Human Pluripotent Stem Cell Lines Research Articles

A new hiPSC-based viral expression system to study virus host-cell interaction in hiPSC-derived cardiomyocytes

Abstract Coxsackievirus B3 (CVB3) belongs to the Picornaviridae family and is a member of the enterovirus genus. CVB3 is the most frequently detected pathogen in viral myocarditis, which leads to acute heart infections in 20-40% of cases and to dilated cardiomyopathy (DCM) in cases of prolonged viral persistence. Complex arrhythmias and sudden deaths due to CVB infections have also been reported, particularly in newborns and children. Observed arrhythmias include bradycardia, AV block and junctional ectopic tachycardia, suggesting that in addition to the functioning myocardium, the pacemaker cells and cardiac conduction system may also be impaired by CVB3 infection. The symptomatic phenotype is highly variable and depends on unclear host factors. It ranges from inconspicuous inflammation and rapid healing to acute congestive heart failure or viral persistence with progressive chronic disease. Despite the increasing and profound knowledge about viral myocarditis, it is still a challenge to identify further host factors that contribute to acute and chronic myocarditis, as science lacks sensitive and controllable disease models. The presented project shows the generation of a controlled, inducible virus-expressing hiPSC line that provides a solution for the beforementioned problems. The new human induced pluripotent stem cell line (hiPSC) i can be used to carry out virologic experiments in a controlled manner and without risk to the user. For this purpose, the modified and non-infectious genome of the enterovirus Coxsackievirus B3 (CVB3) is stably integrated into the genome of a wild-type hiPSC line. The CVB3 genome to be stably integrated was modified to prevent the formation of functional capsids by a DVP0 mutation. In addition, the 3'UTR and 5'UTR regions of the CVB3 genome were removed, which prevents uncontrolled self-replication of viral RNA. As a result, work with this CVB3DVP0-UTR genome is classified as S1 compliant. The expression of the CVB3DP0-UTR virus genome can be controlled by adding doxycycline to the cell culture medium by a TeT-On System. The transfected cells are then cultivated as clones and tested for virus expression. The dose-dependent CVB3DVP0 expression is verified using qPCR and FACS after 1 day of doxycycline application in the cell culture medium. Clones isolated and positively tested for CVB3 expression are used to carry out initial experiments on the interaction of the expressed viral proteins with the cell. Particular focus is placed on the induction of cell death, remodelling of the cytoskeleton and increase of reactive oxygen species (ROS) in mitochondria after CVB3DVP0-UTR induction in order to validate the cell system for application. Differentiation of the CVB3-expressing hiPSC-line into cardiomyocytes with subsequent virus induction results in the elevation of mitochondrial ROS, degradation of the cytoskeleton and arrhythmic behaviour of the cells.

Establishing a human-induced pluripotent stem cell line SMUSHi005-A from a patient with hypophosphatemic vitamin D-resistant rickets carrying the PHEX c.1586–1586+1 delAG mutation

Hypophosphatemic vitamin D-resistant rickets typically presents in infancy or early childhood with skeletal deformities and growth plate abnormalities. In this report, the SMUSHi005-A human induced pluripotent stem cell (hiPSC) line was successfully established from the PBMCs of a female patient carrying the PHEX mutation with c.1586–1586+1 delAG. The iPSC line has been confirmed to have a normal karyotype. The displayed cells clearly exhibit characteristics similar to embryonic stem cells, expressing pluripotency markers and demonstrating the ability to differentiate into three germ layers.

Generation of human induced pluripotent stem cell lines derived from four Rett syndrome patients with MECP2 mutations

Rett syndrome is characterized by severe global developmental impairments with autistic features and loss of purposeful hand skills. Here we show that human induced pluripotent stem cell (hiPSC) lines derived from four Japanese female patients with Rett syndrome are generated from peripheral blood mononuclear cells using Sendai virus vectors. The generated hiPSC lines showed self-renewal and pluripotency and carried heterozygous frameshift, missense, or nonsense mutations in the MECP2 gene. Since the molecular pathogenesis caused by MECP2 dysfunction remains unclear, these cell resources are useful tools to establish disease models and develop new therapies for Rett syndrome.

Telomerase is essential for cardiac differentiation and sustained metabolism of human cardiomyocytes

Telomeres as the protective ends of linear chromosomes, are synthesized by the enzyme telomerase (TERT). Critically short telomeres essentially contribute to aging-related diseases and are associated with a broad spectrum of disorders known as telomeropathies. In cardiomyocytes, telomere length is strongly correlated with cardiomyopathies but it remains ambiguous whether short telomeres are the cause or the result of the disease. In this study, we employed an inducible CRISPRi human induced pluripotent stem cell (hiPSC) line to silence TERT expression enabling the generation of hiPSCs and hiPSC-derived cardiomyocytes with long and short telomeres. Reduced telomerase activity and shorter telomere lengths of hiPSCs induced global transcriptomic changes associated with cardiac developmental pathways. Consequently, the differentiation potential towards cardiomyocytes was strongly impaired and single cell RNA sequencing revealed a shift towards a more smooth muscle cell like identity in the cells with the shortest telomeres. Poor cardiomyocyte function and increased sensitivity to stress directly correlated with the extent of telomere shortening. Collectively our data demonstrates a TERT dependent cardiomyogenic differentiation defect, highlighting the CRISPRi TERT hiPSCs model as a powerful platform to study the mechanisms and consequences of short telomeres in the heart and also in the context of telomeropathies.

Generation and characterization of six human induced pluripotent stem cell lines (hiPSCs) from three individuals with SSADH Deficiency and CRISPR-corrected isogenic controls

Succinic Semialdehyde Dehydrogenase Deficiency (SSADHD) is an ultra-rare autosomal recessive neurometabolic disorder caused by ALDH5A1 mutations presenting with autism and epilepsy. Here, we report the generation and characterization of human induced pluripotent stem cells (hiPSCs) derived from fibroblasts of three unrelated SSADHD patients – one female and two males with the CRISPR-corrected isogenic controls. These individuals are clinically diagnosed and are being followed in a longitudinal clinical study.

Generation of human induced pluripotent stem cell line (XWHNi004-A) from a male with APOE gene mutation

Genetic polymorphism of apolipoprotein E (APOE) confers differential susceptibility to Alzheimer's disease (AD), and APOE ɛ4 variants is the most powerful risk factor for this disease. Here, we report the generation of a human induced pluripotent stem cell (iPSC) line carrying the APOE ɛ4/ɛ4 genotype from peripheral blood mononuclear cells (PBMCs) isolated from a male with a family history of AD utilizing non-integrative Sendai virus vector. The iPSC maintains their original genotype, highly express endogenous pluripotency markers, displays a normal karyotype, and retains the ability to differentiate into cells representative of the three germ layers.

Generation of human-induced pluripotent stem cell line from PBMC of healthy donor using integration-free Sendai virus technology

We developed a well-characterized human induced pluripotent stem cell (iPSC) line obtained from healthy individuals' peripheral blood mononuclear cells (PBMC). The PBMCs were primed and reprogrammed using a non-integrating sendai viral vector, and the iPSC lines demonstrated complete differentiation capacity. This line, YBLi004-A, is available and registered in the human pluripotent stem cell registry. The line's legitimacy was validated using pluripotent marker expression, in vitro differentiation into three germ layers (ectoderm, mesoderm, and endoderm), karyotyping, and STR analysis. This iPSC line could be used as a healthy control for studies involving disease-specific-iPSCs, e.g. drug toxicity and efficacy testing.

Generation of human induced pluripotent stem cell line MHHi029-A from a male Fabry disease patient carrying c.959A > T mutation

Fabry disease (FD) is a rare and inherited monogenetic disease caused by mutations in the X-chromosomal alpha-galactosidase A gene GLA concomitant with accumulation of its substrate globotriaosylceramide (Gb3) and multi-organ symptoms. We derived an induced pluripotent stem cell line, MHHi029-A, from a male FD patient carrying a c.959A > T missense mutation in the GLA gene. The hiPSCs show a normal karyotype, expression of pluripotency markers and trilineage differentiation capacity. Importantly, they present the patient-specific mutation in the GLA gene and are therefore a valuable resource for investigating the FD mechanism and identifying novel therapies.

Engrailed 1 deficiency induces changes in ciliogenesis during human neuronal differentiation

A key pathological feature of Parkinson’s Disease (PD) is the progressive degeneration of dopaminergic neurons (DAns) in the substantia nigra pars compacta. Considering the major role of EN1 in the development and maintenance of these DAns and the implications from En1 mouse models, it is highly interesting to study the molecular and protective effect of EN1 also in a human cellular model. Therefore, we generated EN1 knock-out (ko) human induced pluripotent stem cell (hiPSCs) lines and analyzed these during neuronal differentiation. Although the EN1 ko didn’t interfere with neuronal differentiation and generation of tyrosine hydroxylase positive (TH+) neurons per se, the neurons exhibited shorter neurites. Furthermore, mitochondrial respiration, as well as mitochondrial complex I abundance was significantly reduced in fully differentiated neurons. To understand the implications of an EN1 ko during differentiation, we performed a transcriptome analysis of human neuronal precursor cells (hNPCs) which unveiled alterations in cilia-associated pathways. Further analysis of ciliary morphology revealed an elongation of primary cilia in EN1-deficient hNPCs. Besides, also Wnt signaling pathways were severely affected. Upon stimulating hNPCs with Wnt which drastically increased EN1 expression in WT lines, the phenotypes concerning mitochondrial function and cilia were exacerbated in EN1 ko hNPCs. They failed to enhance the expression of the complex I subunits NDUFS1 and 3, and now displayed a reduced mitochondrial respiration. Furthermore, Wnt stimulation decreased ciliogenesis in EN1 ko hNPCs but increased ciliary length even further. This further highlights the relevance of primary cilia next to mitochondria for the functionality and correct maintenance of human DAns and provides new possibilities to establish neuroprotective therapies for PD.

CRISPR/Cas9-mediated editing of ACTB in induced pluripotent stem cells: A model for investigating human ACTB loss-of-function and genetic adaptive responses

Heterozygous beta-actin (ACTB) indel and nonsense mutations are linked to developmental disorders. We generated two CRISPR/Cas9 human induced pluripotent stem cell (iPSC) lines, WTSIi018-B-19 and WTSIi018-B-20, carrying heterozygous and homozygous indel mutations in ACTB exon 4. Both iPSCs exhibited normal cell morphology, expression of pluripotency markers, and the ability to differentiate into the three primary germ layers. While iPSCs with a heterozygous ACTB mutation maintain genome integrity, homozygous mutants showed a loss of heterozygosity in chromosome three. These mutants provide a powerful model to study the onset, progression, and complex interplay of genetic compensation and phenotypic variation of ACTB-related diseases.

Generation of two human induced pluripotent stem cell lines with BAX and BAK1 double knock-out using CRISPR/Cas9

Bcl-2-associated X protein (BAX) and Blc-2 homologous antagonist killer 1 (BAK) are two pro-apoptotic members of BCL2 family. Here, two BAX/BAK double knock-out human induced pluripotent stem cell lines (iPSC) we generated using CRISPR-Cas9 to generate apoptosis incompetent cell lines. The resulting cell lines were karyotypically normal, had typical morphology and expressed typical markers for the undifferentiated state.

Generation of two patient specific GABRD variants and their isogenic controls for modeling epilepsy

Developmental and epileptic encephalopathies (DEEs) are early-onset conditions that cause intractable seizures and developmental delays. Missense variants in Gamma-aminobutyric acid type A receptor (GABAAR) subunits commonly cause DEEs. Ahring et al. (2022) showed a variant in the gene that encodes the delta subunit (GABRD) is strongly associated with the gain-of-function of extrasynaptic GABAAR. Here, we report the generation of two patient-specific human induced pluripotent stem cells (hiPSC) lines with (i) a de novo variant and (ii) a maternal variant, both for the pathogenic GABRD c.872 C>T, (p.T291I). The variants in the generated cell line were corrected using the CRISPR-Cas9 gene editing technique (respective isogenic control lines).

Good Manufacturing Practice–compliant human induced pluripotent stem cells: from bench to putative clinical products

Background aimsFew human induced pluripotent stem cell (hiPSC) lines are Good Manufacturing Practice (GMP)-compliant, limiting the clinical use of hiPSC-derived products. Here, we addressed this by establishing and validating an in-house platform to produce GMP-compliant hiPSCs that would be appropriate for producing both allogeneic and autologous hiPSC-derived products. MethodsOur standard research protocol for hiPSCs production was adapted and translated into a GMP-compliant platform. In addition to the generation of GMP-compliant hiPSC, the platform entails the methodology for donor recruitment, consent and screening, donor material procurement, hiPSCs manufacture, in-process control, specific QC test validation, QC testing, product release, hiPSCs storage and stability testing. For platform validation, one test run and three production runs were performed. Highest-quality lines were selected to establish master cell banks (MCBs). ResultsTwo MCBs were successfully released under GMP conditions. They demonstrated safety (sterility, negative mycoplasma, endotoxins <5.0 EU/mL and negative adventitious agents), cell identity (>75% of cells expressing markers of undifferentiated state, identical STR profile, normal karyotype in >20 metaphases), purity (negative residual vectors and no plasmid integration in the genome) and potency (expression of at least two of the three markers for each of the three germ layers). In addition, directed differentiation to somitoids (skeletal muscle precursors) and six potential clinical products from all three germ layers was achieved: pancreatic islets (endoderm), kidney organoids and cardiomyocytes (mesoderm), and keratinocytes, GABAergic interneurons and inner-ear organoids (ectoderm). ConclusionsWe successfully developed and validated a platform for generating GMP-compliant hiPSC lines. The two MCBs released were shown to differentiate into clinical products relevant for our own and other regenerative medicine interests.

Establishment of human induced pluripotent stem cell line SDQLCHi029-A from one Type 1 familial glucocorticoid deficiency patient carrying compound heterozygote mutations in MC2R gene

Type 1 familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder due to variation of the melanocortin-2-receptor (MC2R) gene. Induced pluripotent stem cell (iPSC) line SDQLCHi029-A was successfully generated from peripheral blood mononuclear cells obtained from a 5-day-old girl with MC2R mutations (c.428C > T and c.409C > T). The iPSC line showed genetically stable and matched the donor’s PBMCs. displayed a normal karyotype, expressed high pluripotent markers, and exhibited differentiation potential of three germ layers in vitro. The iPSC line could be a good model to study the pathogenesis of FGD type 1 and screen new drugs for the disease.

Generation of an induced pluripotent stem cell line (UMi043-A) from an African American patient with Alzheimer’s disease carrying an ABCA7 deletion (p.Arg578Alafs)

The ATP-binding cassette, subfamily A (ABC1), member 7 (ABCA7) gene is associated with Alzheimer’s disease (AD) risk in populations of African, Asian, and European ancestry1-5. Numerous ABCA7 mutations contributing to risk have been identified, including a 44 base pair deletion (rs142076058) specific to individuals of African ancestry and predicted to cause a frameshift mutation (p.Arg578Alafs) (Cukier et al., 2016). The UMi043-A human induced pluripotent stem cell line was derived from an African American individual with AD who is heterozygous for this deletion and is a resource to further investigate ABCA7 and how this African-specific deletion may influence disease pathology.

Generation of IPi001-A/B/C human induced pluripotent stem cell lines from healthy amniotic fluid cells

Human induced Pluripotent Stem Cells (hiPSCs) represent an invaluable source of primary cells to investigate development, establish cell and disease models, provide material for regenerative medicine and allow more physiological high-content screenings. Here, we generated three healthy hiPSC control lines - IPi001-A/B/C - from primary amniotic fluid cells (AFCs), an infrequently used source of cells, which can be readily obtained from amniocentesis for the prenatal diagnosis of numerous genetic disorders. These AFCs were reprogrammed by non-integrative viral transduction. The resulting hiPSCs displayed normal karyotype and expressed classic pluripotency hallmarks.

Generation of induced pluripotent stem cell line, NIMHi009-A, from PBMCs of an adult healthy male

Human induced pluripotent stem cells provide an exceptional platform for studying pathogenesis in vitro. We, therefore, have generated and characterized human induced pluripotent stem cell (iPSC) line NIMHi009-A derived from peripheral blood mononuclear cells (PBMCs) of healthy adult male control for an epileptic patient carrying voltage gated sodium channel mutation, using Sendai virus-based reprogramming. The generated iPSCs express pluripotency genes and can spontaneously differentiate into three germ layers. These cells display a normal karyotype and are free of mycoplasma. The iPSC line NIMHi009-A can be used as healthy control for modelling various diseases and screening for drugs.

Generation of two human induced pluripotent stem cell lines, IGIBi012-A and IGIBi013-A from Friedreich’s ataxia (FRDA) patients with homozygous GAA repeat expansion in FXN gene

Friedreich’s ataxia is a neurodegenerative disorder caused by the hyper expansion of (GAA-TTC)n triplet repeats in the first intron of the FXN gene. Here, we generated iPSC lines from two individuals with FRDA, both of whom have homozygous GAA repeat expansion in the first intron of FXN gene. Both iPSC lines demonstrated characteristics of pluripotency, including expression of pluripotency markers, stable karyotypes and ability to develop into all three germ layers, and presence of GAA repeat expansion with reduced FXN mRNA expression. These iPSC lines will serve as invaluable tools for investigating the pathophysiology and phenotypes of FRDA.

Generation and characterization of PBMCs-derived human induced pluripotent stem cell (iPSC) line SDQLCHi055-A from a patient with NEDSDV carrying a heterozygote mutation in the CTNNB1 gene

Neurodevelopmental disorder with spastic diplegia and visual defects (NEDSDV, #615075), a rare autosomal dominant genetic disorder caused by heterozygous mutation in the CTNNB1 gene, is characterized by global developmental delay, impaired intellectual development, axial hypotonia, and dysmorphic craniofacial features with microcephaly. Here, we established an iPSC line (SDQLCHi055-A) from a patient with NEDSDV carrying a heterozygote mutation (c.854 T > A, p.L285*) in the CTNNB1 gene. The iPSC line has typical iPSCs characteristics, including pluripotency and trilineage differentiation hallmarks.

Generation of human induced pluripotent stem cell lines from five patients with Myofibrillar myopathy carrying different heterozygous mutations in the DES gene

Myofibrillar myopathy (MFM) is a rare genetic disorder characterized by muscular dystrophy that is often associated with cardiac disease. This disease is caused by mutations in several genes, among them DES (encoding desmin) is the most frequently affected. Peripheral blood mononuclear cells from 5 different MFM patients with different DES mutations were reprogrammed into induced pluripotent stem cells (IPSC) using non-integrative vectors. For each patient, one IPSC clone was selected and demonstrated pluripotency hallmarks without genomic abnormalities. SNP profiles were identical to the cells of origin and all the clones have the capacity to differentiate into all three germ layers.