iPSC Clones Research Articles

A disease-specific iPS cell resource for studying rare and intractable diseases

BackgroundDisease-specific induced pluripotent stem cells (iPSCs) are useful tools for pathological analysis and diagnosis of rare diseases. Given the limited available resources, banking such disease-derived iPSCs and promoting their widespread use would be a promising approach for untangling the mysteries of rare diseases. Herein, we comprehensively established iPSCs from patients with designated intractable diseases in Japan and evaluated their properties to enrich rare disease iPSC resources.MethodsPatients with designated intractable diseases were recruited for the study and blood samples were collected after written informed consent was obtained from the patients or their guardians. From the obtained samples, iPSCs were established using the episomal method. The established iPSCs were deposited in a cell bank.ResultsWe established 1,532 iPSC clones from 259 patients with 139 designated intractable diseases. The efficiency of iPSC establishment did not vary based on age and sex. Most iPSC clones originated from non-T and non-B hematopoietic cells. All iPSC clones expressed key transcription factors, OCT3/4 (range 0.27–1.51; mean 0.79) and NANOG (range 0.15–3.03; mean 1.00), relative to the reference 201B7 iPSC clone.ConclusionsThese newly established iPSCs are readily available to the researchers and can prove to be a useful resource for research on rare intractable diseases.

Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.

Genome-edited human-induced pluripotent stem cells (iPSCs) have broad applications in disease modeling, drug discovery, and regenerative medicine. Despite the development of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, the gene editing process is inefficient and can take several weeks to months to generate edited iPSC clones. We developed a strategy to improve the efficiency of the iPSC gene editing process via application of a small-molecule, trichostatin A (TSA), a Class I and II histone deacetylase inhibitor. We observed that TSA decreased global chromatin condensation and further resulted in increased gene-editing efficiency of iPSCs by twofold to fourfold while concurrently ensuring no increased off-target effects. The edited iPSCs could be clonally expanded while maintaining genomic integrity and pluripotency. The rapid generation of therapeutically relevant gene-edited iPSCs could be enabled by these findings.

Abstract 135: The Noncoding Haplotype Region Of Blood Pressure-associated Single Nucleotide Polymorphism Rs1173771 Regulates The Expression Of Natriuretic Peptide Receptor C

Genome-wide association studies have identified 1,071 loci containing 26,585 single nucleotide polymorphisms (SNPs) as associated with blood pressure (BP). These loci together explain more than 25% of BP heritability. The BP-associated SNP rs1173771 has been associated with NPR3 expression in human tissues. NPR3 encodes natriuretic peptide receptor C (NPR-C). The haplotype in linkage disequilibrium (LD) with rs1173771 contains 11 SNPs spanning a noncoding genomic region of more than 17 kbp. The haplotype region is located 126 kbp from the transcription start site of NPR3 . We used genome editing to examine the effect of the noncoding haplotype region in LD with rs1173771 on gene expression. We employed a CRISPR/Cas9 mediated genetic engineering approach to delete the 17 kbp haplotype region in a human induced pluripotent stem cell (iPSC) line. Two clones of iPSCs with homozygous deletion of the 17 kbp region were obtained after screening 50 single cell clones. The genomic modification was confirmed by PCR, Sanger sequencing, and whole genome sequencing. Immunofluorescence staining for pluripotency markers indicated that pluripotency was retained following the genome editing procedures. Each edited iPSC clone and the unedited iPSC line were differentiated into endothelial cells (iECs) in 3 independent rounds of differentiation. Real-time PCR analysis showed a 1.59±0.17-fold upregulation of NPR3 mRNA in iECs with the haplotype region deletion compared to unedited iECs (n=11 and 7, p<0.05). The expression of other neighboring genes ( SUB1 and TARS ) and a distal gene ( MRPS30 ) was not significantly affected by the deletion. The results provide definitive evidence that the noncoding haplotype region in LD with BP-associated SNP rs1173771 regulates the expression of natriuretic peptide receptor C in iECs.

Generation of an induced pluripotent stem cell line from a patient with epileptic encephalopathy caused by the CYFIP2 R87C variant.

Induced pluripotent stem cells (iPSCs) opened the possibility to use patient cells as a model for several diseases. iPSCs can be reprogrammed from somatic cells collected in a non-invasive way, and then differentiated into any other cell type, while maintaining the donor´s genetic background. CYFIP2 variants were associated with the onset of an early form of epileptic encephalopathy. Studies with patients showed that the R87C variant seems to be one of the variants that causes more severe disease, however, to date there are no studies with a human cell model that allows investigation of the neuronal phenotype of the R87C variant. Here, we generated an iPSC line from a patient with epileptic encephalopathy caused by the CYFIP2 R87C variant. We obtained iPSC clones by reprogramming urinary progenitor cells from a female patient. The generated iPSC line presented a pluripotent stem cell morphology, normal karyotype, expressed pluripotency markers and could be differentiated into the three germ layers. In further studies, this cell line could be used as model for epileptic encephalopathy disease and drug screening studies.

Selection of iPSCs without mtDNA deletion for autologous cell therapy in a patient with Pearson syndrome

Screening for genetic defects in the cells should be examined for clinical application. The Pearson syndrome (PS) patient harbored nuclear mutations in the POLG and SSBP1 genes, which could induce systemic large-scale mitochondrial genome (mtDNA) deletion. We investigated iPSCs with mtDNA deletions in PS patient and whether deletion levels could be maintained during differentiation. The iPSC clones derived from skin fibroblasts (9% deletion) and blood mononuclear cells (24% deletion) were measured for mtDNA deletion levels. Of the 13 skin-derived iPSC clones, only 3 were found to be free of mtDNA deletions, whereas all blood-derived iPSC clones were found to be free of deletions. The iPSC clones with (27%) and without mtDNA deletion (0%) were selected and performed in vitro and in vivo differentiation, such as embryonic body (EB) and teratoma formation. After differentiation, the level of deletion was retained or increased in EBs (24%) or teratoma (45%) from deletion iPSC clone, while, the absence of deletions showed in all EBs and teratomas from deletion-free iPSC clones. These results demonstrated that non-deletion in iPSCs was maintained during in vitro and in vivo differentiation, even in the presence of nuclear mutations, suggesting that deletion-free iPSC clones could be candidates for autologous cell therapy in patients.

Generation of two human NRF2 knockout iPSC clones using CRISPR/Cas9 editing

The nuclear factor erythroid 2-related factor 2 (NFE2L2, known as NRF2) regulates the expression of antioxidative and anti-inflammatory proteins. In order to investigate its impact during viral infections and testing of antiviral compounds, we applied CRISPR/Cas9 editing to eliminate NRF2 in the human iPS cell line MHHi001-A and generated two NRF2 knockout iPSC clones MHHi001-A-6 and MHHi001-A-7. After differentiation into epithelia or endothelial cells, these cells are useful tools to examine the antiviral effects of activators of the NRF2 signaling pathway.

The Establishment of In Vitro Human Induced Pluripotent Stem Cell-Derived Neurons

Induced pluripotent stem cells (iPSCs) have been generated using different reprogramming strategies. Lentiviruses remain a strategic method for cell reprogramming as it is highly efficient in gene transfer. The latest fourth-generation lentiviral packaging systems claimed to be efficient and safe. However, modifications made to enhance safety of lentiviral vectors have been shown to affect vector performance. In this study, we established that the fourth-generation lentiviral packaging system can produce high-titre lentiviruses with high-transduction efficiencies. Subsequently, the robustness and reproducibility of generating iPSCs from adult human dermal fibroblasts were tested using these lentiviruses. The use of fourth-generation lentiviruses consistently generates iPSCs with similar efficiency and quality in different primary cell lines. This study demonstrated that the human-derived iPSCs can be maintained using mitomycin-C inactivated feeder cells. The iPSC clones highly expressed key pluripotency markers and can spontaneously differentiate into cells from the three embryonic germ layers. The iPSCs generated were able to differentiate into neural stem cell lineages, producing cells expressing Nestin and Sox2 as well as able to further differentiate into neurons with more than 70% efficiency. The data demonstrated that the use of the fourth-generation lentiviral packaging to produce lentiviruses for iPSCs generation is robust and reproducible as it can generate iPSCs from different adult dermal fibroblasts with the potential to differentiate into neural stem cells and neurons. The use of safer lentiviral packaging systems combined with established vector plasmids will help to expedite the generation of iPSCs for clinical applications.

Predicting reprogramming-related gene expression from cell morphology in human induced pluripotent stem cells.

Purification is essential before differentiating human induced pluripotent stem cells (hiPSCs) into cells that fully express particular differentiation marker genes. High-quality iPSC clones are typically purified through gene expression profiling or visual inspection of the cell morphology; however, the relationship between the two methods remains unclear. We investigated the relationship between gene expression levels and morphology by analyzing live-cell, phase-contrast images and mRNA profiles collected during the purification process. We employed these data and an unsupervised image feature extraction method to build a model that predicts gene expression levels from morphology. As a benchmark, it was confirmed that the method can predict the gene expression levels from tissue images for cancer genes, performing as well as state-of-the-art methods. We then applied the method to iPSCs and identified two genes that are well predicted from cell morphology. Although strong batch (or possibly donor) effects resulting from the reprogramming process preclude the ability to use the same model to predict across batches, prediction within a reprogramming batch is sufficiently robust to provide a practical approach for estimating expression levels of a few genes and monitoring the purification process.

ACTA1 H40Y mutant iPSC-derived skeletal myocytes display mitochondrial defects in an in vitro model of nemaline myopathy

Nemaline myopathies (NM) are a group of congenital myopathies that lead to muscle weakness and dysfunction. While 13 genes have been identified to cause NM, over 50% of these genetic defects are due to mutations in nebulin (NEB) and skeletal muscle actin (ACTA1), which are genes required for normal assembly and function of the thin filament. NM can be distinguished on muscle biopsies due to the presence of nemaline rods, which are thought to be aggregates of the dysfunctional protein. Mutations in ACTA1 have been associated with more severe clinical disease and muscle weakness. However, the cellular pathogenesis linking ACTA1 gene mutations to muscle weakness are unclear To evaluate cellular disease phenotypes, iPSC-derived skeletal myocytes (iSkM) harboring an ACTA1 H40Y point mutation were used to model NM in skeletal muscle. These were generated by Crispr-Cas9, and include one non-affected healthy control (C) and 2 NM iPSC clone lines, therefore representing isogenic controls. Fully differentiated iSkM were characterized to confirm myogenic status and subject to assays to evaluate nemaline rod formation, mitochondrial membrane potential, mitochondrial permeability transition pore (mPTP) formation, superoxide production, ATP/ADP/phosphate levels and lactate dehydrogenase release. C- and NM-iSkM demonstrated myogenic commitment as evidenced by mRNA expression of Pax3, Pax7, MyoD, Myf5 and Myogenin; and protein expression of Pax4, Pax7, MyoD and MF20. No nemaline rods were observed with immunofluorescent staining of NM-iSkM for ACTA1 or ACTN2, and these mRNA transcript and protein levels were comparable to C-iSkM. Mitochondrial function was altered in NM, as evidenced by decreased cellular ATP levels and altered mitochondrial membrane potential. Oxidative stress induction revealed the mitochondrial phenotype, as evidenced by collapsed mitochondrial membrane potential, early formation of the mPTP and increased superoxide production. Early mPTP formation was rescued with the addition of ATP to media. Together, these findings suggest that mitochondrial dysfunction and oxidative stress are disease phenotypes in the in vitro model of ACTA1 nemaline myopathy, and that modulation of ATP levels was sufficient to protect NM-iSkM mitochondria from stress-induced injury. Importantly, the nemaline rod phenotype was absent in our in vitro model of NM. We conclude that this in vitro model has the potential to recapitulate human NM disease phenotypes, and warrants further study.

PL-3: RECENT PROGRESS IN IPS CELL RESEARCH AND APPLICATION

Induced pluripotent stem cells (iPSCs) can proliferate almost indefinitely and differentiate into multiple lineages, giving them wide medical applications. As a result, they are being used for new cell-based therapies, disease models, and drug development around the world. In our basic research, we are focusing on the mechanisms of protein translational regulation in the proliferation and differentiation of iPSCs. We have previously reported that the translation regulator NAT1 is essential for the self-renewal and neuronal differentiation potential of hiPSCs, which is a “Bench to Bedside” activity for the medical application of iPSCs. As translational research, we are proceeding with an iPSC stock project in which clinical-grade iPSC clones are being established from healthy donors with homologous HLA haplotypes to lower the risk of transplant rejection. We started distributing the iPSC stock to domestic and overseas institutions, and related clinical studies have begun for age-related macular degeneration (AMD), Parkinson's disease, corneal epithelial stem cell deficiency, and other diseases, giving expectation that iPSC-based regenerative medicine will be widely used in the future. However, donors with HLA homozygous are rare. Genome editing technology could be used to reduce the transplant-rejection risk. Indeed, we reported HLA gene-edited iPSCs that could expand the range of patients who benefit from iPSC therapies faster than the homologous HLA haplotype strategy. This technology also has the potential to prevent or treat genetic diseases and gives great hope to patients. Other applications of iPSCs are drug screening, toxicity studies, and disease modeling. In 2017, a new drug screening system using iPS cells for fibrodysplasia ossificans progressiva (FOP) was reported, revealing one drug candidate, Rapamycin, which is now undergoing a clinical trial to treat FOP patients. Additionally, Bosutinib, a drug for leukemia was revealed to be efficacious for amyotrophic lateral sclerosis (ALS) using a disease-specific iPSC model. Accordingly, we initiated a Phase 1 clinical trial of Bosutinib to treat ALS at Kyoto University Hospital and other centers in 2019 and began Phase 2 clinical trials in April 2022. Furthermore, we initiated a Phase I clinical trial of Bromocriptine to treat Alzheimer's disease at Kyoto University Hospital in June 2020. Thus, drug repurposing using iPS cells is progressing at an accelerated pace. Over the past decade, iPSC research has made great progress, moving toward innovative therapeutics for people with intractable diseases by the application of new findings from basic science and reverse translation from clinics.

CRISPR Manipulations in Stem Cell Lines.

Insights into genome engineering in cells have allowed researchers to cultivate and modify cells as organoids that display structural and phenotypic features of human diseases or normal health status. The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. Modified organoids derived from patients' tissue cells are used as models to study diseases and test novel drugs. CRISPR-Cas9 technology has contributed to an explosion of advances that have the ability to edit genomes for the study of monogenic diseases and cancers. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of utmost importance as these cells carry the potential to be differentiated into any cell lineage. We describe recent developments that are broadening our understanding and extend DNA specificity, product selectivity, and fundamental capabilities. Furthermore, fundamental capabilities and remarkable advancements in basic research, biotechnology, and therapeutics development in cell engineering are detailed within this chapter. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter includes detailed protocols for the preparation of reagents to target loci of interest and transfection to genotype single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of ribonucleoprotein (RNP) delivered directly to cells.

LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction.

LMNA-related muscular dystrophy is an autosomal-dominant progressive disorder caused by mutations in LMNA. LMNA missense mutations are becoming correctable with CRISPR/Cas9-derived tools. Evaluating the functional recovery of LMNA after gene editing bears challenges as there is no reported direct loss of function of lamin A/C proteins in patient-derived cells. The proteins encoded by LMNA are lamins A/C, important ubiquitous nuclear envelope proteins but absent in pluripotent stem cells. We induced lamin A/C expression in induced pluripotent stem cells (iPSCs) of two patients with LMNA-related muscular dystrophy, NM_170707.4 (LMNA): c.1366A > G, p.(Asn456Asp) and c.1494G > T, p.(Trp498Cys), using a short three-day, serum-induced differentiation protocol and analyzed expression profiles of co-regulated genes, examples being COL1A2 and S100A6. We then performed precise gene editing of LMNA c.1366A > G using the near-PAMless (PAM: protospacer-adjacent motif) cytosine base editor. We show that the mutation can be repaired to 100% efficiency in individual iPSC clones. The fast differentiation protocol provided a functional readout and demonstrated increased lamin A/C expression as well as normalized expression of co-regulated genes. Collectively, our findings demonstrate the power of CRISPR/Cas9-mediated gene correction and effective outcome measures in a disease with, so far, little perspective on therapies.

Combined Noncoding RNA-mRNA Regulomics Signature in Reprogramming and Pluripotency in iPSCs

Somatic cells are reprogrammed with reprogramming factors to generate induced pluripotent stem cells (iPSCs), offering a promising future for disease modeling and treatment by overcoming the limitations of embryonic stem cells. However, this process remains inefficient since only a small percentage of transfected cells can undergo full reprogramming. Introducing miRNAs, such as miR-294 and miR302/3667, with reprogramming factors, has shown to increase iPSC colony formation. Previously, we identified five transcription factors, GBX2, NANOGP8, SP8, PEG3, and ZIC1, which may boost iPSC generation. In this study, we performed quantitative miRNAome and small RNA-seq sequencing and applied our previously identified transcriptome to identify the potential miRNA-mRNA regulomics and regulatory network of other ncRNAs. From each fibroblast (N = 4), three iPSC clones were examined (N = 12). iPSCs and original fibroblasts expressed miRNA clusters differently and miRNA clusters were compared to mRNA hits. Moreover, miRNA, piRNA, and snoRNAs expression profiles in iPSCs and original fibroblasts were assessed to identify the potential role of ncRNAs in enhancing iPSC generation, pluripotency, and differentiation. Decreased levels of let-7a-5p showed an increase of SP8 as described previously. Remarkably, the targets of identifier miRNAs were grouped into pluripotency canonical pathways, on stemness, cellular development, growth and proliferation, cellular assembly, and organization of iPSCs.

Transient Retention of Photoreceptor Outer Segments in Matrigel-Embedded Retinal Organoids

Retinal organoids (ROs) are three-dimensional retinal tissues, which are differentiated in vitro from induced pluripotent stem cells (iPSC), ultimately forming all main retinal cell types under defined culture conditions. ROs show several highly specialized retinal features, including the outgrowth of photoreceptor outer segments (OSs). In vivo, the photoreceptor OSs are enveloped and maintained by protrusions of retinal pigment epithelium (RPE) cells, the so-called apical microvilli, while ROs fail to recapitulate this critical interaction in culture development. Here, we define specific co-culture conditions aiming to compensate for the missing physical proximity of RPE and OSs in RO development. Accordingly, functional RPE cells and ROs were differentiated simultaneously from the same iPSC clone, the former resulting in byproduct RPE or bRPE cells. While some co-culture approaches indicated a temporary functional interaction between bRPE and RO photoreceptors, they did not improve the photoreceptor histoarchitecture. In contrast, embedding ROs in a basement membrane extract without bRPE cells showed a robust improvement in the rate of photoreceptor OS retention. RO embedding is a quick and easy method that greatly enhances the preservation of photoreceptor OSs, an important structure for modelling retinal diseases with the involvement of photoreceptors.

Germline Loss-of-Function Mutations in MDM4 Cause p53-Dependent Hematopoietic Cell Death in Patients with Variable Bone Marrow Failure Phenotypes

The mouse double minute 4 (MDM4) negatively regulates p53 through the p53/MDM2/MDM4 regulatory network and is commonly overexpressed in several cancers. Here, we describe two unrelated pedigrees with germline MDM4 mutations with clinical features of bone marrow (BM) failure. Patient 1 (P1) presented at the age of 11 months with chronic pure red cell aplasia requiring chronic red blood cell transfusions and BM findings with absent erythroid precursors and dysplastic megakaryocytes. Patient 2 (P2) presented at age of 4 weeks with neutropenia with hypogranulation of neutrophils and a normocellular bone marrow with hypoplastic myelopoiesis and dysplasia of all three lineages. Both patients had normal chromosomal breakage and normal for age telomere length in peripheral blood. Whole genome and panel sequencing identified germline heterozygous variants in MDM4: p.Leu31Thrfs*15 in P1 and p.T454M in P2 as the only relevant candidate variants. Both mutations are absent in >140 thousand individuals in gnomAD database, and the constraint analysis indicates that MDM4 gene is intolerant to missense variation (observed to expected (O/E) ratio of 181/258, Z-score 1.71) and highly intolerant to protein truncating variation (O/E: 0/27 with a pLI score of 1). MDM4 protein consists of four key domains: p53 binding N-terminus, an acidic region, a zinc finger domain, and a C-terminal RING domain, which heterodimerizes with MDM2 to ubiquitinate and promote p53 degradation. MDM4 p.T454M missense mutation found in P2 was previously reported by Toufektchan et al. in 2020 in one patient with BM failure phenotype with short telomeres who carried additional TERT variant. The MDM4 p.T454M mutation was shown to result in lower protein levels (consistent with a loss-of-function effect) and had altered capacity to dimerize with the MDM2 RING domain, causing p53 hyperactivity. MDM4 p.Leu31Thrfs*15 mutation described in P1 is localized in the N-terminal p53 binding domain and results in RNA transcript loss, as confirmed by us in patient cells using RNA sequencing showing a two-fold reduction of the dominant transcript [ENST00000367182.8]. This was corroborated by Western blot demonstrating decreased levels of MDM4 protein. We sought to elucidate the impact of p.Leu31Thrfs*15 and p.T454M mutations on hematopoiesis and regulation of p53 expression. In two human induced pluripotent stem cell (iPSC) lines derived from male and female healthy donors, we knocked-in the MDM4 p.T454M and p.Leu31Thrfs*15 mutations by CRISPR/Cas9 engineering. Of note, MDM4 p.Leu31Thrfs*15 homozygous knock-in line could not be established despite multiple attempts. We discovered statistically significant decreased erythroid and myeloid differentiation capacity from MDM4-mutant iPSCs compared to isogenic control iPSCs (p <0.05). There were statistically significantly more AnnexinV+/DAPI+ mutant iPSC-derived myeloid and erythroid progenitors compared to isogenic control-derived progenitors (p <0.05), pointing to a higher rate of cell death in MDM4-mutant cells. We further demonstrated increased expression of p53 and p21 proteins in MDM4 p.Leu31Thrfs*15 iPSC clones. To better understand the loss-of-function effect of MDM4, we knocked out MDM4 in healthy CD34+ cells using CRISPR/Cas9 editing. Knock out of MDM4 with an efficiency of >80% was achieved in multiple biological replicates, resulting in increased p53 activity and fewer blast-forming erythroid (BFU-E) and myeloid colonies compared to control cells that were Cas9-edited at the AAVS1 safe harbor locus. This impairment was rescued by disruption of TP53 gene. Sequencing of individual BFU-E colonies did not reveal any cells with biallelic loss of MDM4, while similar percentages of unedited colonies and heterozygous disrupted MDM4 colonies were detected. Xenotransplantation studies using immunodeficient NBSGW mice are currently underway to explore the impact of MDM4-edited CD34+ cells in vivo. In summary, we describe two patients with germline loss-of-function MDM4 mutations that cause p53 hyperactivity which causes the unifying feature of hematopoietic defect across a BMF disorder spectrum without telomere shortening. Sharma and Bhoopalan are equal contributors

Abstract 14660: Truncating Variants in MYBPC3 Cause Ubiquitin Proteasome System Dysfunction Related to Hypertrophic Cardiomyopathy in Human Induced Pluripotent Stem Cell Model

Introduction: Hypertrophic cardiomyopathy (HCM) is the most common inherited monogenic cardiomyopathy. Cardiac myosin binding protein C ( MYBPC3 ) is the most frequently mutated gene in this disease, and most pathogenic variants in MYBPC3 are truncating variants including splice site variants. The ubiquitin proteasome system (UPS), a major intracellular protein degradation system, plays an important role in the heart. Although impairment of UPS is reportedly caused by truncated cMyBP-C, the underlying molecular mechanism causing HCM is largely unknown. Hypothesis: Truncating variants in MYBPC3 lead to impaired UPS activity in human iPS cell-derived cardiomyocytes (hiPSC-CMs) and are associated with HCM phenotypes. Methods: Patient derived iPSC clones were generated from HCM patients with MYBPC3 variants (IVS11-9G&gt;A and IVS27+1G&gt;A). Corrected isogenic control of IVS27+1G&gt;A was generated by base editing. To evaluate UPS activity, we employed ubiquitin fusion degradation reporter, Ub-G76V-GFP, which provides fluorescence readout of UPS activity. BNP expression of hiPSC-CMs were evaluated by antibody labeling and fluorescence-activated cell sorting (FACS). Contractility and Ca 2+ transient were measured from single biopatterned hiPSC-CMs. Results: Both MYBPC3 variant lines exhibited UPS impairment as reflected by increased GFP fluorescence compared to isogenic and healthy controls (Fig. A). IVS27+1G&gt;A showed higher expression of BNP (Fig. B and C). Enhanced contractility and Ca 2+ handling abnormalities were observed in MYBPC3 variant iPSC-CM lines as compared to control (Fig. D and E). Conclusions: Impairment of UPS activity may be associated with a marker of cellular hypertrophy, and contractility and Ca 2+ handling abnormalities in hiPSC-CMs with MYBPC3 truncating variants. These findings support prior reports suggesting that decreased UPS activity contribute to the pathophysiological mechanism of HCM caused by truncating MYBPC3 variants.

Dysfunctional pancreatic cells differentiated from induced pluripotent stem cells with mitochondrial DNA mutations

Diabetes mellitus (DM) is a serious disease in which blood sugar levels rise abnormally because of failed insulin production or decreased insulin sensitivity. Although many studies are being conducted for the treatment or early diagnosis of DM, it is not fully understood how mitochondrial genome (mtDNA) abnormalities appear in patients with DM. Here, we induced iPSCs from fibroblasts, PBMCs, or pancreatic cells of three patients with type 2 DM (T2D) and three patients with non-diabetes counterpart. The mtDNA mutations were detected randomly without any tendency among tissues or patients. In T2D patients, 62% (21/34) of iPSC clones harbored multiple mtDNA mutations, of which 37% were homoplasmy at the 100% mutation level compared to only 8% in non-diabetes. We next selected iPSC clones that were a wild type or carried mutations and differentiated into pancreatic cells. Oxygen consumption rates were significantly lower in cells carrying mutant mtDNA. Additionally, the mutant cells exhibited decreased production of insulin and reduced secretion of insulin in response to glucose. Overall, the results suggest that screening mtDNA mutations in iPSCs from patients with T2D is an essential step before pancreatic cell differentiation for disease modeling or autologous cell therapy.

Generation of two TMEM16A knockout iPSC clones each from a healthy human iPSC line, from a Cystic Fibrosis patient specific line with p.Phe508del mutation and from the gene corrected iPSC line

The Transmembrane member 16A (TMEM16A), also known as anoctamin-1 (ANO1), is a calcium-activated chloride channel present in the airway epithelium. It is known to be involved in the apical chloride secretion indicating that TMEM16A could be addressed for the treatment of chloride secretion defects like in Cystic- Fibrosis (CF). In this paper we generated knockout cell lines using CRISPR/Cas9-mediated ablation in a healthy human iPSC line (MHHi001-A), in a CF patient iPSC line (MHHi002-A) and in its corrected counterpart (MHHi002-A-1). These lines can be used for gaining information about the role of TMEM16A for mucus secretion and/or production and evaluating its therapeutic potential.

Myogenesis defects in a patient-derived iPSC model of hereditary GNE myopathy

Hereditary muscle diseases are disabling disorders lacking effective treatments. UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE) myopathy (GNEM) is an autosomal recessive distal myopathy with rimmed vacuoles typically manifesting in late adolescence/early adulthood. GNE encodes the rate-limiting enzyme in sialic acid biosynthesis, which is necessary for the proper function of numerous biological processes. Outside of the causative gene, very little is known about the mechanisms contributing to the development of GNE myopathy. In the present study, we aimed to address this knowledge gap by querying the underlying mechanisms of GNE myopathy using a patient-derived induced pluripotent stem-cell (iPSC) model. Control and patient-specific iPSCs were differentiated down a skeletal muscle lineage, whereby patient-derived GNEM iPSC clones were able to recapitulate key characteristics of the human pathology and further demonstrated defects in myogenic progression. Single-cell RNA sequencing time course studies revealed clear differences between control and GNEM iPSC-derived muscle precursor cells (iMPCs), while pathway studies implicated altered stress and autophagy signaling in GNEM iMPCs. Treatment of GNEM patient-derived iMPCs with an autophagy activator improved myogenic differentiation. In summary, we report an in vitro, iPSC-based model of GNE myopathy and implicate defective myogenesis as a contributing mechanism to the etiology of GNE myopathy.

Establishment of Four Induced Pluripotent Stem Cell Lines from CD34+ Hematopoietic Stem and Progenitor Cells from a Patient Diagnosed with an Invasive Lobular Mammary Carcinoma

CD34+ cells were isolated from peripheral blood of a breast cancer patient. By the introduction of five integration-free episomal vectors, the CD34+ cells were successfully reprogrammed and resulted in four iPSC clones. Flow Cytometry, reverse transcriptase PCR and immunocytochemistry confirm a robust expression of pluripotency factors and the concomitant loss of exogenous reprogramming plasmids. The maintenance of genomic integrity was confirmed by array-based comparative genomic hybridization and iPSCs harbored the capacity to differentiate into all three germ layers. Here, we present the generation and characterization of four iPSC lines that will find application in the field of breast cancer research.