Didem Demirbas

A comparison of non-integrating reprogramming methods

Human induced pluripotent stem cells (hiPSCs) are useful in disease modeling and drug discovery, and they promise to provide a new generation of cell-based therapeutics. To date there has been no systematic evaluation of the most widely used techniques for generating integration-free hiPSCs. Here we compare Sendai-viral (SeV), episomal (Epi) and mRNA transfection mRNA methods using a number of criteria. All methods generated high-quality hiPSCs, but significant differences existed in aneuploidy rates, reprogramming efficiency, reliability and workload. We discuss the advantages and shortcomings of each approach, and present and review the results of a survey of a large number of human reprogramming laboratories on their independent experiences and preferences. Our analysis provides a valuable resource to inform the use of specific reprogramming methods for different laboratories and different applications, including clinical translation.

KCNQ1, KCNE2, and Na(+)-coupled solute transporters form reciprocally regulating complexes that affect neuronal excitability

Complexes of solute transporters and potassium channels that reciprocally regulate each other may contribute to seizure susceptibility. Stopping Seizures The activity of potassium channels limits neuronal excitability, and mutations in the regulatory subunit (KCNE2), which promotes the activity of the potassium-conducting pore (KCNQ1), are associated with increased seizure susceptibility. Abbott et al. found that SMIT1, which transports the molecule myo-inositol, associated with KCNQ1 or KCNQ1-KCNE2 complexes. When complexed with KCNE2, KCNQ1 is constitutively active. SMIT1 activity was increased in the presence of KCNQ1 but was inhibited in the presence of KCNQ1-KCNE2. SMIT1 increased the activity of both KCNQ1 and KCNQ1-KCNE2 complexes. The increased seizure activity of mice deficient in KCNE2 was attenuated by administration of myo-inositol, suggesting that a decrease in SMIT1 activity or alterations in the activity of these molecular complexes may contribute to seizure susceptibility. Na+-coupled solute transport is crucial for the uptake of nutrients and metabolic precursors, such as myo-inositol, an important osmolyte and precursor for various cell signaling molecules. We found that various solute transporters and potassium channel subunits formed complexes and reciprocally regulated each other in vitro and in vivo. Global metabolite profiling revealed that mice lacking KCNE2, a K+ channel β subunit, showed a reduction in myo-inositol concentration in cerebrospinal fluid (CSF) but not in serum. Increased behavioral responsiveness to stress and seizure susceptibility in Kcne2−/− mice were alleviated by injections of myo-inositol. Suspecting a defect in myo-inositol transport, we found that KCNE2 and KCNQ1, a voltage-gated potassium channel α subunit, colocalized and coimmunoprecipitated with SMIT1, a Na+-coupled myo-inositol transporter, in the choroid plexus epithelium. Heterologous coexpression demonstrated that myo-inositol transport by SMIT1 was augmented by coexpression of KCNQ1 but was inhibited by coexpression of both KCNQ1 and KCNE2, which form a constitutively active, heteromeric K+ channel. SMIT1 and the related transporter SMIT2 were also inhibited by a constitutively active mutant form of KCNQ1. The activities of KCNQ1 and KCNQ1-KCNE2 were augmented by SMIT1 and the glucose transporter SGLT1 but were suppressed by SMIT2. Channel-transporter signaling complexes may be a widespread mechanism to facilitate solute transport and electrochemical crosstalk.

Development of a Fission Yeast-Based High-Throughput Screen to Identify Chemical Regulators of cAMP Phosphodiesterases

Cyclic nucleotide phosphodiesterases (PDEs) comprise a superfamily of enzymes that serve as drug targets in many human diseases. There is a continuing need to identify high-specificity inhibitors that affect individual PDE families or even subtypes within a single family. The authors describe a fission yeast-based high-throughput screen to detect inhibitors of heterologously expressed adenosine 3′,5′-cyclic monophosphate (cAMP) PDEs. The utility of this system is demonstrated by the construction and characterization of strains that express mammalian PDE2A, PDE4A, PDE4B, and PDE8A and respond appropriately to known PDE2A and PDE4 inhibitors. High-throughput screens of 2 bioactive compound libraries for PDE inhibitors using strains expressing PDE2A, PDE4A, PDE4B, and the yeast PDE Cgs2 identified known PDE inhibitors and members of compound classes associated with PDE inhibition. The authors verified that the furanocoumarin imperatorin is a PDE4 inhibitor based on its ability to produce a PDE4-specific elevation of cAMP levels. This platform can be used to identify PDE activators, as well as genes encoding PDE regulators, which could serve as targets for future drug screens. (Journal of Biomolecular Screening 2008:62-71)

New classes of PDE7 inhibitors identified by a fission yeast-based HTS

Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor α from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.

A yeast-based chemical screen identifies a PDE inhibitor that elevates steroidogenesis in mouse Leydig cells via PDE8 and PDE4 inhibition.

A cell-based high-throughput screen (HTS) was developed to detect phosphodiesterase 8 (PDE8) and PDE4/8 combination inhibitors. By replacing the Schizosaccharomyces pombe PDE gene with the murine PDE8A1 gene in strains lacking adenylyl cyclase, we generated strains whose protein kinase A (PKA)-stimulated growth in 5-fluoro orotic acid (5FOA) medium reflects PDE8 activity. From our previously-identified PDE4 and PDE7 inhibitors, we identified a PDE4/8 inhibitor that allowed us to optimize screening conditions. Of 222,711 compounds screened, ∼0.2% displayed composite Z scores of >20. Additional yeast-based assays using the most effective 367 compounds identified 30 candidates for further characterization. Among these, compound BC8-15 displayed the lowest IC50 value for both PDE4 and PDE8 inhibition in in vitro enzyme assays. This compound also displays significant activity against PDE10A and PDE11A. BC8-15 elevates steroidogenesis in mouse Leydig cells as a single pharmacological agent. Assays using BC8-15 and two structural derivatives support a model in which PDE8 is a primary regulator of testosterone production by Leydig cells, with an additional role for PDE4 in this process. BC8-15, BC8-15A, and BC8-15C, which are commercially available compounds, display distinct patterns of activity against PDE4, PDE8, PDE10A, and PDE11A, representing a chemical toolkit that could be used to examine the biological roles of these enzymes in cell culture systems.

Succinyl-CoA synthetase (SUCLA2) deficiency in two siblings with impaired activity of other mitochondrial oxidative enzymes in skeletal muscle without mitochondrial DNA depletion

Mutations in SUCLA2 result in succinyl-CoA ligase (ATP-forming) or succinyl-CoA synthetase (ADP-forming) (A-SCS) deficiency, a mitochondrial tricarboxylic acid cycle disorder. The phenotype associated with this gene defect is largely encephalomyopathy. We describe two siblings compound heterozygous for SUCLA2 mutations, c.985A>G (p.M329V) and c.920C>T (p.A307V), with parents confirmed as carriers of each mutation. We developed a new LC-MS/MS based enzyme assay to demonstrate the decreased SCS activity in the siblings with this unique genotype. Both siblings shared bilateral progressive hearing loss, encephalopathy, global developmental delay, generalized myopathy, and dystonia with choreoathetosis. Prior to diagnosis and because of lactic acidosis and low activity of muscle pyruvate dehydrogenase complex (PDC), sibling 1 (S1) was placed on dichloroacetate, while sibling 2 (S2) was on a ketogenic diet. S1 developed severe cyclic vomiting refractory to therapy, while S2 developed Leigh syndrome, severe GI dysmotility, intermittent anemia, hypogammaglobulinemia and eventually succumbed to his disorder. The mitochondrial DNA contents in skeletal muscle (SM) were normal in both siblings. Pyruvate dehydrogenase complex, ketoglutarate dehydrogenase complex, and several mitochondrial electron transport chain (ETC) activities were low or at the low end of the reference range in frozen SM from S1 and/or S2. In contrast, activities of PDC, other mitochondrial enzymes of pyruvate metabolism, ETC and, integrated oxidative phosphorylation, in skin fibroblasts were not significantly impaired. Although we show that propionyl-CoA inhibits PDC, it does not appear to account for decreased PDC activity in SM. A better understanding of the mechanisms of phenotypic variability and the etiology for tissue-specific secondary deficiencies of mitochondrial enzymes of oxidative metabolism, and independently mitochondrial DNA depletion (common in other cases of A-SCS deficiency), is needed given the implications for control of lactic acidosis and possible clinical management.

Inborn Errors of Metabolism with Hepatopathy: Metabolism Defects of Galactose, Fructose, and Tyrosine

The liver is one of the most essential organs in metabolism and is responsible for metabolizing a wide variety of molecules from amino acids to sugars. Although it is responsible for many essential metabolic processes, it is one of the most severely affected by metabolic disease because, in many cases, it is the first to be exposed to the toxic intermediates. The metabolism of galactose, fructose, and tyrosine involve the liver and although there are systemic findings in metabolic disease involved with these substrates, severe hepatopathy is a common presenting aspect of galactosemia, hereditary fructose intolerance, and tyrosinemia type I.

5,10-methenyltetrahydrofolate synthetase deficiency causes a neurometabolic disorder associated with microcephaly, epilepsy, and cerebral hypomyelination

Folate metabolism in the brain is critically important and serves a number of vital roles in nucleotide synthesis, single carbon metabolism/methylation, amino acid metabolism, and mitochondrial translation. Genetic defects in almost every enzyme of folate metabolism have been reported to date, and most have neurological sequelae. We report 2 patients presenting with a neurometabolic disorder associated with biallelic variants in the MTHFS gene, encoding 5,10-methenyltetrahydrofolate synthetase. Both patients presented with microcephaly, short stature, severe global developmental delay, progressive spasticity, epilepsy, and cerebral hypomyelination. Baseline CSF 5-methyltetrahydrolate (5-MTHF) levels were in the low-normal range. The first patient was treated with folinic acid, which resulted in worsening cerebral folate deficiency. Treatment in this patient with a combination of oral L-5-methyltetrahydrofolate and intramuscular methylcobalamin was able to increase CSF 5-MTHF levels, was well tolerated over a 4 month period, and resulted in subjective mild improvements in functioning. Measurement of MTHFS enzyme activity in fibroblasts confirmed reduced activity. The direct substrate of the MTHFS reaction, 5-formyl-THF, was elevated 30-fold in patient fibroblasts compared to control, supporting the hypothesis that the pathophysiology of this disorder is a manifestation of toxicity from this metabolite.

Hyperammonemia in a Child Presenting with Growth Delay, Short Stature, and Diarrhea

A 5-year-old male child was referred to the metabolism clinic with a history of failure to thrive, chronic intermittent emesis, diarrhea, seizures, hypotonia, and growth hormone deficiency. He was also noted to experience frequent falling episodes. Routine biochemistry testing revealed increased plasma ammonia concentration at 184 μmol/L (reference interval, 16–47) and lactate dehydrogenase concentration at 445 U/L (reference interval, 110–295). Plasma free and total carnitine concentrations were decreased at 5 μmol/L (reference interval, 26–60) and 17.1 μmol/L (reference interval, 32–84), respectively. Orotic acid excretion in urine was also markedly increased at >80 mmol/mol creatinine (reference interval, 0.2–1.5). Plasma insulin-like growth factor-I (IGF-I)4 concentration was also decreased at <25 ng/mL (reference interval, 50–286). Paired plasma and urine specimens were collected for amino acid analysis using ultraperformance liquid chromatography. The results are shown in Table 1. View this table: Table 1. Amino acid analysis of paired plasma and urine. This patient has lysinuric protein intolerance (LPI), an autosomal recessive disorder of amino acid transport, caused by mutations in the solute carrier family 7, member 7 ( SLC7A7 ) gene (1, 2, 3). Although the worldwide incidence is not known, LPI is estimated to occur in approximately 1 in 60000 newborns in Finland and Japan (2). The SLC7A7 gene product is the …