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Generation of Isthmic Organizer-Like Cells from Human Embryonic Stem Cells

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Generation of Isthmic Organizer-Like Cells from Human Embryonic Stem Cells Molecules and Cells Minireview Generation of Isthmic Organizer-Like Cells from Human Embryonic Stem Cells Junwon Lee1,2,3,5, Sang-Hwi Choi1,3,5, Dongjin R Lee1,3, Dae-Sung Kim4,*, and Dong-Wook Kim1,3,* 1Department of Physiology, 2Department of Ophthalmology, 3Brain Korea 21 PLUS Project for Medical Science, Yonsei Universi- ty College of Medicine, Seoul 03722, Korea, 4Department of Biotechnology, Brain Korea 21 PLUS Project for Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Korea, 5These authors contributed equally to this work. *Correspondence: [email protected] (DWK); [email protected] (DSK) http://dx.doi.org/10.14348/molcells.2018.2210 www.molcells.org The objective of this study was to induce the production of INTRODUCTION isthmic organizer (IsO)-like cells capable of secreting fibroblast growth factor (FGF) 8 and WNT1 from human embryonic The secondary organizer, a specific group of cells that stem cells (ESCs). The precise modulation of canonical Wnt emerges during early embryonic development, can influence signaling was achieved in the presence of the small molecule the identity of surrounding tissues (Kiecker and Lumsden, CHIR99021 (0.6 μM) during the neural induction of human 2012). These cells have the ability to secret morphogens, ESCs, resulting in the differentiation of these cells into IsO-like thus specifying the fate of adjacent cells in a space- and cells having a midbrain-hindbrain border (MHB) fate in a time-dependent manner, and elaborating the development manner that recapitulated their developmental course in vivo. of a given tissue. The isthmic organizer (IsO) in the verte- Resultant cells showed upregulated expression levels of FGF8 brate central nervous system (CNS) has been comprehen- and WNT1. The addition of exogenous FGF8 further in- sively characterized. Located at the midbrain-hindbrain bor- creased WNT1 expression by 2.6 fold. Gene ontology follow- der (MHB) of the developing neural tube, the IsO influences ing microarray analysis confirmed that IsO-like cells enriched the induction, proliferation, and differentiation of neural the expression of MHB-related genes by 40 fold compared to cells between the midbrain and hindbrain by secreting Wnt1 control cells. Lysates and conditioned media of IsO-like cells and fibroblast growth factor (FGF) 8 (Rhinn and Brand, contained functional FGF8 and WNT1 proteins that could 2001; Wurst and Bally-Cuif, 2001). The development of the induce MHB-related genes in differentiating ESCs. The meth- IsO has been intensively studied in lower vertebrates, such as od for generating functional IsO-like cells described in this chick embryos. IsO development begins with anteroposterior study could be used to study human central nervous system (AP) patterning in the neural plate (Partanen, 2007), during development and congenital malformations of the midbrain which the canonical Wnt signal plays a crucial role in the and hindbrain. establishment of the MHB (Ciani and Salinas, 2005; Kiecker and Lumsden, 2005; Wurst and Bally-Cuif, 2001). It is Keywords: FGF, human pluripotent stem cells, isthmic organ- known that the position of the IsO is determined by the jux- izer, neural differentiation, Wnt taposition of two homeobox domain-containing transcrip- tion factors (Otx2 and Gbx2) (Simeone, 2000), followed by Received 6 September, 2017; revised 16 November, 2017; accepted 17 November, 2017; published online 31 January, 2018 eISSN: 0219-1032 The Korean Society for Molecular and Cellular Biology. All rights reserved. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/. 110 Mol. Cells 2018; 41(2): 110-118 Isthmic Organizer-Like Cells from Human Embryonic Stem Cells Junwon Lee et al. the expression of key transcription factors, such as En1, En2, tion kit (iNtRON Biotechnology, Korea). cDNA was synthe- Pax2, and Pax5, as well as Wnt1 and FGF8 in the MHB in a sized from 1 μg total RNA using the PrimeScript RT Master temporally and spatially controlled manner (Wurst and Bally- Mix (Takara Bio, Japan). Transcript levels of each marker Cuif, 2001). Previous studies have shown that either the gene were quantified by real-time PCR using SYBR Premix Ex targeted ablation of Wnt1 or the aberrant expression of Taq (Takara Bio) and the CFX96 Real-Time System (Bio-Rad, FGF8 will disturb the normal morphogenesis of the midbrain USA). Ct values of target genes were normalized to those of and hindbrain, and may cause cerebellar malformations β-actin. Normalized expression levels of target genes were (Crossley et al., 1996; McMahon et al., 1992), thus substan- compared using the ΔΔCt method (Pfaffl, 2001). Data are tiating their pivotal roles in MHB formation. It has been expressed as the mean relative expression level ± standard shown that convergent Wnt and FGF signals precisely induce error of the mean (SEM) from at least three independent the formation of the IsO (Olander et al., 2006) and that the experiments. Sequences of primers used in qRT-PCR are expression of both Wnt1 and FGF8 in the IsO can maintain listed in Supplementary Table S1. the integrity of the MHB (Canning et al., 2007; Ciani and Salinas, 2005; Kiecker and Lumsden, 2005). On the other Enzyme-linked immunosorbent assay hand, the development of the IsO in higher mammals, like IsO-like cells and control cells (DMSO-treated and/or dorso- humans, remains unexplored, primarily due to the lack of an morphin + SB treated cells) were differentiated from human appropriate model system. Considering several congenital ESCs until day 7. After a thorough rinse with Dulbecco’s developmental defects that are relevant to the IsO (Barko- phosphate-buffered saline (DPBS, Invitrogen) to avoid po- vich et al., 2009; Basson and Wingate, 2013), there is a de- tential contamination with any exogenous factor added to mand for a faithful model system in which to study the cellu- the culture, cells were detached from the culture dish using lar and molecular mechanisms underlying MHB and IsO de- a curved Pasteur pipette to spontaneously form spherical velopment. masses that were then cultured in media devoid of CHIR and In this study, we induced the differentiation of human FGF8. One day later, the same number of spheres were ob- ESCs into cells with characteristics of the IsO. Taking ad- tained from each experimental group and sonicated com- vantage of a neural differentiation technology, we attempt- pletely in lysis buffer. Supernatants were isolated from the ed to regionalize human ESC-derived neural precursors (NPs) cellular debris by centrifugation, and the concentration of to the MHB by the precise control of canonical Wnt signaling. total protein was measured using the Bradford protein assay. We also tested whether exogenous FGF8 was required for Human WNT1 and FGF8 kits (Cat. # CSB-EL026128HU and the generation of IsO-like cells. Finally, we characterized IsO- CSB-E15861h, CUSABIO Life-Science, Baltimore, MD, USA) like cells by microarray analysis and assessed their functionali- were used to detect WNT1 and FGF8 protein levels in the ty using lysates and conditioned media generated from the- cell lysates, following the manufacturer’s protocol. Protein se cells. levels of WNT1 and FGF8 were quantified relative to total protein levels. MATERIALS AND METHODS Microarray analysis Cells and culture conditions Ten micrograms of total RNA from each sample were col- Human ESCs (H9, WiCell Inc., USA) were cultured in human lected and analyzed using a Human HT-12 Expression v.4.0 ESC medium composed of DMEM/F12 medium (Invitrogen, bead array (Macrogen, Korea). For clustering analysis, nor- USA) supplemented with 20% Knockout-Serum Replace- malized data were narrowed down to 14,548 using a cutoff ment (Invitrogen), 1% nonessential amino acids (Invitrogen), value that was based on a fail count of less ≤ 3. Gene On- 0.1 mM beta-mercaptoethanol (Sigma, USA), and 4 ng/mL tology (GO) analysis was performed using DAVID (Database basic FGF (Peprotech, USA). For differentiation, embryoid for Annotation, Visualization and Integrated Discovery). Sig- bodies (EBs) were formed by mechanically detaching ESC nificantly upregulated and downregulated genes were com- colonies and culturing them in DMEM/F12:Neurobasal me- pared against DAVID’s GO FAT database to clarify their bio- dia (Invitrogen) (1:1), 1% N2 supplement (Invitrogen), and logical significance. P values were derived by Fisher’s exact 2% B27 supplement without vitamin A (Invitrogen). On day tests (P < 0.01; fold enrichment ≥ 2.0). Corrected P values 4 of differentiation, these EBs were plated onto Matrigel (BD were applied to multiple testing corrections using the Ben- Biosciences, USA)-coated dishes and cultured in the same jamini-Yekutieli method (Benjamini and Yekutieli, 2001). The medium except that the concentrations of N2 and B27 sup- accession number for the microarray data reported in this plements were reduced by half (0.5%) for five days. During study is found in GEO: GSE104847. the first four days, 5 μM dorsomorphin (Calbiochem, USA) and 10 μM SB431542 (SB) (Sigma) were added to the me- Immunocytochemistry dium to facilitate neural induction. To induce IsO-like cells, Cells were fixed with 4% of paraformaldehyde at day 7, and CHIR99021 (CHIR) at various concentrations (0–1.2 μM) permeablized with 0.1% of triton X-100 DPBS solution for (Calbiochem) and 100 ng/ml FGF8 (Peprotech) were added 10 min and then blocked with 2% bovine serum albumin to the medium as described in Supplementary Fig. S1. solution for at least 1 h. Then, cells were incubated with primary antibodies (mouse anti-WNT1 antibody, Abcam Quantitative real-time reverse transcription-PCR (qRT-PCR) (ab91191), Cambridge, UK, 1:200; mouse anti-FGF8 anti- Total RNA was isolated using the Easy-Spin Total RNA Extrac- body, Novus Biologicals (47109), Littleton, CO, USA, 1:50) Mol.
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