High-Throughput and Cost-Effective Characterization of Induced Pluripotent Stem Cells.

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) offers the possibility of studying the molecular mechanisms underlying human diseases in cell types difficult to extract from living patients, such as neurons and cardiomyocytes. To date, studies have been published that use small panels of iPSC-derived cell lines to study monogenic diseases. However, to study complex diseases, where the genetic variation underlying the disorder is unknown, a sizable number of patient-specific iPSC lines and controls need to be generated. Currently the methods for deriving and characterizing iPSCs are time consuming, expensive, and, in some cases, descriptive but not quantitative. Here we set out to develop a set of simple methods that reduce cost and increase throughput in the characterization of iPSC lines. Specifically, we outline methods for high-throughput quantification of surface markers, gene expression analysis of in vitro differentiation potential, and evaluation of karyotype with markedly reduced cost

Proteomic analyses reveal misregulation of LIN28 expression and delayed timing of glial differentiation in human iPS cells with MECP2 loss-of-function.

Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation

Association of Human iPSC Gene Signatures and X Chromosome Dosage with Two Distinct Cardiac Differentiation Trajectories.

Despite the importance of understanding how variability across induced pluripotent stem cell (iPSC) lines due to non-genetic factors (clone and passage) influences their differentiation outcome, large-scale studies capable of addressing this question have not yet been conducted. Here, we differentiated 191 iPSC lines to generate iPSC-derived cardiovascular progenitor cells (iPSC-CVPCs). We observed cellular heterogeneity across the iPSC-CVPC samples due to varying fractions of two cell types: cardiomyocytes (CMs) and epicardium-derived cells (EPDCs). Comparing the transcriptomes of CM-fated and EPDC-fated iPSCs, we discovered that 91 signature genes and X chromosome dosage differences are associated with these two distinct cardiac developmental trajectories. In an independent set of 39 iPSCs differentiated into CMs, we confirmed that sex and transcriptional differences affect cardiac-fate outcome. Our study provides novel insights into how iPSC transcriptional and X chromosome gene dosage differences influence their response to differentiation stimuli and, hence, cardiac cell fate

Human iPSC differentiation to retinal organoids in response to IGF1 and BMP4 activation is line- and method-dependent

Induced pluripotent stem cell (iPSC)‐derived retinal organoids provide a platform to study human retinogenesis, disease modeling, and compound screening. Although retinal organoids may represent tissue structures with greater physiological relevance to the in vivo human retina, their generation is not without limitations. Various protocols have been developed to enable development of organoids with all major retinal cell types; however, variability across iPSC lines is often reported. Modulating signaling pathways important for eye formation, such as those involving bone morphogenetic protein 4 (BMP4) and insulin‐like growth factor 1 (IGF1), is a common approach used for the generation of retinal tissue in vitro. We used three human iPSC lines to generate retinal organoids by activating either BMP4 or IGF1 signaling and assessed differentiation efficiency by monitoring morphological changes, gene and protein expression, and function. Our results showed that the ability of iPSC to give rise to retinal organoids in response to IGF1 and BMP4 activation was line‐ and method‐dependent. This demonstrates that careful consideration is needed when choosing a differentiation approach, which would also depend on overall project aims

Allele-specific NKX2-5 binding underlies multiple genetic associations with human electrocardiographic traits.

The cardiac transcription factor (TF) gene NKX2-5 has been associated with electrocardiographic (EKG) traits through genome-wide association studies (GWASs), but the extent to which differential binding of NKX2-5 at common regulatory variants contributes to these traits has not yet been studied. We analyzed transcriptomic and epigenomic data from induced pluripotent stem cell-derived cardiomyocytes from seven related individuals, and identified ~2,000 single-nucleotide variants associated with allele-specific effects (ASE-SNVs) on NKX2-5 binding. NKX2-5 ASE-SNVs were enriched for altered TF motifs, for heart-specific expression quantitative trait loci and for EKG GWAS signals. Using fine-mapping combined with epigenomic data from induced pluripotent stem cell-derived cardiomyocytes, we prioritized candidate causal variants for EKG traits, many of which were NKX2-5 ASE-SNVs. Experimentally characterizing two NKX2-5 ASE-SNVs (rs3807989 and rs590041) showed that they modulate the expression of target genes via differential protein binding in cardiac cells, indicating that they are functional variants underlying EKG GWAS signals. Our results show that differential NKX2-5 binding at numerous regulatory variants across the genome contributes to EKG phenotypes

Evaluation of commonly used ectoderm markers in iPSC trilineage differentiation.

Patient-derived induced pluripotent stem cells (iPSCs) have become a promising resource for exploring genetics of complex diseases, discovering new drugs, and advancing regenerative medicine. Increasingly, laboratories are creating their own banks of iPSCs derived from diverse donors. However, there are not yet standardized guidelines for qualifying these cell lines, i.e., distinguishing between bona fide human iPSCs, somatic cells, and imperfectly reprogrammed cells. Here, we report the establishment of a panel of 30 iPSCs from CD34+ peripheral blood mononuclear cells, of which 10 were further differentiated in vitro into all three germ layers. We characterized these different cell types with commonly used pluripotent and lineage specific markers, and showed that NES, TUBB3, and OTX2 cannot be reliably used as ectoderm differentiation markers. Our work highlights the importance of marker selection in iPSC authentication, and the need for the field to establish definitive standard assays

Comprehensive translational assessment of human-induced pluripotent stem cell derived cardiomyocytes for evaluating drug-induced arrhythmias

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) hold promise for assessment of drug-induced arrhythmias and are being considered for use under the comprehensive in vitro proarrhythmia assay (CiPA). We studied the effects of 26 drugs and 3 drug combinations on 2 commercially available iPSC-CM types using high-throughput voltage-sensitive dye and microelectrode-array assays being studied for the CiPA initiative and compared the results with clinical QT prolongation and torsade de pointes (TdP) risk. Concentration-dependent analysis comparing iPSC-CMs to clinical trial results demonstrated good correlation between drug-induced rate-corrected action potential duration and field potential duration (APDc and FPDc) prolongation and clinical trial QTc prolongation. Of 20 drugs studied that exhibit clinical QTc prolongation, 17 caused APDc prolongation (16 in Cor.4U and 13 in iCell cardiomyocytes) and 16 caused FPDc prolongation (16 in Cor.4U and 10 in iCell cardiomyocytes). Of 14 drugs that cause TdP, arrhythmias occurred with 10 drugs. Lack of arrhythmic beating in iPSC-CMs for the four remaining drugs could be due to differences in relative levels of expression of individual ion channels. iPSC-CMs responded consistently to human ether-a-go-go potassium channel blocking drugs (APD prolongation and arrhythmias) and calcium channel blocking drugs (APD shortening and prevention of arrhythmias), with a more variable response to late sodium current blocking drugs. Current results confirm the potential of iPSC-CMs for proarrhythmia prediction under CiPA, where iPSC-CM results would serve as a check to ion channel and in silico modeling prediction of proarrhythmic risk. A multi-site validation study is warranted

ALS mutant FUS proteins are recruited into stress granules in induced pluripotent stem cells- derived motoneurons

Patient-derived induced Pluripotent Stem Cells (iPSCs) provide an opportunity to study human diseases mainly in those cases where no suitable model systems are available. Here we have taken advantage of in vitro iPSCs derived from patients affected by Amyotrophic Lateral Sclerosis and carrying mutations in the RNA-binding proteins FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patients' fibroblasts, while in the case of the severe FUS(P525L) mutation, where fibroblasts were not available, a heterozygous and a homozygous iPSC lines were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, nicely correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis

Pathological progression induced by the frontotemporal dementia-associated R406W tau mutation in patient-derived iPSCs

Mutations in the microtubule-associated protein tau (MAPT) gene are known to cause familial frontotemporal dementia (FTD). The R406W tau mutation is a unique missense mutation whose patients have been reported to exhibit Alzheimer\u27s disease (AD)-like phenotypes rather than the more typical FTD phenotypes. In this study, we established patient-derived induced pluripotent stem cell (iPSC) models to investigate the disease pathology induced by the R406W mutation. We generated iPSCs from patients and established isogenic lines using CRISPR/Cas9. The iPSCs were induced into cerebral organoids, which were dissociated into cortical neurons with high purity. In this neuronal culture, the mutant tau protein exhibited reduced phosphorylation levels and was increasingly fragmented by calpain. Furthermore, the mutant tau protein was mislocalized and the axons of the patient-derived neurons displayed morphological and functional abnormalities, which were rescued by microtubule stabilization. The findings of our study provide mechanistic insight into tau pathology and a potential for therapeutic intervention

Endothelial cells derived from patients' induced pluripotent stem cells for sustained factor VIII delivery and the treatment of hemophilia A.

Hemophilia A (HA) is a bleeding disorder characterized by spontaneous and prolonged hemorrhage. The disease is caused by mutations in the coagulation factor 8 gene (F8) leading to factor VIII (FVIII) deficiency. Since FVIII is primarily produced in endothelial cells (ECs) in a non-diseased human being, ECs hold great potential for development as a cell therapy for HA. We showed that HA patient-specific induced pluripotent stem cells (HA-iPSCs) could provide a renewable supply of ECs. The HA-iPSC-derived ECs were transduced with lentiviral vectors to stably express the functional B domain deleted F8 gene, the luciferase gene, and the enhanced green fluorescent protein gene (GFP). When transplanted intramuscularly into neonatal and adult immune deficient mice, the HA-iPSC-derived ECs were retained in the animals for at least 10-16 weeks and maintained their expression of FVIII, GFP, and the endothelial marker CD31, as demonstrated by bioluminescence imaging and immunostaining, respectively. When transplanted into HA mice, these transduced HA-iPSC-derived ECs significantly reduced blood loss in a tail-clip bleeding test and produced therapeutic plasma levels (11.2%-369.2%) of FVIII. Thus, our studies provide proof-of-concept that HA-iPSC-derived ECs can serve as a factory to deliver FVIII for the treatment of HA not only in adults but also in newborns