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. 2018 Aug 15;11:282. doi: 10.3389/fnmol.2018.00282

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Copyright © 2018 Haenfler, Skariah, Rodriguez, Monteiro da Rocha, Parent, Smith and Todd.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Derivation, expansion and characterization of Fragile X-disease specific human embryonic stem cell (hESC; vFrag-X-ds-hESC; UM139-2 PGD) line. (A) Human blastocyst with FMR1 expansion that was cryopreserved, donated, shipped and warmed prior to attempting hESC derivation. Scale bar represents 30 μm. (B) The inner cell mass (ICM) with surrounding polar trophectoderm (PT) was laser-dissected from the blastocyst and plated/attached on human foreskin fibroblast (HFF). This micrograph represents the early Frag-X-ds-hESC colony before the first passage, 5 days after laser-dissection and plating of the ICM/PT (P0D5). Scale bar represents 15 μm. (C) Expanding undifferentiated Frag-X-ds-hESCs with tight colony borders on a HFF feeder layer (P3D3). Scale bar represents 10 μm. (D) Undifferentiated Frag-X-ds-hESCs expressed pluripotency markers (Oct4, Nanog, Sox2) as assessed by qPCR. Electrophoresis demonstrated anticipated amplicons for each pluripotency marker PCR primer sets. (E) Expanded undifferentiated Frag-X-ds-hESCs with tight colony borders on Martigel (brightfield micrographs) expressed pluripotency marker proteins in the nucleus (same location as Hoechst staining; Oct4, Nanog, Sox2) or cytoplasmic/cell membrane associated (SSEA4 and TRA-1-60). (F) Frag-X-ds-hESCs were differentiated into embryoid bodies for 21 days in culture. Differentiated Frag-X-ds-embryoid bodies expressed linage marker RNA of endoderm [α-fetoprotein (AFP) and GATA4], mesoderm [brachyury (Brachy) and VE-Cadherin (VE-Cad)] and ectoderm [neuron-specific class III beat-tubulin (Tuj-1) and keratin-18 (Krt-18)] with anticipated amplicon size by electrophoresis for each linage marker PCR primer set. Scale bar represents 10 μm. (G) Passage 6 undifferentiated Frag-X-ds-hESCs were sent to Cell Line Genetics (Madison, WI, USA) for G-B and karyotyping and reported to be a 46XY, euploid hESC line.