A functional genomics approach to investigate the differentiation of iPSCs into lung epithelium at air-liquid interface.
ABSTRACT: The availability of robust protocols to differentiate induced pluripotent stem cells (iPSCs) into many human cell lineages has transformed research into the origins of human disease. The efficacy of differentiating iPSCs into specific cellular models is influenced by many factors including both intrinsic and extrinsic features. Among the most challenging models is the generation of human bronchial epithelium at air-liquid interface (HBE-ALI), which is the gold standard for many studies of respiratory diseases including cystic fibrosis. Here, we perform open chromatin mapping by ATAC-seq and transcriptomics by RNA-seq in parallel, to define the functional genomics of key stages of the iPSC to HBE-ALI differentiation. Within open chromatin peaks, the overrepresented motifs include the architectural protein CTCF at all stages, while motifs for the FOXA pioneer and GATA factor families are seen more often at early stages, and those regulating key airway epithelial functions, such as EHF, are limited to later stages. The RNA-seq data illustrate dynamic pathways during the iPSC to HBE-ALI differentiation, and also the marked functional divergence of different iPSC lines at the ALI stages of differentiation. Moreover, a comparison of iPSC-derived and lung donor-derived HBE-ALI cultures reveals substantial differences between these models.
Project description:The airway epithelium forms a barrier between the internal and external environments. Epithelial dysfunction is critical in the pathology of many respiratory diseases, including cystic fibrosis. Ets homologous factor (EHF) is a key member of the transcription factor network that regulates gene expression in the airway epithelium in response to endogenous and exogenous stimuli. EHF, which has altered expression in inflammatory states, maps to the 5' end of an intergenic region on Chr11p13 that is implicated as a modifier of cystic fibrosis airway disease. Here we determine the functions of EHF in primary human bronchial epithelial (HBE) cells and relevant airway cell lines. Using EHF ChIP followed by deep sequencing (ChIP-seq) and RNA sequencing after EHF depletion, we show that EHF targets in HBE cells are enriched for genes involved in inflammation and wound repair. Furthermore, changes in gene expression impact cell phenotype because EHF depletion alters epithelial secretion of a neutrophil chemokine and slows wound closure in HBE cells. EHF activates expression of the SAM pointed domain-containing ETS transcription factor, which contributes to goblet cell hyperplasia. Our data reveal a critical role for EHF in regulating epithelial function in lung disease.
Project description:RATIONALE:Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. OBJECTIVE:Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. METHODS AND RESULTS:Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively. CONCLUSIONS:Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity.
Project description:<h4>Background</h4>High-tidal-volume mechanical ventilation used in patients with acute lung injury (ALI) can induce the release of inflammatory cytokines, as macrophage inflammatory protein-2 (MIP-2), recruitment of neutrophils, and disruption of alveolar epithelial and endothelial barriers. Induced pluripotent stem cells (iPSCs) have been shown to improve ALI in mice, but the mechanisms regulating the interactions between mechanical ventilation and iPSCs are not fully elucidated. Nuclear factor kappa B (NF-?B) and NF-?B repressing factor (NKRF) have been proposed to modulate the neutrophil activation involved in ALI. Thus, we hypothesized intravenous injection of iPSCs or iPSC-derived conditioned medium (iPSC-CM) would decrease high-tidal-volume ventilation-induced neutrophil infiltration, oxidative stress, and MIP-2 production through NF-?B/NKRF pathways.<h4>Methods</h4>Male C57BL/6 mice, aged between 6 and 8 weeks, weighing between 20 and 25 g, were exposed to high-tidal-volume (30 ml/kg) mechanical ventilation with room air for 1 to 4 h after 5×10(7) cells/kg mouse iPSCs or iPSC-CM administration. Nonventilated mice were used as control groups.<h4>Results</h4>High-tidal-volume mechanical ventilation induced the increases of integration of iPSCs into the injured lungs of mice, microvascular permeability, neutrophil infiltration, malondialdehyde, MIP-2 production, and NF-?B and NKRF activation. Lung injury indices including inflammation, lung edema, ultrastructure pathologic changes and functional gas exchange impairment induced by mechanical ventilation were attenuated with administration of iPSCs or iPSC-CM, which was mimicked by pharmacological inhibition of NF-?B activity with SN50 or NKRF expression with NKRF short interfering RNA.<h4>Conclusions</h4>Our data suggest that iPSC-based therapy attenuates high-tidal-volume mechanical ventilation-induced lung injury, at least partly, through inhibition of NF-?B/NKRF pathways. Notably, the conditioned medium of iPSCs revealed beneficial effects equal to those of iPSCs.
Project description:<h4>Background</h4>Thalassemia is the most common genetic disease worldwide; those with severe disease require lifelong blood transfusion and iron chelation therapy. The definitive cure for thalassemia is allogeneic hematopoietic stem cell transplantation, which is limited due to lack of HLA-matched donors and the risk of post-transplant complications. Induced pluripotent stem cell (iPSC) technology offers prospects for autologous cell-based therapy which could avoid the immunological problems. We now report genetic correction of the beta hemoglobin (HBB) gene in iPSCs derived from a patient with a double heterozygote for hemoglobin E and ?-thalassemia (HbE/?-thalassemia), the most common thalassemia syndrome in Thailand and Southeast Asia.<h4>Methods</h4>We used the CRISPR/Cas9 system to target the hemoglobin E mutation from one allele of the HBB gene by homology-directed repair with a single-stranded DNA oligonucleotide template. DNA sequences of the corrected iPSCs were validated by Sanger sequencing. The corrected clones were differentiated into hematopoietic progenitor and erythroid cells to confirm their multilineage differentiation potential and hemoglobin expression.<h4>Results</h4>The hemoglobin E mutation of HbE/?-thalassemia iPSCs was seamlessly corrected by the CRISPR/Cas9 system. The corrected clones were differentiated into hematopoietic progenitor cells under feeder-free and OP9 coculture systems. These progenitor cells were further expanded in erythroid liquid culture system and developed into erythroid cells that expressed mature HBB gene and HBB protein.<h4>Conclusions</h4>Our study provides a strategy to correct hemoglobin E mutation in one step and these corrected iPSCs can be differentiated into hematopoietic stem cells to be used for autologous transplantation in patients with HbE/?-thalassemia in the future.
Project description:Human induced pluripotent stem cells (iPSCs) are a potential source of hepatocytes for liver transplantation to treat end-stage liver disease. In vitro differentiation of human iPSCs into hepatic cells has been achieved using a multistage differentiation protocol, but whether these cells are functional and capable of engrafting and regenerating diseased liver tissue is not clear. We show that human iPSC-derived hepatic cells at various differentiation stages can engraft the liver in a mouse transplantation model. Using the same differentiation and transplantation protocols, we also assessed the ability of human iPSCs derived from each of the three developmental germ layer tissues (that is, ectoderm, mesoderm, and endoderm) to regenerate mouse liver. These iPSC lines, with similar but distinct global DNA methylation patterns, differentiated into multistage hepatic cells with an efficiency similar to that of human embryonic stem cells. Human hepatic cells at various differentiation stages derived from iPSC lines of different origins successfully repopulated the liver tissue of mice with liver cirrhosis. They also secreted human-specific liver proteins into mouse blood at concentrations comparable to that of proteins secreted by human primary hepatocytes. Our results demonstrate the engraftment and liver regenerative capabilities of human iPSC-derived multistage hepatic cells in vivo and suggest that human iPSCs of distinct origins and regardless of their parental epigenetic memory can efficiently differentiate along the hepatic lineage.
Project description:Immunodeficiency, Centromeric Instability, and Facial Anomalies Type I (ICF1) Syndrome is a rare genetic disease caused by mutations in DNMT3B, a de novo DNA methyltransferase. However, the molecular basis of how DNMT3B-deficiency leads to ICF1 pathogenesis is unclear. Induced pluripotent stem cell (iPSC) technology facilitates the study of early human developmental diseases via facile in vitro paradigms. Here, we generate iPSCs from ICF Type 1 Syndrome patient fibroblasts followed by directed differentiation of ICF1-iPSCs to mesenchymal stem cells (MSCs). By performing genome-scale bisulfite sequencing, we find that DNMT3B-deficient iPSCs exhibit global loss of non-CG methylation and select CG hypomethylation at gene promoters and enhancers. Further unbiased scanning of ICF1 iPSC methylomes also identifies large megabase regions of CG hypomethylation typically localized in centromeric and subtelomeric regions. RNA sequencing of ICF1 and control iPSCs reveals abnormal gene expression in ICF1 iPSCs relevant to ICF Syndrome phenotypes, some directly associated with promoter or enhancer hypomethylation. Upon differentiation of ICF1 iPSCs to mesenchymal stem cells (MSCs), we find virtually all CG hypomethylated regions remained hypomethylated when compared to either wild-type iPSC-derived MSCs or primary bone-marrow MSCs. Collectively, our results show specific methylome and transcriptome defects in both ICF1-iPSCs and differentiated somatic cell lineages, providing a valuable stem cell system for further in vitro study of the molecular pathogenesis of ICF1 Syndrome. MethylC-seq and RNA-Seq in ICF Syndrome patient fibroblast derived induced pluripotent stem cells.
Project description:Induced pluripotent stem cells (iPSCs) can differentiate into neural progenitor cells (NPC) under proper conditions. NPC can be used as a model and is a useful tool for disease mechanism exploration and drug screening. However, the characteristics of the cells in various stages from NPC to functional neurons have not been fully described. This study investigated the characteristics of iPSC-derived NPCs during differentiation. Morphological characteristics of the NPCs, including soma area, neurite length, and the number of neurite branches, were examined on selected differentiation days. Physiological functions were assessed by recordings of sodium current, spontaneous excitatory postsynaptic current (sEPSC), and spontaneous inhibitory postsynaptic current (sIPSC). Furthermore, gene expression patterns were assessed with RNA-seq. We found that NPCs derived from iPSCs can be differentiated into glutamatergic and gabaergic neurons. Cell growth peaked during differentiation day 7-12, as the soma area decreased after day 12, growth cone and the number of branches peaked at day 9 and decreased afterwards; whereas a functional synapse formed after day 23. RNA-seq analysis found that a differential expression pattern emerged by day 7. Overall, the study provides a framework for the differentiation process of hiPSC-derived NPCs.
Project description:Induced pluripotent stem cells (iPSCs) are of interest for the study of disease, where these cells can be derived from patients and have the potential to be differentiated into any cell type; however, three-dimensional (3D) culture and differentiation of iPSCs within well-defined synthetic matrices for these applications remains limited. Here, we aimed to establish synthetic cell-degradable hydrogels that allow precise presentation of specific biochemical cues for 3D culture of iPSCs with relevance for hypothesis testing and lineage-specific differentiation. We synthesized poly(ethylene glycol)-(PEG)-peptide-based hydrogels by photoinitiated step growth polymerization and used them to test the hypothesis that the viability of iPSCs within these matrices could be rescued with appropriate biochemical cues inspired by proteins and integrins important for iPSC culture on Matrigel. Specifically, we selected a range of motifs inspired by iPSC binding to Matrigel, including laminin-derived IKVAV and YIGSR, ?5?1-binding PHSRNG10RGDS, ?v?5-binding KKQRFRHRNRKG, and RGDS that is known to bind a variety of integrins for generally promoting cell adhesion. YIGSR and PHSRNG10RGDS resulted in the highest iPSC viability, where binding of ?1 integrin was key, and these permissive compositions also allowed iPSC differentiation into neural progenitor cells (NPCs) (decreased oct4 expression and increased pax6 expression) in response to soluble factors. The resulting NPCs formed clusters of different sizes in response to each peptide, suggesting that matrix biochemical cues affect iPSC proliferation and clustering in 3D culture. In summary, we have established photopolymerizable synthetic matrices for the encapsulation, culture, and differentiation of iPSCs for studies of cell-matrix interactions and deployment in disease models.
Project description:The ocular lens serves as an excellent system to investigate the intricate details of development and differentiation. Generation of lentoid bodies or lens-like structures using pluripotent stem cells is important for understanding the processes critical for lens morphogenesis and the mechanism of cataractogenesis. We previously reported the generation of peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cells (iPSCs). Here, we report generation of lentoid bodies from human embryonic stem cells (hESCs) and (PBMC)-originated, iPSCs employing the "fried egg" method with brief modifications. The ultrastructure analysis of hESC- and iPSC-derived lentoid bodies identified closely packed lens epithelial- and differentiating fiber-like cells. In addition, we performed RNA sequencing (RNA-Seq) based transcriptome profiling of hESC- and iPSC-derived lentoid bodies at differentiation day 25. Next-generation RNA sequencing (RNA-Seq) of hESC- and iPSC-derived lentoid bodies detected expression (?0.659 RPKM) of 13,975 and 14,003 genes, respectively. Comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies revealed 13,563 (>96%) genes common in both datasets. Among the genes common in both transcriptome datasets, 12,856 (~95%) exhibited a quantitatively similar expression profile. Next, we compared the mouse lens epithelial and fiber cell transcriptomes with hESC- and iPSC-derived lentoid bodies transcriptomes and identified?>?96% overlap with lentoid body transcriptomes. In conclusion, we report first-time comparative transcriptome analysis of hESC- and iPSC-derived lentoid bodies at differentiation day 25.
Project description:Optic neuropathy is one of the leading causes of irreversible blindness caused by retinal ganglion cell (RGC) degeneration. The development of induced pluripotent stem cell (iPSC)-based therapy opens a therapeutic window for RGC degeneration, and tissue engineering may further promote the efficiency of differentiation process of iPSCs. The present study was designed to evaluate the effects of a novel biomimetic polybenzyl glutamate (PBG) scaffold on culturing iPSC-derived RGC progenitors. The iPSC-derived neural spheres cultured on PBG scaffold increased the differentiated retinal neurons and promoted the neurite outgrowth in the RGC progenitor layer. Additionally, iPSCs cultured on PBG scaffold formed the organoid-like structures compared to that of iPSCs cultured on cover glass within the same culture period. With RNA-seq, we found that cells of the PBG group were differentiated toward retinal lineage and may be related to the glutamate signaling pathway. Further ontological analysis and the gene network analysis showed that the differentially expressed genes between cells of the PBG group and the control group were mainly associated with neuronal differentiation, neuronal maturation, and more specifically, retinal differentiation and maturation. The novel electrospinning PBG scaffold is beneficial for culturing iPSC-derived RGC progenitors as well as retinal organoids. Cells cultured on PBG scaffold differentiate effectively and shorten the process of RGC differentiation compared to that of cells cultured on coverslip. The new culture system may be helpful in future disease modeling, pharmacological screening, autologous transplantation, as well as narrowing the gap to clinical application.