The incomplete differentiation of human induced pluripotent stem cells (iPSCs) poses a serious safety risk owing to their potential tumorigenicity, hindering their clinical application. Here, we ...explored the potential of phospho-D-peptides as novel iPSC-eliminating agents. Alkaline phosphatases overexpressed on iPSCs dephosphorylate phospho-D-peptides into hydrophobic peptides that aggregate and induce cell death. We isolated a peptide candidate, D-3, that selectively and rapidly induced toxicity in iPSCs within 1 hr but had little influence on various non-iPSCs, including primary hepatocytes and iPSC-derived cardiomyocytes. Two hours of D-3 treatment efficiently eliminated iPSCs from both single cultures and co-cultures spiked with increasing ratios of iPSCs. In addition, D-3 prevented residual iPSC-induced teratoma formation in a mouse tumorigenicity assay. These results suggest the enormous potential of D-3 as a low-cost and effective anti-iPSC agent for both laboratory use and for the safe clinical application of iPSC-derived cells in regenerative medicine.
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•D-3 induces toxicity in iPSCs and ESCs within 1 hr of incubation•D-3 has little influence on various non-iPSCs, including hepatocytes and neurons•D-3 prevents residual iPSC-induced teratoma formation in an animal model•Alkaline phosphatase activity on the cell surface is required for D-3's toxicity
Yi Kuang et al. discovered a phosphor-peptide D-3 that responds to the activity of alkaline phosphatase on the cell surface to selectively and effectively remove iPSCs and ESCs within 1–2 hr. D-3 is a promising low-cost iPSC-eliminating agent and is simple to use.
Abstract
Synthetic messenger RNA (mRNA) tools often use pseudouridine and 5-methyl cytidine as substitutions for uridine and cytidine to avoid the immune response and cytotoxicity induced by ...introducing mRNA into cells. However, the influence of base modifications on the functionality of the RNA tools is poorly understood. Here we show that synthetic mRNA switches containing N1-methylpseudouridine (m1Ψ) as a substitution of uridine substantially out-performed all other modified bases studied, exhibiting enhanced microRNA and protein sensitivity, better cell-type separation ability, and comparably low immune stimulation. We found that the observed phenomena stem from the high protein expression from m1Ψ containing mRNA and efficient translational repression in the presence of target microRNAs or proteins. In addition, synthetic gene circuits with m1Ψ significantly improve performance in cells. These findings indicate that synthetic mRNAs with m1Ψ modification have enormous potentials in the research and application of biofunctional RNA tools.
The efficiency of pluripotent stem cell differentiation is highly variable, often resulting in heterogeneous populations that contain undifferentiated cells. Here we developed a sensitive, ...target-specific, and general method for removing undesired cells before transplantation. MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. We synthesized a new RNA tool, miR-switch, as a live-cell reporter mRNA for miR-302a activity that can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay. Automated and selective hiPSC-elimination was achieved by controlling puromycin resistance using the miR-302a switch. Our system uniquely provides sensitive detection of pluripotent stem cells and partially differentiated cells. In addition to its ability to eliminate undifferentiated cells, miR-302a switch also holds great potential in investigating the dynamics of differentiation and/or reprograming of live-cells based on intracellular information.
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•APP overexpression results in reduced β-catenin nuclear expression.•APP binds to β-catenin in vitro and in vivo.•AD patients show reduced nuclear β-catenin levels.•The expression of ...p-GSK3 is decreased in AD brains.
Accumulating evidence has shown that the processing of the amyloid precursor protein (APP) and the formation of amyloid-β are associated with the canonical Wnt/ β-catenin signalling pathway. It was recently published that the drosophila homologue of APP is a conserved modulator of Wnt PCP signalling, suggesting a potential regulation of this pathway by APP. The aim of this study was to investigate the potential interaction of APP with the canonical Wnt pathway. APP overexpression in N2a cells led to alterations in the subcellular distribution of β-catenin by physically binding to it, preventing its translocation to the nucleus and precluding the transcription of Wnt target genes. In addition, studies in APP transgenic mice and human Alzheimer’s disease (AD) brain tissue showed the cellular co-localization of APP and β-catenin and binding of both proteins, suggesting the formation physical complexes of APP and β-catenin, yet not present in healthy controls. Furthermore, a reduction in the levels of nuclear β-catenin was detected in AD brains compared to controls as well as a decrease in the expression of the inactive phosphorylated Glycogen synthase kinase 3 (GSK3) isoform. Therefore, these findings indicate a reciprocal regulation of Wnt/ β-catenin signalling pathway and APP processing involving a physical interaction between APP and β-catenin.
Non-allelic recombination between homologous repetitive elements contributes to evolution and human genetic disorders. Here, we combine short- and long-DNA read sequencing of repeat elements with a ...new bioinformatics pipeline to show that somatic recombination of Alu and L1 elements is widespread in the human genome. Our analysis uncovers tissue-specific non-allelic homologous recombination hallmarks; moreover, we find that centromeres and cancer-associated genes are enriched for retroelements that may act as recombination hotspots. We compare recombination profiles in human-induced pluripotent stem cells and differentiated neurons and find that the neuron-specific recombination of repeat elements accompanies chromatin changes during cell-fate determination. Finally, we report that somatic recombination profiles are altered in Parkinson’s and Alzheimer’s disease, suggesting a link between retroelement recombination and genomic instability in neurodegeneration. This work highlights a significant contribution of the somatic recombination of repeat elements to genomic diversity in health and disease.
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•Somatic recombination of Alu and L1 elements is widespread in the human genome•Somatic recombination events of Alu and L1 elements exhibit tissue-specific hallmarks•Neuronal differentiation of iPSCs is accompanied by changes in recombination profiles•Somatic recombination profiles are altered in Parkinson’s and Alzheimer’s diseases
Large-scale analysis of human tissue samples unveils the tissue-specific somatic recombination of repeat elements that is widespread and contributes to the genomic diversity underpinning human development and disease.
Protein-responsive ribozymes generated by a streamlined design process will expand the plug-and-play toolbox for synthetic biologists. Endo et al comment on a study by Auslnder et al in which they ...give general guidelines for the design, optimization and validation of RNA devices with the goal of obtaining a protein- responsive switch that functions in both bacteria and mammalian cells.
Mutations in fukutin-related protein (FKRP) underlie a group of muscular dystrophies associated with the hypoglycosylation of α-dystroglycan (α-DG), a proportion of which show central nervous system ...involvement. Our original FKRP knock-down mouse (FKRP(KD)) replicated many of the characteristics seen in patients at the severe end of the dystroglycanopathy spectrum but died perinatally precluding its full phenotyping and use in testing potential therapies. We have now overcome this by crossing FKRP(KD) mice with those expressing Cre recombinase under the Sox1 promoter. Owing to our original targeting strategy, this has resulted in the restoration of Fkrp levels in the central nervous system but not the muscle, thereby generating a new model (FKRP(MD)) which develops a progressive muscular dystrophy resembling what is observed in limb girdle muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) is a bifunctional glycosyltransferase previously shown to hyperglycosylate α-DG. To investigate the therapeutic potential of LARGE up-regulation, we have now crossed the FKRP(MD) line with one overexpressing LARGE and show that, contrary to expectation, this results in a worsening of the muscle pathology implying that any future strategies based upon LARGE up-regulation require careful management.
A growing number of studies are focusing on the associations between human milk (HM) immunological composition and allergic diseases. This scoping review aims to identify statistical methods applied ...in the field and highlight pitfalls and unmet needs. A comprehensive literature search in MEDLINE and Embase retrieved 13,607 unique records. Following title/abstract screening, 29 studies met the selection criteria and were included in this review. We found that definitions of colostrum and mature milk varied across the studies. A total of 17 out of 29 (59%) studies collected samples longitudinally, but only 12% of these used serial (longitudinal) analyses. Multivariable analysis was used in 45% of the studies, but statistical approaches to modelling varied largely across the studies. Types of variables included as potential confounding factors differed considerably between models. Discrimination analysis was absent from all studies and only a single study reported classification measures. Outcomes of this scoping review highlight lack of standardization, both in data collection and handling, which remains one of the main challenges in the field. Improved standardization could be obtained by a consensus group of researchers and clinicians that could recommend appropriate methods to be applied in future prospective studies, as well as already existing datasets.
ABSTRACT
Alterations in the Wnt signaling pathway have been implicated in Alzheimer's disease; however, its role in the processing of the amyloid precursor protein remains unknown. In this study, ...activation of the Wnt pathway by overexpression of the agonist Wnt3a or β‐catenin or by inhibition of glycogen kinase synthase‐3 in N2a cells resulted in a reduction in Aβ levels and in the activity and expression of BACE1 (β‐APP cleaving enzyme). Conversely, inhibition of the pathway by transfection of the antagonists secreted frizzled receptor protein‐1 or dickkopf‐1 produced the opposite effects. Chromatin immunoprecipitation analysis demonstrated that β‐catenin binds specifically to regions within the promoter of BACE1 containing putative T‐cell factor/lymphoid enhancer binding factor‐1 (TCF/LEF) motifs, consistent with canonical Wnt target regulation. Furthermore, cells transfected with β‐catenin mutants incapable of binding to TCF/LEF increased BACE1 gene promoter activity. Interestingly, TCF4 knockdown reversed the effects of Wnt3a activation on BACE1 transcription. We found that TCF4 binds to the same region on BACE1 promoter following Wnt3a stimulation, indicating that TCF4 functions as a transcriptional repressor of BACE1 gene. In conclusion, Wnt/β‐catenin stimulation may repress BACE1 transcription via binding of TCF4 to BACE1 gene, and therefore, activation of the Wnt pathway may hold the key to new treatments of Alzheimer disease.—Parr, C., Mirzaei, N., Christian, M., and Sastre, M. Activation of the Wnt/β‐catenin pathway represses the transcription of the β‐amyloid precursor protein cleaving enzyme (BACE1) via binding of T‐cell factor‐4 to BACE1 promoter. FASEB J. 29, 623‐635 (2015). www.fasebj.org
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