IPSC to endoderm differentiation experiments from the HipSci project
Ontology highlight
ABSTRACT: "Cultures of induced pluripotent stem cells (iPSC) from the HipSci project were differentiated to the endoderm lineage. The iPSC lines were differentiated i) independently as separate cultures, ii) as a co-culture comprising a mixture of cell lines. At various time points, live cells were FACS sorted from these bulk cultures, plated, and frozen. Each sorted cell taken from the mixed co-culture was later identified by its genetics using RNA sequencing."
Project description:For pluripotent stem cell (PSC)-based regenerative therapy against diabetes, the differentiation efficiency to pancreatic lineage cells needs to be improved based on the mechanistic understanding of pancreatic differentiation. Here, we aimed to elucidate the molecular mechanisms underlying pancreatic endoderm differentiation by searching for factors that regulate a crucial pancreatic endoderm marker gene, NKX6.1. Unbiasedly screening an siRNA knockdown library, we identified a candidate transcription factor, HHEX. HHEX knockdown suppressed the expression of another pancreatic endoderm marker gene, PTF1A, as well as NKX6.1, independently of PDX1, a known regulator of NKX6.1 expression. In contrast, the overexpression of HHEX upregulated the expressions of NKX6.1 and PTF1A. RNA-seq analysis showed decreased expressions of several genes related to pancreatic development, such as NKX6.1, PTF1A, ONECUT1 and ONECUT3, in HHEX knockdown pancreatic endoderm. These results suggest that HHEX plays a key role in pancreatic endoderm differentiation.
Project description:In addition to cooperatively driving transcriptional programs, emerging evidence supports transcription factors interacting with one another to modulate the outcome of binding events. As such, transcription factor interactions fine-tune the unique gene expression profiles required for developmental progression. Using human-induced pluripotent stem cells as a model of human endoderm lineage commitment, we reveal that GATA6 transiently co-localizes with EOMES at regions associated with non-endodermal lineages and is required for the repression of chromatin opening at these loci. Our results indicate that GATA6-dependent repression of chromatin remodeling, which is potentially mediated via the recruitment of NCOR1 to the EOMES interactome, contributes to definitive endoderm commitment. We anticipate that similar mechanisms are common during human development, furthering our understanding of the complex mechanisms that define cell fate decisions.
Project description:The endoderm, differentiated from human induced pluripotent stem cells (iPSCs), can differentiate into the small intestine and liver, which are vital for drug absorption and metabolism. The development of human iPSC-derived enterocytes (HiEnts) and hepatocytes (HiHeps) has been reported. However, pharmacokinetic function-deficiency of these cells remains to be elucidated. Here, we aimed to develop an efficient differentiation method to induce endoderm formation from human iPSCs. Cells treated with activin A for 168 h expressed higher levels of endodermal genes than those treated for 72 h. Using activin A (days 0-7), CHIR99021 and PI-103 (days 0-2), and FGF2 (days 3-7), the hiPSC-derived endoderm (HiEnd) showed 97.97% CD-117 and CD-184 double-positive cells. Moreover, HiEnts derived from the human iPSC line Windy had similar or higher expression of small intestine-specific genes than adult human small intestine. Activities of the drug transporter P-glycoprotein and drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5 were confirmed. Additionally, Windy-derived HiHeps expressed higher levels of hepatocyte- and pharmacokinetics-related genes and proteins and showed higher CYP3A4/5 activity than those derived through the conventional differentiation method. Thus, using this novel method, the differentiated HiEnts and HiHeps with pharmacokinetic functions could be used for drug development.
Project description:Experiment was designed to compare induction of human iPS Cells to endoderm and then to hepatocytes using our novel protocol with and without doxycycline. The endpoints where to understand if there were major differences at endoderm or after complete differentiation to hepatocytes with and without doxycycline.
Project description:Human induced pluripotent stem cells (hiPSCs) can serve as an unlimited source to rebuild organotypic tissues in vitro. Successful engineering of functional cell types and complex organ structures outside the human body requires knowledge of the chemical, temporal, and spatial microenvironment of their in vivo counterparts. Despite an increased understanding of mouse and human embryonic development, screening approaches are still required for the optimization of stem cell differentiation protocols to gain more functional mature cell types. The liver, lung, pancreas, and digestive tract originate from the endoderm germ layer. Optimization and specification of the earliest differentiation step, which is the definitive endoderm (DE), is of central importance for generating cell types of these organs because off-target cell types will propagate during month-long cultivation steps and reduce yields. Here, we developed a microfluidic large-scale integration (mLSI) chip platform for combined automated three-dimensional (3D) cell culturing and high-throughput imaging to investigate anterior/posterior patterns occurring during hiPSC differentiation into DE cells. Integration of 3D cell cultures with a diameter of 150 μm was achieved using a U-shaped pneumatic membrane valve, which was geometrically optimized and fluidically characterized. Upon parallelization of 32 fluidically individually addressable cell culture unit cells with a total of 128 3D cell cultures, complex and long-term DE differentiation protocols could be automated. Real-time bright-field imaging was used to analyze cell growth during DE differentiation, and immunofluorescence imaging on optically cleared 3D cell cultures was used to determine the DE differentiation yield. By systematically alternating transforming growth factor β (TGF-β) and WNT signaling agonist concentrations and temporal stimulation, we showed that even under similar DE differentiation yields, there were patterning differences in the 3D cell cultures, indicating possible differentiation differences between established DE protocols. The automated mLSI chip platform with the general analytical workflow for 3D stem cell cultures offers the optimization of in vitro generation of various cell types for cell replacement therapies.
Project description:To investigate the role of HHEX in regulating human pancreatic endoderm differentiation, we knocked down the HHEX gene in 585A1 cell line-derived pancreatic endoderm cells by siRNA. We then performed gene expression profiling analysis using data obtained from RNA-seq of the 3 cell samples.
Project description:Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers, PAX6 and NESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers GFAP and aquaporin 4 (AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.
Project description:Quantitative analysis of human induced pluripotent stem cell (iPSC) lines from healthy donors is a powerful tool for uncovering the relationship between genetic variants and cellular behavior. We previously identified rare, deleterious non-synonymous single nucleotide variants (nsSNVs) in cell adhesion genes that are associated with outlier iPSC phenotypes in the pluripotent state. Here, we generated micropatterned colonies of iPSCs to test whether nsSNVs influence patterning of radially ordered germ layers. Using a custom-built image analysis pipeline, we quantified the differentiation phenotypes of 13 iPSC lines that harbor nsSNVs in genes related to cell adhesion or germ layer development. All iPSC lines differentiated into the three germ layers; however, there was donor-specific variation in germ layer patterning. We identified one line that presented an outlier phenotype of expanded endodermal differentiation, which was associated with a nsSNV in ITGB1. Our study establishes a platform for investigating the impact of nsSNVs on differentiation.
Project description:The generation of induced pluripotent stem cells (iPSCs) provides a novel method to facilitate investigations into the mechanisms that control stem cell pluripotency and self-renewal. Myc has previously been shown to be critical for murine embryonic stem cell (mESC) maintenance, while also enhancing directed reprogramming of fibroblasts by effecting widespread changes in gene expression. Despite several studies identifying in vivo target genes, the precise mechanism by which Myc regulates pluripotency remains unknown. Here we report that codeletion of c- and N-MYC in iPSCs and ESCs results in their spontaneous differentiation to primitive endoderm. We show that Myc sustains pluripotency through repression of the primitive endoderm master regulator GATA6, while also contributing to cell cycle control by regulation of the mir-17-92 miRNA cluster. Our findings demonstrate the indispensable requirement for c- or N-myc in pluripotency beyond proliferative and metabolic control.
Project description:Integrins of the beta1 subfamily are highly expressed in the early mouse embryo and are essential for the formation of primitive germ layers from the inner cell mass (ICM). We investigated the mechanisms by which alphabeta1 integrins regulate ICM morphogenesis by using the embryonic-stem-cell-derived embryoid body (EB), a model for peri-implantation development. Ablation of integrin beta1 in EBs resulted in endoderm detachment and in maturation defects, which were manifested by the mislocalization of GATA4 in the cytoplasm and the markedly reduced synthesis of basement membrane (BM) components and the lineage marker disabled homolog 2. The mutant endoderm cells failed to spread on BM substrates, but could spread on vitronectin, which induced upregulation of alphavbeta3 integrin and integrin-dependent GATA4 nuclear translocation. Forced expression of integrin beta3 in the mutant EBs completely rescued endoderm morphogenesis, suggesting that integrin beta3 can substitute for integrin beta1 in the endoderm. Furthermore, the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 (ERK1/2) and p38 were activated in endoderm in an integrin-dependent fashion. Pharmacological inhibition of ERK1/2 or p38 MAPK blocked vitronectin-induced GATA4 nuclear translocation and endoderm maturation, whereas expression of a constitutively active ERK kinase (MEK1) or p38 MAPK in the mutant cells rescued endoderm maturation in integrin-beta1-null endoderm cells. Collectively, these results suggest that integrins are required for both the stable adhesion and maturation of visceral endoderm, the latter being mediated through the activation of ERK1/2 and p38 MAPK.