An induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells of a 3-day-old boy with 47,XXY and Ornithine Transcarbamylase Deficiency carrying hemizygote mutation (c.663+2T>G (sliping)) in OTC. The iPSCs had original 47,XXY, and mutation in OTC, expressing pluripotency markers and bearing differentiation potential in vitro.

Methylmalonic acidemia and homocystinuria, cblC type is a rare autosomal recessive inheritance disease. Its clinical phenotype involves multiple systems with varying degrees of severity. The disease is caused by the mutations in the MMACHC gene located on chromosome 1p34.1. Here we report the generation of an iPSC line from the PBMCs of a patient with compound heterozygous mutations in the MMACHC gene. This new iPSC line will allow a better understanding of the MMA disease.

Dermal fibroblasts were donated by a 43 year old male patient with clinically diagnosed familial amyotrophic lateral sclerosis (ALS), carrying the SOD1E101G mutation. The induced pluripotent stem cell (iPSC) line UOWi007-A was generated using repeated mRNA transfections for pluripotency transcription factors Oct4, Klf4, Sox2, c-Myc, Lin28 and Nanog. The iPSCs carried the SOD1E101G genotype and had a normal karyotype, expressed expected pluripotency markers and were capable of in vitro differentiation into endodermal, mesodermal and ectodermal lineages. This iPSC line may be useful for investigating familial ALS resulting from a SOD1 E101G mutation.

we generated iPSCs from peripheral blood mononuclear cells of a child with epilepsy carrying heterozygous missense mutation in GRIN2A, using integration free episomal vectors. These iPSCs express pluripotent markers, represent a normal karyotype and have the ability to differentiate into three germ layers.

Here, we describe the generation of an induced pluripotent stem cell (iPSC) line, from a male patient diagnosed with Parkinson's disease (PD). The patient carries a heterozygous variation p.A53T in the SNCA gene. Skin fibroblasts were reprogrammed using the non-integrating Sendai virus technology to deliver OCT3/4, SOX2, c-MYC and KLF4 factors. The generated iPSC line (CSC-32) preserved the mutation, displayed expression of common pluripotency markers, differentiated into derivatives of the three germ layers, and exhibited a normal karyotype. The clone CSC-32B is presented thereafter; it can be used to study the mechanisms underlying PD pathogenesis.

Aicardi-Goutières syndrome (AGS) is a hereditary early onset encephalopathy. AGS patients display variable clinical manifestations including intracranial calcification, cerebral atrophy, white matter abnormalities and characteristic leukocytosis as well as a constitutive upregulation of type I IFN production indicative of a type I interferonopathy. Seven genes (SAMHD1, TREX1, RNASEH2B, RNASEH2C, RNASEH2A, ADAR1, IFIH1) have been associated with the AGS phenotype, up to now. Here, we describe the generation of three induced pluripotent stem cell lines from a patient with a deletion of coding exons 14 and 15 of the SAMHD1 gene.

Pluripotent stem cells (PSCs) offer a promising tool for regenerative medicine. The clinical application of PSCs inevitably requires a large-scale culture in a highly defined environment. The present study aimed to devise defined coating materials for the efficient adhesion and proliferation of human PSCs (hPSCs). We tested the activity of seven fibronectin-derived peptides and three laminin-derived peptides for the attachment and proliferation of hPSCs through their immobilization on the bottom of culture dishes by creating a fusion protein with the mussel adhesion protein. Among the extracellular matrix (ECM) mimetics tested, one fibronectin-derived peptide, PHSRN-GRGDSP, significantly promoted adhesion, enhanced alkaline phosphatase activity, and increased pluripotency-related gene expression in hPSCs compared to Matrigel. Furthermore, co-immobilization of a particular canofin peptide derived from fibroblast growth factor 2 increased pluripotency marker expression, which may offer the possibility of culture without growth factor supplementation. Our findings afford a novel defined condition for the efficient culture of hPSCs and may be utilized in future clinical applications.

Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiovascular disorder. In this study, we generated human induced pluripotent stem cells (iPSC) ZZUNEUi007-A from dermal fibroblasts of an HCM patient with the p. R663H (c. 1988G > A) mutation in MYH7. The generated hiPSC line had normal karyotype, showed robust expression of pluripotency markers and could differentiate into all three germ layers in vivo.

MUSIe001-A cell line was derived from a Southeast Asian (SEA) type deletion α0-thalassemia embryo. The SEA deletion embryo was donated for research with informed consent. This cell line shows normal hESC morphology, expresses all pluripotent markers, and has the potential to differentiate into all three germ layers in vitro and in vivo. The MUSIe001-A line has normal karyotype and is free from mycoplasma contamination. PCR analysis confirmed the MUSIe001-A cell line to be a SEA type deletion. MUSIe001-A is a valuable proof of principle model for gene therapy that will facilitate the development of new treatments for affected foetuses.

Bloom syndrome is characterized by severe pre- and postnatal growth deficiency, immune abnormalities, sensitivity to sunlight, insulin resistance, and a high risk for many cancers that occur at an early age. The diagnosis is established on characteristic clinical features and/or presence of biallelic pathogenic variants in the BLM gene. An increased frequency of sister-chromatid exchanges is also observed and can be useful to diagnose BS patients with weak or no clinical features. For the first time, we derived an induced pluripotent cell line from a Bloom syndrome patient retaining the specific sister-chromatid exchange feature as a unique tool to model the pathology.

Death-associated protein kinase 1 (DAPK1) is a Ca2+/calmodulin regulated Ser/Thr kinase involved in various cellular processes including cell death, autophagy and inflammation. Its dysregulation has been linked to tumour metastasis, anti-viral responses, Alzheimer's disease and other neurological disorders. To further investigate the role of DAPK1 in these processes, we generated a DAPK1 knockout first (conditional ready) human embryonic stem (hES) cell line in which the endogenous DAPK1 can be easily restored with expression of FLPe. This cell line provides an ideal model to study the role of DAPK1 in human development and various pathologies related to DAPK1 dysregulation in vitro.

Human induced pluripotent stem cells (hiPSCs) from individual patient basis are considered a powerful resource to model human diseases. However, to study complex multigenic diseases such as Congenital Heart Disease, it is crucial to generate perfect isogenic controls to understand gene singularity and contribution. Here, we report the engendering of an isogenic hiPSC line with homozygous correction of c.455G > A alteration in the DAND5 gene, using CRISPR/Cas9 technology. The characterization of a clone of this cell line demonstrates normal karyotype, pluripotent state, and potential to differentiate in vitro towards endoderm, mesoderm, and ectoderm.

To develop an iPSC SOX9 reporter line for monitoring differentiation into SOX9 expressing cells such as chondrocytes, cranial neural crest and Sertoli cells, we used gene editing to introduce sequences encoding the tdTomato fluorescent protein into the SOX9 locus. The gene-edited line had a normal karyotype, expressed pluripotency markers and differentiated into cells representative of the three embryonic germ layers. Endogenous SOX9 expression was undisturbed and the tdTomato fluorescent reporter mirrored SOX9 mRNA expression. This iPSC line will be useful for assessing iPSC differentiation into SOX9-expressing cells and enrichment by cell sorting.

Hearing loss is the most common disorder in the sensory system. Mutations in GJB2 have been reported to be very common in sensorineural hearing loss patients. In this report, we generated an induced pluripotent stem cell (iPSC) line, MMCi001-A, from the peripheral blood mononuclear cells of a 4-year-old male hearing loss patient carrying GJB2 pV37I mutation by using the Sendai virus delivery system. The generated iPSCs were demonstrated to express pluripotent markers and be differentiated into three germ layers in vitro and in vivo. This GJB2-pV37I iPSCs is valuable for studying the pathogenic mechanisms and drug discovery of hearing loss.

The human induced pluripotent stem cell line NCCDFWi001-A was derived from peripheral blood mononuclear cells (PBMC) of a 26-year-old female Marfan syndrome patient carrying two compound heterozygous variants FBN1c.2613A>C, (p.Leu871Phe) and c.684_736+4del. The established patient-derived iPSC showed expression of pluripotent stem cell markers and had the ability to differentiate into all of the three germ layers and possessed a normal karyotype.

Human endothelial cells (ECs) are important tools in research and development of new therapies in the fields of angiogenesis, vasculogenesis, engineering organoids and multicellular tissues, drug discovery, and disease modeling. Efficient and robust induction of ECs from human pluripotent stem cells (hPSCs) serve as a renewable and indefinite cell sources. However, individual lines of embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) are distinct and can often respond very differently to the same microenvironmental cues. Therefore, we set out to develop a differentiation methodology specifically designed for robustness across multiple human iPSC lines. In general, the key soluble signals remain consistent across cell lines, but because the differentiation and proliferation kinetics can differ slightly in hESC and iPSC cell lines, the time point for KDR+ cell sorting must be pre-determined for each cell line. This three-stage induction method uses three different chemically defined medium formulations and generates highly purified populations of actively proliferating and functional VE-cadherin+ ECs within 30 days.

Three induced pluripotent stem cells (iPSC) clones NCCSi007-A, NCCSi007-B and NCCSi007-C were generated from CD4+T cells of a 38 years old male patient suffering from liver cirrhosis- alcoholic and minimal hepatic encephalopathy of Indian origin. The CD4+T cells of the patient were reprogrammed using integration free, Sendai viral vector system. Each of the three iPSC clones showed high alkaline phosphatase (ALP) activity, expressed pluripotency markers OCT4, SOX2, NANOG, KLF4, SSEA-4, TRA-1-60, showed normal male karyotype (46, XY) and exhibited multi-lineage differentiation.

The human induced pluripotent stem cell (iPSC) line YAHKMUi001-A was derived from the dermal fibroblasts of a patient with Tetralogy of Fallot (TOF), with a mutation in the TBX1 gene (c.928G > A). The skin fibroblasts were obtained from a 4-year-old boy, and were infected with Sendai virus expressing the Yamanaka factors. The YAHKMUi001-A iPSC line expresses pluripotent stem cell markers, displays a normal karyotype, and has the capacity to differentiate into 3 germ layers. This cell line model can be a good tool to study the pathological mechanism of the TBX1 gene mutations associated with TOF.

Muscle stem cells (MuSCs) are involved in homeostatic maintenance of skeletal muscle and play a central role in muscle regeneration in response to injury. Thus, understanding MuSC autonomous properties is of fundamental importance for studies of muscle degenerative diseases and muscle plasticity. Rat, as an animal model, has been widely used in the skeletal muscle field, however rat MuSC isolation through fluorescence-activated cell sorting has never been described. This work validates a protocol for effective MuSC isolation from rat skeletal muscles. Tibialis anterior was harvested from female rats and digested for isolation of MuSCs. Three protocols, employing different cell surface markers (CD106, CD56, and CD29), were compared for their ability to isolate a highly enriched MuSC population. Cells isolated using only CD106 as a positive marker showed high expression of Pax7, ability to progress through myogenic lineage while in culture, and complete differentiation in serum-deprived conditions. The protocol was further validated in gastrocnemius, diaphragm, and the individual components of the pelvic floor muscle complex (coccygeus, iliocaudalis, and pubocaudalis), proving to be reproducible. CD106 is an efficient marker for reliable isolation of MuSCs from a variety of rat skeletal muscles.

Here, we report the establishment of the human iPS cell line N1-FiPS4F#7 generated from skin cells of a patient with no rheumatic diseases, thus obtaining an appropriate control iPS cell line for researchers working in the field of rheumatic diseases. The reprogramming factors Oct4, Sox2, Klf4 and c-Myc were introduced using a non-integrating reprogramming strategy involving Sendai Virus.

Human induced pluripotent stem cells (iPSCs) that express stable and robust fluorescent proteins have proven to be indispensable in basic and translational research. These reporter iPSC lines can greatly facilitate cell imaging, sorting, and tracking in in vitro and in vivo studies. Here, we document two reporter human iPSC lines generated by gene-editing technologies that precisely integrated one-copy of a tdTomato transgene driven by strong CAG promoter into the AAVS1 human safe harbor locus.

Induced pluripotent stem cells (iPSCs) are a useful tool to investigate pathomechanistic and cellular processes due to their differentiation potential into different somatic cell types in vitro. Here, we have generated iPSCs from an apparently healthy male individual using an integration-free reprogramming method. The resulting iPSCs are pluripotent and display a normal karyotype. Furthermore, we demonstrate that this iPSC line can be differentiated into all three germ layers.

Fabry disease is an X-linked inherited disease caused by a mutation in the galactosidase alpha (GLA) gene. Here, we generated a GLA knock-out cell line (GLA-KO hiPSCs) from normal human-induced pluripotent stem cells (hFSiPS1) using the CRISPR-Cas9 genome-editing tool. The GLA-KO hiPSCs maintained normal morphology, karyotypes, expression of stemness markers, and trilineage differentiation potential. Furthermore, the GLA-KO hiPSCs exhibited dissipation of GLA activity and abnormal Globotriaosylceramide (Gb3) accumulation. Our GLA-KO hiPSC line represents a valuable tool for studying the mechanisms involved in Fabry disease and the development of novel therapeutic alternatives to treat this rare condition.

The gap junction beta-2 (GJB2) gene is the most common genetic cause of hereditary deafness worldwide. Especially, V37I mutation in GJB2 is most prevalent in Southeast Asia including Thailand, Malaysia, and Indonesia. Furthermore, it is the second most prevalent cause in Japan and China, and exhibits an audiometric phenotype of mild-to-moderate hearing loss. In this study, we generated induced pluripotent stem cells (iPSC) from peripheral blood mononuclear cells (PBMCs) of patient with homozygous V37I mutation. This iPSC line will be a powerful tool for investigating the pathogenesis and for developing a treatment for GJB2-related hearing loss.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by death of motor neurons. To date, neither etiology nor pathogenesis of ALS are known, which leads to the absence of an effective treatment strategy. ALS patient-specific induced pluripotent stem cells (iPSCs) represent an excellent tool for the disease study. We obtained iPSCs line from peripheral blood mononuclear cells of the patient with homozygous Asp90Ala mutation in the SOD1 gene using non-integrating episomal vectors. The iPSCs line retained pathological genotype and expressed pluripotency markers. It also displayed a normal karyotype and the ability to differentiate into derivatives of three germ layers.

Autosomal dominant polycystic kidney disease (ADPKD) is the most prevalent inherited renal disease, characterized by multiple cysts that can lead to kidney failure resulting in end-stage renal disease. ADPKD is mainly caused by mutations in either the PKD1 and PKD2 genes, encoding for polycystin-1 and polycystin-2, respectively. In order to clarify the disease mechanisms, here we describe the generation of two isogenic induced pluripotent stem cell (iPSC) lines in which the PKD2 gene was deleted using CRISPR/Cas9 technology. The PKD2−/- iPSCs expressed the main pluripotency markers, were able to differentiate into the three germ layers and had a normal karyotype.

Retinitis Pigmentosa (RP) is an inherited disorder of retinal degeneration with progressive loss of rod and cone photoreceptors. RPE65 is a gene encoding the trans-cis isomerase which is essential for the classical visual cycle. While most RPE65 mutations associated with RP have been reported as autosomal recessive, an Irish c.1430A>G (p.D477G) mutation is the first case reported to cause dominantly inherited RP. In this study, we used the non-integrational Sendai virus to generate induced pluripotent stem cell (iPSC) lines carrying the c.1430A>G (p.D477G) mutation from three familial RP patients.

Summary The German Stem Cell Network (GSCN) aims at creating synergies between all areas of basic and applied stem cell research and to provide an interface between science, education, politics and society as a whole. The central task of the GSCN is to pool the expertise in stem cell research in Germany and develop synergies between basic research, regenerative medicine and pharmacology. The initiative promotes innovative research activities on a national and international level. In addition, targeted information and events are offered to encourage the public discourse on stem cell research. The objectives of the network are: • To maintain an organizational structure for a German network for basic and applied stem cell research; • To organize joint annual conferences on stem cell research to be rotated among German cities: • To coordinate scientific and strategic working groups; • To provide a platform for communication on stem cell research, enabling exchange of important news, discussions and networking between scientists, institutions, policy-makers and the general public (in German and English); • To publish documents about basic and applied stem cell research in Germany and help to organize public meetings and outreach programs on these topics.

The Hadassah hESC Research Center's aim is to be a supplier of clinical and research-grade human embryonic stem cell (hESC) lines. In 2012, we derived the first three entirely GMP-compliant and xeno-free, fully-characterised, feeder-dependent (human umbilical cord) hESC lines developed under cleanroom conditions. In 2018, we established four new GMP and xeno-free, feeder-independent MCB hESCs under GMP conditions using commercially available reagents, media and matrix. All cell lines were derived under Israeli Ministry of Health's National Ethics Committee for Genetic Research in Humans and the ethical considerations that guided the development of the hESCs strictly followed Israeli law. Hadassah has provided its clinical-grade hESC lines to commercial entities of which two are already in clinical trials, establishing Hadassah as a key provider of clinical-grade hESC lines.

Induced pluripotent stem cells (iPSCs) can be used to generate different types of somatic cells in vitro, including neuronal cells. Here, a human iPSC line was generated from the peripheral blood mononuclear cells of a healthy 39-year-old individual. The resulting iPSCs were integration-free, maintained the normal karyotype, expressed pluripotency stem cell markers, and were demonstrated to be capable of differentiating into cells representative of the three embryonic germ layers. Furthermore, we showed that this iPSC line could be differentiated into neural stem cells. Taken together, this generated iPSC line could be useful to test multiple differentiation protocols, and also serve as a control for investigating drug development and disease mechanisms.

Here, we present the characterization of three iPSC lines derived from dermal fibroblasts of old healthy subjects. Fibroblasts were reprogrammed using Sendai viral vectors encoding OCT4, SOX2, KLF4 and c-MYC. The iPSCs expressed endogenous pluripotency markers, could generate the three germ layers (ectoderm, mesoderm and endoderm), maintained a stable karyotype, and were free from Sendai vectors and reprogramming factors. These integration-free iPSCs can serve for establishing control cell cultures in studies searching for phenotypes and mechanisms that could potentially be dysregulated in degenerative diseases.

Duchenne muscular dystrophy (DMD), an X-linked genetic disorder characterized by progressive muscle weakness and atrophies affecting skeletal and cardiac muscles, is caused by mutations in dystrophin (DMD) gene that spans 79 exons. Here, we generated iPSCs from a Chinese patient with 49 to 50 exons deletion in DMD gene by reprogramming peripheral blood mononuclear cells with non-integrating vectors. The generated iPSCs line (SDQLCHi007-A) carrying the identical deletion of 49-50 exons, expresses pluripotency markers, presents a normal karyotype and is able to differentiate into three germ layers.

Human IPSC Line, ZZUNEUi003-A, was generated from a 32-year-old male patient with Wilson's Disease carrying a homozygous R778L mutation in ATP7B gene, using non-integrative reprogramming method. This cell line shows pluripotency both in vitro and vivo, and has a normal karyotype.

Autosomal recessive osteopetrosis (ARO) is a rare inherited disorder leading to increased bone density with impairment in bone resorption. Among the genes responsible for ARO, the TCIRG1 gene, coding for the a3 subunit of the osteoclast proton pump, is mutated in more than 50% of the cases, increasing the importance of TCIRG1-iPSCs as disease model. We generated 3 iPSC clones derived from Peripheral Blood Mononuclear Cells (PBMCs) of a patient carrying the heterozygous mutations p.Y512X and c.2236+1G>A. A Sendai virus-based vector was used and the iPSCs were characterized for genetic identity to parental cells, genomic integrity, pluripotency, and differentiation ability.

We generated human induced pluripotent stem cells (hiPSCs) from dermal fibroblasts of a 40 years old female patient homozygous for the mutation c.535 G>A p.G179S on the KCNQ1 gene, causing a severe form of autosomal recessive Long QT Syndrome type 1 (AR-LQT1). The hiPSCs, generated using classical approach of the four retroviruses enconding the reprogramming factors OCT4, SOX2, cMYC and KLF4, display pluripotent stem cell characteristics, and differentiate into cell lineages of all three germ layers.

Tumor protein p63 (p63) encodes for a transcription factor of the p53 family and is a marker for respiratory basal cells. Based on a NKX2.1 knock-in reporter cell line from human induced pluripotent stem cells (hiPSCs) (MHHi06-A-2) we established a NKX2.1/p63 double transgenic knock-in reporter cell line using TALEN technology. The reporter enables the optimization and monitoring of hiPSC differentiation towards NKX2.1/p63 double positive cells as well as enrichment for single or double positive cells.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) have evolved into widely used and reliable cell sources for modeling cardiovascular channelopathies and for drug safety pharmacology. However, the electrophysiological and pharmacological applications of hiPSC-CMs are hampered by manual patch-clamp technique, which is labor-intensive and generates a low-output. The automated patch-clamp technique is showing potential to overcome this problem. Here, we describe a new dissociation method, with which we can harvest a vast number of single relaxed hiPSC-CMs with smooth membrane suited for automated patch-clamp. Using the automated whole-cell patch-clamp technology, we report a high success rate for cell capture and whole-cell access (around 70%). We are able to identify and record several currents and paced action potentials (APs) with different success rates, including Na+ current (INa), L-type Ca2+ current (ICaL), two specific K+ currents, the transient outward K+ current (Ito) and the inward rectifier K+ current (IK1). Moreover, we successfully applied dynamic current-clamp to virtually increase IK1 for AP recordings. Our study suggests that automated patch-clamp technology could be used to investigate the relevant ionic currents and APs in hiPSC-CMs. The combination of automated patch-clamp and hiPSC-CM technologies promises a wide range of applications in the future.

Peripheral blood mononuclear cells (PBMCs) were collected from a 6-year-old female child who was clinically diagnosed as primary nephrotic syndrome (NS) with hormone resistance. An iPSC line was successfully established by the Sendai-virus (SeV) delivery system. The iPS-19 (GSPHi001-A) expressed pluripotent markers, exhibited a normal karyotype and differentiated towards three germ layers. The iPSC line might offer a potentially useful tool for investigating mechanisms of primary NS, drug testing and gene therapy studies.

In this study, we describe the generation and characterization of induced pluripotent stem cell (iPSC) lines from familial long QT syndrome type 1 (LQT1) patients carrying the KCNQ1 c.1201dupC (p.Arg401fs) frame shift mutation by using non-integrational Sendai reprogramming method. The patient-specific iPSC lines harbouring the c.1201dupC mutation on KCNQ1 gene expressed pluripotency markers and had the capacity to differentiate into three germ layers.

We generated eight induced pluripotent stem cell (iPSC) lines from Parkinson's disease (PD) patients with different familial mutations using non-integrating episomal plasmids. All iPSC lines have a normal karyotype, express pluripotent genes including POU5F1, NANOG, and show alkaline phosphatase activity, as well as the ability to differentiate into all three germ layers. These PD iPSC lines can be used for disease modeling to identify PD mechanisms and for the development or stratification of new drugs.

NRXN1 copy number variation is a rare genetic factor commonly shared among autism spectrum disorder (ASD), schizophrenia, intellectual disability, epilepsy and developmental delay. Human induced pluripotent stem cells (iPSCs) are essential for disease modelling and drug discovery, but familial cases are particularly rare. We report here the derivation of familial iPSC lines from two controls and three ASD patients carrying NRXN1α+/−, using a non-integrating Sendai viral kit. The genotype and karyotype of the resulting iPSCs were validated by whole genome SNP array. All iPSC lines expressed comparable levels of pluripotency markers and could be differentiated into three germ layers.

Studying Parkinson's disease (PD), one of the most common neurodegenerative disorders worldwide, requires different model systems, including patient-specific induced pluripotent stem cell lines. With the help of non-integrating episomal vectors the IPSC lines ICGi015-A and ICGi015-B were generated from blood mononuclear cells of PD patient, carrying three SNPs, associated with PD development. The obtained iPSC lines express pluripotency markers and demonstrate the ability to in vitro differentiate into the three germ layers. These cell lines may be useful for studying molecular mechanisms of PD and for drug screening.

Pluripotent stem cells are considered to be the ideal candidates for cell-based therapies in humans. In this regard, both nuclear transfer embryonic stem (ntES) cells and induced pluripotent stem (iPS) cells are particularly advantageous because patient-specific autologous ntES and iPS cells can avoid immunorejection and other side effects that may be present in the allogenic pluripotent stem cells derived from unrelated sources. However, they have been found to contain deleterious genetic and epigenetic changes that may hinder their therapeutic applications. Indeed, deregulation of genomic imprinting has been frequently observed in reprogrammed ntES and iPS cells. We will survey the recent studies on genomic imprinting in pluripotent stem cells, particularly in iPS cells. In a previous study published about six years ago, genomic imprinting was found to be variably lost in mouse iPS clones. Intriguingly, de novo DNA methylation also occurred at the previously unmethylated imprinting control regions (ICRs) in a high percentage of iPS clones. These unexpected results were confirmed by a recent independent study with a similar approach. Since dysregulation of genomic imprinting can cause many human diseases including cancer and neurological disorders, these recent findings on genomic imprinting in reprogramming may have some implications for therapeutic applications of pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) have become indispensable for disease modelling. They are an important resource to access patient cells harbouring disease-causing mutations. Derivation of midbrain dopaminergic (DAergic) neurons from hiPSCs of PD patients represents the only option to model physiological processes in a cell type that is not otherwise accessible from human patients. However, differentiation does not produce a homogenous population of DA neurons and contaminant cell types may interfere with the readout of the in vitro system. Here, we use CRISPR/Cas9 to generate novel knock-in reporter lines for DA neurons, engineered with an endogenous fluorescent tyrosine hydroxylase – enhanced green fluorescent protein (TH-eGFP) reporter. We present a reproducible knock-in strategy combined with a highly specific homologous directed repair (HDR) screening approach using digital droplet PCR (ddPCR). The knock-in cell lines that we created show a functioning fluorescent reporter system for DA neurons that are identifiable by flow cytometry.

Induced pluripotent stem cells (iPSCs) were generated from peripheral blood-derived erythroid progenitor cells obtained from a presymptomatic female carrying the heterozygous R418X progranulin (GRN) nonsense mutation, known to cause autosomal dominant frontotemporal lobar degeneration. Erythroid progenitor cells were reprogrammed into iPSCs using integration free episomal plasmids which enables exogenous expression of the Yamanaka factors. The pluripotent potential of the iPSCs was validated through expression of pluripotency factors and their capacity to differentiate into the three primary germ layers. The cells were confirmed to carry the described mutation and shown to have a normal karyotype.

Lymphoblast cells from four individuals of a family of two genetically unrelated parents and their monozygotic twins were used to generate integration-free human induced pluripotent stem cells (hiPSCs). Reprogramming factors were delivered by co-electroporation of three episomal-based plasmids expressing OCT3/4, SOX2, KLF4, L-MYC and LIN28. The hiPSCs showed a normal karyotype, expressed pluripotency-associated markers, displayed the capacity for in vitro differentiation into the three germ layers and were Epstein Barr virus-free. These hiPSC lines offer the possibility to compare genetically unrelated and genetically identical tissues from different individuals and to study genotype-specific effects, which are particularly relevant for toxicology testing.

Familial partial lipodystrophy type 2 (FPLD2) is a rare autosomal dominant metabolic disorder caused by heterozygous mutations in the LMNA gene, which encodes for the lamin A/C. A human induced pluripotent stem cell (iPSC) line was generated from peripheral blood mononuclear cells (PBMCs) of a 30 year-old male patient with FPLD2 who had a heterozygous p.R349W (c.1045C>T) mutation in the LMNA gene using non-integrating episomal vector technique. This iPSC line offers a useful resource to investigate pathogenic mechanisms in FPLD2, as well as a cell-based model for drug development to treat FPLD2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) gene (LRRK2 G2019S) is a representative autosomal dominant mutation that can cause Parkinson's disease (PD). A bacterial artificial chromosome-based homologous recombination (BAC-based HR) system was utilized for gene therapy of LRRK2 G2019S-mutant induced pluripotent stem cells (iPSCs) produced by reprogramming episomal vectors. The gene-corrected iPSCs retained typical pluripotency required for their spontaneous differentiation into differentiated cells. The iPSCs had a normal karyotype and were confirmed to have no off-target sites by melting curve analysis.

Human ERF gene is a transcription factor involved in development, trophoblast differentiation, apoptosis, and cancer progress. To further understand the exact roles of ERF in these processes, here we report that establishment of two ERF knockout human embryonic stem cell (hESC) lines by CRISPR/Cas9 mediated gene targeting. These cell lines exhibited classical hESC morphology and normal karyotype, and highly expressed pluripotent markers, and had differentiation potential in vitro. These cell lines provide good materials to understand the roles of ERF in development, trophoblast differentiation and craniosynostosis for further studies.

The last several years have witnessed renewed interest regarding the contribution of cancer stem cells in tumorigenesis and neoplastic heterogeneity. It has been reported that patients who undergo bone marrow transplantation are more prone to develop a malignancy during their life time; usually hematological tumors, but solid neoplasms may also develop, which in certain instances are donor-derived. It has also been well documented that multipotent bone marrow derived cells can migrate to diverse organs, differentiating into various histological lineages. The present study reports an experimental syngeneic transplantation model, using fluorescently tagged bone marrow cells from p53 null male mice into female wild-type counterparts. We found that transplanted non-neoplastic mutant bone marrow cells can generate tumors of distinct histogenesis, including thymic lymphomas, sarcomas, and carcinomas after carcinogen induction, providing evidence that multipotent cancer-prone stem cells can reside in the bone marrow and are transplantable.

Fibrodysplasia ossificans progressiva (FOP) is a very rare devastating heterotopic ossification disorder, classically caused by a heterozygous single point mutation (c.617G>A) in the ACVR1 gene, encoding the Bone morphogenetic protein (BMP) type I receptor, also termed activin receptor-like kinase (ALK)2. FOP patients develop heterotopic ossification episodically in response to inflammatory insults, thereby compromising tissue sampling and the development of in vitro surrogate models for FOP. Here we describe the generation and characterization of a control and a classical FOP induced pluripotent stem cell (iPSC) line derived from periodontal ligament fibroblast cells using Sendai virus vectors.

Human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts of a patient affected with an autosomal dominant retinal dystrophy carrying the mutation c.782A>C, p.Glu261Ala in ITM2B and from an unaffected brother. Three different iPSC lines were generated and characterized from primary dermal fibroblasts of the affected subject and two from the unaffected brother. All iPSC lines expressed the pluripotency markers, were able to differentiate into the three germ layers and presented normal karyotypes. This cellular model will provide a powerful tool to study this retinal dystrophy and better understand the role of ITM2B.

Bardet-Biedl syndrome (BBS), an autosomal recessive disease, is associated with non-functional primary cilia. BBS5 is part of the protein complex termed the BBSome. The BBSome associates with intra flagellar transport (IFT) particles and mediates trafficking of membrane proteins in the cilium, a process important for cilia-mediated signal transduction. Here we describe the generation of three induced pluripotent stem cell (iPSC) lines, KCi003-A, KCi003-B and KCi003-C from a patient with BBS and homozygous for the disease causing variant c.214G>A, p.(Gly72Ser) in BBS5. The iPSC lines can be used for investigation of IFT in different cell types differentiated from the iPSC line.

We describe generation of human induced pluripotent stem cell (hiPSC) lines of three unrelated idiopathic late onset Parkinson disease patients and two healthy controls above 60 years of age without neurological diseases nor Ashkenazi ancestry. Human iPSC were derived from peripheral blood-erythroblasts using integration free episomal plasmids carrying four reprogramming factors OCT4, SOX2, c-MYC, KLF4 and BCL-XL. The hiPSC lines were characterized according to established criteria.

The coronary slow flow phenomenon (CSFP) is characterized by delayed progression of the injected contrast medium through the coronary tree during coronary angiography due to unknown mechanisms. Here, a human induced pluripotent stem cell (iPSC) line (SYSUi002-A) was established using the Sendai-virus delivery system from dermal fibroblasts of a CSFP patient. This cell line may represent a valuable tool for investigating the pathogenesis and therapeutic strategies of CSFP.

Autism spectrum disorder (ASD) is a childhood-onset neurodevelopmental disorder challenged in social reciprocity and restrictive repetitive behaviors. Here, we generated an induced pluripotent stem cell (iPSC) line SDQLCHi014-A from a patient with ASD and hyperactivity, carrying a 303 kb de novo deletion at chr3p26.1 implicating GRM7 gene by reprogramming urine cells with non-integrating vectors. SDQLCHi014-A have shown full pluripotency, differentiation capacity and genetic stability. This iPSC line provides a valuable resource to study the molecular mechanisms underlying ASD.

Prader–Willi syndrome (PWS) is a neurodevelopmental disorder caused by loss of paternally expressed genes in an imprinted region of 15q11.2-q13. We established a human-induced pluripotent stem cell (hiPSC) line, KSCBi007-A, from the peripheral blood mononuclear cells of a 5-month-old girl with PWS that retained maternal uniparental disomy (UPD). Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) of genomic DNA revealed the maternal UPD in the hiPSCs. The generated hiPSC line expressed pluripotency markers and showed the ability to differentiate into three germ layers in vitro. This hiPSC line could be used as a cellular model of an imprinting disorder in humans.

Overexpression of NEUROG2 and NEUROG1 (NEUROG2/1) in human embryonic stem cells (hESCs) rapidly produces functional networks of excitatory and inhibitory neurons. To facilitate the use of this efficient inducible human neuron model in neuroscience research, we generated hESCs with doxycycline-inducible NEUROG2/1 via lentivirus and a tdTomato fluorescent reporter knock-in at the MAP2 locus using the CRISPR nuclease Cas9. Upon doxycycline-driven induction of NEUROG2/1, these hESCs differentiate within days into cells that are uniformly MAP2 immunoreactive and tdTomato fluorescent.

Senior-Loken syndrome (SLS) is a rare disorder primarily associated with kidney and retinal dysfunction. We generated a human induced pluripotency stem cell (hiPSC) line, designated DKHi005-A, from peripheral blood mononuclear cells of a patient with SLS using a Sendai virus reprogramming method. We confirmed that DKHi005-A cells harbor the same mutation as the patient and show a normal karyotype. DKHi005-A also has pluripotency and the capacity for differentiation into the three germ layers. This cell line is registered and available at the National Stem Cell Bank, Korea National Institute of Health.