Patient-specific iPSC Lines Research Articles

Generation of an induced pluripotent stem cell (iPSC) line from a 40-year-old patient with the A8344G mutation of mitochondrial DNA and MERRF (myoclonic epilepsy with ragged red fibers) syndrome

Mitochondrial defects are associated with clinical manifestations from common diseases to rare genetic disorders. Myoclonus epilepsy associated with ragged-red fibers (MERRF) syndrome results from an A to G transition at nucleotide position 8344 in the tRNALys gene of mitochondrial DNA (mtDNA) and is characterized by myoclonus, myopathy and severe neurological symptoms. In this study, Sendai reprogramming method was used to generate an iPS cell line carrying the A8344G mutation of mtDNA from a MERRF patient. This patient-specific iPSC line expressed pluripotent stem cell markers, possessed normal karyotype, and displayed the capability to differentiate into mature cells in three germ layers.

Generation of Urine Cell-Derived Non-integrative Human iPSCs and iNSCs: A Step-by-Step Optimized Protocol.

Objective: Establishing a practical procedure to generate induced pluripotent stem cells (iPSCs) and induced neural stem cells (iNSCs) from human urine cells (UCs). In this report, we optimized a non-integrative protocol to generate patient-specific iPSC and iNSC lines with high reprogramming efficiency.Methods: UCs were electroporated with the pEP4-EO2S-ET2K and pEP4-M2L plasmids containing the OCT4, SOX2, KLF4, SV40LT, c-MYC, and LIN28 genes, and then cultured with N2B27 medium plus four small molecule compounds (A83-01, PD0325901, Thiazovivin, and CHIR99021). When iPSC or iNSC clones emerged, the medium was replaced with mTeSR1 or neural growth medium. Morphological changes were seen at day 4–7. After day 10, the clones were picked up when the clone diameter exceeded 1 mm.Results: iPSCs and iNSCs were successfully derived from UCs with up to 80 clones/well. These iPSCs and iNSCs showed typical hESC or NSC morphology and were self-renewable. The iPSCs had pluripotency to differentiate into the three germinal layers and displayed high levels of expression of pluripotency markers SOX2, NANOG, OCT4, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase (AP). They maintained normal karyotype and had no transgene expression or genomic integration. The iNSCs were positive for NSC markers NESTIN, PAX6, SOX2, and OLIG2.Conclusion: The optimized protocol is an easy and fast procedure to yield both iPSC and iNSC lines from a convenient source of human urine in a single experiment.

Derivation of a disease-specific human induced pluripotent stem cell line from a biliary atresia patient

Biliary atresia (BA) is a common cause of pediatric end-stage liver disease. While its etiology is not yet clear, evidence has suggested that BA results from interactions between genetic susceptibility and environmental factors. Disease relevant human cellular models of BA will facilitate identification of both genetic and environmental factors that are important for disease prevention and treatment. Here we report the generation of a human induced pluripotent stem cell line from a BA patient using episomal vectors. Patient-specific BA iPSC lines provide valuable tools for disease mechanism study and drug development.

Generation of induced pluripotent stem cells (ARO-iPSC1-11) from a patient with autosomal recessive osteopetrosis harboring the c.212 + 1G > T mutation in SNX10 gene

Pathogenic sequence variants in the Sorting Nexin 10 (SNX10) gene have been associated with autosomal recessive osteopetrosis (ARO) in human. In this study, an induced pluripotent stem cell (iPSC) line (ARO-iPSC1-11) was generated from an ARO patient carrying the homozygous c.212+1G>T mutation in SNX10, using a retroviral-based reprogramming protocol. Characterization confirmed that the generated iPSCs expressed pluripotency markers, displayed normal karyotype, showed pluripotent differentiation capacity and retained the targeted mutation. Disease modeling with this ARO patient-specific iPSC line will shed further light on the critical role of the SNX10 mutation in ARO development.

A PATIENT-DERIVED IPSC MODEL OF A RARE TTC3 MUTATION

While 20+ loci have been associated with late onset Alzheimer's disease (LOAD), a large number of genetic factors still have yet to be revealed. We recently performed whole exome sequencing on a non-Hispanic white LOAD family with 11 AD individuals with a range of onset between 70–85 years old (average 75.7 years) that was negative for alterations in known AD candidate genes (Kohli, et al, 2016). A single rare, nonsynonymous variant was identified in all affected individuals in the tetratricopeptide repeat domain 3 (TTC3) gene. This missense alteration, rs377155188 (p.S1038C), is predicted to be deleterious by five in silico algorithms and extremely rare in the ExAC database (1.96x10-5). Studies have reported that TTC3 expression is reduced in LOAD patients and negatively correlated with AD neuropathology, making this gene an interesting LOAD candidate to further investigate. To understand the mechanism by which the TTC3 alteration may be acting to contribute to LOAD risk, induced pluripotent stem cells (iPSC) lines were developed to examine cellular consequences in neuronal tissue, which can otherwise only be collected postmortem. Peripheral blood mononuclear cells (PBMCs) were extracted from the whole blood of two LOAD individuals bearing the TTC3 change, as well as two aged, and ethnically matched control individuals. iPSC reprogramming was performed using Sendai virus. Each iPSC line generated was validated for pluripotency through immunocytochemical staining and shown to be negative for any large scale chromosomal abnormalities via karyotyping. These iPSC lines will be differentiated into forebrain neurons to generate a disease relevant tissue to assess. There is evidence that modulation of TTC3 affects neurite growth (Berto, et al, 2007); therefore, morphological measures of axon and synapse formation will be assessed in differentiating neuronal cultures using live cell imaging. Alzheimer-specific phenotypes such as the levels of secreted beta amyloid and intercellular whole and phosphorylated tau will also be measured. Utilizing patient-specific iPSC lines will permit assessing the cellular and molecular consequences of genetic alterations which may contribute to LOAD risk. We will present the morphological and cellular phenotypes of these lines carrying a potential risk factor for AD.

Decreased calcium flux in Niemann-Pick type C1 patient-specific iPSC-derived neurons due to higher amount of calcium-impermeable AMPA receptors.

Niemann-Pick disease type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene, resulting mainly in the accumulation of cholesterol and the ganglioside GM2. Recently, we described accumulations of these lipids in neuronal differentiated cells derived from NPC1 patient-specific induced pluripotent stem cells (iPSCs). As these lipids are essential for proper cell membrane composition, we were interested in the expression and function of voltage-gated ion channels and excitatory AMPA receptors (AMPARs) in neurons derived from three patient-specific iPSC lines. By means of patch clamp recordings and microfluorimetric measurements of calcium (Ca2+), we examined the expression of voltage-gated ion channels and AMPARs. Cells of the three used cell lines carrying the c.1836A>C/c.1628delC, the c.1180T>C or the c.3182T>C mutation demonstrated a significantly reduced AMPA-induced Ca2+-influx, suggesting an altered expression profile of these receptors. RT-qPCR revealed a significant upregulation of mRNA for the AMPA receptor subunits GluA1 and GluA2 and western blot analysis showed increased protein level of GluA2. Thus, we conclude that the observed reduced Ca2+-influx is based on an increase of GluA2 containing Ca2+-impermeable AMPARs. An attenuated function of GluRs in neurons potentially contributes to the progressive neurodegeneration observed in NPC1 and might represent an objective in regard of the development of new therapeutic approaches in NPC1.

Generation of six multiple sclerosis patient-derived induced pluripotent stem cell lines

Multiple sclerosis (MS) is considered a chronic autoimmune disease of the central nervous system that leads to gliosis, demyelination, axonal damage and neuronal death. The MS disease aetiology is unknown, though a polymorphism of the TNFRSF1A gene, rs1800693, is known to confer an increased risk for MS. Using retroviral delivery of reprogramming transgenes, we generated six MS patient-specific iPSC lines with two distinct genotypes, CC or TT, of the polymorphism rs1800693. iPSC lines had normal karyotype, expressed pluripotency genes and differentiated into the three germ layers. These lines offer a good tool to study MS pathomechanisms and for drug testing.

High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

SummaryReprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost.

Blast Crisis in a Dish: Generation of a Blast Crisis Model in Chronic Myeloid Leukemia (CML) Using Patient-Specific Induced Pluripotent Stem Cells ( iPSC)

Despite the major progress obtained in prognosis with the use of tyrosine kinase inhibitors (TKI), the great majority of patients with CML remain on long-term therapy and progression occurs in patients with either primary or secondary resistance. The mechanisms of progression towards accelerated phase (AP) and blast crisis (BC) have been studied so far only in primary patient samples in BC. Currently, there is no in vitro model to study sequentially the molecular events leading from CP to BC as only some primary sequential samples are amenable to analysis. Using induced Pluripotent Stem Cell (iPSC) technology, it is now possible to reprogram the primary leukemic cells to pluripotency and generate a major source of stem cells. To determine the feasibility of studying progression of CML towards AP and BC, we have used a patient-specific iPSC line that we have generated from the primary leukemic cells of a patient who later showed a TKI-resistance requiring an allogeneic stem cell transplant. These iPSC expressed BCR-ABL, exhibited all pluripotency markers and after injection in NSG mice, generated teratoma with differentiation into three germ layers. In hematopoietic differentiation assays using day 19-embryoid bodies (EB), increased numbers of hematopoietic progenitors were found as compared to control iPSC (5-fold increase n= 3). We have then treated leukemic iPSC with the mutagenic agent N-ethyl-N-nitrosourea (ENU) during regular medium changes. After 61 days in ENU cultures, day-19 derived embryoid bodies generated hematopoietic cells (>90% CD45+, CD43+) which proliferated in liquid cultures with myeloid and some blast cell morphology. Cytogenetic analyses of iPSC revealed chromosomal abnormalities such as loss of Y and loss of der q9+, both alterations known to occur in CML during progression. They exhibited increased numbers of micronuclei (MN) as compared to leukemic iPSC without ENU (X 3 Fold increase) suggesting acquisition of a progressive chromosomal instability. CGH array analyses were performed using ENU-treated iPSC-derived hematopoietic cells in two different timepoints as compared to leukemic iPSC cultured without ENU. Genomic aberrations were analyzed by Agilent Cytogenomics software with Mosaicism workflow on HG19 genome. 249 gene loci alterations were detected after polymorphism filtration on European population. These analyses showed DNA losses and DNA gains in genes implicated in mesoderm development and hematopoietic lineage as well as genes implicated in DNA damage response. Several loci of transcription factors were found to be involved such as IKZF1 described in imatinib-refractory chronic myeloid leukemia (Bolton-Gillespie et al. 2013). The aberrations included SIRT1and BLM which is implicated in DNA repair. Several cancer genes were found to be involved, some known to be altered in leukemia (BLM, IKZF1, NCOA2, ALK, EP300, ERG, MKL1, PHF6 and TET1). Remarkably, transcriptome geodataset GSE4170 (Radich et al. 2006) allowed us to associate 125 of 249 of the aberrations that we detected in CML iPSC, with the CML progression genes already described during progression from chronic and AP to BC (p-value =9.43E-32, after ANOVA with 1000 permutations). 38 most predictive aberrations allowed perfect reclassification of BC and chronic phase samples by unsupervised classification. Among these candidates, eleven of them have been described in CML physiopathology and connected to TKI resistance and genomic instability. Majority of them ( 7/11) are connected to chronic phase (FAS, ACTB, TRIM21, ANPEP, MLK1, CSF2RA, and MAGEC2) whereas a minority of them (4/11) are connected to BC (ACP1, SH3YL1, FHL1, IL3RA). Interestingly, these experiments also allowed us to discover the connection of a new multidrug resistance molecule associated to BC and having the ability to modify interferon pathway connected to the TKI sensitivity. Thus, genomic instability pattern that we have generated using a single leukemic iPSC allowed duplication of genomic abnormalities described in CML progression and allowed identification of some novel genes. Overall, these results demonstrated that we have generated for the first time to our knowledge, an in vitro BC model, reproducing genomic events described in patients with BC. This "blast crisis in a dish" tool using patient-derived iPSC will be of major interest to study CML progression and eventually to test novel therapies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Finding Expandable Induced Cardiovascular Progenitor Cells.

Expandable Cardiovascular Progenitor Cells Reprogrammed From Fibroblasts Zhang et al Cell Stem Cell . 2016;18:368–381. Lineage Reprogramming of Fibroblasts Into Proliferative Induced Cardiac Progenitor Cells by Defined Factors Lalit et al Cell Stem Cell . 2016;18:354–367. Cardiovascular progenitor cells (CPCs) are a promising cell source for cardiac regenerative therapy because of their ability for self-renewal and differentiation into various cardiovascular cell types beneficial for myocardial repair: cardiomyocytes, smooth muscle cells, and endothelial cells. Previous evaluations of exogenously derived CPCs have focused mainly on their capacity for trilineage differentiation rather than self-renewal because of the lack of an effective protocol to maintain and expand CPCs long-term in culture. In a recent issue of Cell Stem Cell , 2 groups of investigators independently reported their success in isolating, maintaining, and expanding mouse CPCs in culture for >18 to 20 passages (1010- to 1015-fold expansion), with faithful preservation of progenitor phenotype and ability for trilineage-restricted differentiation in both cell culture and mouse models of myocardial infarction (MI). This commentary will discuss the unique findings of these 2 studies, highlight the strengths and weaknesses of each CPC derivation/expansion technique, and propose additional steps necessary to accelerate their clinical translation. The past 2 decades have witnessed some of the most dramatic developments in the field of cardiac regeneration. Central to numerous controversies is the debate on whether certain stem cell types can differentiate into functional cardiomyocytes at a clinically relevant efficiency after implantation for repair of MI. Bone marrow–derived hematopoietic stem cells1,2 and c-kit+ resident cardiac stem cells3 are 2 notable cell types at the center of contention. Although many adult stem cells can still confer modest therapeutic benefits via paracrine effects, the search for a bona fide progenitor cell type that can generate a significant …

Generation and characterization of integration-free induced pluripotent stem cells from patients with autoimmune disease.

Autoimmune diseases (AIDs), a heterogeneous group of immune-mediated disorders, are a major and growing health problem. Although AIDs are currently treated primarily with anti-inflammatory and immunosuppressive drugs, the use of stem cell transplantation in patients with AIDs is becoming increasingly common. However, stem cell transplantation therapy has limitations, including a shortage of available stem cells and immune rejection of cells from nonautologous sources. Induced pluripotent stem cell (iPSC) technology, which allows the generation of patient-specific pluripotent stem cells, could offer an alternative source for clinical applications of stem cell therapies in AID patients. We used nonintegrating oriP/EBNA-1-based episomal vectors to reprogram dermal fibroblasts from patients with AIDs such as ankylosing spondylitis (AS), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). The pluripotency and multilineage differentiation capacity of each patient-specific iPSC line was validated. The safety of these iPSCs for use in stem cell transplantation is indicated by the fact that all AID-specific iPSCs are integrated transgene free. Finally, all AID-specific iPSCs derived in this study could be differentiated into cells of hematopoietic and mesenchymal lineages in vitro as shown by flow cytometric analysis and induction of terminal differentiation potential. Our results demonstrate the successful generation of integration-free iPSCs from patients with AS, SS and SLE. These findings support the possibility of using iPSC technology in autologous and allogeneic cell replacement therapy for various AIDs, including AS, SS and SLE.

Activity and High-Order Effective Connectivity Alterations in Sanfilippo C Patient-Specific Neuronal Networks

SummaryInduced pluripotent stem cell (iPSC) technology has been successfully used to recapitulate phenotypic traits of several human diseases in vitro. Patient-specific iPSC-based disease models are also expected to reveal early functional phenotypes, although this remains to be proved. Here, we generated iPSC lines from two patients with Sanfilippo type C syndrome, a lysosomal storage disorder with inheritable progressive neurodegeneration. Mature neurons obtained from patient-specific iPSC lines recapitulated the main known phenotypes of the disease, not present in genetically corrected patient-specific iPSC-derived cultures. Moreover, neuronal networks organized in vitro from mature patient-derived neurons showed early defects in neuronal activity, network-wide degradation, and altered effective connectivity. Our findings establish the importance of iPSC-based technology to identify early functional phenotypes, which can in turn shed light on the pathological mechanisms occurring in Sanfilippo syndrome. This technology also has the potential to provide valuable readouts to screen compounds, which can prevent the onset of neurodegeneration.

Optimized Generation of Functional Neutrophils and Macrophages from Patient-Specific Induced Pluripotent Stem Cells: Ex Vivo Models of X(0)-Linked, AR22(0)- and AR47(0)- Chronic Granulomatous Diseases.

Chronic granulomatous disease (CGD) is an inherited orphan disorder caused by mutations in one of the five genes encoding reduced nicotinamide-adenine-dinucleotide-phosphate oxidase subunits, which subsequently lead to impairment in the production of microbicidal reactive oxygen species (ROS). In order to offer several cell line models of CGD and therefore support research on pathophysiology and new therapeutic approaches, we optimized protocols to differentiate induced pluripotent stem cells (iPSCs) from wild-type, X0-, AR220- and AR470-CGD patient's fibroblasts into neutrophils and into macrophages. Aberrant genetic clones were discarded after chromosome karyotyping and array-comparative genomic hybridization analysis. All remaining iPSC lines showed human embryonic stem cell–like morphology, expressed all tested pluripotency markers and formed embryoid bodies that contained cells originating from all three primary germ layers. Furthermore, each CGD patient-specific iPSC line retained the gp91phox, p47phox, and p22phox mutations found in the corresponding patient's neutrophils. The average production of CD34+ progenitors was of 1.5×106 cells after 10 days of differentiation of 10×106 iPSCs. They were terminally differentiated into about 3×105 neutrophils or into 3×107 macrophages. Based on morphological, phenotypical, and functional criteria both phagocyte types were mature and indistinguishable from the native human neutrophils and macrophages. However, neutrophils and macrophages derived from X0-, AR220-, and AR470-CGD patient-specific iPSC lines lacked ROS production and the corresponding mutated proteins. To simplify the phagocytes' production upon request, progenitors can be cryopreserved. In conclusion, we describe a reproducible, simple, and efficient way to generate neutrophils and macrophages from iPSCs and provide a new cellular model for the AR220-CGD genetic form that has not been described before.

Modeling of human cardiomyopathy with induced pluripotent stem cells.

Human inherited cardiomyopathies are one of the major etiologies for heart failure which are associated with significant mortality and morbidity. Unfortunately, there are lack of effective specific therapies for human cardiomyopathies due to the limited understanding on their pathophysiology. Currently, most of the mechanistic studies of human cardiomyopathy are based on transgenic mouse models and invasive collection of limited amount of myocardial biopsy specimen. Disease-specific stem-cells are already available for studying single-gene mutation related diseases, such as cystic fibrosis and fragile X syndrome. The possibility of obtaining stem-cells using induced pluripotent stem cell (iPSC) technology provides the opportunity to generate stem cells carrying an inherited disease phenotype that will then serve as an invaluable model to study the disease biology and treatment of human cardiomyopathies. Here, we review the major strategies and workflow of using the patient-specific iPSCs derived cardiomyocytes to model inherited human cardiomyopathies. The creation of patient-specific iPSC lines in patients with hypertrophic cardiomyopathy and dilated cardiomyopathy have been reported and served as important human models of inherited diseases to improve our understanding of the disease mechanisms and enable drug screening.

Modeling Liver Diseases Using Induced Pluripotent Stem Cell (Ipsc)-Derived Hepatocytes

The induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells in a stem state. The iPSCs can give rise to cells of all three germ layers and provide an unlimited supply of tissue-specific differentiated cell types for disease modeling and cell therapy. The generation of patient-specific iPSC lines and studying disease phenotype in a dish using differentiated hepatocytes open up new avenue towards personalized medicine. There has been active investigation on generating homogenous functional human hepatocytes from iPSCs. Liver carries out secretory and metabolic functions. Recent studies showed that iPSC derived-human hepatocytes are useful for in vitro investigation of genetic liver disorders, drug screening and metabolism, hepatitis C viral infection and assessing efficacy of cell therapy. Inherited metabolic disorders, including α1-antitrypsin deficiency (A1AD), familial hypercholesterolemia, glycogen storage disease type 1a and Wilson’s disease have been modeled using disease-specific iPSC lines. The iPSC hepatocytes derived from patients with A1AD were used for drug screening. Advancement made in precise genetic engineering technology using designer nucleases provides a new tool for gene correction, and reverse genetic engineering of disease causing genotype in pluripotent stem cells. Moreover, iPSC-hepatocytes from various genetic backgrounds are valuable resource for evaluating drug interactions and drug metabolism. In this review, we summarize the recent developments on the various applications of iPSC-derived human hepatocytes for disease modeling.

Targeted correction Of RUNX1 Mutation In The FPD Patient-Specific Induced Pluripotent Stem Cells Restores Megakaryocytic Differentiation

Familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disease caused by RUNX1 mutations. RUNX1 encodes a transcription factor, which is frequently mutated in leukemias. It is also essential in early hematopoiesis and megakaryocytic differentiation. FPD patients have a mild to moderate bleeding disorder, thrombocytopenia, and over 35% of patients will develop AML during their lifetime. Currently there are no suitable animal models for the disease, making it necessary to develop alternative approaches for research studies.To develop an in vitro model of FPD, we established induced pluripotent stem cell (iPSC) lines using episomal, non-integrating vectors from skin fibroblasts from a family of FPD patients harboring a nonsense mutation in the C-terminal region of the RUNX1 (Y260X). The derived iPSC lines showed typical characteristics of pluripotency such as teratoma formation and expressions of pluripotency markers (Oct4, Sox2, Nanog, and Tra-1-81). In order to correct the mutation in the patient-specific iPSCs by homologous recombination, we designed a pair of RUNX1 zinc finger nucleases (ZFNs) and a targeting vector. We then performed ZFN-mediated gene targeting in one of the FPD iPSC lines and confirmed correction of the mutant RUNX1 allele in at least two iPSC clones by Southern, FISH and sequence analyses. RUNX1 mRNA expression analysis of the FPD iPSC line and the corrected iPSC lines showed expression of only wildtype RUNX1 transcripts in the corrected iPSC lines, but not in the uncorrected lines. Furthermore, we directed hematopoietic differentiation of the FPD iPSCs and the corrected iPSC lines using the spin embryoid body formation method. We found defects in hematopoietic differentiation of FPD iPSCs, particularly towards megakaryopoiesis. Importantly, ZFN-mediated mutation correction rescued the hematopoietic differentiation defects with almost double the number of CD41a+/CD42a+ megakaryocytes produced from the corrected iPSCs.Our results collectively demonstrate successful in vitro modeling of FPD using iPSC lines with an innovative approach to correct the RUNX1 mutation by gene targeting. Mutation correction rescues the hematopoietic defects, indicating that hematopoietic stem cell transplantation using patient-specific iPSC lines has great potential to treat the FPD patients in the future. Disclosures:Horwitz:United States Government: Consultancy, United States ex rel. Horwitz v. Amgen Inc., Civil Action No. C07-0248 (W.D. Wash.) Other.

Human Induced Pluripotent Stem Cells As a Tool to Model a Form of Leber Congenital Amaurosis

Our purpose was to investigate genes and molecular mechanisms involved in patients with Leber congenital amaurosis (LCA) and to model this type of LCA for drug screening. Fibroblasts from two unrelated clinically identified patients with a yet undetermined gene mutation were reprogrammed to pluripotency by retroviral transduction. These human induced pluripotent stem cells (hiPSCs) were differentiated into neural stem cells (NSCs) that mimicked the neural tube stage and retinal pigmented epithelial (RPE) cells that could be targeted by the disease. A genome-wide transcriptome analysis was performed with Affymetrix Exon Array GeneChip(®), comparing LCA-hiPSCs derivatives to controls. A genomic search for alteration in all genes known to be involved in LCA revealed a common polymorphism on the GUCY2D gene, referenced as the LCA type I (OMIM *600179 and #204000), but the causative gene remained unknown. The hiPSCs expressed the key pluripotency factors and formed embryoid bodies in vitro containing cells originating from all three germ layers. They were successfully differentiated into NSC and RPE cells. One gene, NNAT, was upregulated in LCA cell populations, and three genes were downregulated, GSTT1, TRIM61 and ZNF558, with potential correlates for molecular mechanisms of this type of LCA, in particular for protein degradation and oxidative stress. The two LCA patient-specific iPSC lines will contribute to modeling LCA phenotypes and screening candidate drugs.

Modeling Long-QT Syndromes with iPS Cells

The generation of induced pluripotent stem cells (iPSC) from human somatic cells bears the possibility to generate patient-specific stem cell lines which can serve as a theoretically unlimited source of somatic cells carrying the genotype of the patients. Different types of the long-QT syndrome have been studied by analyzing the phenotype of cardiomyocytes generated from patient-specific iPSC lines. Major aspects of the pathophysiology of long-QT syndrome, like prolonged action potentials, arrhythmia, and the effects of pro- and antiarrhythmic drugs could be recapitulated in these cells. In the future, patient-specific iPSC-derived cardiomyocytes might be used to screen for new drugs, to avoid unwanted drug side effects, and to deepen our understanding on the pathophysiology of long-QT syndromes.

Small Molecules Greatly Improve Conversion of Human-Induced Pluripotent Stem Cells to the Neuronal Lineage

Efficient in vitro differentiation into specific cell types is more important than ever after the breakthrough in nuclear reprogramming of somatic cells and its potential for disease modeling and drug screening. Key success factors for neuronal differentiation are the yield of desired neuronal marker expression, reproducibility, length, and cost. Three main neuronal differentiation approaches are stromal-induced neuronal differentiation, embryoid body (EB) differentiation, and direct neuronal differentiation. Here, we describe our neurodifferentiation protocol using small molecules that very efficiently promote neural induction in a 5-stage EB protocol from six induced pluripotent stem cells (iPSC) lines from patients with Parkinson's disease and controls. This protocol generates neural precursors using Dorsomorphin and SB431542 and further maturation into dopaminergic neurons by replacing sonic hedgehog with purmorphamine or smoothened agonist. The advantage of this approach is that all patient-specific iPSC lines tested in this study were successfully and consistently coaxed into the neural lineage.