue slice cultures, obtained from mice with implanted syngeneic tumors (MBT2 bladder tumors in C3H mice, Renca kidney, and CT26 colon tumors in BALB/c mice), as well as from patients with cancer, revealed that tumor-associated hyaluronan (HA) supports the development of immunosuppressive PD-L1+ macrophages. Using genetically modified tumor cells, we identified epithelial tumor cells and cancer-associated mesenchymal fibroblast-like cells as a major source of HA in the tumor microenvironment. These HA-producing tumor cells, and particularly the vimentin-positive fibroblast-like cells of bone marrow origin, directly interact with tumor-recruited myeloid cells to form large stromal congregates/clusters that are highly enriched for both HA and PD-L1. Furthermore, similar cell clusters composed of HA-producing fibroblast-like cells and PD-L1+ macrophages were detected in tumor-draining, but not in distant, lymph nodes. Collectively, our findings indicate that the formation of multiple large HA-enriched stromal clusters that support the development of PD-L1-expressing APCs in the tumor microenvironment and draining lymph nodes could contribute to the immune escape and resistance to immunotherapy in cancer.

Angiopoietin-like protein 8 (betatrophin) is a stress-response protein that down-regulates expression of adipocyte triglyceride lipase.

AIM: Atypical angiopoietin-like 8 (ANGPTL8), also known as betatrophin, is known to regulate lipid metabolism. However, its mechanism of action remains elusive. METHODS: HepG2, 3T3-L1, and NIT-1 cells were cultured in amino acid-complete MEM or histidine-free MEM to detect ANGPTL8 expression. The three cell types were treated with or without recombinant ANGPTL8 to investigate its role in lipid metabolism. Hydrodynamic tail vein gene delivery was also used to examine the role of ANGPTL8 in mice. RESULTS: ANGPTL8 is significantly up-regulated in amino acid-deprived cultured cells in vitro. The activation of ANGPTL8 gene transcription was mediated through the RAS/c-RAF/MAPK signaling pathway rather than the general GCN2/ATF4 pathways. ANGPTL8 activated the ERK signal transduction pathway in hepatocytes, adipocytes, and pancreatic ß-cells, up-regulating early growth response transcription factor (Egr1) and down-regulating adipose triglyceride lipase (ATGL). CONCLUSION: ANGPTL8 is a stress-response protein that regulates fat metabolism by suppressing ATGL expression, revealing a mechanistic connection between ANGPTL8 and lipid homeostasis in mammalian cells.

MAPK signaling triggers transcriptional induction of cFOS during amino acid limitation of HepG2 cells.

Amino acid (AA) deprivation in mammalian cells activates a collection of signaling cascades known as the AA response (AAR), which is characterized by transcriptional induction of stress-related genes, including FBJ murine osteosarcoma viral oncogene homolog (cFOS). The present study established that the signaling mechanism underlying the AA-dependent transcriptional regulation of the cFOS gene in HepG2 human hepatocellular carcinoma cells is independent of the classic GCN2-eIF2-ATF4 pathway. Instead, a RAS-RAF-MEK-ERK cascade mediates AAR signaling to the cFOS gene. Increased cFOS transcription is observed from 4-24 h after AAR-activation, exhibiting little or no overlap with the rapid and transient increase triggered by the well-known serum response. Furthermore, serum is not required for the AA-responsiveness of the cFOS gene and no phosphorylation of promoter-bound serum response factor (SRF) is observed. The ERK-phosphorylated transcription factor E-twenty six-like (p-ELK1) is increased in its association with the cFOS promoter after activation of the AAR. This research identified cFOS as a target of the AAR and further highlights the importance of AA-responsive MAPK signaling in HepG2 cells.

A mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)-dependent transcriptional program controls activation of the early growth response 1 (EGR1) gene during amino acid limitation.

Amino acid (AA) limitation in mammalian cells triggers a collection of signaling cascades jointly referred to as the AA response (AAR). In human HepG2 hepatocellular carcinoma, the early growth response 1 (EGR1) gene was induced by either AA deprivation or endoplasmic reticulum stress. AAR-dependent EGR1 activation was discovered to be independent of the well characterized GCN2-ATF4 pathway and instead dependent on MEK-ERK signaling, one of the MAPK pathways. ChIP showed that constitutively bound ELK1 at the EGR1 proximal promoter region was phosphorylated after AAR activation. Increased p-ELK1 binding was associated with increased de novo recruitment of RNA polymerase II to the EGR1 promoter. EGR1 transcription was not induced in HEK293T cells lacking endogenous MEK activity, but overexpression of exogenous constitutively active MEK in HEK293T cells resulted in increased basal and AAR-induced EGR1 expression. ChIP analysis of the human vascular endothelial growth factor A (VEGF-A) gene, a known EGR1-responsive gene, revealed moderate increases in AAR-induced EGR1 binding within the proximal promoter and highly inducible binding to a site within the first intron. Collectively, these data document a novel AA-activated MEK-ERK-ELK1 signaling mechanism.

The reprogrammed pancreatic progenitor-like intermediate state of hepatic cells is more susceptible to pancreatic beta cell differentiation.

Induced pluripotent stem cells (iPSCs) hold great promise for cell therapy. However, their low efficiency of lineage-specific differentiation and tumorigenesis severely hinder clinical translation. We hypothesized that reprogramming of somatic cells into lineage-specific progenitor cells might allow for large-scale expansion, avoiding the tumorigenesis inherent with iPSCs and simultaneously facilitating lineage-specific differentiation. Here we aimed at reprogramming rat hepatic WB cells, using four Yamanaka factors, into pancreatic progenitor cells (PPCs) or intermediate (IM) cells that have characteristics of PPCs. IM clones were selected based on their specific morphology and alkaline phosphatase activity and stably passaged under defined culture conditions. IM cells did not have iPSC properties, could be stably expanded in large quantity, and expressed all 14 genes that are used to define the PPC developmental stage. Directed differentiation of IM and WB cells by Pdx1-Ngn3-MafA (PNM) into pancreatic beta-like cells revealed that the IM cells are more susceptible to directed beta cell differentiation because of their open chromatin configuration, as demonstrated by expression of key pancreatic beta cell genes, secretion of insulin in response to glucose stimulation, and easy access to exogenous PNM proteins at the rat insulin 1 and Pdx1 promoters. This notion that IM cells are superior to their parental cells is further supported by the epigenetic demonstration of accessibility of Pdx1 and insulin 1 promoters. In conclusion, we have developed a strategy to derive and expand PPC cells from hepatic WB cells using conventional cell reprogramming. This proof-of-principal study may offer a novel, safe and effective way to generate autologous pancreatic beta cells for cell therapy of diabetes.

Expression of recombinant human IL-4 in Pichia pastoris and relationship between its glycosylation and biological activity.

Secretory human interleukin 4 (hIL4) is an N-glycosylated pleiotropic cytokine. It is unknown if these N-linked glycans are required and essential for hIL4 protein stability, expression, secretion, and activity in vivo, and hIL4 expressed from Pichia pastoris yeast has not been tested to date. In this study, we successfully expressed human hIL4 in P. pastoris, the methylotrophic yeast, with a yield of 15.0mg/L. Using the site-directed mutagenesis technique, we made two mutant hIL4 cDNA clones (N38A and N105L) and subsequently expressed them in P. pastoris to analyze the relevant function of each N-glycosylation site on hIL4. Our results demonstrate that the glycosylation only occurs at position Asn38, but not Asn105. The glycosylated form of hIL4 unexpectedly has lower biological activity and lower stability when compared to its non-glycosylated form. The implications of this are discussed.

Preparation of lactose-free pasteurized milk with a recombinant thermostable ß-glucosidase from Pyrococcus furiosus.

BACKGROUND: Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions. RESULTS: A thermostable ß-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this ß-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose. CONCLUSIONS: The thermostablity of this recombinant ß-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization.

Tissue Slice Culture and Analysis of Tumor-Associated Hyaluronan in Urothelial Carcinoma.

Hyaluronan is a major component of the extracellular matrix in both normal and tumor tissue. Many solid cancers, including bladder cancer, are characterized by deregulated hyaluronan metabolism. It is postulated that the deregulated metabolism in cancer tissue is characterized by elevated hyaluronan synthesis and degradation. This results in the accumulation of small hyaluronan fragments in the tumor microenvironment which promotes cancer-related inflammation, stimulates tumor cell proliferation and angiogenesis, and contributes to immune-associated immune suppression. For a better understanding of the complex mechanisms of hyaluronan metabolism in cancer, it has been proposed to use precision-cut tissue slice cultures prepared using freshly excised cancer tissue. Here we describe the protocol for establishing tissue slice cultures and analysis of tumor-associated hyaluronan in human urothelial carcinoma.

Hyaluronan Metabolism in Urologic Cancers.

Hyaluronan (HA) is one of the major components of the extracellular matrix in tumor tissue. Recent reports have made it clear that the balance of HA synthesis and degradation is critical for tumor progression. HA is synthesized on the cytoplasmic surface of the plasma membrane by hyaluronan synthases (HAS) and extruded into the extracellular space. Excessive HA production in cancer is associated with enhanced HA degradation in the tumor microenvironment, leading to the accumulation of HA fragments with small molecular weight. These perturbations in both HA synthesis and degradation may play important roles in tumor progression. Recently, it has become increasingly clear that small HA fragments can induce a variety of biological events, such as angiogenesis, cancer-promoting inflammation, and tumor-associated immune suppression. Progression of urologic malignancies, particularly of prostate and bladder cancers, as well as of certain types of kidney cancer show markedly perturbed metabolism of tumor-associated HA. This review highlights the recent research findings regarding HA metabolism in tumor microenvironments with a special focus on urologic cancers. It also will discuss the potential implications of these findings for the development of novel therapeutic interventions for the treatment of prostate, bladder, and kidney cancers.

Expression and secretion of glycosylated barley oxalate oxidase in Pichia pastoris.

Oxalate oxidase is an enzyme that degrades oxalate and is used in commercial urinary assays to measure oxalate levels. The objective of this study was to establish an enhanced expression system for secretion and purification of oxalate oxidase using Pichia pastoris. A codon optimized synthetic oxalate oxidase gene derived from Hordeum vulgare (barley) was generated and cloned into the pPICZα expression vector downstream of the N-terminal alpha factor secretion signal peptide sequence and used for expression in P. pastoris X-33 strain. A novel chimeric signal peptide consisting of the pre-OST1 sequence fused to pro-αpp8 containing several amino acid substitutions was also generated to enhance secretion. Active enzyme was purified to greater than 90% purity using Q-Sepharose anion exchange chromatography. The purified oxalate oxidase enzyme had an estimated Km value of 256µM, and activity was determined to be 10U/mg. We have developed an enhanced oxalate oxidase expression system and method for purification.

Irisin protected hemopoietic stem cells and improved outcome of severe bone marrow failure.

Acquired aplastic anemia (AA) is a bone marrow failure (BMF) disease, characterized by fatty bone marrow (BM) and BM hypocellularity resulted from auto-immune dysregulated T cells-mediated destruction of BM haemopoietic stem cells (HPSC). The objective of this study was to investigate potential therapeutic effect of irisin, a molecule involved in adipose tissue transition, on AA mouse model. Our results showed that the concentration of irisin in serum was lower in AA patients than in healthy controls, suggesting a role of irisin in the pathogenesis of AA. In the AA mice, irisin administration prolonged the survival rate, prevented or attenuated peripheral pancytopenia, and preserved HPSC in the BM. Moreover, irisin also markedly reduced BM adipogenesis. In vitro results showed that irisin increased both cell proliferation and colony numbers of HPSC. Furthermore, our results demonstrated that irisin upregulated the expression of mitochondrial ATPase Inhibitory Factor 1 (IF1) in HPSC, inhibited the activation of mitochondrial fission protein (DRP1) and enhanced aerobic glycolysis. Taken together, our findings indicate novel roles of irisin in the pathogenesis of AA, and in the protection of HPSC through stimulation of proliferation and regulation of mitochondria function, which provides a proof-of-concept for the application of irisin in AA therapy.

Deregulated hyaluronan metabolism in the tumor microenvironment drives cancer inflammation and tumor-associated immune suppression.

Hyaluronan (HA) is known to be a prominent component of the extracellular matrix in tumors, and many solid cancers are characterized by aberrant HA metabolism resulting in increased production in tumor tissue. HA has been implicated in regulating a variety of cellular functions in tumor cells and tumor-associated stromal cells, suggesting that altered HA metabolism can influence tumor growth and malignancy at multiple levels. Importantly, increased HA production in cancer is associated with enhanced HA degradation due to high levels of expression and activity of hyaluronidases (Hyal). Understanding the complex molecular and cellular mechanisms involved in abnormal HA metabolism and catabolism in solid cancers could have important implications for the design of future cancer therapeutic approaches. It appears that extensive crosstalk between immune cells and HA-enriched stroma contributes to tumor growth and progression in several ways. Specifically, the interaction of tumor-recruited Hyal2-expressing myeloid-derived suppressor cells (MDSCs) of bone marrow origin with HA-producing cancer-associated fibroblasts and epithelial tumor cells results in enhanced HA degradation and accumulation of small pro-inflammatory HA fragments, which further drives cancer-related inflammation. In addition, hyaluronan-enriched stroma supports the transition of tumor-recruited Hyal2+MDSCs to the PD-L1+ tumor-associated macrophages leading to the formation of an immunosuppressive and tolerogenic tumor microenvironment. In this review, we aim to discuss the contribution of tumor-associated HA to cancer inflammation, angiogenesis, and tumor-associated immune suppression. We also highlight the recent findings related to the enhanced HA degradation in the tumor microenvironment.

Expression of Recombinant Rat Secretable FNDC5 in Pichia Pastoris and Detection of Its Biological Activity.

FNDC5 is the precursor of the myokine irisin proposed to exhibit favorable metabolic activity, including anti-obesity and anti-diabetes effects. The diversity of FNDC5 transcripts has been reported by several studies, but the role and existence of these transcripts are not well defined. In our previous study, a novel secretable FNDC5 (sFNDC5) isoform lacking the transmembrane region was found in rat INS-1 cells and multiple rat tissues. In the current study, we established a high-yield system for the expression and purification of sFNDC5 in Pichia pastoris, and functional investigations were undertaken using 3T3-L1 cells. We discovered that this new isoform has similar and even stronger biological functions than irisin, which may be due to its more complete structure without cleavage. Hence, we believe that sFNDC5, as the first identified readily secretable derivative, can better induce lipolysis and can potentially prevent obesity and related metabolic diseases.

Initial experience: ex-vivo perfused pig kidney to study urinary oxalate excretion.

Existing animal models of renal oxalate excretion utilize either gut or peritoneal cavity for oxalate absorption. Ex vivo renal perfusion is an established tool for graft preservation. We sought to repurpose this concept to study the early pathogenesis of urinary lithiasis. Juvenile female Yorkshire porcine kidneys were removed laparoscopically and placed on an ex vivo cardiopulmonary bypass circuit utilizing whole-blood based perfusate. Pre-defined goals were identified for each attempt (n = 5) with plans to increase physiologic model complexity. Tissue perfusion and oxygenation were monitored by serial perfusate iSTAT testing. Once steady urine production was achieved, aqueous oxalate was injected into the perfusate. Renal outcomes were assessed by histology and blood/urinary assays. After demonstrating proof-of-concept in early trials, normothermic (37 °C) ex vivo whole-blood perfusion with Steen Solution™ was performed exceeding three hours at physiologic mean arterial pressures. Circuit parameters remained in the physiologic range for electrolytes, temperature, mean arterial pressure, lactate, and pH. Urine was produced in three experiments. Urinary filtrate demonstrated consistently higher urine creatinine compared to perfusate, and arterial perfusate oxalate boluses lead to urinary oxalate spikes followed by continuous oxalate clearance. Histopathologic analysis with H&E and Pizzolato's method staining demonstrated formation of calcium oxalate crystals. In light of these promising metabolite clearances, ex vivo porcine renal perfusion appears to be a feasible alternative to study oxalate excretion. Longer validation studies are necessary to establish this technique as a model for kidney stone pathogenesis.

Distinct regulation of hepatic nuclear factor 1alpha by NKX6.1 in pancreatic beta cells.

Hepatic nuclear factor 1alpha (HNF1alpha) is a key regulator of development and function in pancreatic beta cells and is specifically involved in regulation of glycolysis and glucose-stimulated insulin secretion. Abnormal expression of HNF1alpha leads to development of MODY3 (maturity-onset diabetes of the young 3). We report that NK6 homeodomain 1 (NKX6.1) binds to a cis-regulatory element in the HNF1alpha promoter and is a major regulator of this gene in beta cells. We identified an NKX6.1 recognition sequence in the distal region of the HNF1alpha promoter and demonstrated specific binding of NKX6.1 in beta cells by electrophoretic mobility shift and chromatin immunoprecipitation assays. Site-directed mutagenesis of the NKX6.1 core-binding sequence eliminated NKX6.1-mediated activation and substantially decreased activity of the HNF1alpha promoter in beta cells. Overexpression or small interfering RNA-mediated knockdown of the Nkx6.1 gene resulted in increased or diminished HNF1alpha gene expression, respectively, in beta cells. We conclude that NKX6.1 is a novel regulator of HNF1alpha in pancreatic beta cells. This novel regulatory mechanism for HNF1alpha in beta cells may provide new molecular targets for the diagnosis of MODY3.

GLP-1 Induces the Expression of FNDC5 Derivatives That Execute Lipolytic Actions.

Multiple GLP-1-derived therapeutics are clinically used to treat type 2 diabetes and obesity. However, the underlying mechanism of how these drugs regulate the body weight of obese patients remains incompletely understood. Here, we report that the lipolysis effects of GLP-1 on ß cells can depend on its induced expression of fibronectin type III domain containing 5 (FNDC5). The transmembrane FNDC5 is a precursor of the recently identified hormone irisin that possesses a range of bioactivities, including anti-obesity and anti-diabetes. We revealed that GLP-1 upregulates the expression and secretion of FNDC5 in ß cells, while GLP-1 itself fails to activate the lipolysis genes in FNDC5-knockout ß cells. In addition, liraglutide, a clinically used GLP-1 receptor agonist, induced the expression of FNDC5 in mouse pancreas and brain tissues and increased the serum level of secreted FNDC5. Furthermore, we observed the expression of the well-known membrane-associated FNDC5 and a novel, secretable FNDC5 (sFNDC5) isoform in ß cells and multiple rat tissues. Recombinant sFNDC5 stimulated lipolysis of wild type and FNDC5-knockout ß cells. This new isoform further induced lipolysis and browning of adipocytes, and similar to irisin, executed potent anti-obesity activities in an obese mouse model. Overall, our studies provided new mechanistic insights into GLP-1's anti-obesity actions in which GLP-1 induces the secretion of FNDC5 derivatives from its responsive organs that then mediate its anti-obesity activities.

Hyal2 Expression in Tumor-Associated Myeloid Cells Mediates Cancer-Related Inflammation in Bladder Cancer.

The increased presence of myeloid-derived suppressor cells (MDSC) and tumor-associated macrophages (TAM) in tumor tissue has been extensively reported. However, their role in the regulation of hyaluronan (HA) metabolism in the tumor microenvironment has not been established. Here we describe a novel function of tumor-associated myeloid cells related to the enhanced breakdown of extracellular HA in human bladder cancer tissue, leading to the accumulation of small HA fragments with molecular weight (MW) <20 kDa. Increased fragmentation of extracellular HA and accumulation of low molecular weight HA (LMW-HA) in tumor tissue was associated with elevated production of multiple inflammatory cytokines, chemokines, and angiogenic factors. The fragmentation of HA by myeloid cells was mediated by the membrane-bound enzyme hyaluronidase 2 (Hyal2). Increased numbers of Hyal2+CD11b+ myeloid cells were detected in the tumor tissue as well as in the peripheral blood of patients with bladder cancer. Coexpression of CD33 suggested that these cells belong to monocytic myeloid-derived suppressor cells. The HA-degrading function of Hyal2-expressing MDSCs could be enhanced by exposure to tumor-conditioned medium, and IL1ß was identified as one of the factors involved in the stimulation of Hyal2 activity. CD44-mediated signaling played an important role in the regulation of HA-degrading activity of Hyal2-expressing myeloid cells, as the engagement of CD44 receptor with specific mAb triggered translocation of Hyal2 enzyme to the cellular surface and stimulated secretion of IL1ß. Taken together, this work identifies Hyal2-expressing tumor-associated myeloid cells as key players in the accumulation of LMW-HA in the tumor microenvironment and cancer-related inflammation and angiogenesis. SIGNIFICANCE: This study identifies Hyal2-expressing tumor-associated myeloid cells of monocyte-macrophage lineage as contributors to hyaluronan degradation in bladder cancer tissue, leading to accumulation of inflammatory and proangiogenic low molecular weight hyaluronan fragments.

Pancreatic duodenal homeobox 1 protein is a novel beta-cell-specific autoantigen for type I diabetes.

Pancreatic duodenal homeobox 1 (Pdx1) protein is a key transcription factor involved in the regulation of insulin gene expression that is expressed at high levels in the beta-cells of the pancreatic islets. We asked whether Pdx1 is a target of anti-islet autoimmunity in type I diabetes (T1D). Pdx1 autoantibodies (PAAs) were detected in non-obese diabetic (NOD) mice using ELISA, western blotting, and radioimmunoprecipitation of [(35)S]-labeled insulinoma cell line-derived Pdx1 protein. PAAs were detected as early as at 5 weeks of age, and generally peaked before the onset of clinically overt diabetes in diabetes-prone female NOD mice. Levels declined substantially after the onset of diabetes. PAAs were not detected in the sera of NOD-scid, C57BL/6, or BALB/c mice. The titers of PAAs in NOD mouse sera were as high as 1/93 750 by ELISA. The fine specificity of PAAs was determined by western blotting using a series of truncated recombinant Pdx1 proteins. The immunodominant epitopes were located to the C-terminus of the Pdx1 (p200-283) in NOD mice. PAAs also were detected in sera from human T1D patients, but the major epitopes were localized to amino acids 159-200 as well as the same region (p200-283) recognized by PAAs from NOD mice. Using [(3)H]thymidine incorporation, the p83 fragment of Pdx1 specifically stimulated proliferation of splenic T cells from recent-onset diabetic NOD mice. The presence of PAAs in prediabetic NOD mice and human T1D patients, and Pdx1-specific T-cell proliferation in NOD mice provide a strong rationale for further investigation of the pathogenic role of immune responses against Pdx1 in T1D.

Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris.

Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.

Real-time observation of pancreatic beta cell differentiation from human induced pluripotent stem cells.

Directed differentiation of human pluripotent stem cells (hPSCs) into functional insulin-producing cells (IPCs) holds great promise for cell therapy for diabetic patients. Despite recent advances in developing beta cell differentiation protocols, it is becoming clear that the hPSC-derived beta-like cells are functionally immature, and the efficiencies of differentiation can be variable depending on the hPSC lines used. Therefore, advanced methodologies are highly desirable for the development and refinement of beta cell differentiation protocols from hPSCs. In this report, we first derived and validated a Pdx1-mRFP/insulin-hrGFP dual-reporter cell line from MRC5-iPSCs. Then, using this dual-reporter cell line, we developed and optimized an in vitro beta cell differentiation protocol through real-time monitoring expression of Pdx1 and insulin. We demonstrated that DNA demethylation could increase the efficiency of beta cell differentiation. Furthermore, three-dimensional induction not only significantly increased the efficiency of pancreatic progenitor specification and the yield of IPCs, but also produced more mature IPCs. The current study indicates that this dual-reporter cell line is of great value for developing and optimizing the beta cell differentiation protocols. It will facilitate the development of novel protocols for generating IPCs from hPSCs and the investigation of beta cell differentiation mechanisms.