Cranial neural crest cells are multipotent cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into various craniofacial organ derivatives. Therefore, migrating cranial neural crest cells are considered one of the most attractive candidate cell sources in regenerative medicine. We generated cranial neural crest like cell (cNCCs) using mouse-induced pluripotent stem cells cultured in neural crest-inducing medium for 14 days. Subsequently, we conducted RNA sequencing experiments to analyze gene expression profiles of cNCCs at different time points after induction. cNCCs expressed several neural crest specifier genes; however, some previously reported specifier genes such as paired box 3 and Forkhead box D3, which are essential for embryonic neural crest development, were not expressed. Moreover, ETS protooncogene 1, transcription factor and sex-determining region Y-box 10 were only expressed after 14 days of induction. Finally, cNCCs expressed multiple protocadherins and a disintegrin and metalloproteinase with thrombospondin motifs enzymes, which may be crucial for their migration.

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38from miPS cells. Eventually, these cNCCs comprised a broad spectrum of protocadherin (Pcdh) 39 and a disintegrin and metalloproteinase with thrombospondin motifs (Adamts) family proteins, 40 which may be crucial in their migration. 3 41 54 pharyngeal glands (thymus, thyroid, and parathyroid) [12]. Consequently, presumably cNCCs 55 may represent a new treatment strategy for diseases in the craniofacial region [13]. 56 Development from the premigratory to migratory stage proceeds swiftly [14], making it 57 difficult to isolate and characterize a pure cNCC population from the embryo [15]. A recent 58 transcriptome analysis of pure populations of sex determining region Y-box 10 (Sox10) + 4 59 migratory cNCCs from chicks [16] has greatly improved our understanding of the characteristics 60 of cNCCs, and methods for deriving NCCs from the embryonic stem (ES) cells have also been 61 reported [17-30]; however, it remains unclear whether these cells are in the migratory stage and 62 how long it takes to promote ES cell-derived NCCs from the pre-migratory to migratory stage. 63 In recent years, the use of induced pluripotent stem (iPS) cells as a revolutionary approach 64 to the treatment of various medical conditions has gained immense attention [31,32] and iPS cells 65 have several clear advantages over ES cells and primary cultured cNCCs as a cell source in 66 regenerative medicine [16]. NCCs have been generated from iPS cells in numerous ways [24,33-67 38], with two reports having examined the differentiation of NCCs from ES or iPS cells [24,39] 68 and two articles having described the protocol for differentiating NCCs from mouse iPS (miPS) 69 cells [33,34]; however, few studies have investigated the changes in the properties of these NCCs 70 overtime during the dynamic differentiation processes in the NC, in particular, during the 71 migratory stage. Embryonic NC development depends on several environmental factors that 72 influence the NC progenitors, regulation, and the timing of differentiation, making the elucidation 73 of the gene regulatory network and expression profiles of miPS cell-derived cNCCs important. 74 Recent advances in the next-generation RNA sequencing technology (RNA-seq) have made 75 it possible to analyze the gene expression profiles comprehensively [40-42]. Therefore, here, we 76 used RNA-seq to investigate the gene expression landscape of cNCCs induced from miPS cells. 5 77 We treated the iPS-derived cells with cNCC induction medium for 14 days and performed 78 triplicate RNA-seq experiments. We found that standard NC markers such as nerve growth factor 79 receptor (Ngfr), snail family transcriptional repressor 1 (Snai1), and Snai2 were remarkably 80 increased at 7 days after cNCC induction; whereas, the expression of the cNCC markers ETS 81 proto-oncogene 1, transcription factor (Ets1), and Sox5, -8, -9, and -10 characteristically increased 82 at 14 days after cNCC induction. Nestin (Nes) was upregulated throughout cNCC differentiation, 83 as described previously [23]. In contrast, the hom...