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.

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.

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.

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.

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.

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.

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.

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.

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.

Effects of irisin on the differentiation and browning of human visceral white adipocytes.

BACKGROUND: The aim of this study was to explore the effects of irisin on human visceral adipose tissue and adipocytes functions. METHODS: Fresh human visceral white adipose tissues derived from 11 donors were used to examine the effects of irisin on browning, adipogenesis and osteogenesis gene expression, and anti-inflammatory properties. Preadipocytes were also used to examine the effects of irisin on mitochondrial respiration, adipogenic differentiation, and osteogenic differentiation. KEY RESULTS: Irisin significantly increased cellular mitochondrial energy metabolism in differentiated visceral adipocytes. Irisin also increased mRNA levels of transcriptional regulators of brite/beige adipocytes (UCP-1, PGC1α, PRDM16, TMEM26, and CD137) in subcutaneous white adipose tissue but not in visceral/brown adipose tissue or their derived mature adipocytes. In parallel, irisin increased the protein levels of UCP-1 in subcutaneous white adipose tissue, but had no effect on the expression of this protein in visceral white adipose tissue and perirenal brown adipose tissue. However, irisin inhibited adipogenic differentiation, promoted osteogenic differentiation in visceral adipocytes, down-regulated adipogenesis, and upregulated osteogenesis genes expression in visceral fat tissue. Moreover, administration of irisin reduced the expression of proinflammatory marker mRNAs in both visceral and subcutaneous white adipose tissue. CONCLUSIONS: Our data suggest that (1) irisin may increase mitochondrial respiration and glycolysis in visceral adipocytes by a UCP-1 independent pathway; (2) irisin promotes anti-inflammatory activity on fat tissue.

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.

Pancreatic and duodenal homeobox gene 1 (Pdx1) down-regulates hepatic transcription factor 1 alpha (HNF1α) expression during reprogramming of human hepatic cells into insulin-producing cells.

Ectopic expression of Pdx1 triggers rapid hepatocyte dedifferentiation by down-regulating liver-enriched transcription factors and liver-specific functional genes such as hepatic nuclear factor-1α (HNF1α), albumin, and AAT. However, the links between Pdx1 over-expression and hepatic gene down-regulation are incompletely understood. HNF1α and HNF4α are important transcription factors that establish and maintain the hepatocyte phenotype. The human HNF4α gene contains two promoters (P1 and P2) that drive expression of P1-(HNF4α 1-6) or P2-(HNF4α 7-9)-derived isoforms, which are used in different tissues and at different times during development. We hypothesized that the relative expression of HNF1α and HNF4α following ectopic Pdx1 expression may promote hepatic cell dedifferentiation and transdifferentiation toward pancreatic beta-cells. We produced lentiviruses expressing Pdx1, Pdx1-VP16, and Ngn3, along with dual-color reporter genes to indicate hepatic and pancreatic beta-cell phenotype changes. Using these PTF alone or in combinations, we demonstrated that Pdx1 not only activates specific beta-cell genes but down-regulates HNF1α. Pdx1-mediated reduction of HNF1α is accompanied by altered expression of its major activator, HNF4α isoforms, down-regulating hepatic genes ALB and AAT. Pdx1 up-regulates HNF4α via the P2 promoter. These P2-driven isoforms compete with P1-driven isoforms to suppress target gene transcription. In Huh7 cells, the AF-1 activation domain is more important for transactivation, whereas in INS1 cells, the F inhibitory domain is more important. The loss and gain of functional activity strongly suggests that Pdx1 plays a central role in reprogramming hepatocytes into beta-cells by suppressing the hepatic phenotype.

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.

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.

Irisin stimulates browning of white adipocytes through mitogen-activated protein kinase p38 MAP kinase and ERK MAP kinase signaling.

The number and activity of brown adipocytes are linked to the ability of mammals to resist body fat accumulation. In some conditions, certain white adipose tissue (WAT) depots are readily convertible to a ''brown-like'' state, which is associated with weight loss. Irisin, a newly identified hormone, is secreted by skeletal muscles into circulation and promotes WAT "browning" with unknown mechanisms. In the current study, we demonstrated in mice that recombinant irisin decreased the body weight and improved glucose homeostasis. We further showed that irisin upregulated uncoupling protein-1 (UCP-1; a regulator of thermogenic capability of brown fat) expression. This effect was possibly mediated by irisin-induced phosphorylation of the p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-related kinase (ERK) signaling pathways. Inhibition of the p38 MAPK by SB203580 and ERK by U0126 abolished the upregulatory effect of irisin on UCP-1. In addition, irisin also promoted the expression of betatrophin, another newly identified hormone that promotes pancreatic ß-cell proliferation and improves glucose tolerance. In summary, our data suggest that irisin can potentially prevent obesity and associated type 2 diabetes by stimulating expression of WAT browning-specific genes via the p38 MAPK and ERK pathways.

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.

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.