Transcriptional landscape changes during human embryonic stem cell derivation

Molecular Human Reproduction, Vol.24, No.11 pp. 543–555, 2018 Advanced Access publication on September 17, 2018 doi:10.1093/molehr/gay039 ORIGINAL ARTICLE Transcriptional landscape changes during human embryonic stem cell derivation 1 Ghent-Fertility and Stem cell Team (G-FaST), Department for Reproductive Medicine, Ghent University Hospital, 9000 Ghent, Belgium Laboratory for Pharmaceutical Biotechnology, Faculty of Pharmaceutical Sciences, Ghent University, 9000 Ghent, Belgium 3Nuffield Department of Clinical Neurosciences, John Radcliffe Hospital, University of Oxford, Oxford, OX3 9DS, UK 4Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium 5Department of Nutrition, Genetics and Ethology, Faculty of Veterinary Medicine, Ghent University, 9820 Merelbeke, Belgium 6Department of Anatomy and Embryology, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands 2 *Correspondence address. E-mail: orcid.org/0000-0003-0630-8420 Submitted on January 31, 2018; resubmitted on August 22, 2018; editorial decision on September 4, 2018; accepted on September 14, 2018 STUDY QUESTION: What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? SUMMARY ANSWER: We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. WHAT IS KNOWN ALREADY: It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. STUDY DESIGN, SIZE, DURATION: To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC trans- ition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). MAIN RESULTS AND THE ROLE OF CHANCE: Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI © The Author(s) 2018. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: S. Warrier 1, J. Taelman1, L. Tilleman 2, M. Van der Jeught1, G. Duggal1,3, S. Lierman1, M. Popovic1, A. Van Soom4, L. Peelman5, F. Van Nieuwerburgh2, D. Deforce2, S.M. Chuva de Sousa Lopes1,6, P. De Sutter1, and B. Heindryckx 1,* 544 Warrier et al. also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. LARGE-SCALE DATA: Gene Expression Omnibus (GEO) Accession number GSE119378. LIMITATIONS, REASONS FOR CAUTION: Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. STUDY FUNDING/COMPETING INTEREST(S): This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest. Key words: inner cell mass / post-inner cell mass-intermediate stage / primed / naïve / pluripotency / signaling / transcriptional profiling Introduction The in vitro derivation of human embryonic stem cells (hESCs) from the inner cell mass (ICM) is preceded by an epiblast-like intermediate stage called the ‘post-inner cell mass-intermediate’, or PICMI (O’Leary et al., 2012). The derivation rate of PICMIs is predictive for hESCs isolation efficiency (O’Leary et al., 2011), which has been reported to be around 12% (O’Leary et al., 2011). The PICMI also has a mixed expression profile of both early- and late-epiblast markers after analysis of a limited number of genes, suggestive of inducing a primed state of pluripotency in hESCs (O’Leary et al., 2012; Van der Jeught et al., 2015). Despite these findings, the current knowledge of transcriptional changes during hES (...truncated)