N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm.
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ABSTRACT:
SUBMITTER: Mallanna SK
PROVIDER: S-EPMC5340625 | biostudies-literature | 2017 Mar
REPOSITORIES: biostudies-literature
Publications
N-glycoprotein surfaceome of human induced pluripotent stem cell derived hepatic endoderm.
Mallanna Sunil K SK Waas Matthew M Duncan Stephen A SA Gundry Rebekah L RL
Proteomics 20170201 5
Using cell surface capture technology, the cell surface N-glycoproteome of human-induced pluripotent stem cell derived hepatic endoderm cells was assessed. Altogether, 395 cell surface N-glycoproteins were identified, represented by 1273 N-glycopeptides. This study identified N-glycoproteins that are not predicted to be localized to the cell surface and provides experimental data that assist in resolving ambiguous or incorrectly annotated transmembrane topology annotations. In a proof-of-concept ...[more]
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Project description:UnlabelledWith the advent of induced pluripotent stem cell (iPSC) technology, it is now feasible to generate iPSCs with a defined genotype or disease state. When coupled with direct differentiation to a defined lineage, such as hepatic endoderm (HE), iPSCs would revolutionize the way we study human liver biology and generate efficient "off the shelf" models of human liver disease. Here, we show the "proof of concept" that iPSC lines representing both male and female sexes and two ethnic origins can be differentiated to HE at efficiencies of between 70%-90%, using a method mimicking physiological relevant condition. The iPSC-derived HE exhibited hepatic morphology and expressed the hepatic markers albumin and E-cadherin, as assessed by immunohistochemistry. They also expressed alpha-fetoprotein, hepatocyte nuclear factor-4a, and a metabolic marker, cytochrome P450 7A1 (Cyp7A1), demonstrating a definitive endodermal lineage differentiation. Furthermore, iPSC-derived hepatocytes produced and secreted the plasma proteins, fibrinogen, fibronectin, transthyretin, and alpha-fetoprotein, an essential feature for functional HE. Additionally iPSC-derived HE supported both CYP1A2 and CYP3A4 metabolism, which is essential for drug and toxicology testing.ConclusionThis work is first to demonstrate the efficient generation of hepatic endodermal lineage from human iPSCs that exhibits key attributes of hepatocytes, and the potential application of iPSC-derived HE in studying human liver biology. In particular, iPSCs from individuals representing highly polymorphic variants in metabolic genes and different ethnic groups will provide pharmaceutical development and toxicology studies a unique opportunity to revolutionize predictive drug toxicology assays and allow the creation of in vitro hepatic disease models.
| S-EPMC2799548 | biostudies-literature
Project description:UnlabelledRecent advances in induced pluripotent stem (iPS) cell research have significantly changed our perspective on regenerative medicine. Patient-specific iPS cells have been derived not only for disease modeling but also as sources for cell replacement therapy. However, there have been insufficient data to prove that iPS cells are functionally equivalent to human embryonic stem (hES) cells or are safer than hES cells. There are several important issues that need to be addressed, and foremost are the safety and efficacy of human iPS cells of different origins. Human iPS cells have been derived mostly from cells originating from mesoderm and in a few cases from ectoderm. So far, there has been no report of endoderm-derived human iPS cells, and this has prevented comprehensive comparative investigations of the quality of human iPS cells of different origins. Here we show for the first time reprogramming of human endoderm-derived cells (i.e., primary hepatocytes) to pluripotency. Hepatocyte-derived iPS cells appear indistinguishable from hES cells with respect to colony morphology, growth properties, expression of pluripotency-associated transcription factors and surface markers, and differentiation potential in embryoid body formation and teratoma assays. In addition, these cells are able to directly differentiate into definitive endoderm, hepatic progenitors, and mature hepatocytes.ConclusionThe technology to develop endoderm-derived human iPS cell lines, together with other established cell lines, will provide a foundation for elucidating the mechanisms of cellular reprogramming and for studying the safety and efficacy of differentially originated human iPS cells for cell therapy. For the study of liver disease pathogenesis, this technology also provides a potentially more amenable system for generating liver disease-specific iPS cells.
| S-EPMC2925460 | biostudies-literature
Project description:Cell surface capture technology data from human pluripotent stem cell derived hepatic endoderm at day 10 of differentiation
2016-10-03 | MSV000080215 | MassIVE
Project description:The human developmental processes during the early post-implantation stage instruct the specification and organization of the lineage progenitors into a body plan. These processes, which include patterning, cell sorting, and establishment of the three germ layers, have been classically studied in non-human model organisms and only recently, through micropatterning technology, in a human-specific context. Micropatterning technology has unveiled mechanisms during patterning and germ layer specification; however, cell sorting and their segregation in specific germ layer combinations have not been investigated yet in a human-specific in vitro system. Here, we developed an in vitro model of human ectodermal patterning, in which human pluripotent stem cells (hPSCs) self-organize to form a radially regionalized neural and non-central nervous system (CNS) ectoderm. We showed that by using micropatterning technology and by modulating BMP and WNT signals, we can regulate the appearance and spatial distribution of the different ectodermal populations. This pre-patterned ectoderm can be used to investigate the cell sorting behavior of hPSC-derived meso-endoderm cells, with an endoderm that segregates from the neural ectoderm. Thus, the combination of micro-technology with germ layer cross-mixing enables the study of cell sorting of different germ layers in a human context.
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Project description:Progression of fibrosis and the development of cirrhosis are responsible for the liver related morbidity and mortality associated with chronic liver diseases. There is currently a great unmet need for effective anti-fibrotic strategies. Stem cells play a central role in wound healing responses to restore liver homeostasis following injury. Here we tested the hypothesis that extracellular vesicles (EVs) isolated from induced pluripotent stem cells (iPSC) modulate hepatic stellate cell (HSCs) activation and may have anti-fibrotic effects. Human iPSCs were generated by reprogramming primary skin fibroblasts. EVs were isolated by differential centrifugation, quantified by flow cytometry (FACS) and characterized by dynamic light scattering (DLS) and electron microscopy (TEM). Primary human HSCs were activated with TGFβ (10 ng/mL) and exposed to iPSC-EVs. Efficacy of iPSC-EVs was tested on HSC in vitro and in two murine models of liver injury (CCl4 and bile duct ligation). Characterization of iPSC-derived EVs by flow cytometry identified a large population of EVs released by iPSC, primarily with a diameter of 300 nm and that could be visualized by TEM as round, cup-shaped objects. Fluorescent tracing assays detected iPSC-EVs in HSC cytosol after a short incubation and EV uptake by HSCs resulted in both decrease of pro-fibrogenic markers αSMA, CollagenIα1, Fibronectin and TIMP-1 and HSC pro-fibrogenic responses such as chemotaxis and proliferation. Genomics analyses of iPSC-EV miRNA cargo revealed 22 highly expressed miRNAs, among which miR-92a-3p resulted the most abundant. Transcriptome analysis identified 60 genes down-modulated and 235 up-regulated in TGF-β-primed HSC in presence or absence of iPSC-EVs. Intravenous injection of iPSC-EVs in CCl4 and bile duct ligation-induced liver fibrosis resulted in anti-fibrotic effects at protein and gene levels. Results of this study identify iPSC-EVs as a novel anti-fibrotic approach that may reduce or reverse liver fibrosis in patients with chronic liver disease.
| S-EPMC6675559 | biostudies-literature
Project description:The crosstalk between cells is important for differentiation of cells. Murine-derived feeder cells, SNL76/7 feeder cells (SNLs) or mouse primary embryonic fibroblast feeder cells (MEFs) are widely used for culturing undifferentiated human induced pluripotent stem cells (hiPSCs). It is still unclear whether different culture conditions affect the induction efficiency of definitive endoderm (DE) differentiation from hiPSCs. Here we show that the efficiency of DE differentiation from hiPSCs cultured on MEFs was higher than that of hiPSCs cultured on SNLs. The qPCR, immunofluorescent and flow cytometry analyses revealed that the expression levels of mRNA and/or proteins of the DE marker genes, SOX17, FOXA2 and CXCR4, in DE cells differentiated from hiPSCs cultured on MEFs were significantly higher than those cultured on SNLs. Comprehensive RNA sequencing and molecular network analyses showed the alteration of the gene expression and the signal transduction of hiPSCs cultured on SNLs and MEFs. Interestingly, the expression of non-coding hXIST exon 4 was up-regulated in hiPSCs cultured on MEFs, in comparison to that in hiPSCs cultured on SNLs. By qPCR analysis, the mRNA expression of undifferentiated stem cell markers KLF4, KLF5, OCT3/4, SOX2, NANOG, UTF1, and GRB7 were lower, while that of hXIST exon 4, LEFTY1, and LEFTY2 was higher in hiPSCs cultured on MEFs than in those cultured on SNLs. Taken together, our finding indicated that differences in murine-feeder cells used for maintenance of the undifferentiated state alter the expression of pluripotency-related genes in hiPSCs by the signaling pathways and affect DE differentiation from hiPSCs, suggesting that the feeder cells can potentiate hiPSCs for DE differentiation.
| S-EPMC6062072 | biostudies-literature
Project description:Pluripotent stem cells present an extraordinary powerful tool to investigate embryonic development in humans. Essentially, they provide a unique platform for dissecting the distinct mechanisms underlying pluripotency and subsequent lineage commitment. Modest information currently exists about the expression and the role of ion channels during human embryogenesis, organ development, and cell fate determination. Of note, small and intermediate conductance, calcium-activated potassium channels have been reported to modify stem cell behaviour and differentiation. These channels are broadly expressed throughout human tissues and are involved in various cellular processes, such as the after-hyperpolarization in excitable cells, and also in differentiation processes. To this end, human induced pluripotent stem cells (hiPSCs) generated from plucked human hair keratinocytes have been exploited in vitro to recapitulate endoderm formation and, concomitantly, used to map the expression of the SK channel (SKCa) subtypes over time. Thus, we report the successful generation of definitive endoderm from hiPSCs of ectodermal origin using a highly reproducible and robust differentiation system. Furthermore, we provide the first evidence that SKCas subtypes are dynamically regulated in the transition from a pluripotent stem cell to a more lineage restricted, endodermal progeny.
| S-EPMC3654369 | biostudies-literature
Project description:Hepatic stem/progenitor cells in liver development have a high proliferative potential and the ability to differentiate into both hepatocytes and cholangiocytes. In this study, we focused on the cell surface molecules of human induced pluripotent stem (iPS) cell-derived hepatic progenitor-like cells (HPCs) and analyzed how these molecules modulate expansion of these cells. Human iPS cells were differentiated into immature hepatic lineage cells by cytokines. In addition to hepatic progenitor markers (CD13 and CD133), the cells were coimmunostained for various cell surface markers (116 types). The cells were analyzed by flow cytometry and in vitro colony formation culture with feeder cells. Twenty types of cell surface molecules were highly expressed in CD13(+)CD133(+) cells derived from human iPS cells. Of these molecules, CD221 (insulin-like growth factor receptor), which was expressed in CD13(+)CD133(+) cells, was quickly downregulated after in vitro expansion. The proliferative ability was suppressed by a neutralizing antibody and specific inhibitor of CD221. Overexpression of CD221 increased colony-forming ability. We also found that inhibition of CD340 (erbB2) and CD266 (fibroblast growth factor-inducible 14) signals suppressed proliferation. In addition, both insulin-like growth factor (a ligand of CD221) and tumor necrosis factor-like weak inducer of apoptosis (a ligand of CD266) were provided by feeder cells in our culture system. This study revealed the expression profiles of cell surface molecules in human iPS cell-derived HPCs and that the paracrine interactions between HPCs and other cells through specific receptors are important for proliferation.
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Project description:A human cell-based liver model capable of long-term expansion and mature hepatic function is a fundamental requirement for pre-clinical drug development. We previously established self-renewing and functionally mature human pluripotent stem cell-derived liver organoids as an alternate to primary human hepatocytes. In this study, we tested long-term prolonged culture of organoids to increase their maturity. Organoid growing at the edge of Matrigel started to deteriorate two weeks after culturing, and the expression levels of the functional mature hepatocyte marker ALB were decreased at four weeks of culture. Replating the organoids weekly at a 1：2 ratio in fresh Matrigel, resulted in healthier morphology with a thicker layer compared to organoids maintained on the same Matrigel and significantly increased ALB expression until three weeks, although, it decreased sharply at four weeks. The levels of the fetal hepatocyte marker AFP were considerably increased in long-term cultures of organoids. Therefore, we performed serial passaging of organoids, whereby they were mechanically split weekly at a 1：3∼1：5 ratio in fresh Matrigel. The organoids expanded so far over passage 55, or 1 year, without growth retardation and maintained a normal karyotype after long-term cryopreservation. Differentiation potentials were maintained or increased after long-term passaging, while AFP expression considerably decreased after passaging. Therefore, these data demonstrate that organoids can be exponentially expanded by serial passaging, while maintaining long-term functional maturation potential. Thus, hepatic organoids can be a practical and renewable cell source for human cell-based and personalized 3D liver models.
| S-EPMC7378903 | biostudies-literature
Project description:BackgroundThe use of a personalized liver organoid derived from human-induced pluripotent stem cells (HuiPSCs) is advancing the use of in vitro disease models for the design of specific, effective therapies for individuals. Collecting patient peripheral blood cells for HuiPSC generation is preferable because it is less invasive; however, the capability of blood cell-derived HuiPSCs for hepatic differentiation and liver organoid formation remains uncertain. Moreover, the currently available methods for liver organoid formation require a multistep process of cell differentiation or a combination of hepatic endodermal, endothelial and mesenchymal cells, which is a major hurdle for the application of personalized liver organoids in high-throughput testing of drug toxicity and safety. To demonstrate the capability of blood cell-derived HuiPSCs for liver organoid formation without support from endothelial and mesenchymal cells.MethodsThe peripheral blood-derived HuiPSCs first differentiated into hepatic endoderm (HE) in two-dimensional (2D) culture on Matrigel-coated plates under hypoxia for 10 days. The HE was then collected and cultured in 3D culture using 50% Matrigel under ambient oxygen. The maturation of hepatocytes was further induced by adding hepatocyte growth medium containing HGF and oncostatin M on top of the 3D culture and incubating the culture for an additional 12-17 days. The function of the liver organoids was assessed using expression analysis of hepatocyte-specific gene and proteins. Albumin (ALB) synthesis, glycogen and lipid storage, and metabolism of indocyanine were evaluated. The spatial distribution of albumin was examined using immunofluorescence and confocal microscopy.ResultsCD34+ hematopoietic cell-derived HuiPSCs were capable of differentiating into definitive endoderm expressing SOX17 and FOXA2, hepatic endoderm expressing FOXA2, hepatoblasts expressing AFP and hepatocytes expressing ALB. On day 25 of the 2D culture, cells expressed SOX17, FOXA2, AFP and ALB, indicating the presence of cellular heterogeneity. In contrast, the hepatic endoderm spontaneously formed a spherical, hollow structure in a 3D culture of 50% Matrigel, whereas hepatoblasts and hepatocytes could not form. Microscopic observation showed a single layer of polygonal-shaped cells arranged in a 3D structure. The hepatic endoderm-derived organoid synthesis ALB at a higher level than the 2D culture but did not express definitive endoderm-specific SOX17, indicating the greater maturity of the hepatocytes in the liver organoids. Confocal microscopic images and quantitative ELISA confirmed albumin synthesis in the cytoplasm of the liver organoid and its secretion. Overall, 3D culture of the hepatic endoderm is a relatively fast, simple, and less laborious way to generate liver organoids from HuiPSCs that is more physiologically relevant than 2D culture.
| S-EPMC7580584 | biostudies-literature