Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Stage-specific signaling through TGFß family members and WNT regulates patterning and pancreatic specification of human pluripotent stem cells.

The generation of insulin-producing ß-cells from human pluripotent stem cells is dependent on efficient endoderm induction and appropriate patterning and specification of this germ layer to a pancreatic fate. In this study, we elucidated the temporal requirements for TGFß family members and canonical WNT signaling at these developmental stages and show that the duration of nodal/activin A signaling plays a pivotal role in establishing an appropriate definitive endoderm population for specification to the pancreatic lineage. WNT signaling was found to induce a posterior endoderm fate and at optimal concentrations enhanced the development of pancreatic lineage cells. Inhibition of the BMP signaling pathway at specific stages was essential for the generation of insulin-expressing cells and the extent of BMP inhibition required varied widely among the cell lines tested. Optimal stage-specific manipulation of these pathways resulted in a striking 250-fold increase in the levels of insulin expression and yielded populations containing up to 25% C-peptide+ cells.

Rag mutations reveal robust alternative end joining.

Mammalian cells repair DNA double-strand breaks (DSBs) through either homologous recombination or non-homologous end joining (NHEJ). V(D)J recombination, a cut-and-paste mechanism for generating diversity in antigen receptors, relies on NHEJ for repairing DSBs introduced by the Rag1-Rag2 protein complex. Animals lacking any of the seven known NHEJ factors are therefore immunodeficient. Nevertheless, DSB repair is not eliminated entirely in these animals: evidence of a third mechanism, 'alternative NHEJ', appears in the form of extremely rare V(D)J junctions and a higher rate of chromosomal translocations. The paucity of these V(D)J events has suggested that alternative NHEJ contributes little to a cell's overall repair capacity, being operative only (and inefficiently) when classical NHEJ fails. Here we find that removing certain portions of murine Rag proteins reveals robust alternative NHEJ activity in NHEJ-deficient cells and some alternative joining activity even in wild-type cells. We propose a two-tier model in which the Rag proteins collaborate with NHEJ factors to preserve genomic integrity during V(D)J recombination.

Simple, Fast, and Efficient Method for Derivation of Dermal Fibroblasts From Skin Biopsies.

Primary fibroblasts are a precious resource in the field of translational regenerative medicine. Dermal fibroblasts derived from human subject biopsies are being used as donor tissues for the derivation of patient-specific iPSC lines, which in turn are used for disease modeling, drug screening, tissue engineering, and cell transplantation. We developed a fast and simple protocol to grow dermal fibroblasts from skin biopsies. Using this protocol, we simply and firmly fix the biopsy piece on the surface of a tissue culture-treated plate and allow the fibroblasts to grow. This novel method eliminates any need for enzymatic digestion or mechanical dissociation of the biopsy piece. By using this newly developed protocol, we have successfully established around 100 fibroblast lines characterized by the expression of specific markers [Serpin H1 (Hsp-47), F-actin, and Vimentin]. Finally, we have used many of these fibroblast lines as donor tissues to successfully derive iPSC lines. We have developed a method that is simple, fast, convenient, efficient, and gentle on the cells to derive dermal fibroblasts from human skin biopsies. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Skin biopsy collection and fibroblast derivation Support Protocol 1: Culturing, freezing, and thawing dermal fibroblasts derived from a skin biopsy Support Protocol 2: Characterization of dermal fibroblasts by immunocytochemistry.

iPSC-based modeling of helicase deficiency reveals impaired cell proliferation and increased apoptosis after NK cell lineage commitment.

Cell proliferation is a ubiquitous process required for organismal development and homeostasis. However, individuals with partial loss-of-function variants in DNA replicative helicase components often present with immunodeficiency due to specific loss of natural killer (NK) cells. Such lineage-specific disease phenotypes raise questions on how the proliferation is regulated in cell type-specific manner. We aimed to understand NK cell-specific proliferative dynamics and vulnerability to impaired helicase function using iPSCs from individuals with NK cell deficiency (NKD) due to hereditary compound heterozygous GINS4 variants. We observed and characterized heterogeneous cell populations that arise during the iPSC differentiation along with NK cells. While overall cell proliferation decreased with differentiation, early NK cell precursors showed a short burst of cell proliferation. GINS4 deficiency induced replication stress in these early NK cell precursors, which are poised for apoptosis, and ultimately recapitulate the NKD phenotype.

Derivation and characterization of the induced pluripotent stem cell line CUIMCi004-A from a patient with a novel frameshift variant in exon 18a of OCRL.

OCRL encodes for an inositol polyphosphate 5-phosphatase, located in the trans-Golgi network, endosomes, endocytic clathrin-coated pits, primary cilia. Mutations in OCRL causes Lowe syndrome (LS), a rare and complex disorder characterized by congenital cataracts, renal tubular dysfunction, and mental retardation. Here we generated an induced pluripotent stem cell (iPSC) line from Peripheral Blood Mononuclear Cell (PBMCs) of a 5-year-old boy with severe obesity carrying a novel pathogenic variant in the brain-expressed isoform of OCRL. The Sendai virus approach was used for reprogramming. The iPSC line CUIMCi004-A may serve as a useful resource to further investigate the tissue-specific function of OCRL.

CRISPR-AsCas12a Efficiently Corrects a GPR143 Intronic Mutation in Induced Pluripotent Stem Cells from an Ocular Albinism Patient.

Mutations in the GPR143 gene cause X-linked ocular albinism type 1 (OA1), a disease that severely impairs vision. We recently generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of an OA1 patient carrying a point mutation in intron 7 of GPR143. This mutation activates a new splice site causing the incorporation of a pseudoexon. In this study, we present a high-performance CRISPR-Cas ribonucleoprotein strategy to permanently correct the GPR143 mutation in these patient-derived iPSCs. Interestingly, the two single-guide RNAs available for SpCas9 did not allow the cleavage of the target region. In contrast, the cleavage achieved with the CRISPR-AsCas12a system promoted homology-directed repair at a high rate. The CRISPR-AsCas12a-mediated correction did not alter iPSC pluripotency or genetic stability, nor did it result in off-target events. Moreover, we highlight that the disruption of the pathological splice site caused by CRISPR-AsCas12a-mediated insertions/deletions also rescued the normal splicing of GPR143 and its expression level.

Efficient Cas9-based Genome Editing Using CRISPR Analysis Webtools in Severe Early-onset-obesity Patient-derived iPSCs.

The CRISPR system is an adaptive defense mechanism used by bacteria and archaea against viruses and plasmids. The discovery of the CRISPR-associated protein Cas9 and its RNA-guided cleavage mechanism marked the beginning of a new era in genomic engineering by enabling the editing of a target region in the genome. Gene-edited cells or mice can be used as models for understanding human diseases. Given its high impact in functional genomic experiments on different model systems, several CRISPR/Cas9 protocols have been generated in the past years. The technique uses a straightforward "cut and stitch" mechanism, but requires an accurate step-by-step design. One of the key points is the use of an efficient programmable guide RNA to increase the rate of success in obtaining gene-specific edited clones. Here, we describe an efficient editing protocol using a ribonucleotide protein (RNP) complex for homology-directed repair (HDR)-based correction of a point mutation in an induced pluripotent stem cell (iPSC) line generated from a 14-year-old patient with severe early-onset obesity carrying a de novo variant of ARNT2. The resulting isogenic iPSC line, named CUIMCi003-A-1, has a normal karyotype, expresses stemness markers, and can be differentiated into progenies from all three germ layers. We provide a detailed workflow for designing a single guide RNA and donor DNA, and for isolating clonal human iPSCs edited with the desired modification. This article also focuses on parameters to consider when selecting reagents for CRISPR/Cas9 gene editing after testing their efficiency with in silico tools. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of sgRNAs and PCR primers Basic Protocol 2: Testing the efficiency of sgRNAs Basic Protocol 3: Design of template or donor DNA Basic Protocol 4: Targeted gene editing Basic Protocol 5: Selection of positive clones Basic Protocol 6: Freezing, thawing, and expansion of cells Basic Protocol 7: Characterization of edited cell lines.

Establishment of the iPSC line CUIMCi005-A from a patient with Stargardt disease for retinal organoid culture.

Pathogenic variation in the ABCA4 gene is the underlying cause of Stargardt disease, the most common inherited retinal degeneration. We established an induced pluripotent stem cell line for retinal organoid research from a patient with mild disease features who is compound heterozygous for the frequent c.5882G>A (p.Gly1961Glu) missense variant and a c.4947delC (p.Glu1650Argfs*12) frameshift variant. Peripheral blood mononuclear cells were reprogrammed using a non-integrating Sendai virus approach. G-banded karyotyping was normal (46, XY) and mycoplasma testing was negative. Immunohistochemistry and RT-qPCR were performed to verify the expression of pluripotency and stemness markers (LIN28, NANOG, OCT4 and SOX2) and trilineage differentiation.

Fibroblast growth factor homologous factors serve as a molecular rheostat in tuning arrhythmogenic cardiac late sodium current.

Voltage-gated sodium (Nav1.5) channels support the genesis and brisk spatial propagation of action potentials in the heart. Disruption of NaV1.5 inactivation results in a small persistent Na influx known as late Na current (I Na,L), which has emerged as a common pathogenic mechanism in both congenital and acquired cardiac arrhythmogenic syndromes. Here, using low-noise multi-channel recordings in heterologous systems, LQTS3 patient-derived iPSCs cardiomyocytes, and mouse ventricular myocytes, we demonstrate that the intracellular fibroblast growth factor homologous factors (FHF1-4) tune pathogenic I Na,L in an isoform-specific manner. This scheme suggests a complex orchestration of I Na,L in cardiomyocytes that may contribute to variable disease expressivity of NaV1.5 channelopathies. We further leverage these observations to engineer a peptide-inhibitor of I Na,L with a higher efficacy as compared to a well-established small-molecule inhibitor. Overall, these findings lend insights into molecular mechanisms underlying FHF regulation of I Na,L in pathophysiology and outline potential therapeutic avenues.

Generation of the iPSC line CUIMCi003-A derived from a patient with severe early onset obesity.

Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) is a basic helix-loop-helix (bHLH/PAS) transcription factor involved in the development of paraventricular nucleus of the hypothalamus (PVH) through the heterodimerization with Single-minded 1 (SIM1) (Michaud et al., 2000). Using a Sendai virus-based approach, the four reprogramming factors OCT3/4, SOX2, KLF4 and C-MYC were delivered into Peripheral Blood Mononuclear Cell (PBMCs) from a 14-year-old girl with early onset obesity carrying a de novo variant (p.P130A) in ARNT2. The resulting iPSC line CUIMCi003-A had a normal karyotype, showed pluripotency and three germ layer differentiation capacity in vitro and was heterozygous for the de novo ARNT2 variant.

Depression patient-derived cortical neurons reveal potential biomarkers for antidepressant response.

Major depressive disorder is highly prevalent worldwide and has been affecting an increasing number of people each year. Current first line antidepressants show merely 37% remission, and physicians are forced to use a trial-and-error approach when choosing a single antidepressant out of dozens of available medications. We sought to identify a method of testing that would provide patient-specific information on whether a patient will respond to a medication using in vitro modeling. Patient-derived lymphoblastoid cell lines from the Sequenced Treatment Alternatives to Relieve Depression study were used to rapidly generate cortical neurons and screen them for bupropion effects, for which the donor patients showed remission or non-remission. We provide evidence for biomarkers specific for bupropion response, including synaptic connectivity and morphology changes as well as specific gene expression alterations. These biomarkers support the concept of personalized antidepressant treatment based on in vitro platforms and could be utilized as predictors to patient response in the clinic.

Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder.

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.

Molecular Features of the Measles Virus Viral Fusion Complex That Favor Infection and Spread in the Brain.

Measles virus (MeV) bearing a single amino acid change in the fusion protein (F)-L454W-was isolated from two patients who died of MeV central nervous system (CNS) infection. This mutation in F confers an advantage over wild-type virus in the CNS, contributing to disease in these patients. Using murine ex vivo organotypic brain cultures and human induced pluripotent stem cell-derived brain organoids, we show that CNS adaptive mutations in F enhance the spread of virus ex vivo. The spread of virus in human brain organoids is blocked by an inhibitory peptide that targets F, confirming that dissemination in the brain tissue is attributable to F. A single mutation in MeV F thus alters the fusion complex to render MeV more neuropathogenic. IMPORTANCE Measles virus (MeV) infection can cause serious complications in immunocompromised individuals, including measles inclusion body encephalitis (MIBE). In some cases, MeV persistence and subacute sclerosing panencephalitis (SSPE), another severe central nervous system (CNS) complication, develop even in the face of a systemic immune response. Both MIBE and SSPE are relatively rare but lethal. It is unclear how MeV causes CNS infection. We introduced specific mutations that are found in MIBE or SSPE cases into the MeV fusion protein to test the hypothesis that dysregulation of the viral fusion complex-comprising F and the receptor binding protein, H-allows virus to spread in the CNS. Using metagenomic, structural, and biochemical approaches, we demonstrate that altered fusion properties of the MeV H-F fusion complex permit MeV to spread in brain tissue.

Establishment and characterization of two iPSC lines derived from healthy controls.

We have generated two iPSC lines from skin biopsies of two healthy individuals. Skin fibroblasts were derived and reprogrammed using a Sendai virus-based approach. The resulting iPSC lines have normal karyotype, express stemness markers and can generate endoderm, mesoderm and ectoderm in vitro. These iPSC lines can be used as healthy controls in differentiation paradigms as well as backbone for gene editing experiments.

Standardized Reporter Systems for Purification and Imaging of Human Pluripotent Stem Cell-derived Motor Neurons and Other Cholinergic Cells.

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.

Small Molecules Restore Bestrophin 1 Expression and Function of Both Dominant and Recessive Bestrophinopathies in Patient-Derived Retinal Pigment Epithelium.

Purpose: Bestrophinopathies are a group of untreatable inherited retinal dystrophies caused by mutations in the retinal pigment epithelium (RPE) Cl- channel bestrophin 1. We tested whether sodium phenylbutyrate (4PBA) could rescue the function of mutant bestrophin 1 associated with autosomal dominant and recessive disease. We then sought analogues of 4PBA with increased potency and determined the mode of action for 4PBA and a lead compound 2-naphthoxyacetic acid (2-NOAA). Lastly, we tested if 4PBA and 2-NOAA could functionally rescue bestrophin 1 function in RPE generated from induced pluripotent stem cells (iPSC-RPEs) derived from patients with a dominant or recessive bestrophinopathy. Methods: Global and plasma membrane expression was determined by Western blot and immunofluorescent microscopy, respectively. The effect of 4PBA and 2-NOAA on transcription was measured by quantitative RT-PCR and the rate of protein turnover by cycloheximide chase and Western blot. Channel function was measured by whole-cell patch clamp. Results: 4PBA and 2-NOAA can rescue the global and membrane expression of mutant bestrophin 1 associated with autosomal dominant disease (Best vitelliform macular dystrophy [BVMD]) and autosome recessive bestrophinopathy (ARB), and these small molecules have different modes of action. Both 4PBA and 2-NOAA significantly increased the channel function of mutant BVMD and ARB bestrophin 1 in HEK293T and iPSC-RPE cells derived from patients with BVMD and ARB. For 4PBA, the increased mutant channel function in BVMD and ARB iPSC-RPE was equal to that of wild-type iPSC-RPE bestrophin 1. Conclusions: The restoration of bestrophin 1 function in patient-derived RPE confirms the US Food and Drug Administration-approved drug 4PBA as a promising therapeutic treatment for bestrophinopathies.

Generation of two iPS cell lines (FRIMOi003-A and FRIMOi004-A) derived from Stargardt patients carrying ABCA4 compound heterozygous mutations.

Recessive Stargardt disease (STGD1) is an autosomal recessive retinal dystrophy, caused by mutations in the retina-specific ATP-binding cassette transporter (ABCA4) gene, which plays a role as a retinaldehyde flippase in the photoreceptor outer segments. In this work, two human induced pluripotent stem cell (iPSC) lines were generated from STGD1 patients carrying compound heterozygous mutations in ABCA4. Skin fibroblasts were reprogrammed with the Yamanaka factors using a non-integrating, Sendai virus-based approach. Both iPSC lines displayed typical embryonic stem cell morphology, had normal karyotype, expressed several pluripotency markers and were able to differentiate into all three germ layers. Resource table.

Establishment and characterization of an iPSC line (FRIMOi001-A) derived from a retinitis pigmentosa patient carrying PDE6A mutations.

Retinitis pigmentosa (RP) refers to a clinical and genetic heterogeneous group of inherited retinal degenerations characterized by photoreceptor cell death. In this work, we have generated an induced pluripotent stem cell (iPSC) line derived from a RP patient with two heterozygous mutations in the cGMP-specific phosphodiesterase 6A alpha subunit (PDE6A) gene. Skin fibroblasts were generated and reprogrammed by using a Sendai virus-based approach. The iPSC line had a normal karyotype, carried the two PDE6A mutations, expressed pluripotency markers and could generate endoderm, mesoderm and ectoderm in vitro. Resource table.

Generation of an induced pluripotent stem cell line (FRIMOi002-A) from a retinitis pigmentosa patient carrying compound heterozygous mutations in USH2A gene.

A human induced pluripotent stem cell (iPSC) line was generated from a female patient affected by autosomal recessive retinitis pigmentosa with two mutations in the USH2A gene: c.2209C > T (p.Arg737Ter) and c.8693A > C (p.Tyr2898Ser). Skin fibroblasts were infected with Sendai virus containing the Yamanaka factors and the resulting cells were fully characterized to confirm successful reprogramming. The iPSC line expressed several pluripotency markers, could generate the three germ layers, had a normal karyotype, carried the two USH2A mutations and was free of Sendai virus. This cell line will serve as a model to unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration.