MAGE-TAB Version 1.1 Investigation Title RNA-seq of transplanted pluripotent stem cells-derived human intestinal organoids upon mechanical lengthening and human jejunum tissues Experimental Design organism part comparison design stimulus or stress design Experimental Design Term Source REF EFO EFO Experimental Design Term Accession Number EFO_0001750 EFO_0001762 Experimental Factor Name developmental stage organism part stimulus Experimental Factor Type developmental stage organism part stimulus Experimental Factor Term Source REF EFO EFO Experimental Factor Term Accession Number EFO_0000399 EFO_0000635 Person Last Name Poling Mahe Person First Name Holly Maxime Person Mid Initials M M Person Email holly.poling@cchmc.org maxime.mahe@inserm.fr Person Phone 15136026899 Person Fax Person Affiliation Division of Pediatric General and Thoracic Surgery, Cincinnati Childrens Hospital Medical Center 1-Division of Pediatric General and Thoracic Surgery, Cincinnati Childrens Hospital Medical Center 2- Person Address Cincinnati Childrens Hospital Medical Center, Cincinnati, OH 45229 USA 1-Cincinnati Childrens Hospital Medical Center, Cincinnati, OH 45229 USA 2-Inserm 1235 Unit, 1 Rue Gaston Veil, 44035 Nantes Cedex 1, France Person Roles experiment performer investigator;submitter Person Roles Term Source REF Person Roles Term Accession Number Quality Control Type Quality Control Term Source REF Quality Control Term Accession Number Replicate Type Replicate Term Source REF Replicate Term Accession Number Normalization Type Normalization Term Source REF Normalization Term Accession Number Date of Experiment 2016-08-19 Public Release Date 2018-03-30 PubMed ID Publication DOI Publication Author List Publication Title Publication Status Publication Status Term Source REF Publication Status Term Accession Number Experiment Description Human Intestinal Organoids (HIOs) generated from embryonic and/or induced pluripotent stem cell lines offer an avenue to study both developmental and human specific disease states. Recently, progress has been made in scaling and maturing these inherently immature tissues through transplanting them in vivo. However, these resultant grafts best approximate fetal intestinal tissue thus limiting their utility. To induce growth and maturation of HIOs we used a nitinol spring device to mechanically induce enterogenesis of HIO in vivo. HIOs are cultured prior to implantation within the mesentery of immunodeficient mice. They are allowed to grow, vascularize, and mature before a second procedure is performed wherein a compressed nitinol spring is implanted within the lumen of the transplanted HIO (tHIO). Next Generation RNA sequencing was performed across transplanted samples as well as on human surgical samples to highlight the transcriptional similarities and differences between groups. Transcriptionally, the tHIO+S samples were more similar to human tissues than the tHIO. With these initial experiments, we concluded that the application of an intraluminal uniaxial force is a practical method to induce maturation of tHIOs in vivo without concomitant architectural disruptions. While our current system does lack certain complexities, we have demonstrated enterogenesis by means of mechanical manipulation. Protocol Name P-MTAB-67923 P-MTAB-67924 P-MTAB-67921 P-MTAB-67922 P-MTAB-67925 P-MTAB-67918 P-MTAB-67919 P-MTAB-67920 Protocol Type nucleic acid sequencing protocol high throughput sequence alignment protocol nucleic acid extraction protocol nucleic acid library construction protocol normalization data transformation protocol growth protocol treatment protocol sample collection protocol Protocol Term Source REF EFO EFO EFO EFO EFO EFO EFO EFO Protocol Term Accession Number EFO_0004170 EFO_0004917 EFO_0002944 EFO_0004184 EFO_0003816 EFO_0003789 EFO_0003969 EFO_0005518 Protocol Description Completed libraries were sequenced on an Illumina HiSeq2000, generating around 20 million high quality 50 base long reads per sample. High throughput sequence alignment protocol was based on the TopHat/Cufflinks pipeline. First, sequences were aligned to the reference genome (Hg19) with TopHat. Total RNA was extracted using an RNeasy Plus Micro Kit (Qiagen) according to manufacturer guidelines. The libraries were constructed according to manufacturer guidelines. (Illumina). Briefly, 350 to 900 ng of total RNA determined by Qbit high sensitivity spectrofluorometric measurement (Invitrogen) was poly-A selected and reverse transcribed using Illuminas TruSeq RNA library preparation kit V2. Each sample was fitted with a unique adapter containing a 6 base molecular barcode for high level multiplexing. Prior sequencing, a 12 cycles of PCR amplification was performed on the samples. Each sample was independently processed with Cufflinks in order to generate an initial transcriptome. We used the Cuffmerge tool to merge the private transcriptomes into a single reference, and at the same time annotated known genes and extended partial transcripts. This common transcriptome was used in a second pass with Cufflinks, which quantified each transcript and gene (known or novel) in each sample. The reference annotation used was based on the UCSC knownGenes table. Human Intestinal Organoids (HIOs) were generated and maintained as previously described in Spence, J.R., Mayhew, C.N., Rankin, S.A., Kuhar, M.F., Vallance, J.E., Tolle, K., Hoskins, E.E., Kalinichenko, V.V., Wells, S.I., Zorn, A.M., et al. (2011). Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105-109. Briefly, line H1 embryonic stem cells (WiCell Research Institute, Inc.) were grown in feeder-free conditions in Matrigel (BD Biosciences) coated six-well Nunclon surface plates (Nunc) and maintained in mTESR1 media (Stem Cell Technologies). For induction of definitive endoderm (DE), cells were passaged with Accutase (Stem Cell Technologies) and plated at a density of 65,000 cells per well in 24-well Nunc plates. Cells were allowed to grow in mTESR1 media for two days before treatment with 100 ng/ml of Activin A for three days as previously described. DE was then treated with hindgut induction medium (RPMI 1640, 100x NEAA, 2% dFCS,) for four days with 100 ng/ml FGF4 (R&D) and 3 M Chiron 99021 (Tocris) to induce formation of mid-hindgut spheroids. Spheroids were then plated in Growth Factor Reduced (GFR) Matrigel and maintained in intestinal growth medium (Advanced DMEM/F-12, N2 supplement, B27 supplement, 15 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin) supplemented with 100 ng/ml EGF (R&D) to generate human intestinal organoids (HIOs). Media was changed twice weekly thereafter. HIOs were replated in fresh Matrigel every 14 days. HIOs were prepared for transplantation as previously described in Watson, C.L., Mahe, M.M., Munera, J., Howell, J.C., Sundaram, N., Poling, H.M., Schweitzer, J.I., Vallance, J.E., Mayhew, C.N., Sun, Y., et al. (2014). An in vivo model of human small intestine using pluripotent stem cells. Nat Med 20, 1310-1314. Briefly, single matrigel embedded HIOs were transplanted into the mesentery of the mice. Mice were anesthetized with 2% inhaled isoflurane (Butler Schein), and the abdomen of the mouse was then shaved, and prepped in sterile fashion using isopropyl alcohol and povidine-iodine. A midline incision of approximately 2 cm was made in order for the intestine to be accessed. A small pocket was created in the mesentery and the HIO placed within. The skin was closed in a double layer and the mice were given a subcutaneous injection of Buprenex (0.05 mg/kg; Midwest Veterinary Supply) for pain management. Ten to twelve weeks following engraftment, the mice then underwent a secondary surgery with similar preparations. During this procedure, the engrafted tHIO was cut and briefly flushed before insertion of the gelatin capsule within the lumen of the tHIO. Mice were sacrificed and tissue harvested 14d postoperatively. Prior RNA extraction, total tissue collected in RLT Plus buffer were lysed using a FastPrep machine. Total tissue was collected in RLT Plus buffer (Quiagen) supplemented with 0.1% 2-Mercaptoethanol in FastPrep lysing Matrix D tubes. Protocol Parameters Protocol Hardware Illumina HiSeq 2000 Protocol Software TopHat Cufflinks/Cuffmerge Protocol Contact Term Source Name EFO ArrayExpress Term Source File http://www.ebi.ac.uk/efo/ http://www.ebi.ac.uk/arrayexpress/ Term Source Version 2.38 SDRF File E-MTAB-6017.sdrf.txt Comment [Submitted Name] RNA-seq of transplanted pluripotent stem cells-derived human intestinal organoids upon mechanical lengthening and human jejunum tissues Comment [SecondaryAccession] ERP104301 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119467-ERR2119468 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119461-ERR2119462 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119458-ERR2119459 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119464-ERR2119465 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119458-ERR2119458 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119462-ERR2119463 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119466-ERR2119467 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119465-ERR2119466 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119468-ERR2119468 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119459-ERR2119460 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119460-ERR2119461 Comment [SequenceDataURI] http://www.ebi.ac.uk/ena/data/view/ERR2119463-ERR2119464 Comment [AEExperimentType] RNA-seq of coding RNA Comment[ArrayExpressAccession] E-MTAB-6017 Comment [AEMINSEQEScore] 4