Addition Of Nicotinamide Research Articles

Activated NAD+ biosynthesis pathway induces olaparib resistance in BRCA1 knockout pancreatic cancer cells.

PARP inhibitors have been developed as anti-cancer agents based on synthetic lethality in homologous recombination deficient cancer cells. However, resistance to PARP inhibitors such as olaparib remains a problem in clinical use, and the mechanisms of resistance are not fully understood. To investigate mechanisms of PARP inhibitor resistance, we established a BRCA1 knockout clone derived from the pancreatic cancer MIA PaCa-2 cells, which we termed C1 cells, and subsequently isolated an olaparib-resistant C1/OLA cells. We then performed RNA-sequencing and pathway analysis on olaparib-treated C1 and C1/OLA cells. Our results revealed activation of cell signaling pathway related to NAD+ metabolism in the olaparib-resistant C1/OLA cells, with increased expression of genes encoding the NAD+ biosynthetic enzymes NAMPT and NMNAT2. Moreover, intracellular NAD+ levels were significantly higher in C1/OLA cells than in the non-olaparib-resistant C1 cells. Upregulation of intracellular NAD+ levels by the addition of nicotinamide also induced resistance to olaparib and talazoparib in C1 cells. Taken together, our findings suggest that upregulation of intracellular NAD+ is one of the factors underlying the acquisition of PARP inhibitor resistance.

Abstract CT252: Nicotinamide in combination with EGFR-TKIs for the treatment of stage IV lung adenocarcinoma with EGFR mutations: A randomized double-blind (Phase IIb) trial

Abstract Background: RUNX3 is a tumor suppressor gene, which is inactivated in approximately 70% of lung adenocarcinomas. Nicotinamide, a sirtuin inhibitor, has demonstrated potential in re-activating epigenetically silenced RUNX3 in cancer cells. This study assessed the therapeutic benefits of combining nicotinamide with first-generation EGFR-TKIs for patients with stage IV lung cancer carrying EGFR mutations. Patients and Methods: We assessed the impact of nicotinamide on carcinogen-induced lung adenocarcinomas in mice and observed that nicotinamide increased RUNX3 levels and inhibited lung cancer growth. Subsequently, 110 consecutive patients with stage IV lung cancer who had EGFR mutations were recruited: 70 females (63.6%) and 84 never-smokers (76.4%). The patients were randomly assigned to receive either nicotinamide (1 g/day, n = 55) or placebo (n = 55). The primary and secondary endpoints were progression-free survival (PFS) and overall survival (OS), respectively. Results: After a median follow-up of 54.3 months, the nicotinamide group exhibited a median PFS of 12.7 months (95% CI, 10.4-18.3), while the placebo group had a PFS of 10.9 months (9.0-13.2; p = 0.2). The median OS was similar in the two groups (31.0 months with nicotinamide vs. 29.4 months with placebo; p = 0.2). Notably, subgroup analyses revealed a significant reduction in mortality risk for females (p = 0.01) and never-smokers (p = 0.03) treated with nicotinamide. Conclusion: The addition of nicotinamide with EGFR-TKIs demonstrated potential improvements in PFS and OS, with notable survival benefits for female patients and those who had never smoked. (ClinicalTrials.gov Identifier: NCT02416739) Citation Format: Cheol-Kyu Park, Hyung-Joo Oh, Suk-Chul Bae, In-Jae Oh, Kyoung-Mi Jung, Da-Mi Kim, Jung-Won Lee, Chang Kyun Kang, Il Yeong Park, Young-Chul Kim. Nicotinamide in combination with EGFR-TKIs for the treatment of stage IV lung adenocarcinoma with EGFR mutations: A randomized double-blind (Phase IIb) trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT252.

GTP-dependent regulation of heterochromatin fluctuations at subtelomeric regions in Saccharomyces cerevisiae.

In eukaryotes, single cells in a population display different transcriptional profiles. One of the factors regulating this heterogeneity is the chromatin state in each cell. However, the mechanisms of epigenetic chromatin regulation of specific chromosomal regions remain unclear. Therefore, we used single-cell tracking system to analyze IMD2. IMD2 is located at the subtelomeric region of budding yeast, and its expression is epigenetically regulated by heterochromatin fluctuations. Treatment with mycophenolic acid, an inhibitor of de novo GTP biosynthesis, triggered a decrease in GTP, which caused heterochromatin fluctuations at the IMD2 locus. Interestingly, within individually tracked cells, IMD2 expression state underwent repeated switches even though IMD2 is positioned within the heterochromatin region. We also found that 30% of the cells in a population always expressed IMD2. Furthermore, the addition of nicotinamide, a histone deacetylase inhibitor, or guanine, the GTP biosynthesis factor in salvage pathway of GTP biosynthesis, regulated heterogeneity, resulting in IMD2 expression being uniformly induced or suppressed in the population. These results suggest that gene expression heterogeneity in the IMD2 region is regulated by changes in chromatin structure triggered by slight decreases in GTP.

Nicotinamide potentiates amphotericin B activity against Candida albicans

ABSTRACT Amphotericin B (AmB) is a widely used antifungal agent especially for the therapy of systemic fungal infections. However, the severe side effects of AmB often leads to the premature termination of the treatment. So it is imperative to find the drugs that can both reduce the dosage and enhance the antifungal efficacy of AmB. Here we demonstrated that Nicotinamide (NAM), a cheap and safe vitamin, could enhance the antifungal activities of AmB. We demonstrated the synergistic interaction of NAM and AmB against Candida albicans as well as other Candida spp. and Cryptococcus neoformans. Moreover, NAM could enhance of the activity of AmB against biofilm. This enhancement was also observed in disseminated candidiasis in vivo. Our further study revealed that AmB could induce oxidative damage through the modification of histone acetylation. AmB could inhibit the expression of HST3, an H3K56 deacetylase in C. albicans. The immunoblotting test revealed excessive H3K56ac in AmB-treated fungal cells. Consistantly, the hst3Δ mutant displayed high sensitivity to AmB, while addition of NAM, an H3K56 deacetylation inhibitor, resulted in an even severe inhibition in the growth of this strain. These results indicated that AmB could execute antifungal activity via boosting H3K56ac which was mediated by HST3, and the mechanism for the synergistic interaction of NAM and AmB was based on exacerbating this process, which led to even excessive H3K56ac and oxidative damage. This finding provided theoretical basis for better understanding the antifungal mechanisms of AmB and clinical application of this drug.

Metabolic engineering of Escherichia coli for improved d-pantothenic acid biosynthesis by enhancing NADPH availability

Microbial production of D-pantothenic acid from renewable carbon sources has attracted significant attention in recent years. Cofactor NADPH is involved in a variety of synthetic metabolic reactions, and its availability is considered to play a key role in biotransformation. In this study, nicotinamide and niacin, the precursors of NADPH, were supplemented to the growth medium of a wild-type Escherichia coli strain DPA11A01. Results showed that the addition of nicotinamide and niacin to the medium led to a significant increase in the levels of D-pantothenic acid, which were 22.76 % and 29.84 % higher than those of without precursor, respectively. Furthermore, the essential genes (zwf, icd, maeB, pntAB, sthA, yfjB, ppnk or pos5) for NADPH de novo synthesis were selected and co-overexpressed to increase the intracellular NADPH pool. The recombinant strain DPA11A14 carrying pos5 and ppnk co-expression plasmid reached 2.45 μmol NADPH /g DCW, while the control strain yielded 0.38 μmol NADPH /g DCW. Increased NADPH supply in the cells significantly enhanced D-pantothenic acid accumulation. The D-pantothenic acid titer of the co-overexpression strain DPA11A14 could reach 6.32 g/L in shake flask cultivation, which increased 53.4 % than that of the control. Fed-batch fermentation in 5-L bioreactor employing strain DPA11A14 allowed production of 63.58 g/L D-pantothenic acid with productivity of 0.73 g/L/h and yield of 0.33 g/g glucose. This study provides a productive D-pantothenic acid fermentation strain and an effective cofactor manipulating strategy for enhancing of NADPH-dependent products.

Nicotinamide Inhibits T Cell Exhaustion and Increases Differentiation of CD8 Effector T Cells.

Simple SummaryT cells that undergo repeated stimulation undergo a change called T cell exhaustion (TEx) that limits their ability to kill cancer cells and infections with microorganisms. The factors involved in the initiation and kinetics of these changes are poorly understood. In this research the authors have used an in-vitro model of repeated stimulation to investigate whether changes of TEx can be prevented by providing nicotinamide that is known to be integral in energy supply to cells. The model was found to be a good replicate of TEx changes such as upregulation of inhibitory receptors and down regulation of cytokines involved in killing of cancer. The results provide strong support for the notion that TEx results from poor energy supply from mitochondria due to generation of reactive oxygen species and that these changes can be prevented and reversed by treatment with nicotinamide. These findings appear to have important implications for treatment of cancers by immunotherapy.One of the limitations of immunotherapy is the development of a state referred to as T cell exhaustion (TEx) whereby T cells express inhibitory receptors (IRs) and lose production of effectors involved in killing of their targets. In the present studies we have used the repeated stimulation model with anti CD3 and anti CD28 to understand the factors involved in TEx development and treatments that may reduce changes of TEx. The results show that addition of nicotinamide (NAM) involved in energy supply to cells prevented the development of inhibitory receptors (IRs). This was particularly evident for the IRs CD39, TIM3, and to a lesser extent LAG3 and PD1 expression. NAM also prevented the inhibition of IL-2 and TNFα expression in TEx and induced differentiation of CD4+ and CD8 T cells to effector memory and terminal effector T cells. The present results showed that effects of NAM were linked to regulation of reactive oxygen species (ROS) consistent with previous studies implicating ROS in upregulation of TOX transcription factors that induce TEx. These effects of NAM in reducing changes of TEx and in increasing the differentiation of T cells to effector states appears to have important implications for the use of NAM supplements in immunotherapy against cancers and viral infections and require further exploration in vivo.

Silymarin modulates catabolic cytokine expression through Sirt1 and SOX9 in human articular chondrocytes

BackgroundSilymarin (SMN), a polyphenolic flavonoid, is involved in multiple bioactive functions including anti-inflammation. Pretreatment with SMN demonstrated chondroprotection against tumour necrosis factor-alpha (TNF-α) stimulation in a chondrocyte cell line. However, pre- and posttreatment with phytochemicals have varying effects on osteoarthritis (OA) chondrocytes, and the therapeutic potential of SMN after catabolic cytokine stimulation is not fully elucidated.MethodsThe cytotoxicity of SMN (12.5, 25, 50 and 100 μM) was evaluated in human primary chondrocytes. The chondrocytes were supplemented with SMN (25 and 50 μM) after interleukin-1beta (IL-1β) stimulation. The mRNA expression and protein production of catabolic/anabolic cytokines as well as extracellular matrix (ECM) components were evaluated.ResultsHigh-dose SMN (100 μM) impaired the mitochondrial activity in chondrocytes, and 50 μM SMN further caused cell death in IL-1β-stimulated cells. The addition of 25 μM SMN ameliorated cell senescence; downregulated the catabolic genes of inducible nitric oxide synthase, IL-1β, TNF-α, matrix metalloproteinase-3 (MMP-3), MMP-9 and MMP-13; upregulated the anabolic genes of tissue inhibitor of metalloproteinase-1 (TIMP-1) and collagen type II alpha 1; and restored the expression of chondrogenic phenotype genes SOX9 and sirtuin-1 (Sirt1). In addition, the production of IL-1β, MMP-3 and MMP-9 decreased with an increase in TIMP-1 secretion. However, the mRNA levels of IL-6, IL-8 and IL-10 and protein production remained high. The addition of nicotinamide, a Sirt1 inhibitor, downregulated SOX9 and attenuated the therapeutic effects of SMN on IL-1β-stimulated chondrocytes.ConclusionSMN regulates the chondrocyte phenotype through Sirt1 and SOX9 to improve ECM homeostasis and may serve as a complementary therapy for early-stage knee OA.

Nicotinamide Metabolism Modulates the Proliferation/Differentiation Balance and Senescence of Human Primary Keratinocytes

Nicotinamide (NAM) is the main precursor of nicotinamide adenine dinucleotide (NAD+), a coenzyme essential for DNA repair, glycolysis, and oxidative phosphorylation. NAM has anti-aging activity on human skin, but theunderlying mechanisms of action are unclear. Using 3-dimensional organotypic skin models, we show thatNAM inhibits differentiation of the upper epidermal layers and maintains proliferation in the basal layer. In2-dimensional culture, NAM reduces the expression of early and late epidermal differentiation markers andincreases the proliferative capacity of human primary keratinocytes. This effect is characterized by elevated clonogenicity and an increased proportion of human primary keratinocyte stem cell (holoclones) compared tocontrols. By contrast, preventing the conversion of NAM to NAD+ using FK866 leads to premature humanprimary keratinocyte differentiation and senescence, together with a dramatic drop in glycolysis and cellular adenosine triphosphate levels while oxidative phosphorylation is moderately affected. All these effectsare rescued by addition of NAM, known to compete with FK866, which suggests that conversion to NAD+ is part of the mechanistic response. These data provide insights into the control of differentiation, proliferation, and senescence by NAM and NAD+ in skin. They may lead to new therapeutic advances for skin conditions characterized by dysregulated epidermal homeostasis and premature skin aging, such as photoaging.

Magnesium but not nicotinamide prevents vascular calcification in experimental uraemia.

Optimal phosphate control is an unmet need in chronic kidney disease (CKD). High serum phosphate increases calcification burden and is associated with mortality and cardiovascular disease in CKD. Nicotinamide (NA) alone or in combination with calcium-free phosphate binders might be a strategy to reduce phosphate levels and calcification and thus impact cardiovascular disease in CKD. We studied the effect of NA alone and in combination with magnesium carbonate (MgCO3) as a potential novel treatment strategy. CKD was induced in dilute brown non-agouti/2 mice by subtotal nephrectomy followed by a high-phosphate diet (HP) and 7 weeks of treatment with NA, MgCO3 or their combination. Control mice underwent subtotal nephrectomy and received an HP or underwent sham surgery and received standard chow plus NA. CKD mice showed increased serum fibroblast growth factor 23 and calcium-phosphate product that was normalized by all treatment regimes. NA alone increased soft tissue and vascular calcification, whereas any treatment with MgCO3 significantly reduced calcification severity in CKD. While MgCO3 supplementation alone resulted in decreased calcification severity, it resulted in increased intestinal expression of the phosphate transporters type II sodium-dependent phosphate transporter 1 (Pit-1). Combined therapy of MgCO3 and NA reduced tissue calcification and normalized expression levels of intestinal phosphate transporter proteins. In conclusion, the data indicate that NA increases while MgCO3 reduces ectopic calcification severity. Augmented expression of intestinal phosphate transporters by MgCO3 treatment was abolished by the addition of NA. However, the clinical relevance of the latter remains to be explored. Importantly, the data suggest no benefit of NA regarding treatment of calcification in addition to MgCO3.

Excess Nicotinamide Induces Reactive Oxygen Species Generation, Insulin Resistance, and Epigenetic Change in Rats and Humans

The implementation of grain fortification with niacin, commonly in the form of nicotinamide, has significantly increased niacin per capita consumption in U.S. from 1930s. Nicotinamide is the precursor of NAD and NADP, which participate in many biological reactions. Therefore, nicotinamide homeostasis is vitally important for the body. This study is to investigate the effects of excess nicotinamide on ROS generation, insulin resistance, and epigenetic change, which are hallmarks of type 2 diabetes. We found acute nicotinamide overload increased ROS generation, insulin resistance, and reduced muscle glycogen level in adult rats. Besides these effects, long-term nicotinamide overload led to oxidative stress in kidney and liver, increased their DNA damage, and decreased their global DNA methylation profile. We further demonstrated that decreasing nicotinamide degradation and excretion level was crucial for the ROS generation- and insulin resistance-inducing effects using an impaired skin detoxification rat model. Using oral glucose tolerance test (OGTT), we observed that in healthy volunteers, nicotinamide addition induced higher levels of ROS generation and insulin resistance in the early phase, followed by hypoglycemia in the late phase. Compared to controls, diabetic patients showed a disturbed nicotinamide degradation pattern, characterized by higher plasma N1-methylnicotinamide (NMN) levels and lower urinary degraded metabolite, N1-methyl-2-pyridone-5-carboxamide (2-Py). Mechanistically nicotinamide degradation process was associated with ROS generation; excess nicotinamide led to higher ROS generation causing kidney and liver damage. We also found that excess nicotinamide disturbed the monoamine neurotransmitters degradation and DNA methylation by competing for the biological methyl-donor, betaine in the body. In summary, our results indicate that excess nicotinamide may play an important role in type 2 diabetes. Disclosure Y. Zhou: None. N. Chen: None. D. Li: None. W. Sun: None. S. Zhou: None.

Nicotinamide alone accelerates the conversion of mouse embryonic stem cells into mature neuronal populations.

IntroductionVitamin B3 has been shown to play an important role during embryogenesis. Specifically, there is growing evidence that nicotinamide, the biologically active form of vitamin B3, plays a critical role as a morphogen in the differentiation of stem cells to mature cell phenotypes, including those of the central nervous system (CNS). Detailed knowledge of the action of small molecules during neuronal differentiation is not only critical for uncovering mechanisms underlying lineage-specification, but also to establish more effective differentiation protocols to obtain clinically relevant cells for regenerative therapies for neurodegenerative conditions such as Huntington’s disease (HD). Thus, this study aimed to investigate the potential of nicotinamide to promote the conversion of stem cells to mature CNS neurons.MethodsNicotinamide was applied to differentiating mouse embryonic stem cells (mESC; Sox1GFP knock-in 46C cell line) during their conversion towards a neural fate. Cells were assessed for changes in their proliferation, differentiation and maturation; using immunocytochemistry and morphometric analysis methods.ResultsResults presented indicate that 10 mM nicotinamide, when added at the initial stages of differentiation, promoted accelerated progression of ESCs to a neural lineage in adherent monolayer cultures. By 14 days in vitro (DIV), early exposure to nicotinamide was shown to increase the numbers of differentiated βIII-tubulin-positive neurons. Nicotinamide decreased the proportion of pluripotent stem cells, concomitantly increasing numbers of neural progenitors at 4 DIV. These progenitors then underwent rapid conversion to neurons, observed by a reduction in Sox 1 expression and decreased numbers of neural progenitors in the cultures at 14 DIV. Furthermore, GABAergic neurons generated in the presence of nicotinamide showed increased maturity and complexity of neurites at 14 DIV. Therefore, addition of nicotinamide alone caused an accelerated passage of pluripotent cells through lineage specification and further to non-dividing mature neurons.ConclusionsOur results show that, within an optimal dose range, nicotinamide is able to singly and selectively direct the conversion of embryonic stem cells to mature neurons, and therefore may be a critical factor for normal brain development, thus supporting previous evidence of the fundamental role of vitamins and their metabolites during early CNS development. In addition, nicotinamide may offer a simple effective supplement to enhance the conversion of stem cells to clinically relevant neurons.

Inhibition effect of nicotinamide and its Mannich base derivatives on mild steel corrosion in HCl

Mannich base derivatives of nicotinamide (NA), namely N-(morpholino(phenyl)methyl)nicotinamide (NMB) and N-(phenyl(piperidin-1-yl)methyl) nicotinamide (NPB), were synthesized, characterized and studied as corrosion inhibitors for mild steel (MS) in 1.0 M hydrochloric acid using gravimetric, electrochemical experiments and theoretical calculations. Nicotinamide was also subjected for all these studies. The adsorption of NA, NMB and NPB molecules on the mild steel surface in the presence of 1.0 M HCl was found to be spontaneous and obey the Langmuir adsorption isotherm as well as Dubinin–Radushkevich isotherm. Tafel polarization measurements evidenced that all the studied inhibitors are of mixed type. EIS measurements showed that the addition of NA, NMB and NPB decreases the double-layer capacitance (C dl) and consequently increases the charge transfer resistance (R CT) of the corrosion process. FT-IR analysis, SEM micrographs and AFM analysis confirmed the presence of inhibitor film and its role on the MS surface. Quantum chemical calculations at the DFT level were achieved to correlate electronic structure parameters of NA, NMB and NPB molecules with the experimental results.

Kinetics and Mechanism of oxidation of Maltose By N-Bromonicotin amide (NBN)

Kinetics study of oxidation of maltose, by aqueous alkaline solution of N-Bromonicotinamide (NBN) has been carried out in the temperature range 308-323K.The reaction exhibits first order in [KOH] and [Maltose] and zero order about oxidant. Addition of nicotinamide (NA) has no effect. Increase in ionic strength of the medium does not change the rate. Effect of temperature on the rate of oxidation has been followed to show the validity of Arrehenius equation and various activation parameters have been computed. The stoichiometry of the reaction was found to be 1:1. 1,2-enediol is found to be the reactive intermediate. Maltobionic acid is the product of oxidation.

Effects of Sirtuin 1 on the proliferation and osteoblastic differentiation of periodontal ligament stem cells and stem cells from apical papilla.

The function of SIRT1 in the proliferation and osteoblastic differentiation of dental stem cells is unclear. The aim of this study was to assess the roles of SIRT1 in these processes using periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAPs). A defined concentration of resveratrol, an SIRT1 activator, or nicotinamide, an SIRT1 inhibitor, was administered to PDLSCs, SCAPs, and a mixed group of the two cell lines, and their effects on these processes analyzed. Cell proliferation was tested using microtitration with a tetrazolium dye (MTT). Alkaline phosphatase (ALP) activity, mineralization ability, and the expression of osteoblastic differentiation-associated genes were assessed as well. These studies demonstrated that resveratrol could promote cell proliferation of all three groups in a gradually increasing trend over time. In contrast, nicotinamide suppressed the proliferation of the three cell lines. The results also showed that the markers of osteoblastic differentiation: ALP activity, mineralization ability, and the expression levels of the osteoblastic genes ALP, osteopontin, osteocalcin, and bone sialoprotein, were enhanced in the groups with resveratrol treatment. In contrast, following addition of nicotinamide, ALP activity, mineralization ability, and the expression levels of the osteoblastic genes were down-regulated in the cells. Together, these results suggest that the SIRT1 activator and inhibitor compounds, resveratrol and nicotinamide, function at high efficiency in adjusting cell proliferation, and that SIRT1 is a powerful regulator of osteoblastic differentiation of PDLSCs and SCAPs. In addition, co-culture of the two cell lines could promote their abilities of proliferation and osteogenic differentiation.

Kinetics and Mechanism of Oxidation of Sucrose by N-Bromonicotinamide NBN

Kinetics study of oxidation of sucrose, by aqueous alkaline solution of N-Bromonicotinamide (NBN) has been carried out in the temperature range 308-323K.The reaction exhibits first order in [NaOH] and [Sucrose] and zero order about oxidant. Addition of nicotinamide (NA) has no effect. Increase in ionic strength of the medium does not change the rate. Effect of temperature on the rate of oxidation has been followed to show the validity of Arrehenius equation and various activation parameters have been computed. The stoichiometry of the reaction was found to be 1:1. 1,2-enediol is found to be the reactive intermediate. Arabinonic acid, glycolic acid and formic acid are the products of oxidation.

Kinetics and Mechanism of Oxidation of Sucrose by N-Bromonicotinamide NBN

Kinetics study of oxidation of sucrose, by aqueous alkaline solution of N-Bromonicotinamide (NBN) has been carried out in the temperature range 308-323K.The reaction exhibits first order in [NaOH] and [Sucrose] and zero order about oxidant. Addition of nicotinamide (NA) has no effect. Increase in ionic strength of the medium does not change the rate. Effect of temperature on the rate of oxidation has been followed to show the validity of Arrehenius equation and various activation parameters have been computed. The stoichiometry of the reaction was found to be 1:1. 1,2-enediol is found to be the reactive intermediate. Arabinonic acid, glycolic acid and formic acid are the products of oxidation.

In Vitro Expansion of Human Gastric Epithelial Stem Cells and Their Responses to Bacterial Infection

We previously established long-term, 3-dimensional culture of organoids from mouse tissues (intestine, stomach, pancreas, and liver) and human intestine and pancreas. Here we describe conditions required for long-term 3-dimensional culture of human gastric stem cells. The technology can be applied to study the epithelial response to infection with Helicobacter pylori. We generated organoids from surgical samples of human gastric corpus. Culture conditions were developed based on those for the mouse gastric and human intestinal systems. We used microinjection to infect the organoids with H pylori. Epithelial responses were measured using microarray and quantitative polymerase chain reaction analyses. Human gastric cells were expanded indefinitely in 3-dimensional cultures. We cultured cells from healthy gastric tissues, single-sorted stem cells, or tumor tissues. Organoids maintained many characteristics of their respective tissues based on their histology, expression of markers, and euploidy. Organoids from healthy tissue expressed markers of 4 lineages of the stomach and self-organized into gland and pit domains. They could be directed to specifically express either lineages of the gastric gland, or the gastric pit, by addition of nicotinamide and withdrawal of WNT. Although gastric pit lineages had only marginal reactions to bacterial infection, gastric gland lineages mounted a strong inflammatory response. We developed a system to culture human gastric organoids. This system can be used to study H pylori infection and other gastric pathologies.

Su1928 Comparison of Effects of Sleeve Gastrectomy, Duodenal-Jejunal Bypass and Ileal Transposition for the Treatment of Type II Diabetes

Background: Porcine models are important tools for preclinical studies of stem cell transplantation. However, methods for the long-term culture and genetic manipulation of small intestinal crypt stem cells from adult pigs are lacking. Methods: Two-cm segments of jejunum were procured from 10-14 week-old Yorkshire pigs (n=17). Crypts were isolated by EDTA chelation, suspended in Matrigel and grown in various culture media. We first used murine media containing only epidermal growth factor, noggin, and R-spondin 1 (ENR medium) and then tested supplementation of Wnt3a, nicotinamide and small molecule inhibitors of GSK3 (CHIR99021), p160ROCK (Y27632), p38MAP kinase (SB202190), and TGFβ receptor (LY2157299). We also isolated intestinal subepithelial myofibroblasts (ISEMFs), cultured them in DMEM with 10% FBS and added ISEMF or Wnt3a-conditioned medium (CM) to crypt cultures in a 1:1 ratio. We evaluated all media for enterosphere-forming efficiency and assessed cell lineage differentiation by immunohistology and fluorescence microscopy (FM). After we found that enteroids could be grown long-term in ENR medium with nicotinamide, Wnt3a-CM, SB202190 and LY2157299, we passaged enteroids while adding various compounds, e.g., PGE2 and the Notch ligand Jagged1, and testedmedia combinations to produce transducible cells. When crypts were grown in ENR medium with ISEMF-CM, CHIR99021 and Y27632, spheroids formed in culture that could be transduced using a lentiviral vector encoding a GFP reporter. Spheroids were maintained in this medium for 2 days and reporter activity was assessed by FM. Results: ENR medium did not sustain adult porcine enterospheres beyond 5 days in culture. Addition of nicotinamide and small molecule inhibitors drastically improved 2-5 day survival of enterospheres but did not support long-term culture. Crypts grown in presence of porcine ISEMF-CM or co-cultured with porcine ISEMFs formed large, thin-walled spheroids. In contrast, Wnt3a-CM resulted in complex enteroids with budding extensions. Passaged enteroids continued to proliferate well for up to 5 weeks in culture when PGE2 was added at time of passage to enhance growth whereas Jagged1 had no discernible effect. Successful lentiviral transfection of spheroids was demonstrated with reporter activity present in >90% of transduced spheroids compared to controls. Discussion: We describe a novel method to maintain adult porcine crypt cells in culture over several weeks. Porcine crypts can be induced by specific supplements to form enteroids which can be repeatedly passaged in vitro. Porcine ISEMFs produce factors that induce formation of spheroids which can be successfully transduced with >90% efficiency using lentiviral vectors. Acknowledgment: This work was conducted by the Intestinal Stem Cell Consortium. The ISCC is supported by the NIDDK and NIAID (grant #DK085535).

Su1927 Generation of Porcine Intestinal Enteroids and Transducible Spheroids

Background: Porcine models are important tools for preclinical studies of stem cell transplantation. However, methods for the long-term culture and genetic manipulation of small intestinal crypt stem cells from adult pigs are lacking. Methods: Two-cm segments of jejunum were procured from 10-14 week-old Yorkshire pigs (n=17). Crypts were isolated by EDTA chelation, suspended in Matrigel and grown in various culture media. We first used murine media containing only epidermal growth factor, noggin, and R-spondin 1 (ENR medium) and then tested supplementation of Wnt3a, nicotinamide and small molecule inhibitors of GSK3 (CHIR99021), p160ROCK (Y27632), p38MAP kinase (SB202190), and TGFβ receptor (LY2157299). We also isolated intestinal subepithelial myofibroblasts (ISEMFs), cultured them in DMEM with 10% FBS and added ISEMF or Wnt3a-conditioned medium (CM) to crypt cultures in a 1:1 ratio. We evaluated all media for enterosphere-forming efficiency and assessed cell lineage differentiation by immunohistology and fluorescence microscopy (FM). After we found that enteroids could be grown long-term in ENR medium with nicotinamide, Wnt3a-CM, SB202190 and LY2157299, we passaged enteroids while adding various compounds, e.g., PGE2 and the Notch ligand Jagged1, and testedmedia combinations to produce transducible cells. When crypts were grown in ENR medium with ISEMF-CM, CHIR99021 and Y27632, spheroids formed in culture that could be transduced using a lentiviral vector encoding a GFP reporter. Spheroids were maintained in this medium for 2 days and reporter activity was assessed by FM. Results: ENR medium did not sustain adult porcine enterospheres beyond 5 days in culture. Addition of nicotinamide and small molecule inhibitors drastically improved 2-5 day survival of enterospheres but did not support long-term culture. Crypts grown in presence of porcine ISEMF-CM or co-cultured with porcine ISEMFs formed large, thin-walled spheroids. In contrast, Wnt3a-CM resulted in complex enteroids with budding extensions. Passaged enteroids continued to proliferate well for up to 5 weeks in culture when PGE2 was added at time of passage to enhance growth whereas Jagged1 had no discernible effect. Successful lentiviral transfection of spheroids was demonstrated with reporter activity present in >90% of transduced spheroids compared to controls. Discussion: We describe a novel method to maintain adult porcine crypt cells in culture over several weeks. Porcine crypts can be induced by specific supplements to form enteroids which can be repeatedly passaged in vitro. Porcine ISEMFs produce factors that induce formation of spheroids which can be successfully transduced with >90% efficiency using lentiviral vectors. Acknowledgment: This work was conducted by the Intestinal Stem Cell Consortium. The ISCC is supported by the NIDDK and NIAID (grant #DK085535).

Sirtainties in pancreatic cancer?

Sirtainties in pancreatic cancer?