Patient-derived Induced Pluripotent Stem Cells Research Articles

5'-UTR SNP of FGF13 causes translational defect and intellectual disability.

The congenital intellectual disability (ID)-causing gene mutations remain largely unclear, although many genetic variations might relate to ID. We screened gene mutations in Chinese Han children suffering from severe ID and found a single-nucleotide polymorphism (SNP) in the 5'-untranslated region (5'-UTR) of fibroblast growth factor 13 (FGF13) mRNA (NM_001139500.1:c.-32c>G) shared by three male children. In both HEK293 cells and patient-derived induced pluripotent stem cells, this SNP reduced the translation of FGF13, which stabilizes microtubules in developing neurons. Mice carrying the homologous point mutation in 5'-UTR of Fgf13 showed delayed neuronal migration during cortical development, and weakened learning and memory. Furthermore, this SNP reduced the interaction between FGF13 5'-UTR and polypyrimidine-tract-binding protein 2 (PTBP2), which was required for FGF13 translation in cortical neurons. Thus, this 5'-UTR SNP of FGF13 interferes with the translational process of FGF13 and causes deficits in brain development and cognitive functions.

Effects of CSF3R mutations on Myeloid Differentiation and Proliferation of Hematopoietic Cells of Congenital Neutropenia Patients

Over the last 25 years, with the clinical approval of recombinant human granulocyte colony-stimulating factor (rhG-CSF) remarkable progress has been achieved in the therapy of severe congenital neutropenia (CN). Nevertheless, CN patients have a high rate of transformation to myelodysplasia (MDS) or acute myeloid leukemia (AML). This risk is especially high in the group of patients who harbor acquired G-CSFR mutations, suggesting that these mutations are involved in the leukemogenesis. Deep sequencing of the intracellular part of G-CSFR allowed us to identify a subset of CN patients with a high mutant allele frequency (MAF) of G-CSFR mutations in the granulocytic compartment. We performed CSF3R mutation analysis of myeloid colonies from colony forming unit (CFU) assay of BM and CD34+ samples of 3 ELANE -CN patients, in which the percentage of granulocytes with G-CSFR mutation varied between 22.6% and 81%. Average number of sequenced colonies per CFU sample was 18. Intriguingly, we found that only cell clones with WT G-CSFR were grown in CFU assay. We hypothesized that lack of G-CSFR mutant clones in CFU assay is due to abnormal signaling downstream of truncated G-CSFR. To compare myeloid differentiation of G-CSFR mutant and WT G-CSFR hematopoietic cells, we generated ELANE -CN patient-derived induced pluripotent stem cells (CN-iPSCs) and performed CRISPR/Cas9 genome editing of CSF3R . Using 2 different sgRNAs specific for the intracellular region of G-CSFR, we introduced nucleotide indels at amino acid positions 740 and 735 (NP_000751.1) in CN-iPSCs. Subsequently, homozygous and heterozygous CN-iPSCs clones with truncated distal part of cytoplasmic domain of G-CSF receptor and subsequent loss of 3 out of 4 conserved tyrosine residues were generated.We compared myeloid differentiation of healthy donor iPSCs (HD-iPSCs), CN-iPSCs and CN-iPSCs with heterozygous and homozygous truncated G-CSF receptor (G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs, respectively). Absolute cell count during myeloid differentiation (18-32 day) of HD-iPSCs, G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs didn't differ significantly (p>0.05). In contrast, absolute cell count was found to be elevated from day 25 to day 32 of differentiation when G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs hematopoietic progenitors were compared with CN-iPSCs-derived hematopoietic cells (p<0.05). FACS analysis of CN-iPSCs derived hematopoietic progenitors with and without truncated G-CSF receptor using a panel of hematopoietic markers (KDR, CD34, CD235a, CD41a, CD45 and CD33) showed no difference in percentage of early progenitors (CD34+/KDR+), E/MK precursors (CD41a+CD235a+/ CD45-), CD34+/CD45+ and CD34+/CD43+ cells. Intriguingly, we observed increase in percentage of CD34+/CD43+ (p<0.01), CD33+/CD45+ (p<0.05) and CD41a+CD235a+/ CD45- (p<0.05) cells at day 14 of myeloid differentiation of G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs versus HD-iPSc. As expected, relative number CD14+ and CD16+ cells were not changed upon myeloid differentiation of hematopoietic progenitors with mutant G-CSFR but relative levels of CD15+ and CD15+/CD16+ were negatively affected by homozygous and heterozygous G-CSFR mutations. Next, CFU assay of hematopoietic progenitors derived from G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs produced markedly less colonies than HD- and CN-iPSCs derived hematopoietic cells. Cell morphology analysis also confirmed the reduced number of mature granulocytes seen after myeloid differentiation of G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs. Finally, reprogramming of BM cells from CN patients into iPSCs followed by mutation analysis of single cell derived iPSCs clones allowed us to identify iPSCs with naturally acquired G-CSFR mutations. Preliminary results revealed reduced myeloid differentiation of these cells, which is in line with data generated from G-CSFR*HET CN-iPSCs and G-CSFR*HOM CN-iPSCs.Using a combination of patient CN-iPSCs and CRISPR/Cas9 gene editing we were able to recapitulate main features of G-CSFR mutant versus G-CSFR WT CN cells - defective neutrophil differentiation and increased proliferation of human hematopoietic progenitors. Since virtually all studies of truncated G-CSFR receptor signaling have been performed in murine cytokine-responsive myeloid cell lines, our approach is more suitable to explore human aberrant G-CSF receptor signaling in depth. DisclosuresNo relevant conflicts of interest to declare.

Impaired neuronal activity and differential gene expression in STXBP1 encephalopathy patient iPSC-derived GABAergic neurons.

Syntaxin-binding protein 1 (STXBP1; also called MUNC18-1), encoded by STXBP1, is an essential component of the molecular machinery that controls synaptic vesicle docking and fusion. De novo pathogenic variants of STXBP1 cause a complex set of neurological disturbances, namely STXBP1 encephalopathy (STXBP1-E) that includes epilepsy, neurodevelopmental disorders and neurodegeneration. Several animal studies have suggested the contribution of GABAergic dysfunction in STXBP1-E pathogenesis. However, the pathophysiological changes in GABAergic neurons of these patients are still poorly understood. Here, we exclusively generated GABAergic neurons from STXBP1-E patient-derived induced pluripotent stem cells (iPSCs) by transient expression of the transcription factors ASCL1 and DLX2. We also generated CRISPR/Cas9-edited isogenic iPSC-derived GABAergic (iPSC GABA) neurons as controls. We demonstrated that the reduction in STXBP1 protein levels in patient-derived iPSC GABA neurons was slight (approximately 20%) compared to the control neurons, despite a 50% reduction in STXBP1 mRNA levels. Using a microelectrode array-based assay, we found that patient-derived iPSC GABA neurons exhibited dysfunctional maturation with reduced numbers of spontaneous spikes and bursts. These findings reinforce the idea that GABAergic dysfunction is a crucial contributor to STXBP1-E pathogenesis. Moreover, gene expression analysis revealed specific dysregulation of genes previously implicated in epilepsy, neurodevelopment and neurodegeneration in patient-derived iPSC GABA neurons, namely KCNH1, KCNH5, CNN3, RASGRF1, SEMA3A, SIAH3 and INPP5F. Thus, our study provides new insights for understanding the biological processes underlying the widespread neuropathological features of STXBP1-E.

Brain cells derived from Alzheimer\u2019s disease patients have multiple specific innate abnormalities in energy metabolism

Altered energy metabolism has been implicated both in aging and the pathogenesis of late-onset Alzheimer’s disease (LOAD). However, it is unclear which anomalies are acquired phenotypes and which are inherent and predispose to disease. We report that neural progenitor cells and astrocytes differentiated from LOAD patient-derived induced pluripotent stem cells exhibit multiple inter-related bioenergetic alterations including: changes in energy production by mitochondrial respiration versus glycolysis, as a consequence of alterations in bioenergetic substrate processing and transfer of reducing agents, reduced levels of NAD/NADH, diminished glucose uptake and response rates to insulin (INS)/IGF-1 signaling, decreased INS receptor and glucose transporter 1 densities, and changes in the metabolic transcriptome. Our data confirm that LOAD is a “multi-hit” disorder and provide evidence for innate inefficient cellular energy management in LOAD that likely predisposes to neurodegenerative disease with age. These processes may guide the development and testing of diagnostic procedures or therapeutic agents.

PIAS1 modulates striatal transcription, DNA damage repair, and SUMOylation with relevance to Huntington’s disease

DNA damage repair genes are modifiers of disease onset in Huntington's disease (HD), but how this process intersects with associated disease pathways remains unclear. Here we evaluated the mechanistic contributions of protein inhibitor of activated STAT-1 (PIAS1) in HD mice and HD patient-derived induced pluripotent stem cells (iPSCs) and find a link between PIAS1 and DNA damage repair pathways. We show that PIAS1 is a component of the transcription-coupled repair complex, that includes the DNA damage end processing enzyme polynucleotide kinase-phosphatase (PNKP), and that PIAS1 is a SUMO E3 ligase for PNKP. Pias1 knockdown (KD) in HD mice had a normalizing effect on HD transcriptional dysregulation associated with synaptic function and disease-associated transcriptional coexpression modules enriched for DNA damage repair mechanisms as did reduction of PIAS1 in HD iPSC-derived neurons. KD also restored mutant HTT-perturbed enzymatic activity of PNKP and modulated genomic integrity of several transcriptionally normalized genes. The findings here now link SUMO modifying machinery to DNA damage repair responses and transcriptional modulation in neurodegenerative disease.

Binding of Gtf2i-β/δ transcription factors to the ARMS2 gene leads to increased circulating HTRA1 in AMD patients and in vitro

The disease-initiating molecular events for age-related macular degeneration (AMD), a multifactorial retinal disease affecting many millions of elderly individuals worldwide, are still unknown. Of the over 30 risk and protective loci so far associated with AMD through whole genome-wide association studies (GWAS), the Age-Related Maculopathy Susceptibility 2 (ARMS2) gene locus represents one of the most highly associated risk regions for AMD. A unique insertion/deletion (in/del) sequence located immediately upstream of the High Temperature Requirement A1 (HTRA1) gene in this region confers high risk for AMD. Using electrophoretic mobility shift assay (EMSA), we identified that two Gtf2i-β/δ transcription factor isoforms bind to the cis-element 5′- ATTAATAACC-3′ contained in this in/del sequence. The binding of these transcription factors leads to enhanced upregulation of transcription of the secretory serine protease HTRA1 in transfected cells and AMD patient-derived induced pluripotent stem cells (iPSCs). Overexpression of Htra1 in mice using a CAG-promoter demonstrated increased blood concentration of Htra1 protein, caused upregulation of vascular endothelial growth factor (VEGF), and produced a choroidal neovascularization (CNV)-like phenotype. Finally, a comparison of 478 AMD patients to 481 healthy, age-matched controls from Japan, India, Australia, and the USA showed a statistically increased level of secreted HTRA1 blood concentration in AMD patients compared with age-matched controls. Taken together, these results suggest a common mechanism across ethnicities whereby increased systemic blood circulation of secreted serine protease HTRA1 leads to subsequent degradation of Bruch's membrane and eventual CNV in AMD.

The M1311V variant of ATP7A is associated with impaired trafficking and copper homeostasis in models of motor neuron disease

Disruption in copper homeostasis causes a number of cognitive and motor deficits. Wilson's disease and Menkes disease are neurodevelopmental disorders resulting from mutations in the copper transporters ATP7A and ATP7B, with ATP7A mutations also causing occipital horn syndrome, and distal motor neuropathy. A 65 year old male presenting with brachial amyotrophic diplegia and diagnosed with amyotrophic lateral sclerosis (ALS) was found to harbor a p.Met1311Val (M1311V) substitution variant in ATP7A. ALS is a fatal neurodegenerative disease associated with progressive muscle weakness, synaptic deficits and degeneration of upper and lower motor neurons. To investigate the potential contribution of the ATP7AM1311V variant to neurodegeneration, we obtained and characterized both patient-derived fibroblasts and patient-derived induced pluripotent stem cells differentiated into motor neurons (iPSC-MNs), and compared them to control cell lines. We found reduced localization of ATP7AM1311V to the trans-Golgi network (TGN) at basal copper levels in patient-derived fibroblasts and iPSC-MNs. In addition, redistribution of ATP7AM1311V out of the TGN in response to increased extracellular copper was defective in patient fibroblasts. This manifested in enhanced intracellular copper accumulation and reduced survival of ATP7AM1311V fibroblasts. iPSC-MNs harboring the ATP7AM1311V variant showed decreased dendritic complexity, aberrant spontaneous firing, and decreased survival. Finally, expression of the ATP7AM1311V variant in Drosophila motor neurons resulted in motor deficits. Apilimod, a drug that targets vesicular transport and recently shown to enhance survival of C9orf72-ALS/FTD iPSC-MNs, also increased survival of ATP7AM1311V iPSC-MNs and reduced motor deficits in Drosophila expressing ATP7AM1311V. Taken together, these observations suggest that ATP7AM1311V negatively impacts its role as a copper transporter and impairs several aspects of motor neuron function and morphology.

YIPF5 mutations cause neonatal diabetes and microcephaly through endoplasmic reticulum stress.

Neonatal diabetes is caused by single gene mutations reducing pancreatic β cell number or impairing β cell function. Understanding the genetic basis of rare diabetes subtypes highlights fundamental biological processes in β cells. We identified 6 patients from 5 families with homozygous mutations in the YIPF5 gene, which is involved in trafficking between the endoplasmic reticulum (ER) and the Golgi. All patients had neonatal/early-onset diabetes, severe microcephaly, and epilepsy. YIPF5 is expressed during human brain development, in adult brain and pancreatic islets. We used 3 human β cell models (YIPF5 silencing in EndoC-βH1 cells, YIPF5 knockout and mutation knockin in embryonic stem cells, and patient-derived induced pluripotent stem cells) to investigate the mechanism through which YIPF5 loss of function affects β cells. Loss of YIPF5 function in stem cell–derived islet cells resulted in proinsulin retention in the ER, marked ER stress, and β cell failure. Partial YIPF5 silencing in EndoC-βH1 cells and a patient mutation in stem cells increased the β cell sensitivity to ER stress–induced apoptosis. We report recessive YIPF5 mutations as the genetic cause of a congenital syndrome of microcephaly, epilepsy, and neonatal/early-onset diabetes, highlighting a critical role of YIPF5 in β cells and neurons. We believe this is the first report of mutations disrupting the ER-to-Golgi trafficking, resulting in diabetes.

IPSC-derived endothelial cells reflect accelerated senescence and increased inflammatory response in a CVD-risk stratified manner

Abstract Background Cardiovascular disease (CVD) remains the leading cause of death worldwide and its multifactorial nature is recognized, with both an individual's genetic background and lifestyle strongly impacting on its course. On a molecular level, accelerated cellular aging and increased inflammation interact to drive the severity of the disease. We have used patient-derived induced pluripotent stem cells (iPSC) to investigate the impact of a patients' genetic background on (epi-)genetically determined functionality, telomere attrition and inflammatory response. Purpose The purpose of this study is to investigate differences on the cellular level between iPSC-derived endothelial cells from risk-stratified CAD patients (both ACS and stable CAD) in both functionality, inflammatory response and ageing rate. Methods iPSCs were generated from participants with similar lifestyle but different disease progression (healthy &amp;gt;65 y/o, stable CAD &amp;gt;65 y/o, ACS &amp;lt;65y/o) and differentiated into endothelial cells (iPS-EC). After quality control, iPS-EC were exposed to inflammatory stimulation via TNF-α under static and pro- vs. anti-atherogenic flow conditions (laminar vs. disturbed flow, high vs. low shear stress) and their gene and protein expression of adhesion molecules was assessed by qPCR and flow cytometry. Telomere length was determined by a qPCR-based assay in iPSC-ECs after four (p4) and eleven population doublings [KN1] (p11) in fully supplemented growth medium. Results ACS-iPS-ECs showed a significantly stronger upregulation of E-selectin in response to inflammatory stimulation with TNF-a than healthy-iPS-ECs on mRNA level (ACS: 532.1-fold±48.7 vs. healthy: 322.3-fold±55.7, p=0.02). Similarly, ICAM1 protein upregulation was stronger in ACS-iPS-ECs than in healthy iPSC-ECs as assessed by flow cytometry (ACS: 1.4-fold±0.01 vs. healthy: 1.1-fold±0.0007; p&amp;lt;0.001) whereas VCAM1 upregulation was weaker in ACS-iPS-ECs compared to healthy controls (ACS: 1.8-fold±0.3 vs. healthy: 4.2-fold±0.9, p&amp;lt;0.001). Telomere shortening from p4 to p11 was significantly stronger in ACS-iPS-ECs compared to healthy controls (ACS: 35%±10 vs. healthy: 10%±5, p&amp;lt;0.05). Conclusion The more pronounced upregulation of specific adhesion molecules (E-Selectin, ICAM-1, but not VCAM-1) in response to inflammatory stimulation together with a stronger telomere attrition in ACS-iPS-EC hints at cells entering faster into a senescent state. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Research scholarship from the German Society of Cardiology. Allocated research funds from University hospital.

Investigating Developmental and Epileptic Encephalopathy Using Drosophila melanogaster.

Developmental and epileptic encephalopathies (DEEs) are the spectrum of severe epilepsies characterized by early-onset, refractory seizures occurring in the context of developmental regression or plateauing. Early infantile epileptic encephalopathy (EIEE) is one of the earliest forms of DEE, manifesting as frequent epileptic spasms and characteristic electroencephalogram findings in early infancy. In recent years, next-generation sequencing approaches have identified a number of monogenic determinants underlying DEE. In the case of EIEE, 85 genes have been registered in Online Mendelian Inheritance in Man as causative genes. Model organisms are indispensable tools for understanding the in vivo roles of the newly identified causative genes. In this review, we first present an overview of epilepsy and its genetic etiology, especially focusing on EIEE and then briefly summarize epilepsy research using animal and patient-derived induced pluripotent stem cell (iPSC) models. The Drosophila model, which is characterized by easy gene manipulation, a short generation time, low cost and fewer ethical restrictions when designing experiments, is optimal for understanding the genetics of DEE. We therefore highlight studies with Drosophila models for EIEE and discuss the future development of their practical use.

Hereditary motor neuropathies.

Hereditary motor neuropathies (HMN) comprise a broad genotypic and phenotypic spectrum of rare, progressively disabling diseases manifesting with length-dependent muscle weakness and atrophy. To date, more than half of the cases cannot be genetically explained. To provide symptomatic and disease-modifying treatments in the future, a better understanding of disease mechanisms is required. By whole exome and genome sequencing, the discovery of several novel genes (SCO2, TDRKH, SPTAN1, CADM3, and SORD) involved in the pathogenesis of HMN has now relevantly changed the pathophysiological knowledge. This recent success in causative understanding has mainly been driven by the development of functional models including cell culture, animal, and patient-derived induced pluripotent stem cell platforms. These models have an important impact on therapeutic advances including broader approaches to prevent or reverse axonal degeneration and individualized gene silencing attempts using sequence-specific RNA degradation mechanisms. In rare diseases such as HMN, the recent development of genetic sequencing and data interpretation methods has enabled a broader diagnostic approach, whereas treatment strategies are becoming more individualized. Significant milestones have been reached in the discovery of new genes, the establishment of functional disease models, and the preclinical development of mechanistic-based therapies.

Generation of three Duchenne muscular dystrophy patient-derived induced pluripotent stem cell (iPSC) lines ICGi002-A, ICGi002-B and ICGi002-C.

Duchenne muscular dystrophy (DMD) is a severe and rapidly progressive hereditary muscular disease with X-linked recessive inheritance, occurring mainly in males. A complete loss of dystrophin resulted from out-of-frame deletion mutations in the DMD gene leads to Duchenne muscular dystrophy. DMD induced pluripotent stem cells (iPSCs) are a suitable cell model to study muscle development and disease mechanisms underlying muscular dystrophy and to screen novel compounds with potential therapeutic effects. We generated iPSCs from a DMD patient using non-integrating episomal plasmid vectors. The obtained iPSC lines showed ESC-like morphology, expression pluripotency markers, displayed a normal karyotype and possessed trilineage differentiation potential.

CRISPR/Cas9-Mediated Gene Correction to Understand ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the death of motor neurons in the spinal cord and brainstem. ALS has a diverse genetic origin; at least 20 genes have been shown to be related to ALS. Most familial and sporadic cases of ALS are caused by variants of the SOD1, C9orf72, FUS, and TARDBP genes. Genome editing using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9) can provide insights into the underlying genetics and pathophysiology of ALS. By correcting common mutations associated with ALS in animal models and patient-derived induced pluripotent stem cells (iPSCs), CRISPR/Cas9 has been used to verify the effects of ALS-associated mutations and observe phenotype differences between patient-derived and gene-corrected iPSCs. This technology has also been used to create mutations to investigate the pathophysiology of ALS. Here, we review recent studies that have used CRISPR/Cas9 to understand the genetic underpinnings of ALS.

Optic Atrophy 1 Controls Human Neuronal Development by Preventing Aberrant Nuclear DNA Methylation.

SummaryOptic atrophy 1 (OPA1), a GTPase at the inner mitochondrial membrane involved in regulating mitochondrial fusion, stability, and energy output, is known to be crucial for neural development: Opa1 heterozygous mice show abnormal brain development, and inactivating mutations in OPA1 are linked to human neurological disorders. Here, we used genetically modified human embryonic and patient-derived induced pluripotent stem cells and reveal that OPA1 haploinsufficiency leads to aberrant nuclear DNA methylation and significantly alters the transcriptional circuitry in neural progenitor cells (NPCs). For instance, expression of the forkhead box G1 transcription factor, which is needed for GABAergic neuronal development, is repressed in OPA1+/− NPCs. Supporting this finding, OPA1+/− NPCs cannot give rise to GABAergic interneurons, whereas formation of glutamatergic neurons is not affected. Taken together, our data reveal that OPA1 controls nuclear DNA methylation and expression of key transcription factors needed for proper neural cell specification.

IPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients Acquire CFTR Activity upon TALEN-Mediated Repair of the p.F508del Mutation

Cystic fibrosis (CF) is the main genetic cause of death among the Caucasian population. The disease is characterized by abnormal fluid and electrolyte mobility across secretory epithelia. The first manifestations occur within hours of birth (meconium ileus), later extending to other organs, generally affecting the respiratory tract. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR encodes a cyclic adenosine monophosphate (cAMP)-dependent, phosphorylation-regulated chloride channel required for transport of chloride and other ions through cell membranes. There are more than 2,000 mutations described in the CFTR gene, but one of them, phenylalanine residue at amino acid position 508 (p.F508del), a recessive allele, is responsible for the vast majority of CF cases worldwide. Here, we present the results of the application of genome-editing techniques to the restoration of CFTR activity in p.F508del patient-derived induced pluripotent stem cells (iPSCs). Gene-edited iPSCs were subsequently used to produce intestinal organoids on which the physiological activity of the restored gene was tested in forskolin-induced swelling tests. The seamless restoration of the p.F508del mutation resulted in normal expression of the mature CFTR glycoprotein, full recovery of CFTR activity, and a normal response of the repaired organoids to treatment with two approved CF therapies: VX-770 and VX-809.

Recapitulating the human segmentation clock with pluripotent stem cells.

Pluripotent stem cells are increasingly used to model different aspects of embryogenesis and organ formation1. Despite recent advances in in vitro induction of major mesodermal lineages and cell types2,3, experimental model systems that can recapitulate more complex features of human mesoderm development and patterning are largely missing. Here we used induced pluripotent stem cells for the stepwise in vitro induction of presomitic mesoderm and its derivatives to model distinct aspects of human somitogenesis. We focused initially on modelling the human segmentation clock, a major biological concept believed to underlie the rhythmic and controlled emergence of somites, which give rise to the segmental pattern of the vertebrate axial skeleton. We observed oscillatory expression of core segmentation clock genes, including HES7 and DKK1, determined the period of the human segmentation clock to be around five hours, and demonstrated the presence of dynamic travelling-wave-like gene expression in in vitro-induced human presomitic mesoderm. Furthermore, we identified and compared oscillatory genes in human and mouse presomitic mesoderm derived from pluripotent stem cells, which revealed species-specific and shared molecular components and pathways associated with the putative mouse and human segmentation clocks. Using CRISPR-Cas9-based genome editing technology, we then targeted genes for which mutations in patients with segmentation defects of the vertebrae, such as spondylocostal dysostosis, have been reported (HES7, LFNG, DLL3 and MESP2). Subsequent analysis of patient-like and patient-derived induced pluripotent stem cells revealed gene-specific alterations in oscillation, synchronization or differentiation properties. Our findings provide insights into the human segmentation clock as well as diseases associated with human axial skeletogenesis.

Advances of Usher syndrome and USH2A gene

Usher syndrome (USH) is the most common cause of deaf-blindness diseases characterized by sensorineural hearing loss and retinitis pigmentosa. Patients are clinically and genetically heterogeneous, however, there are no convincing methods for prevention and treatment. USH2A is the most common disease-causing gene among 14 genes related to Usher syndrome. Great progress has been achieved in the pathogenic mechanism, animal models studies, diagnosis, and treatments based on gene therapy, cells transplantation and antisense oligonucleotide-based splice correction. Mutations in USH2A result in defects in USH complex proteins which involved in the transport function of the peripheral cilia region. There is respective limitations in established mouse and zebrafish animal models. Two promising treatments of this disease are introduced. One is clinical transplantation of visual organs which induced from corrected patient-derived induced pluripotent stem cells by the CRISPR/Cas9 system and another one is the RNA splicing therapy based on antisense oligonucleotides. Key words: Usher syndromes/genetics; Usher syndromes/etiology; Genes; Mutation; Review

PPP2R5D-Related Intellectual Disability and Neurodevelopmental Delay: A Review of the Current Understanding of the Genetics and Biochemical Basis of the Disorder.

Protein Phosphatase 2 Regulatory Subunit B′ Delta (PPP2R5D)-related intellectual disability (ID) and neurodevelopmental delay results from germline de novo mutations in the PPP2R5D gene. This gene encodes the protein PPP2R5D (also known as the B56 delta subunit), which is an isoform of the subunit family B56 of the enzyme serine/threonine-protein phosphatase 2A (PP2A). Clinical signs include intellectual disability (ID); autism spectrum disorder (ASD); epilepsy; speech problems; behavioral challenges; and ophthalmologic, skeletal, endocrine, cardiac, and genital malformations. The association of defective PP2A activity in the brain with a wide range of severity of ID, along with its role in ASD, Alzheimer’s disease, and Parkinson’s-like symptoms, have recently generated the impetus for further research into mutations within this gene. PP2A, together with protein phosphatase 1 (PP1), accounts for more than 90% of all phospho-serine/threonine dephosphorylations in different tissues. The specificity for a wide variety of substrates is determined through nearly 100 different PP2A holoenzymes that are formed by at least 23 types of regulatory B subunits, and two isoforms each of the catalytic subunit C and the structural subunit A. In the mammalian brain, PP2A-mediated protein dephosphorylation plays an important role in learning and memory. The PPP2R5D subunit is highly expressed in the brain and the PPP2A–PPP2R5D holoenzyme plays an important role in maintaining neurons and regulating neuronal signaling. From 2015 to 2017, 25 individuals with PPP2R5D-related developmental disorder were diagnosed. Since then, Whole-Exome Sequencing (WES) has helped to identify more unrelated individuals clinically diagnosed with a neurodevelopmental disorder with pathological variants of PPP2R5D. In this review, we discuss the current understanding of the clinical and genetic aspects of the disorder in the context of the known functions of the PP2A–PPP2R5D holoenzyme in the brain, as well as the pathogenic mutations in PPP2R5D that lead to deficient PP2A–PPP2R5D dephosphorylation and their implications during development and in the etiology of autism, Parkinson’s disease, Alzheimer’s disease, and so forth. In the future, tools such as transgenic animals carrying pathogenic PPP2R5D mutations, and patient-derived induced pluripotent stem cell lines need to be developed in order to fully understand the effects of these mutations on different neural cell types.

Inhibition of cyclooxygenase-1 by nonsteroidal anti-inflammatory drugs demethylates MeR2 enhancer and promotes Mbnl1 transcription in myogenic cells

Muscleblind-like 1 (MBNL1) is a ubiquitously expressed RNA-binding protein, which is highly expressed in skeletal muscle. Abnormally expanded CUG-repeats in the DMPK gene cause myotonic dystrophy type 1 (DM1) by sequestration of MBNL1 to nuclear RNA foci and by upregulation of another RNA-binding protein, CUG-binding protein 1 (CUGBP1). We previously reported that a nonsteroidal anti-inflammatory drug (NSAID), phenylbutazone, upregulates MBNL1 expression in DM1 mouse model by demethylation of MeR2, an enhancer element in Mbnl1 intron 1. NSAIDs inhibit cyclooxygenase (COX), which is comprised of COX-1 and COX-2 isoforms. In this study, we screened 29 NSAIDs in C2C12 myoblasts, and found that 13 NSAIDs enhanced Mbnl1 expression, where COX-1-selective NSAIDs upregulated Mbnl1 more than COX-2-selective NSAIDs. Consistently, knockdown of COX-1, but not of COX-2, upregulated MBNL1 expression in C2C12 myoblasts and myotubes, as well as in myotubes differentiated from DM1 patient-derived induced pluripotent stem cells (iPSCs). Luciferase assay showed that COX-1-knockdown augmented the MeR2 enhancer activity. Furthermore, bisulfite sequencing analysis demonstrated that COX-1-knockdown suppressed methylation of MeR2. These results suggest that COX-1 inhibition upregulates Mbnl1 transcription through demethylation of the MeR2 enhancer. Taken together, our study provides new insights into the transcriptional regulation of Mbnl1 by the COX-1-mediated pathway.

Disease Model of Atherosclerosis Using Werner Syndrome Patient-Derived Induced Pluripotent Stem Cells Reveals Excessive Inflammatory Response Against Ox-LDL

Atherosclerosis is a major cause of various fatal diseases, such as myocardial infarction, stroke, and ischemic heart failure worldwide. The risk factors of atherosclerosis are well known, including hypertension, diabetes mellitus, dyslipidemia, smoking and obesity. Growing evidence also suggests that aging is the strongest determinant of stroke, which is less common before 40 years old (Soler and Ruiz 2010). Werner syndrome (WS) is a rare human inherited disorder characterized by the appearance of premature aging followed by early onset of aging-associated diseases including atherosclerosis. Myocardial infarction and cancer are being the most common causes of death in patients with WS (Oshima et al. 1993). However, despite being one of the most common causes of death, the etiology of atherosclerosis in WS patients have not been well documented yet. Therefore, the aim of the study was to assess in vitro model of atherosclerosis using WS patient-derived induced pluripotent stem cells (iPSC) to better understand the etiology and pathogenesis of atherosclerosis in WS patients. Atherosclerosis is a chronic inflammatory disease in which vascular endothelial cells (VEC), vascular smooth muscle cells (VSMC) and macrophages (Mφ) play interactive roles in the progression of the disease. To obtain these cells in vitro, we induced VEC, VSMC and Mφ from the progenitor cell populations, KDR+/CD34+/TRA-1-60-, KDR+/CD34-/TRA-1-60- and CD43+/CD34+ cells respectively, derived from iPSCs by the iPS-sac method we developed previously (Takayama et al. 2008, 2010). We found by co-culture with C3H10T1/2 mouse mesenchymal stromal cell line, KDR+/CD34+ and KDR+/CD34- cell populations were able to efficiently induce VEC and VSMC respectively on day 17. Especially, VEC induction efficiency was ~ 4 and ~ 21 fold as compared with the previously established method with collagen IV and laminin-411 respectively (Ohta et al. 2016; Sone et al. 2007; Taura et al. 2009). Furthermore, to induce Mφ, iPS-sac-like structures in culture were maintained for two weeks to get CD43+ hematopoietic cells. CD43+ cells were then cultured for an additional 7 days with a cytokine cocktail on C3H10T1/2 feeder layer. After 3 weeks of initiating culture, ~ 54% of CD11b+/CD33+ Mφ-like cells were obtained. To examine the responses of the induced VEC- and Mφ-like cells in atherosclerotic conditions, we applied oxidized low-density lipoprotein (Ox-LDL) at the concentration of 10, 50 and 100 μg/mL, and performed qRT-PCR analysis. It is well known that inflammatory response is triggered with the uptake of LDL both in VEC and Mφ, thereby contribute to local inflammation, cell necrosis, apoptosis, VSMC proliferation and fibrosis. Accordingly, in Ox-LDL treated iPSC-derived VEC and Mφ, mRNA expression of IL-1β, IL-6 and TNF-α were increased in a dose dependent manner. A critical event in the early stages of atherosclerosis is the internalization of lipid particles by VEC, and then, these lipids particles are oxidized to form Ox-LDL in the endothelium, leading to an increase in inflammatory cell adhesion molecules (ICAM-1 and VCAM-1) on the endothelium. Subsequently, monocytes are then recruited and differentiated into Mφ and start to uptake Ox-LDL particles ultimately forming foam-cells (Wu et al. 2017). Next, we were enthusiastic to compare the effect of Ox-LDL for inflammatory response in WS iPSC-derived VEC and Mφ with healthy iPSC-derived VEC and Mφ. Although no differences were observed in the induction efficiency, cell proliferation and mean fluorescent intensity of Ox-LDL uptake between WS- and healthy-iPSC-derived VEC and Mφ, qRT-PCR analysis revealed that, VEC derived from WS-iPSC shows much higher mRNA expression of cell adhesion molecule gene (ICAM-1; 2.5 fold and VCAM-1; 3.6 fold), inflammatory cytokine gene (IL-6; 12.5 fold and TGF-β; 4.3 fold) and endothelial dysfunction related gene (ET-1; 5.9 fold and PAI-1; 25.2 fold) compared with healthy-iPSC-derived VEC. Consequently, our results suggest that the higher risk of atherosclerosis in WS patients might be due to excessive inflammation response against Ox-LDL in WS-iPSC-derived cells, which is independent of other risk factors frequently observed in WS, such as diabetes and hyperlipidemia, and might be implicated to understand the pathogenesis and therapeutic strategy against WS and general atherosclerosis. Disclosures Eto: Megakaryon Co. Ltd.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.