Human iPSC Line Research Articles

Elucidating an Essential Role of Promoting Endothelial-to-Hematopoietic Transition By a Key EMT-Regulator ZEB2 Using a Human iPSC-Based Model

ZEB2 (Zinc finger E-box binding homeobox 2; also known as SMAD interacting protein-1 or SIP1) is a DNA-binding transcriptional regulator associated with epithelial-to-mesenchymal-transition (EMT). It has been reported that it is important in many other developmental processes, including mesoderm formation during gastrulation and neural crest formation. More recently, a study reported that inactivation of ZEB2 in mice results in differentiation defects in multiple hematopoietic cell lineages (1). In order to examine the roles of ZEB2 in human developmental process and hematopoiesis, we decided to apply human induced pluripotent stem cells (iPSCs) as a model with a pair of isogenic iPSCs with or without functional ZEB2 expression. We used a validated iPSC-based hematopoietic differentiation under a feeder- and xeno-free culture condition was used to study the role of ZEB2 in hematopoietic cell formation and to avoid other EMT-inducing factors in serum. We found that ZEB2 expression is abundant in either cord blood cells or human iPSCs-derived hematopoietic stem/progenitor cells (HSPCs), indicating that ZEB2 deficiency may hinder human hematopoietic differentiation. To knock out the ZEB2 gene and generate ZEB2 deficient human iPSCs, we used two different CRISPR guide RNAs as well as SpCas9 in a human iPSC line BC1. An engineered isogenic BC1 iPSC line that has both alleles 155-bp deletion in ZEB2 exon 1 (ZEB2 -/-) was successfully isolated and expanded, resulting in the frameshift and the consequent disappearance of ZEB2 protein expression. The complete ZEB2 knockout had little effects on human iPSCs expansion. However, we observed that the differentiation of ZEB-/- BC1 into CD34+CD45+ HSPCs was decreased. The number of ZEB-/- BC1-derived HSPCs was 5-fold lower than that from wide type BC1 control. Moreover, ZEB2-deleted HSPCs were defected in hematopoietic colony formation. To figure out at which hematopoietic differentiating stage the defects occurred, we applied inducible iPSC's endothelium-hematopoietic transformation (EHT) system to mimic hemogenic endothelial cells (ECs) giving rise to HSPCs. By using this established EHT system, ZEB2 deficient iPSCs were differentiated in order into CD34+CD31+CD144+ ECs, and then to HSPCs (2). We found that ZEB2 depletion did not adversely affect the formation of ECs from human iPSCs in this EHT experimental system but did impact on HSPCs generation from ECs. This study is unique in several ways to elucidate the roles of ZEB2 in human hematopoietic development. This prospective genetic model allowed us to pinpoint ZEB2 is critical at the stage of EHT during human hematopoietic cell formation. Our results indicate that contribution of ZEB2 deficiency in blood disorders might result from the inhibition of HSPC formation. The human iPSC-based hematopoietic differentiation system coupled with precisely edited isogenic iPSC lines also provide a more controlled genetic model for studying functions of others gene in their regulation of human hematopoiesis.

Inhibition of mTOR Signaling Enhances Maturation of Cardiomyocytes Derived From Human-Induced Pluripotent Stem Cells via p53-Induced Quiescence.

Current differentiation protocols to produce cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations as determined by expression of cardiac troponin T. However, these cardiomyocytes remain immature, more closely resembling the fetal state, with a lower maximum contractile force, slower upstroke velocity, and immature mitochondrial function compared with adult cardiomyocytes. Immaturity of iPSC-derived cardiomyocytes may be a significant barrier to clinical translation of cardiomyocyte cell therapies for heart disease. During development, cardiomyocytes undergo a shift from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR)-signaling pathway plays a key role in nutrient sensing and growth. We hypothesized that transient inhibition of the mTOR-signaling pathway could lead cardiomyocytes to a quiescent state and enhance cardiomyocyte maturation. Cardiomyocytes were differentiated from 3 human iPSC lines using small molecules to modulate the Wnt pathway. Torin1 (0 to 200 nmol/L) was used to inhibit the mTOR pathway at various time points. We quantified contractile, metabolic, and electrophysiological properties of matured iPSC-derived cardiomyocytes. We utilized the small molecule inhibitor, pifithrin-α, to inhibit p53 signaling, and nutlin-3a, a small molecule inhibitor of MDM2 (mouse double minute 2 homolog) to upregulate and increase activation of p53. Torin1 (200 nmol/L) increased the percentage of quiescent cells (G0 phase) from 24% to 48% compared with vehicle control (P<0.05). Torin1 significantly increased expression of selected sarcomere proteins (including TNNI3 [troponin I, cardiac muscle]) and ion channels (including Kir2.1) in a dose-dependent manner when Torin1 was initiated after onset of cardiomyocyte beating. Torin1-treated cells had an increased relative maximum force of contraction, increased maximum oxygen consumption rate, decreased peak rise time, and increased downstroke velocity. Torin1 treatment increased protein expression of p53, and these effects were inhibited by pifithrin-α. In contrast, nutlin-3a independently upregulated p53, led to an increase in TNNI3 expression and worked synergistically with Torin1 to further increase expression of both p53 and TNNI3. Transient treatment of human iPSC-derived cardiomyocytes with Torin1 shifts cells to a quiescent state and enhances cardiomyocyte maturity.

Establishment of a human iPSC line (SDQLCHi010-A) from a patient with optic nerve malformation carrying a heterozygous mutation in PAX6 gene.

We established an induced pluripotent stem cell (iPSC) line (SDQLCHi010-A) from peripheral blood mononuclear cells isolated from a 4-year-old boy with optic nerve malformation and intellectual disability carrying a heterozygous mutation (c.220A>G (p.S74G)) in PAX6 gene. Non-integrating episomal vectors containing OCT4, SOX2, KLF4, BCL-XL and MYC were used for reprogramming. The established iPSC line showed normal karyotype, expressed pluripotency markers, exhibited differentiation potential in vitro and kept PAX6 gene mutation.

Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies.

In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models is time-consuming and challenging. Therefore, human induced pluripotent stem cells (iPSCs) carrying specific cardiomyopathy-associated mutations are a promising alternative. Since the original discovery that pluripotency can be artificially induced by the expression of different transcription factors, various patient-specific-induced pluripotent stem cell lines have been generated to model non-ischemic, genetic cardiomyopathies in vitro. In this review, we describe the genetic landscape of non-ischemic, genetic cardiomyopathies and give an overview about different human iPSC lines, which have been developed for the disease modeling of inherited cardiomyopathies. We summarize different methods and protocols for the general differentiation of human iPSCs into cardiomyocytes. In addition, we describe methods and technologies to investigate functionally human iPSC-derived cardiomyocytes. Furthermore, we summarize novel genome editing approaches for the genetic manipulation of human iPSCs. This review provides an overview about the genetic landscape of inherited cardiomyopathies with a focus on iPSC technology, which might be of interest for clinicians and basic scientists interested in genetic cardiomyopathies.

Derivation of a human DOA iPSC line, IISHDOi006-A, with a mutation in the ACO2 gene: c.1999G&gt;A; p.Glu667Lys

Human iPSC line, IISHDOi006-A, was obtained from fibroblasts of a patient with Dominant Optic Atrophy (DOA) carrying a heterozygous mutation in the gene ACO2: c.1999G>A; p.Glu667Lys. Reprogramming factors Oct3/4, Sox2, Klf4, and c-Myc were delivered using a non-integrative methodology that involves the use of Sendai virus.

Generation of a CFTR knock-in reporter cell line (MHHi006-A-1) from a human induced pluripotent stem cell line.

CFTR encodes for a chloride ion channel expressed primarily in secretory epithelia in the airways, intestine, liver and other tissues. Mutations in the CFTR gene have been identified in people suffering from Cystic Fibrosis. Here, we established a CFTR knock-in reporter cell line from a human iPSC line (MHHi006-A) using TALEN technology. The reporter enables the monitoring and optimization of the differentiation of pluripotent stem cells into CFTR expressing epithelia on a single cell level, as well as the enrichment of CFTR positive cells, which represent an excellent tool for Cystic Fibrosis disease modelling, drug screening and ultimately cellular therapies.

Generation of a Purified iPSC-Derived Smooth Muscle-like Population forCell Sheet Engineering.

SummaryInduced pluripotent stem cells (iPSCs) provide a potential source for the derivation of smooth muscle cells (SMCs); however, current approaches are limited by the production of heterogeneous cell types and a paucity of tools or markers for tracking and purifying candidate SMCs. Here, we develop murine and human iPSC lines carrying fluorochrome reporters (Acta2hrGFP and ACTA2eGFP, respectively) that identify Acta2+/ACTA2+ cells as they emerge in vitro in real time during iPSC-directed differentiation. We find that Acta2hrGFP+ and ACTA2eGFP+ cells can be sorted to purity and are enriched in markers characteristic of an immature or synthetic SMC. We characterize the resulting GFP+ populations through global transcriptomic profiling and functional studies, including the capacity to form engineered cell sheets. We conclude that these reporter lines allow for generation of sortable, live iPSC-derived Acta2+/ACTA2+ cells highly enriched in smooth muscle lineages for basic developmental studies, tissue engineering, or future clinical regenerative applications.

A Large Panel of Isogenic APP and PSEN1 Mutant Human iPSC Neurons Reveals Shared Endosomal Abnormalities Mediated by APP β-CTFs, Not Aβ

Familial Alzheimer's disease (fAD) results from mutations in the amyloid precursor protein (APP) and presenilin (PSEN1 and PSEN2) genes. Here we leveraged recent advances in induced pluripotent stem cell (iPSC) and CRISPR/Cas9 genome editing technologies to generate a panel of isogenic knockin human iPSC lines carrying APP and/or PSEN1 mutations. Global transcriptomic and translatomic profiling revealed that fAD mutations have overlapping effects on the expression of AD-related and endocytosis-associated genes. Mutant neurons also increased Rab5+ early endosome size. APP and PSEN1 mutations had discordant effects on Aβ production butsimilar effects on APP β C-terminal fragments (β-CTFs), which accumulate in all mutant neurons. Importantly, endosomal dysfunction correlated with accumulation of β-CTFs, not Aβ, and could be rescued by pharmacological modulation of β-secretase (BACE). These data display the utility of our mutant iPSCs in studying AD-related phenotypes in a non-overexpression human-based system and support mounting evidence that β-CTF may be critical in AD pathogenesis.

Generation of integration free human iPSC line SORMi002-A from neonatal foreskin fibroblasts.

Circumcision is a common surgical procedure usually performed on neonates worldwide. Skin removed after circumcision is abundant and often discarded. Neonatal foreskin fibroblasts (NFFs) can be easily isolated from it for application in biomedical research including induced pluripotent stem cell (iPSC) generation. In this study NFFs were reprogrammed to iPSCs using non-integrating episomal reprogramming plasmids. The iPSCs generated express pluripotency markers. They form embryoid bodies and differentiate in vitro into cells of endoderm, mesoderm and ectoderm. They have a normal male karyotype, are genetically identical to the parent fibroblasts and are transgene free.

Mowat-Wilson syndrome: Generation of two human iPS cell lines (UUIGPi004A and UUIGPi005A) from siblings with a truncating ZEB2 gene variant

Mowat-Wilson syndrome (MWS) is a complex developmental syndrome caused by heterozygous mutations in the Zinc finger E-box-binding homeobox 2 gene (ZEB2). We generated the first human iPSC lines from primary fibroblasts of two siblings with MWS carrying a heterozygous ZEB2 stop mutation (c.1027C > T; p.Arg343*) using the Sendai virus reprogramming system. Both iPSC lines were free from reprogramming vector genes, expressed pluripotency markers and showed potential to differentiate into the three germ layers. Genetic analysis confirmed normal karyotypes and a preserved stop mutation. These iPSC lines will provide a useful resource to study altered neural lineage fate and neuropathophysiology in MWS.

Generation and characterization of a human iPSC line (UAMi005-A) from a patient with nonketotic hyperglycinemia due to mutations in the GLDC gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery.

ENHANCED FUNCTION OF PLCG2 VARIANT P522R IN MACROPHAGES AND MICROGLIA IS ASSOCIATED WITH PROTECTION FROM ALZHEIMER’S DISEASE

Evidence from GWAS suggests the P522R variant in the enzyme PLCg2 is protective against Alzheimer's disease (AD). This variant lies in the auto-inhibitory component of the PLCg2 gene, and such mutations could possibly alter the activation of PLCg2. Expression of PLCg2 in the brain is predominantly found in microglia and microglia have been implicated in the risk of AD. PLCg2 catalyses the hydrolysis of PIP2 to IP3 and DAG, with IP3 causing intracellular Ca2+ release. Intracellular Ca2+ release causes multiple cellular responses, such as phagocytosis. This suggests that the P522R variant may alter the responsiveness of microglia in conditions such as AD. Several techniques were utilized to investigate function of PLCg2 within the variant. This included molecular dynamic stimulation (GROMACS), run in the NPT ensemble and resulting structures were analysed using RMSD and visualized for structural differences using VMD. In addition, we investigated cytoplasmic Ca2+ increase (Fura2 and Fluo-8), indicative of a signalling response, following activation of PLCg2 in human (using anti-CD32) and murine (using anti-CD16/32 and Ab) models. Novel human P522R KOLF2 iPSC lines and P522R knockin mice were generated using CRISPR-assisted gene-targeting. We used iPSC-derived human microglia and primary murine cortical and hippocampal microglia alongside macrophages derived from conditionally-immortalized mouse macrophage precursor cell lines. The requirement for PLCg2 in the intracellular Ca2+ changes were investigated using specific PLCg inhibitors (edelfosine, U73122) and a specific GapmeR knockdown system. Computational models suggest that structural changes in the P522R mutant would reduce binding of inhibitors. In addition, Ca2+ signalling assays in all the cellular models consistently demonstrated increased cytosolic Ca2+ release in P522R variants compared to controls following activation of PLCg2. Our results are the first to show that the protective P522R variant exhibits increased activity in microglia and macrophages in response to physiological stimuli. This protection most likely results from alteration of microglia response after exposure to the environmental cues present in AD. PLCg2 represents a strong pharmacological target suggesting the possibility of a new intervention site for AD.

Generation of two human iPSC lines (MDCUi001-A and MDCUi001-B) from dermal fibroblasts of a Thai patient with X-linked osteogenesis imperfecta using integration-free Sendai virus

Two clones of human induced pluripotent stem cells (iPSCs) were generated from dermal fibroblasts isolated from a one-year-old Thai patient with X-linked osteogenesis imperfecta. The patient harbored a mutation, p.N459S, in the MBTPS2 gene. The cells were reprogrammed using an integration-free Sendai virus containing KLF4, c-MYC, OCT4 and SOX2. Both of the established iPSC lines (MDCUi001-A and MDCUi001-B) maintained normal karyotype, expressed pluripotent markers and differentiated into all three germ layers.

Early prediction of the differentiation potential during the formation of human iPSC-derived embryoid bodies

Induced pluripotent stem cells (iPSCs) show huge variations in their differentiation potential, even in the same condition. However, methods for predicting these differentiation tendencies, especially in the early stage of differentiation, are still scarce. This study aimed to establish a simple and practical system to predict the differentiation tendency of iPSC lines using embryoid bodies (EBs) with identified parameters in the early stage. We compared four human iPSC lines in terms of the morphology and maintenance of EBs and their gene expression levels of specific markers for three germ-layers. Furthermore, the differentiation potentials of these iPSC lines into melanocytes, which are ectoderm-derived cells, were also compared and correlated with the above parameters. The results showed that iPSC lines forming regular, smooth, and not cystic EBs, which could be maintained in culture for a relatively longer time, also expressed higher levels of ectoderm-specific markers and lower levels of mesoderm/endoderm markers. Additionally, these iPSC lines showed greater potential in melanocyte differentiation using EB-based protocol, and the induced melanocytes expressed melanocytic markers and presented characteristics that were similar to those of normal human melanocytes. By contrast, iPSC lines that formed cystic EBs with bright or dark cavities and expressed relatively lower levels of ectoderm-specific markers failed in the melanocyte differentiation. Collectively, the differentiation tendency of human iPSC lines may be predicted by specific parameters in the EB stage. The formation and maintenance of optimal EBs and the expression of germ layer-specific markers are particularly important and practical for the prediction assay in the early stage.

Generation of a human iPSC line, INMi003-A, with a missense mutation in CRX associated with autosomal dominant cone-rod dystrophy

We generated an induced pluripotent stem cell (iPSC) line using dermal fibroblasts from a 53 year-old patient with autosomal dominant cone-rod dystrophy (CRD) caused by a missense mutation, c.121C > T, in the CRX gene. Patient fibroblasts were reprogrammed using the non-integrative Sendai virus reprogramming system and the human OSKM transcription factor cocktail. The generated iPSCs contained the congenital mutation in exon 3 of CRX and were pluripotent and genetically stable. This iPSC line will be an important tool for retinal differentiation studies to better understand the CRD phenotype caused by the mutant p.Arg41Trp CRX protein.

Generation of a human iPSC line, INMi004-A, with a point mutation in CRX associated with autosomal dominant Leber congenital amaurosis

The human induced pluripotent stem cell (iPSC) line, INMi004-A, was generated using dermal fibroblasts from a 6 year-old patient with autosomal dominant Leber Congenital Amaurosis (LCA) caused by the point mutation c.695delC (p.Pro232Argfs*139) in the CRX gene. We used non-integrative Sendai virus vectors containing the human OSKM transcription factor cocktail to reprogram patient fibroblasts. The generated iPSC line contained the congenital deletion c.695delC in exon 4 of CRX, had a normal karyotype, and was capable of differentiation into all three germ layers. This cell line represents an important tool to study the pathophysiology of CRX-associated LCA.

112-OR: Critical Role of Tyrosine Kinase 2 (TYK2) in Human Pancreatic Differentiation

Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic beta cells. Genome wide association and fine mapping studies to identify causal variants predicted several loci in the human genome that are associated with T1D risk. One such candidate gene is a member of the JAK (Janus Kinase) family of tyrosine kinases, TYK2, which plays a critical role in intracellular signaling stimulated by cytokines through STATs. Loss-of-function variants of TYK2 are associated with protection against T1D. In line with this, silencing of TYK2 in beta cells attenuated inflammatory signaling pathways and apoptosis. To study the role of TYK2 in human beta-cell development, we now generated TYK2- knockout (KO) human iPSC lines using CRISPR-Cas9 genome editing and induced them to differentiate to pancreatic endocrine lineage. Our results suggest that absence of TYK2 does not compromise the pluripotency or the early pancreatic endoderm differentiation potential of iPSC. However, in the later stages of differentiation, the mRNA levels of key pancreatic TFs (PDX1(55±17%, p=0.03), NGN3(65±13%, p=0.008)), RFX6(58±14%, p=0.014), NKX6.1(39±19%, p=0.114) and NKX2.2(68±7%, p=0.0008)) were significantly reduced in the TYK2-KO cells. In the final stages of controlled induction to endocrine lineage TYK2-KO lines expressed lower levels of INS(53±9%, p=0.004), GCG(70±10.5%, p=0.002) and SST(62±8.6%, p=0.002) compared to control lines (mean ± SEM of 3 independent experiments). Furthermore immunostaining for INS, GCG and SST confirmed a lower number of endocrine hormone positive cells in TYK2-KO lines. In contrast to the wild type controls, the TYK2-KO iPSC-derived pancreatic cells displayed no activation of STAT1 and STAT2 and showed impaired STAT3 activation in response to IFNαor stimulation by polyinosinic-polycitidilic acid treatment, a viral infection mimetic. The present study suggests that TYK2 plays an important early role in pancreatic endocrine differentiation. Disclosure V. Chandra: None. H. Ibrahim: None. D. Balboa: None. T. Otonkoski: None.

Generation of human induced pluripotent stem cell (iPSC) lines from three patients with von Hippel-Lindau syndrome carrying distinct VHL gene mutations

Von Hippel-Lindau (VHL) syndrome is a familial cancer syndrome caused by mutations in the tumor suppressor gene VHL. We generated human iPSC lines from primary dermal fibroblasts of three VHL syndrome patients carrying distinct VHL germ line mutations (c.194C>G, c.194C>T and nt440delTCT, respectively). Characterization of the iPSC lines confirmed expression of pluripotency markers, trilineage differentiation potential and absence of exogenous vector expression. The three hiPSC lines were genetically stable and retained the VHL mutation of each donor. These iPSC lines, the first derived from VHL syndrome patients, offer a useful resource to study disease pathophysiology and for anti-cancer drug development.

Generation and characterization of a human iPSC line (UAMi004-A) from a patient with propionic acidemia due to defects in the PCCB gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with propionic acidemia that has a homozygous mutation (c.1218_1231del14ins12 (p.G407 fs)) in the PCCB gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. The generated iPSC line represents a useful tool to study the pathomechanisms underlying the deficiency.

Development of Human in vitro Brain-blood Barrier Model from Induced Pluripotent Stem Cell-derived Endothelial Cells to Predict the in vivo Permeability of Drugs.

An in vitro blood-brain barrier (BBB) model is critical for enabling rapid screening of the BBB permeability of the drugs targeting on the central nervous system. Though many models have been developed, their reproducibility and renewability remain a challenge. Furthermore, drug transport data from many of the models do not correlate well with the data for in vivo BBB drug transport. Induced-pluripotent stem cell (iPSC) technology provides reproducible cell resources for in vitro BBB modeling. Here, we generated a human in vitro BBB model by differentiating the human iPSC (hiPSC) line GM25256 into brain endothelial-type cells. The model displayed BBB characteristics including tight junction proteins (ZO-1, claudin-5, and occludin) and endothelial markers (von Willebrand factor and Ulex), as well as high trans-endothelial electrical resistance (TEER) (1560 Ω.cm2 ± 230 Ω.cm2) and γ-GTPase activity. Co-culture with primary rat astrocytes significantly increased the TEER of the model (2970 Ω.cm2 to 4185 Ω.cm2). RNAseq analysis confirmed the expression of key BBB-related genes in the hiPSC-derived endothelial cells in comparison with primary human brain microvascular endothelial cells, including P-glycoprotein (Pgp) and breast cancer resistant protein (BCRP). Drug transport assays for nine CNS compounds showed that the permeability of non-Pgp/BCRP and Pgp/BCRP substrates across the model was strongly correlated with rodent in situ brain perfusion data for these compounds (R2 = 0.982 and R2 = 0.9973, respectively), demonstrating the functionality of the drug transporters in the model. Thus, this model may be used to rapidly screen CNS compounds, to predict the in vivo BBB permeability of these compounds and to study the biology of the BBB.