Human iPSC Line Research Articles

Generation of the human iPSC line AKOSi010-A from fibroblasts of a female FAHN patient, carrying the compound heterozygous mutation p.Gly45Arg/p.His319Arg

Fatty acid hydroxylase-associated neurodegeneration (FAHN) is a rare childhood onset neurodegenerative disease caused by mutations in the FA2H gene. Patients display abnormal myelination, cerebellar atrophy and some have iron deposition in the central nervous system. Here we describe the generation of AKOSi010-A, a human induced pluripotent stem cell (hiPSC) line derived from fibroblasts of a female patient carrying the compound heterozygous p.Gly45Arg/p.His319Arg, using non-integrating Sendai virus. The generated iPSCs express pluripotency markers, can differentiate into cell types of the three germ layers and show a normal karyotype. This cell line displays a unique source to study the pathophysiology of FAHN.

Development of a human iPSC line (SMBCi017-A) from a healthy donor

The human induced pluripotent stem cell (iPSC) line SMBCi017-A was derived from urine cells of a 20 year old male healthy control donor. The generated iPSCs had normal karyotype, expressed chromosomal stability pluripotency hallmarks and differentiated into the three primary germ layers.

Huntington's disease iPSC models-using human patient cells to understand the pathology caused by expanded CAG repeats.

A major advance in the study of Huntington’s disease (HD) has been the development of human disease models employing induced pluripotent stem cells (iPSCs) derived from patients with HD. Because iPSCs provide an unlimited source of cells and can be obtained from large numbers of HD patients, they are a uniquely valuable tool for investigating disease mechanisms and for discovering potential disease-modifying therapeutics. Here, we summarize some of the important findings in HD pathophysiology that have emerged from studies of patient-derived iPSC lines. Because they retain the genome and actual disease mutations of the patient, they provide a cell source to investigate genetic contributions to the disease. iPSCs provide advantages over other disease models. While iPSC-based technology erases some epigenetic marks, newly developed transdifferentiation methods now let us investigate epigenetic factors that control expression of mutant huntingtin (mHTT). Human HD iPSC lines allow us to investigate how endogenous levels of mHTT affect cell health, in contrast to other models that often rely on overexpressing the protein. iPSCs can be differentiated into neurons and other disease-related cells such as astrocytes from different brain regions to study brain regional differences in the disease process, as well as the cell-cell dependencies involved in HD-associated neurodegeneration. They also serve as a tissue source to investigate factors that impact CAG repeat instability, which is involved in regional differences in neurodegeneration in the HD brain. Human iPSC models can serve as a powerful model system to identify genetic modifiers that may impact disease onset, progression, and symptomatology, providing novel molecular targets for drug discovery.

Establishment of a human iPSC line (SUTCMi001-A) derived from a healthy donor

This study describes the characterization of one induced pluripotent stem cell line (iPSC) from a healthy female. It is crucial to use iPSCs derived from healthy individuals as controls in genetic disease studies. Thus, we established a human iPSC cell line derived from healthy people. The iPSC cell line was generated in our lab from the peripheral blood mononuclear cells (PBMCs) of a 28-year-old girl. The generated hiPSC line is free of episomal vectors, has a normal karyotype, expresses pluripotency markers and can differentiate into three germ layers in vivo.

Generation of a human iPSC line (MPIi008-A) from a patient with Denys-Drash syndrome

An induced pluripotent stem cell (hiPSC) line (MPIi008-A) was generated from fibroblasts of a 1-year-old male patient with Denys-Drash syndrome using lentiviral delivery of reprogramming factors OCT4, SOX2, KLF4 and c-MYC. The MPIi008-A iPSC line exhibited typical iPSC morphology and normal karyotype, expressed pluripotent stem cell markers, and showed developmental potential to differentiate into derivatives of all three germ layers in vivo. The hiPSC line harbours a heterozygous missense mutation (R394L) in exon 9 of the WT1 gene.

Generation of hypoimmunogenic induced pluripotent stem cells by CRISPR-Cas9 system and detailed evaluation for clinical application

In order to expand the promise of regenerative medicine using allogeneic induced pluripotent stem cells (iPSCs), precise and efficient genome editing of human leukocyte antigen (HLA) genes would be advantageous to minimize the immune rejection caused by mismatches of HLA type. However, clinical-grade genome editing of multiple HLA genes in human iPSC lines remains unexplored. Here, we optimized the protocol for good manufacturing practice (GMP)-compatible CRISPR-Cas9 genome editing to deplete the three gene locus (HLA-A, HLA-B, and CIITA genes) simultaneously in HLA homozygous iPSCs. The use of HLA homozygous iPSCs has one main advantage over heterozygous iPSCs for inducing biallelic knockout by a single gRNA. RNA-seq and flow cytometry analyses confirmed the successful depletion of HLAs, and lineage-specific differentiation into cardiomyocytes was verified. We also confirmed that the pluripotency of genome-edited iPSCs was successfully maintained by the three germ layers of differentiation. Moreover, whole-genome sequencing, karyotyping, and optical genome mapping analyses revealed no evident genomic abnormalities detected in some clones, whereas unexpected copy number losses, chromosomal translocations, and complex genomic rearrangements were observed in other clones. Our results indicate the importance of multidimensional analyses to ensure the safety and quality of the genome-edited cells. The manufacturing and assessment pipelines presented here will be the basis for clinical-grade genome editing of iPSCs.

Establishment and Characterization of MUi027-A: A Novel Patient-Derived Cell Line of Polycystic Kidney Disease with PKD1 Mutation.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent genetic diseases affecting the kidneys. A genetically specific mutation model is required to comprehend its pathophysiology and to develop a drug treatment. In this study, we successfully developed human induced pluripotent stem cells (hiPSCs) named MUi027-A from skin fibroblasts of a patient diagnosed with ADPKD and carrying the PKD1 frameshift mutation (c.7946_7947delCT). MUi027-A cells showed the same genetic fingerprints as the parental cells, including the presence of the PKD1 mutation. MUi027-A hiPSCs displayed embryonic stem cell-like characteristics with the capability of differentiating into the three germ layers. Upon directed differentiation, MUi027-A hiPSCs could be differentiated into tubular organoids with the expression of renal cell markers. Furthermore, we compared the efficiency of cyst formation in two human iPSC lines with different PKD1 mutations. When cyst formation was induced by either forskolin or blebbistatin, MUi027-A hiPSC-derived kidney organoids displayed higher frequencies of cyst formation when compared to organoids generated from an iPSC cell line with non-truncating PKD1 mutation genotype (c.5878C > T), suggesting the presence of physiological differences in the mechanism of cyst formation between different PKD1 mutants. Overall, we generated and characterized a novel human iPSC line with a specific PKD mutation and demonstrated its potential as a disease model to study the pathophysiology of genetic determinants in the development of ADPKD disease.

Overlapping and unique substrate specificities of ST3GAL1 and 2 during hematopoietic and megakaryocytic differentiation

AbstractAlthough the sialyltransferases ST3GAL1 and ST3GAL2 are known to transfer sialic acid to the galactose residue of type III disaccharides (Galβ1,3GalNAc) in vitro, sialylation of O-linked glycosylated proteins in living cells has been largely attributed to ST3GAL1. To examine the role of ST3GAL2 in O-sialylation, we examined its expression during differentiation of human-induced pluripotent stem cells (iPSCs) into hematopoietic progenitor cells (HPCs) and megakaryocytes (MKs). ST3GAL1 and ST3GAL2 each became highly expressed during the differentiation of iPSCs to HPCs but decreased markedly in their expression upon differentiation into MKs, suggesting coordination of expression during megakaryopoiesis. To further delineate their role in these processes, we generated ST3GAL1-, ST3GAL2-, and doubly deficient human iPSC lines. Binding of the peanut agglutinin lectin, which reports the presence of unsialylated Galβ1,3GalNAc glycan chains, was strongly increased in HPCs and MKs derived from double-knockout iPSCs and remained moderately increased in cells lacking either one of these sialyltransferases, demonstrating that both can serve as functional cellular O-glycan sialyltransferases. Interestingly, the HPC markers CD34 and CD43, as well as MK membrane glycoprotein (GP) GPIbα, were identified as major GP substrates for ST3GAL1 and ST3GAL2. In contrast, O-sialylation of GPIIb relied predominantly on the expression of ST3GAL2. Finally, although disruption of ST3GAL1 and ST3GAL2 had little impact on MK production, their absence resulted in dramatically impaired MK proplatelet formation. Taken together, these data establish heretofore unknown physiological roles for ST3GAL1 and ST3GAL2 in O-linked glycan sialylation in hemato- and megakaryocytopoiesis.

Generation of a human iPSC line CIBi010-A with a reporter for ASGR1 using CRISPR/Cas9

ASGR1 is a liver-specific surface marker that has been used to purify human pluripotent stem cell (PSC)-derived hepatocytes (iHeps). Furthermore, ASGR1+ iHeps represents a more mature subpopulation of iHeps. To utilize this marker for optimizing iHep differentiation and purification, we substituted the stop coden of ASGR1 with a fluorescent reporter protein mCherry in a human iPSC line iPSN0052 via CRISPR/Cas9-mediated homologus recombination. The generated CIBi010-A enableds us to monitor ASGR1 expression during hepatic differentiation and thus can be used to optimize our hepatic differentiation procedures.

Generation of homozygous PRKN, PINK1 and double PINK1/PRKN knockout cell lines from healthy induced pluripotent stem cells using CRISPR/Cas9 editing

Autosomal recessive mutations in either PRKN or PINK1 are associated with early-onset Parkinson’s disease. The corresponding proteins, PRKN, an E3 ubiquitin ligase, and the mitochondrial serine/threonine-protein kinase PINK1 play a role in mitochondrial quality control. Using CRISPR/CAS9 technology we generated three human iPSC lines from the well characterized AIW002-02 control line. These isogenic iPSCs contain homozygous knockouts of PRKN (PRKN-KO, CBIGi001-A-1), PINK1 (PINK1-KO, CBIGi001-A-2) or both PINK1 and PRKN (PINK1-KO/PRKN-KO, CBIGi001-A-3). The knockout lines display normal karyotypes, express pluripotency markers and upon differentiation into relevant brain cells or midbrain organoids may be valuable tools to model Parkinson’s disease.

Generation of a human iPSC line CIBi011-A from amniocytes of a healthy fetus

Human induced pluripotent stem cells (iPSC) resemble human embryonic stem cells with potential to differentiate into cells of all adult tissues. Nonetheless human iPSCs may have an epigenetic memory of their donor tissue origin, are easier to differentiate to those lineages, and their potential to other cell fates can be controlled. We generated a human iPSC line CIBi011-A from amniocytes of a healthy fetus. CIBi011-A serves as a useful source to investigate the epigenetic memory of iPSCs. As an iPSC line from a healthy donor, this line can also serve as a potential cell source from which to develop stem cell-based cell therapies.

PINK1‐Mediated Neuroprotection Against Mutant Tau Pathology

Protein accumulation, mislocalization and dendritic atrophy are pathological features of neurodegenerative diseases, such as Alzheimer’s disease (AD) and Frontotemporal lobar dementia (FTLD). Postmortem samples from patients with AD or FTLD reveal Tau pathology, wherein aberrant Tau accumulates as oligomers and hyperphosphorylated species. Our preliminary data show that mutant Tau (Tau‐A152T), which is linked to increased risk of AD and FTLD, causes dendritic simplification in cultured neurons. While aberrant Tau accumulation is linked to disease severity in AD and FTLD, endogenous factors that can prevent Tau toxicity remain unknown. We and others have shown that PTEN‐induced Kinase 1 (PINK1) is protective in a wide range of genetic and toxin‐based models of neurodegeneration. Elevating PINK1 levels in vitro enhances dendrite length, while loss of PINK1 causes dendrite simplification. Moreover, using our own cohort of AD, FTD and 4R‐tauopathies, we confirmed published observations of decreased PINK1 mRNA and protein expression in the brains of patients with AD. In this work, we test the potential of elevating PINK1 to protect against mutant Tau pathology. We hypothesize that elevated PINK1 expression can protect against dendrite simplification caused by mutant Tau. To test this hypothesis, mouse primary cortical neurons and cortical neurons differentiated from control human iPSC lines (ic‐neurons) were transfected with GFP only or co‐transfected with Tau‐A152T. To elevate PINK1 levels, cultures were co‐transfected with PINK1‐GFP or treated with a pharmacological inhibitor of PINK1 degradation (BC1464). Through neurite length and Sholl analysis, we found that increased PINK1 levels protects neuronal cultures against mutant Tau‐mediated dendritic simplification. To determine the mechanism of PINK1‐mediated protection, we sought to test whether PINK1 elevation promotes Tau clearance. SH‐SY5Y cells were transfected with Tau‐A152T and treated with either DMSO (vehicle) or BC1464. A six‐hour cycloheximide chase assay was performed, and we found that Tau‐A152T levels decreased with BC1464 treatment, but not in vehicle‐treated cultures. Endogenous, wild type Tau levels were not significantly affected. We recently discovered that PINK1 interacts with Valosin‐Containing Protein (VCP, p97), a critical regulator of proteostasis pathways, to modulate dendritic morphology. Ongoing work is aimed at studying the underlying mechanism(s) of PINK1‐mediated Tau‐A152T clearance and identify the signaling partners with which PINK1 interacts to promote neuroprotection.

Generation of two human iPSC lines, HMGUi003-A and MRIi028-A, carrying pathogenic biallelic variants in the PPCS gene

Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the de novo coenzyme A (CoA) synthesis starting from pantothenate. Mutations in PPCS cause autosomal-recessive dilated cardiomyopathy, often fatal, without apparent neurodegeneration, whereas pathogenic variants in PANK2 and COASY, two other genes involved in the CoA synthesis, cause Neurodegeneration with Brain Iron Accumulation (NBIA). PPCS-deficiency is a relatively new disease with unclear pathogenesis and no targeted therapy. Here, we report the generation of induced pluripotent stem cells from fibroblasts of two PPCS-deficient patients. These cellular models could represent a platform for pathophysiological studies and testing of therapeutic compounds for PPCS-deficiency.

Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase.

Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH+) dopaminergic neurons (DNs) is a powerful method for creating patient-specific in vitro models to elucidate mechanisms underlying Parkinson’s disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (<50%) of the PD-relevant TH+ DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a “floor plate protocol” generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH+ cells (≈30%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH+ neurons compared to the TH– cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH+ and TH– neuronal cell populations and that the “floor plate protocol” is particularly efficient in generating electrophysiologically mature TH+ DNs, which are the most vulnerable neuronal subtype in PD.

Generation of four iPSC lines from four patients with Leigh syndrome carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6

We report the generation of four human iPSC lines (8993-A12, 8993-B12, 8993-C11, and 8993-D7) from fibroblasts of four patients affected by maternally inherited Leigh syndrome (MILS) carrying homoplasmic mutations m.8993T > G or m.8993T > C in the mitochondrial gene MT-ATP6. We used Sendai viruses to deliver reprogramming factors OCT4, SOX2, KLF4, and c-MYC. The established iPSC lines expressed pluripotency markers, exhibited a normal karyotype, were capable to form cells of the three germ layers in vitro, and retained the MT-ATP6 mutations at the same homoplasmic level of the parental fibroblasts.

Generation of human induced pluripotent stem cell (iPSC) lines derived from five patients carrying the pathogenic phospholamban-R14del (PLN-R14del) variant and three non-carrier family members

The R14del pathogenic variant in the phospholamban (PLN) gene (PLN-R14del), has been identified in families with hereditary cardiomyopathy, including dilated and arrhythmogenic cardiomyopathies. Here we have generated human iPSC lines from five PLN-R14del carriers and three non-carrier family members. Peripheral blood mononuclear cells (PBMC) were obtained from the eight individuals and reprogrammed using Sendai viral vector system carrying the Yamanaka factors. All eight lines show typical iPSC morphology, normal karyotype, high expression of pluripotency markers, and possess the ability to differentiate into all three germ layers. These lines represent valuable resources for studying the pathophysiological mechanisms of PLN-R14del associated cardiomyopathy.

Generation of a gene corrected human isogenic iPSC line (CPGHi001-A-1) from a hearing loss patient with the TMC1 p.M418K mutation using CRISPR/Cas9

TMC1 p.M418K mutation is homologous to that in Beethoven mice, which may induce autosomal dominant non-syndromic progressive hearing loss. Previously, we generated an induced pluripotent stem cells (iPSCs) line (CPGHi001-A) from a hearing loss patient with the TMC1 c.1253 T > A (p.M418K) mutation. Here we genetically corrected the TMC1 c.1253 T > A mutation using CRISPR/Cas9 technology to generate an isogenic control, CPGHi001-A-1. The resulting iPSCs had a normal karyotype, showed pluripotency by immunofluorescence staining, and differentiated into the three germ layers in vitro.

Generation of a human iPSC line, INMi005-A, from a patient with non-syndromic USH2A-associated retinitis pigmentosa

We report here the generation of the human iPSC line INMi005-A from a patient with non-syndromic autosomal recessive retinitis pigmentosa caused by compound heterozygous mutations in the USH2A gene. The reprogramming of primary human dermal fibroblasts was performed using the non-integrative Sendai virus method and the OSKM transcription factor cocktail. The generated INMi005-A iPSC line is pluripotent and genetically stable, and will represent a valuable tool for understanding the pathophysiology associated with USH2A mutations.

Generation of heterozygous (MRli003-A-3) and homozygous (MRli003-A-4) TRPM4 knockout human iPSC lines

TRPM4 is a Ca2+-activated channel mediating the transport of monovalent cations across the cell membrane. Mutations in the TRPM4 gene have been associated with cardiac arrhythmias in humans. Using CRISPR/Cas9 gene editing technology, we established two TRPM4 knockout human iPSC lines – one heterozygous (MRli003-A-3) and one homozygous (MRli003-A-4) – by inserting a frameshift mutation in exon 2 of the TRPM4 gene. Both lines maintained pluripotency, a normal karyotype, parental cell morphology, and the ability to differentiate into the three germ layers.

Generation of a human iPSC line (UUIGPi015-A) from a patient with Dravet syndrome and a 2.9 Mb deletion spanning SCN1A on chromosome 2

Dravet syndrome is an early onset devastating epilepsy syndrome usually caused by heterozygous mutations in SCN1A. We generated a human iPSC line (UUIGPi015-A) from dermal fibroblasts of a patient with Dravet syndrome carrying a deletion on chromosome 2 encompassing SCN1A and 9 flanking genes. Characterization of the iPSC line confirmed expression of pluripotency markers, tri-lineage differentiation capacity and absence of exogenous reprogramming factors. The iPSC line retained the deletion and was genomically stable. The iPSC line UUIGPi015-A provides a useful resource for studies on the pathophysiology of Dravet syndrome and seizures caused by haploinsufficiency of SCN1A and flanking gene products.