Use of Induced Pluripotent Stem Cells to Build Isogenic Systems and Investigate Type 1 Diabetes.

Type 1 diabetes (T1D) is a disease that arises due to complex immunogenetic mechanisms. Key cell-cell interactions involved in the pathogenesis of T1D are activation of autoreactive T cells by dendritic cells (DC), migration of T cells across endothelial cells (EC) lining capillary walls into the islets of Langerhans, interaction of T cells with macrophages in the islets, and killing of ß-cells by autoreactive CD8+ T cells. Overall, pathogenic cell-cell interactions are likely regulated by the individual's collection of genetic T1D-risk variants. To accurately model the role of genetics, it is essential to build systems to interrogate single candidate genes in isolation during the interactions of cells that are essential for disease development. However, obtaining single-donor matched cells relevant to T1D is a challenge. Sourcing these genetic variants from human induced pluripotent stem cells (iPSC) avoids this limitation. Herein, we have differentiated iPSC from one donor into DC, macrophages, EC, and ß-cells. Additionally, we also engineered T cell avatars from the same donor to provide an in vitro platform to study genetic influences on these critical cellular interactions. This proof of concept demonstrates the ability to derive an isogenic system from a single donor to study these relevant cell-cell interactions. Our system constitutes an interdisciplinary approach with a controlled environment that provides a proof-of-concept for future studies to determine the role of disease alleles (e.g. IFIH1, PTPN22, SH2B3, TYK2) in regulating cell-cell interactions and cell-specific contributions to the pathogenesis of T1D.

ENTPD3 Marks Mature Stem Cell-Derived ß-Cells Formed by Self-Aggregation In Vitro.

Stem cell-derived ß-like cells (sBC) carry the promise of providing an abundant source of insulin-producing cells for use in cell replacement therapy for patients with diabetes, potentially allowing widespread implementation of a practical cure. To achieve their clinical promise, sBC need to function comparably with mature adult ß-cells, but as yet they display varying degrees of maturity. Indeed, detailed knowledge of the events resulting in human ß-cell maturation remains obscure. Here we show that sBC spontaneously self-enrich into discreet islet-like cap structures within in vitro cultures, independent of exogenous maturation conditions. Multiple complementary assays demonstrate that this process is accompanied by functional maturation of the self-enriched sBC (seBC); however, the seBC still contain distinct subpopulations displaying different maturation levels. Interestingly, the surface protein ENTPD3 (also known as nucleoside triphosphate diphosphohydrolase-3 [NDPTase3]) is a specific marker of the most mature seBC population and can be used for mature seBC identification and sorting. Our results illuminate critical aspects of in vitro sBC maturation and provide important insights toward developing functionally mature sBC for diabetes cell replacement therapy.

Generation of Induced Pluripotent Stem Cells from a Female Patient with a Xq27.3-q28 Deletion to Establish Disease Models and Identify Therapies.

Since it is extremely difficult to establish an animal model for human chromosomal abnormalities, induced pluripotent stem cells (iPSCs) provide a powerful alternative to study underlying mechanisms of these disorders and identify potential therapeutic interventions. In this study we established iPSCs from a young girl with a hemizygous deletion of Xq27.3-q28 who exhibited global developmental delay and intellectual disability from early in infancy. The deletion site on the X chromosome includes Fragile X Mental Retardation 1 (FMR1), the gene responsible for fragile X syndrome, which likely contributes to the patient's neurodevelopmental abnormalities. The FMR1 gene was expressed in approximately half of the iPSC clones we generated while it was absent in the other half due to the random inactivation of normal and abnormal X chromosomes. The normal or absent expression pattern of the FMR1 gene was not altered when the iPSCs were differentiated into neural progenitor cells (NPCs). Moreover, chromosome reactivating reagents such as 5-aza-2-deoxycytidine, trichostatin A, and UNC0638, were tested in an attempt to reactivate the suppressed FMR1 gene in affected iPSC-NPCs. The affected and control isogenic iPSCs developed in this study are ideal models with which to identify downstream consequences caused by the Xq27.3-q28 deletion and also to provide tools for high-throughput screening to identify compounds potentially improving the well-being of this patient population.

Selective serotonin reuptake inhibitors ameliorate MEGF10 myopathy.

MEGF10 myopathy is a rare inherited muscle disease that is named after the causative gene, MEGF10. The classic phenotype, early onset myopathy, areflexia, respiratory distress and dysphagia, is severe and immediately life-threatening. There are no disease-modifying therapies. We performed a small molecule screen and follow-up studies to seek a novel therapy. A primary in vitro drug screen assessed cellular proliferation patterns in Megf10-deficient myoblasts. Secondary evaluations were performed on primary screen hits using myoblasts derived from Megf10-/- mice, induced pluripotent stem cell-derived myoblasts from MEGF10 myopathy patients, mutant Drosophila that are deficient in the homologue of MEGF10 (Drpr) and megf10 mutant zebrafish. The screen yielded two promising candidates that are both selective serotonin reuptake inhibitors (SSRIs), sertraline and escitalopram. In depth follow-up analyses demonstrated that sertraline was highly effective in alleviating abnormalities across multiple models of the disease including mouse myoblast, human myoblast, Drosophila and zebrafish models. Sertraline also restored deficiencies of Notch1 in disease models. We conclude that SSRIs show promise as potential therapeutic compounds for MEGF10 myopathy, especially sertraline. The mechanism of action may involve the Notch pathway.

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.

CRISPR/Cas9 knockout of USP18 enhances type I IFN responsiveness and restricts HIV-1 infection in macrophages.

The IFN-stimulated gene ubiquitin-specific proteinase 18 (USP18) encodes a protein that negatively regulates T1 IFN signaling via stearic inhibition of JAK1 recruitment to the IFN-α receptor 2 subunit (IFNAR2). Here, we demonstrate that USP18 expression is induced by HIV-1 in a T1 IFN-dependent manner. Experimental depletion of USP18 by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene editing results in a significant restriction of HIV-1 replication in an induced pluripotent stem cell (iPSC)-derived macrophage model. In the absence of USP18, macrophages have increased responsiveness to stimulation with T1 IFNs with prolonged phosphorylation of STAT1 and STAT2 and increased expression of IFN-stimulated genes that are key for antiviral responses. Interestingly, HIV-1 requires some signaling through the T1 IFN receptor to replicate efficiently because a neutralizing antibody that inhibits T1 IFN activity reduces HIV-1 replication rate in monocyte-derived macrophages. USP18 induction by HIV-1 tunes the IFN response to optimal levels allowing for efficient transcription from the HIV-1 LTR promoter while minimizing the T1 IFN-induced antiviral response that would otherwise restrict viral replication and spread. Finally, iPSC and CRISPR/Cas9 gene targeting offer a powerful tool to study host factors that regulate innate immune responses.

Isogenic Cellular Systems Model the Impact of Genetic Risk Variants in the Pathogenesis of Type 1 Diabetes.

At least 57 independent loci within the human genome confer varying degrees of risk for the development of type 1 diabetes (T1D). The majority of these variants are thought to contribute to overall genetic risk by modulating host innate and adaptive immune responses, ultimately resulting in a loss of immunological tolerance to ß cell antigens. Early efforts to link specific risk variants with functional alterations in host immune responses have employed animal models or genotype-selected individuals from clinical bioresource banks. While some notable genotype:phenotype associations have been described, there remains an urgent need to accelerate the discovery of causal variants and elucidate the molecular mechanisms by which susceptible alleles alter immune functions. One significant limitation has been the inability to study human T1D risk loci on an isogenic background. The advent of induced pluripotent stem cells (iPSCs) and genome-editing technologies have made it possible to address a number of these outstanding questions. Specifically, the ability to drive multiple cell fates from iPSC under isogenic conditions now facilitates the analysis of causal variants in multiple cellular lineages. Bioinformatic analyses have revealed that T1D risk genes cluster within a limited number of immune signaling pathways, yet the relevant immune cell subsets and cellular activation states in which candidate risk genes impact cellular activities remain largely unknown. In this review, we summarize the functional impact of several candidate risk variants on host immunity in T1D and present an isogenic disease-in-a-dish model system for interrogating risk variants, with the goal of expediting precision therapeutics in T1D.

A pathologist's perspective on induced pluripotent stem cells.

Induced pluripotent stem cell (iPSC) technology was originally developed in 2006. Essentially, it converts somatic cells into pluripotent stem cells by transiently expressing a few transcriptional factors. Once generated, these iPSCs can differentiate into all the cell types of our body, theoretically, which has attracted great attention for clinical research including disease pathobiology studies. Could this technology then become an additional research or diagnostic tool widely available to practicing pathologists? Here we summarize progress in iPSC research toward disease pathobiology studies, its future potential, and remaining problems from a pathologist's perspective. A particular focus will be on introducing the effort to recapitulate disease-related morphological changes through three-dimensional culture of stem cells such as organoid differentiation.

Disulfide bond disrupting agents activate the unfolded protein response in EGFR- and HER2-positive breast tumor cells.

Many breast cancer deaths result from tumors acquiring resistance to available therapies. Thus, new therapeutic agents are needed for targeting drug-resistant breast cancers. Drug-refractory breast cancers include HER2+ tumors that have acquired resistance to HER2-targeted antibodies and kinase inhibitors, and "Triple-Negative" Breast Cancers (TNBCs) that lack the therapeutic targets Estrogen Receptor, Progesterone Receptor, and HER2. A significant fraction of TNBCs overexpress the HER2 family member Epidermal Growth Factor Receptor (EGFR). Thus agents that selectively kill EGFR+ and HER2+ tumors would provide new options for breast cancer therapy. We previously identified a class of compounds we termed Disulfide bond Disrupting Agents (DDAs) that selectively kill EGFR+ and HER2+ breast cancer cells in vitro and blocked the growth of HER2+ breast tumors in an animal model. DDA-dependent cytotoxicity was found to correlate with downregulation of HER1-3 and Akt dephosphorylation. Here we demonstrate that DDAs activate the Unfolded Protein Response (UPR) and that this plays a role in their ability to kill EGFR+ and HER2+ cancer cells. The use of breast cancer cell lines ectopically expressing EGFR or HER2 and pharmacological probes of UPR revealed all three DDA responses: HER1-3 downregulation, Akt dephosphorylation, and UPR activation, contribute to DDA-mediated cytotoxicity. Significantly, EGFR overexpression potentiates each of these responses. Combination studies with DDAs suggest that they may be complementary with EGFR/HER2-specific receptor tyrosine kinase inhibitors and mTORC1 inhibitors to overcome drug resistance.

Concise Review: Induced Pluripotent Stem Cell Research in the Era of Precision Medicine.

Recent advances in DNA sequencing technologies are revealing how human genetic variations associate with differential health risks, disease susceptibilities, and drug responses. Such information is now expected to help evaluate individual health risks, design personalized health plans and treat patients with precision. It is still challenging, however, to understand how such genetic variations cause the phenotypic alterations in pathobiologies and treatment response. Human induced pluripotent stem cell (iPSC) technologies are emerging as a promising strategy to fill the knowledge gaps between genetic association studies and underlying molecular mechanisms. Breakthroughs in genome editing technologies and continuous improvement in iPSC differentiation techniques are particularly making this research direction more realistic and practical. Pioneering studies have shown that iPSCs derived from a variety of monogenic diseases can faithfully recapitulate disease phenotypes in vitro when differentiated into disease-relevant cell types. It has been shown possible to partially recapitulate disease phenotypes, even with late onset and polygenic diseases. More recently, iPSCs have been shown to validate effects of disease and treatment-related single nucleotide polymorphisms identified through genome wide association analysis. In this review, we will discuss how iPSC research will further contribute to human health in the coming era of precision medicine. Stem Cells 2017;35:545-550.

Vascular Smooth Muscle Cells From Hypertensive Patient-Derived Induced Pluripotent Stem Cells to Advance Hypertension Pharmacogenomics.

UNLABELLED: Studies in hypertension (HTN) pharmacogenomics seek to identify genetic sources of variable antihypertensive drug response. Genetic association studies have detected single-nucleotide polymorphisms (SNPs) that link to drug responses; however, to understand mechanisms underlying how genetic traits alter drug responses, a biological interface is needed. Patient-derived induced pluripotent stem cells (iPSCs) provide a potential source for studying otherwise inaccessible tissues that may be important to antihypertensive drug response. The present study established multiple iPSC lines from an HTN pharmacogenomics cohort. We demonstrated that established HTN iPSCs can robustly and reproducibly differentiate into functional vascular smooth muscle cells (VSMCs), a cell type most relevant to vasculature tone control. Moreover, a sensitive traction force microscopy assay demonstrated that iPSC-derived VSMCs show a quantitative contractile response on physiological stimulus of endothelin-1. Furthermore, the inflammatory chemokine tumor necrosis factor α induced a typical VSMC response in iPSC-derived VSMCs. These studies pave the way for a large research initiative to decode biological significance of identified SNPs in hypertension pharmacogenomics. SIGNIFICANCE: Treatment of hypertension remains suboptimal, and a pharmacogenomics approach seeks to identify genetic biomarkers that could be used to guide treatment decisions; however, it is important to understand the biological underpinnings of genetic associations. Mouse models do not accurately recapitulate individual patient responses based on their genetics, and hypertension-relevant cells are difficult to obtain from patients. Induced pluripotent stem cell (iPSC) technology provides a great interface to bring patient cells with their genomic data into the laboratory and to study hypertensive responses. As an initial step, the present study established an iPSC bank from patients with primary hypertension and demonstrated an effective and reproducible method of generating functional vascular smooth muscle cells.

Efficient Gene Editing in Pluripotent Stem Cells by Bacterial Injection of Transcription Activator-Like Effector Nuclease Proteins.

The type III secretion system (T3SS) of Pseudomonas aeruginosa is a powerful tool for direct protein delivery into mammalian cells and has successfully been used to deliver various exogenous proteins into mammalian cells. In the present study, transcription activator-like effector nuclease (TALEN) proteins have been efficiently delivered using the P. aeruginosa T3SS into mouse embryonic stem cells (mESCs), human ESCs (hESCs), and human induced pluripotent stem cells (hiPSCs) for genome editing. This bacterial delivery system offers an alternative method of TALEN delivery that is highly efficient in cleavage of the chromosomal target and presumably safer by avoiding plasmid DNA introduction. We combined the method of bacterial T3SS-mediated TALEN protein injection and transfection of an oligonucleotide template to effectively generate precise genetic modifications in the stem cells. Initially, we efficiently edited a single-base in the gfp gene of a mESC line to silence green fluorescent protein (GFP) production. The resulting GFP-negative mESC was cloned from a single cell and subsequently mutated back to a GFP-positive mESC line. Using the same approach, the gfp gene was also effectively knocked out in hESCs. In addition, a defined single-base edition was effectively introduced into the X-chromosome-linked HPRT1 gene in hiPSCs, generating an in vitro model of Lesch-Nyhan syndrome. T3SS-mediated TALEN protein delivery provides a highly efficient alternative for introducing precise gene editing within pluripotent stem cells for the purpose of disease genotype-phenotype relationship studies and cellular replacement therapies.

A practical guide to induced pluripotent stem cell research using patient samples.

Approximately 3 years ago, we assessed how patient induced pluripotent stem cell (iPSC) research could potentially impact human pathobiology studies in the future. Since then, the field has grown considerably with numerous technical developments, and the idea of modeling diseases 'in a dish' is becoming increasingly popular in biomedical research. Likely, it is even acceptable to include patient iPSCs as one of the standard research tools for disease mechanism studies, just like knockout mice. However, as the field matures, we acknowledge there remain many practical limitations and obstacles for their genuine application to understand diseases, and accept that it has not been as straightforward to model disorders as initially proposed. A major practical challenge has been efficient direction of iPSC differentiation into desired lineages and preparation of the large numbers of specific cell types required for study. Another even larger obstacle is the limited value of in vitro outcomes, which often do not closely represent disease conditions. To overcome the latter issue, many new approaches are underway, including three-dimensional organoid cultures from iPSCs, xenotransplantation of human cells to animal models and in vitro interaction of multiple cell types derived from isogenic iPSCs. Here we summarize the areas where patient iPSC studies have provided truly valuable information beyond existing skepticism, discuss the desired technologies to overcome current limitations and include practical guidance for how to utilize the resources. Undoubtedly, these human patient cells are an asset for experimental pathology studies. The future rests on how wisely we use them.

E2f6-mediated repression of the meiotic Stag3 and Smc1ß genes during early embryonic development requires Ezh2 and not the de novo methyltransferase Dnmt3b.

The E2f6 transcriptional repressor is an E2F-family member essential for the silencing of a group of meiosis-specific genes in somatic tissues. Although E2f6 has been shown to associate with both polycomb repressive complexes (PRC) and the methyltransferase Dnmt3b, the cross-talk between these repressive machineries during E2f6-mediated gene silencing has not been clearly demonstrated yet. In particular, it remains largely undetermined when and how E2f6 establishes repression of meiotic genes during embryonic development. We demonstrate here that the inactivation of a group of E2f6 targeted genes, including Stag3 and Smc1ß, first occurs at the transition from mouse embryonic stem cells (ESCs) to epiblast stem cells (EpiSCs), which represent pre- and post-implantation stages, respectively. This process was accompanied by de novo methylation of their promoters. Of interest, despite a clear difference in DNA methylation status, E2f6 was similarly bound to the proximal promoter regions both in ESCs and EpiSCs. Neither E2f6 nor Dnmt3b overexpression in ESCs decreased meiotic gene expression or increased DNA methylation, indicating that additional factors are required for E2f6-mediated repression during the transition. When the SET domain of Ezh2, a core subunit of the PRC2 complex, was deleted, however, repression of Stag3 and Smc1ß during embryoid body differentiation was largely impaired, indicating that the event required the enzymatic activity of Ezh2. In addition, repression of Stag3 and Smc1ß occurred in the absence of Dnmt3b. The data presented here suggest a primary role of PRC2 in E2f6-mediated gene silencing of the meiotic genes.

Generation of neural cells from DM1 induced pluripotent stem cells as cellular model for the study of central nervous system neuropathogenesis.

Dystrophia myotonica type 1 (DM1) is an autosomal dominant multisystem disorder. The pathogenesis of central nervous system (CNS) involvement is poorly understood. Disease-specific induced pluripotent stem cell (iPSC) lines would provide an alternative model. In this study, we generated two DM1 lines and a normal iPSC line from dermal fibroblasts by retroviral transduction of Yamanaka's four factors (hOct4, hSox2, hKlf4, and hc-Myc). Both DM1 and control iPSC clones showed typical human embryonic stem cell (hESC) growth patterns with a high nuclear-to-cytoplasm ratio. The iPSC colonies maintained the same growth pattern through subsequent passages. All iPSC lines expressed stem cell markers and differentiated into cells derived from three embryonic germ layers. All iPSC lines underwent normal neural differentiation. Intranuclear RNA foci, a hallmark of DM1, were detected in DM1 iPSCs, neural stem cells (NSCs), and terminally differentiated neurons and astrocytes. In conclusion, we have successfully established disease-specific human DM1 iPSC lines, NSCs, and neuronal lineages with pathognomonic intranuclear RNA foci, which offer an unlimited cell resource for CNS mechanistic studies and a translational platform for therapeutic development.

Fibroblast growth factor receptor 2 homodimerization rapidly reduces transcription of the pluripotency gene Nanog without dissociation of activating transcription factors.

Nanog or Gata6-positive cells co-exist and are convertible within the inner cell mass of murine blastocysts and embryonic stem (ES) cells. Previous studies demonstrate fibroblast growth factor receptor 2 (FGFR2) triggers Nanog gene down-regulation and differentiation to primitive endoderm (PE); however, the underlying mechanisms responsible for reversible and fluctuating cell fate are poorly understood. Using an inducible FGFR2 dimerization system in ES cells, we demonstrate that FGFR2 activation rapidly down-regulated Nanog gene transcription through activation of the Mek pathway and subsequently differentiated ES cells into PE cells. FGFR2 rather selectively repressed the Nanog gene with minimal effect on other pluripotency genes, including Oct4 and Sox2. We determined the Nanog promoter region containing minimum Oct4/Sox2 binding sites was sufficient for this transcriptional down-regulation by FGFR2, when the reporter transgenes were integrated with insulators. Of interest, FGFR2-mediated Nanog transcriptional reduction occurred without dissociation of RNA polymerase II, p300, Oct4, Sox2, and Tet1 from the Nanog proximal promoter region and with no increase in repressive histone methylation marks or DNA methylation, implying the gene repression is in the early and transient phase. Furthermore, addition of a specific FGFR inhibitor readily reversed this Nanog repression status. These findings illustrate well how FGFR2 induces rapid but reversible Nanog repression within ES cells.