Chemically defined and xeno-free culture condition for human extended pluripotent stem cells

Extended pluripotent stem (EPS) cells have shown great applicative potentials in generating synthetic embryos, directed differentiation and disease modeling. However, the lack of a xeno-free culture condition has significantly limited their applications. Here, we report a chemically defined and xeno-free culture system for culturing and deriving human EPS cells in vitro. Xeno-free human EPS cells can be long-term and genetically stably maintained in vitro, as well as preserve their embryonic and extraembryonic developmental potentials. Furthermore, the xeno-free culturing system also permits efficient derivation of human EPS cells from human fibroblast through reprogramming. Our study could have broad utility in future applications of human EPS cells in biomedicine.

The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
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Data analysis
For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub R v3.6.1 and Seurat3 was used to analyze the sequencing data,R package clusterProfiler and ComplexHeatmap were used for GSEA analysis . DESeq2 and g:Profiler bioinformatics tool were used for transcriptome analysis.Graphpad Prism 8 was used to analyze statistical data and draw graphs in this study. iMaris 9.7 was used to export the Dragonfly data. Flowjo V10 was used to analyze the flow cytometry data. Bowtie2 version 2.3.5,samtools version 1.10,MACS2-2.2.6,Homer version 4.10.3, clusterProfiler,deepTools-3.5.0 were used to analyze ATAC sequencing data.Genome Analysis Toolkit version 4, Control-FREEC,Fastqc and Trimmomatic-0.39,Picard-tools were used to analyze whole genome sequencing data.

October 2018
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability Field-specific reporting Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection. We independently performed all experiments at least 3 times, allowing to judge the degree of variability between replicates. The sample sizes and number of repeats are defined in each figure legends.
No data was excluded from the analysis.
All experiments prsented here been repeated several times with similar results, the number of repeated experiments is indicated in the figure legends.
For chimeric experiments, mice and embryos were randomly distributed amongst groups at the start of each experiment. For in vitro experiments using cells, cells were randomly assigned to either experiment or control groups.
The investigators were blinded to allocation during experiments and outcome assessment.
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MRI-based neuroimaging
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Describe any disturbance caused by the study and how it was minimized. Antibodies were validated according to manufacturer's instruction.
Human EPS cell lines H1-EPS, ES1-EPS and ES2-EPS were published previously in reference 6. Human ES cell lines H1 was obtained from WiCell and authenticated by karyotype analysis. Human iPSC line was estabilished in our laboratory.OT H1-EPS, iPS-EPS cell lines were established in this study.
H1 was obtained directly from the WiCell.All the cell lines used in the paper were authenticated by karyotyping.
All cultured cells were tested negative for mycoplasma contamination by PCR.
No commonly misidentified lines were used in this study.
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Mice was used in this study. The immunodeficient NPG mice were 6-8 weeks old. The male and female ICR mice were 6-8 weeks old. The mice were housed with a 12 hr light/dark cycle between 06:00 and 18:00 in a temperature controlled room (22 ± 1°C) with free access to water and food.
This study did not involve wild animals.
This study did not involve field-collected samples.
The Ethics Committee of Peking University Health Science Center approved the protocols used in this study.
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