Stage-Dependent Differential Gene Expression Profiles of Cranial Neural Crest Cells Derived from Mouse Induced Pluripotent Stem Cells

Cranial neural crest cells (cNCCs) comprise a multipotent population of cells that migrate into the pharyngeal arches of the vertebrate embryo and differentiate into a broad range of derivatives of the craniofacial organs. Consequently, migrating cNCCs are considered as one of the most attractive candidate sources of cells for regenerative medicine. In this study, we analyzed the gene expression profiles of cNCCs at different time points after induction by conducting three independent RNA sequencing experiments. We successfully induced cNCC formation from mouse induced pluripotent stem (miPS) cells by culturing them in neural crest inducing media for 14 days. We found that these cNCCs expressed several neural crest specifier genes but were lacking some previously reported specifiers, such as paired box 3 (Pax3), msh homeobox 1 (Msx1), and Forkhead box D3 (FoxD3), which are presumed to be essential for neural crest development in the embryo. Thus, a distinct molecular network may the control gene expression in miPS-derived cNCCs. We also found that c-Myc, ETS proto-oncogene 1, transcription factor (Ets1), and sex determining region Y-box 10 (Sox10) were only detected at 14 days after induction. Therefore, we assume that these genes would be useful markers for migratory cNCCs induced from miPS cells. Eventually, these cNCCs comprised a broad spectrum of protocadherin (Pcdh) and a disintegrin and metalloproteinase with thrombospondin motifs (Adamts) family proteins, which may be crucial in their migration.


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Abstract 24 Cranial neural crest cells (cNCCs) comprise a multipotent population of cells that migrate into 25 the pharyngeal arches of the vertebrate embryo and differentiate into a broad range of derivatives 26 of the craniofacial organs. Consequently, migrating cNCCs are considered as one of the most 27 attractive candidate sources of cells for regenerative medicine. In this study, we analyzed the gene 28 expression profiles of cNCCs at different time points after induction by conducting three 29 independent RNA sequencing experiments. We successfully induced cNCC formation from 30 mouse induced pluripotent stem (miPS) cells by culturing them in neural crest inducing media for 31 14 days. We found that these cNCCs expressed several neural crest specifier genes but were 32 lacking some previously reported specifiers, such as paired box 3 (Pax3), msh homeobox 1 33 (Msx1), and Forkhead box D3 (FoxD3), which are presumed to be essential for neural crest 34 development in the embryo. Thus, a distinct molecular network may the control gene expression 35 in miPS-derived cNCCs. We also found that c-Myc, ETS proto-oncogene 1, transcription factor 36 (Ets1), and sex determining region Y-box 10 (Sox10) were only detected at 14 days after induction. 37 Therefore, we assume that these genes would be useful markers for migratory cNCCs induced the pharyngeal arches to form skeletal elements of the face and teeth and contribute to the Japan) and were passaged in 60-mm cell culture plates at a density of 1 × 10 5 cells/plate. The cells 116 were grown in 5% CO 2 at 95% humidity and the culture medium was changed each day.

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Embryoid body (EB) formation and cNCC differentiation 119 We obtained cultured cNCC cells following a previously described procedure

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The strongest immunofluorescent staining was detected in d7 cells for Snai1 and d14 cells 206 for Sox10 ( Fig 2B).     We found that the transcription factor AP-2 alpha (Ap2) along with Pax3 and zinc finger 254 protein of the cerebellum 1 (Zic1), both of which are regulated by Ap2, were most highly 255 expressed in d7 cells (Fig 3A). Pax6, which has been reported in human ES and iPS-derived NC 256 cells (Tables 2 and 3), was detected in both d7 and d14 cells, whereas Pax7, which has not 257 previously been reported in the mouse NC, was also detected in the d7 cells ( Fig 3A). In contrast, 258 the homeobox genes gastrulation brain homeobox 2 (Gbx2), Msx1, distal-less homeobox 3 259 (Dlx3), Zic2, and Zic3 were not detected in d7 or d14 cells, and the homeobox genes Zic1 and 260 Dlx5 were only expressed in d7 cells, despite these having been reported in the NC of a range of 261 species (Table 2); however, Meis homeobox 2 (Meis2) was expressed in both d7 and d14 cells.

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Both MYCN proto-oncogene, bHLH transcription factor (N-myc) and c-Myc have been 263 reported in NCCs (Table 3); however, we did not observe N-myc expression in d7 or d14 cells 20 264 and detected c-Myc expression in the d7 and d14 group (Fig 3A). Furthermore, we observed 265 substantial downregulation of the winged-helix transcription factor FoxD3 over time (Fig 3A), 266 which is an important factor for maintaining the pluripotency of ES cells and a key NC specifier 267 that has been implicated in multiple steps of NC development and NCC migration in the embryo 268 of various species (Table 2).

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Groups were compared using ANOVA followed by the Bonferroni test: *p < 0.05.

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As expected, we found that several Adam and Adamts genes were upregulated, with most of the 393 latter increasing significantly. The Adam genes that increased in the cNCCs were the membrane-394 bound type; whereas, the Adamts genes were secreted proteinases, indicating that the expression 395 of various Adamts may allow the matrix to be digested more efficiently, as each may be capable 396 of digesting a different type of extracellular matrix protein [45].Thus, the secretion of a variety of 397 Adamts and Pcdh proteins may play a crucial role in the migration ability of cNCCs.

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In summary, we successfully induced the formation of cNCCs from miPS cells by placing 399 them in NC inducing media for 14 days. We found that although the resulting cNCCs had several 400 NC specifiers, some were lacking, indicating that a distinct molecular network may control the 401 gene expression in miPS-derived cNCCs. Our results also indicated that cMyc; Ets1; Sox10; 402 Adamts2 and -8; Pcdha2, -5, -7, -11, and -12; Pcdhac1; andPcdhgc3 may represent appropriate 403 markers for migratory cNCCs induced from miPS cells. Eventually, these cNCCs produced a 404 broad spectrum of Adamts family proteins that may play an important role in their migration.