Date Published: January 20, 2017
Publisher: Public Library of Science
Author(s): Tomoko Horikiri, Hiromi Ohi, Mitsuaki Shibata, Makoto Ikeya, Morio Ueno, Chie Sotozono, Shigeru Kinoshita, Takahiko Sato, Irina Kerkis.
The neural crest is a source to produce multipotent neural crest stem cells that have a potential to differentiate into diverse cell types. The transcription factor SOX10 is expressed through early neural crest progenitors and stem cells in vertebrates. Here we report the generation of SOX10-Nano-lantern (NL) reporter human induced pluripotent stem cells (hiPS) by using CRISPR/Cas9 systems, that are beneficial to investigate the generation and maintenance of neural crest progenitor cells. SOX10-NL positive cells are produced transiently from hiPS cells by treatment with TGFβ inhibitor SB431542 and GSK3 inhibitor CHIR99021. We found that all SOX10-NL-positive cells expressed an early neural crest marker NGFR, however SOX10-NL-positive cells purified from differentiated hiPS cells progressively attenuate their NL-expression under proliferation. We therefore attempted to maintain SOX10-NL-positive cells with additional signaling on the plane and sphere culture conditions. These SOX10-NL cells provide us to investigate mass culture with neural crest cells for stem cell research.
The neural crest cell is a unique, transient part of ectodermal derivatives in developing vertebrates and has multi-ability to migrate and differentiate into numerous cells including peripheral neurons, glia, craniofacial cartilage, cornea and so on . Initial neural crest cells are raised at the edge of the neural plate and the non-neural ectoderm. According to the formation of the neural folds, neural crest cells subsequently occur epithelial mesenchymal transition to delaminate from dorsal neural tube and migrate through several pathways to reach target tissues and differentiate into various cell types as above [2–4].
Here we present successful generation of SOX10-NL knock-in hiPS cells by the CRISPR/Cas9 and In-Fusion technologies, which are valuable to monitor SOX10 expression by both of fluorescent and luciferase activities. Nano-lantern enable us to perform not only real-time imaging in living cells as other fluorescent proteins, but also in vivo imaging by sensitive luminescent detection. This SOX10-NL hiPS cell lines can be also used for evaluation of directed differentiation toward neural crest lineage using chemical screening as well as to study temporal emergence of neural crest stem cells during in vitro differentiation. Moreover, our approach shown here is simple, efficient and versatile to be applicable to generate other reporter knock-in cells, and ideal gene targeting for predominant genotype like SOX10 because Haploinsufficiency of hetelozygous SOX10 causes pigmentation and megacolon defect in Hirschsprung disease . Therefore, we chose not to generate SOX10 heterozygotes or BAC transgenic cells, which might affect to intact SOX10 or other gene expression, but to target 3’UTR sequence of SOX10 just with sequences of 2A signal peptide and Nano-lantern instead of SOX10 stop codon to function intact SOX10 coding sequence.