scholarly journals High-Throughput Functional Analysis of CFTR and Other Apically Localized Proteins in iPSC-Derived Human Intestinal Organoids

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3419
Author(s):  
Sunny Xia ◽  
Zoltán Bozóky ◽  
Michelle Di Paola ◽  
Onofrio Laselva ◽  
Saumel Ahmadi ◽  
...  
Keyword(s):  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Channel Activity ◽  
Mutant Proteins ◽  
Pharmacological Modulation ◽  
Intestinal Organoids ◽  
Relevant Context ◽  
Induced Pluripotent

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis (CF)-causing mutant proteins. However, to date, CF-iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease-relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. Specifically, we show how this assay can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This phenotypic platform promises to expand CF therapy discovery to include strategies that target multiple determinants of epithelial fluid transport.

scholarly journals High-throughput functional analysis of CFTR and other apically localized channels in iPSC derived intestinal organoids

2021 ◽  
Author(s):  
Sunny Xia ◽  
Zoltan Bozoky ◽  
Onofrio Laselva ◽  
Michelle DiPaola ◽  
Saumel Ahmadi ◽  
...  
Keyword(s):  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Fluid Transport ◽  
Apical Membrane ◽  
Proof Of Concept ◽  
Channel Activity ◽  
Mutant Proteins ◽  
Intestinal Organoids ◽  
Relevant Context ◽  
Induced Pluripotent

Induced Pluripotent Stem Cells (iPSCs) can be differentiated into epithelial organoids that recapitulate the relevant context for CFTR and enable testing of therapies targeting Cystic Fibrosis-causing mutant proteins. However, to date, CF- iPSC-derived organoids have only been used to study pharmacological modulation of mutant CFTR channel activity and not the activity of other disease relevant membrane protein constituents. In the current work, we describe a high-throughput, fluorescence-based assay of CFTR channel activity in iPSC-derived intestinal organoids and describe how this method can be adapted to study other apical membrane proteins. In these proof-of-concept studies, we show how this fluorescence-based assay of apical membrane potential can be employed to study CFTR and ENaC channels and an electrogenic acid transporter in the same iPSC-derived intestinal tissue. This multiparameter phenotypic platform promises to expand CF therapy discovery to include strategies to target multiple determinants of epithelial fluid transport.

Automated, high-throughput derivation, characterization and differentiation of induced pluripotent stem cells

2015 ◽  
Vol 12 (9) ◽  
pp. 885-892 ◽  
Author(s):  
Daniel Paull ◽  
Ana Sevilla ◽  
Hongyan Zhou ◽  
Aana Kim Hahn ◽  
Hesed Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

scholarly journals Multiplex High-Throughput Targeted Proteomic Assay To Identify Induced Pluripotent Stem Cells

2017 ◽  
Vol 89 (4) ◽  
pp. 2440-2448 ◽  
Author(s):  
Anna Baud ◽  
Frank Wessely ◽  
Francesca Mazzacuva ◽  
James McCormick ◽  
Stephane Camuzeaux ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent

scholarly journals High-throughput propagation of human prostate tissue from induced-pluripotent stem cells

2019 ◽  
Author(s):  
AC Hepburn ◽  
EL Curry ◽  
M Moad ◽  
RE Steele ◽  
OE Franco ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Neuroendocrine Cells ◽  
Prostate Tissue ◽  
Urogenital Sinus ◽  
Human Prostate ◽  
Human Prostate Tissue ◽  
Induced Pluripotent

AbstractPrimary culture of human prostate organoids is slow, inefficient and laborious. To overcome this, we demonstrate a new high-throughput model where rapidly proliferating and easily handled induced pluripotent stem cells, for the first time, enable generation of human prostate tissue in vivo and in vitro. Using a co-culture technique with urogenital sinus mesenchyme, we recapitulated the in situ prostate histology, including the stromal compartment and the full spectrum of epithelial differentiation. This approach overcomes major limitations in primary cultures of human prostate stem, luminal and neuroendocrine cells, as well as the stromal microenvironment. These models provide new opportunities to study prostate development, homeostasis and disease.

scholarly journals Interaction of Salmonella enterica Serovar Typhimurium with Intestinal Organoids Derived from Human Induced Pluripotent Stem Cells

2015 ◽  
Vol 83 (7) ◽  
pp. 2926-2934 ◽  
Author(s):  
Jessica L. Forbester ◽  
David Goulding ◽  
Ludovic Vallier ◽  
Nicholas Hannan ◽  
Christine Hale ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Salmonella Enterica ◽  
Salmonella Enterica Serovar Typhimurium ◽  
Enteric Pathogens ◽  
Content Type ◽  
Intestinal Organoids ◽  
Serovar Typhimurium ◽  
Induced Pluripotent

The intestinal mucosa forms the first line of defense against infections mediated by enteric pathogens such as salmonellae. Here we exploited intestinal “organoids” (iHOs) generated from human induced pluripotent stem cells (hIPSCs) to explore the interaction ofSalmonella entericaserovar Typhimurium with iHOs. Imaging and RNA sequencing were used to analyze these interactions, and clear changes in transcriptional signatures were detected, including altered patterns of cytokine expression after the exposure of iHOs to bacteria.S. Typhimurium microinjected into the lumen of iHOs was able to invade the epithelial barrier, with many bacteria residing withinSalmonella-containing vacuoles. AnS. TyphimuriuminvAmutant defective in theSalmonellapathogenicity island 1 invasion apparatus was less capable of invading the iHO epithelium. Hence, we provide evidence that hIPSC-derived organoids are a promising model of the intestinal epithelium for assessing interactions with enteric pathogens.

scholarly journals Single-cell phenotyping of human induced pluripotent stem cells by high-throughput imaging

2015 ◽  
Author(s):  
Hans Christian Volz ◽  
Florian Heigwer ◽  
Tatjana Wuest ◽  
Marta Galach ◽  
Jochen Utikal ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Single Cell ◽  
High Throughput ◽  
Pluripotent Stem Cells ◽  
Single Cells ◽  
Phenotypic Variability ◽  
Screening Experiments ◽  
Induced Pluripotent ◽  
High Throughput Imaging

Single-cell phenotyping promises to yield insights into biological responses in heterogeneous cell populations. We developed a method based on single-cell analysis to phenotype human induced pluripotent stem cells (hIPSC) by high-throughput imaging. Our method uses markers for morphology and pluripotency as well as social features to characterize perturbations using a meta-phenotype based on mapping single cells to distinct phenotypic classes. Analysis of perturbations on a single cell level enhances the applicability of human induced pluripotent stem cells (hIPSC) for screening experiments taking the inherently increased phenotypic variability of these cells into account. We adapted miniaturized culture conditions to allow for the utilization of hIPSC in RNA interference (RNAi) high-throughput screens and single cell phenotyping by image analysis. We identified key regulators of pluripotency in hIPSC masked in a population-averaged analysis and we confirmed several candidate genes (SMG1, TAF1) and assessed their effect on pluripotency.

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