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Generation of neural crest cells by direct lineage conversion

ABSTRACT: Neural crest cells (NC cells) are multipotent cells that emerge from the dorsal region of a fusing neural tube and migrate throughout the developing embryo. Due to their multipotency and divergent roles during embryogenesis, the generation and differentiation of NC cells should be elucidated in more detail. However, difficulties have been associated with isolating and manipulating NC cells for investigation because a limited number of NC cells are generated in a short period of embryogenesis. To overcome this issue, we attempted to directly convert mouse fibroblasts into NC cells using transcription factors specifically expressed in NC cells. We analyzed the molecular profiles of NC cells and neural tube cells in 9.5-day mouse embryos, and identified the transcription factors that were strongly expressed in NC cells. Among these transcription factors, Sox10 together with c-Myc and Klf4 directly converted mouse fibroblasts into NC cells. The converted NC cells differentiated into neurons, glial cells, smooth muscle cells, adipocytes, and osteoblasts, exhibiting differentiation abilities similar to those of in vivo NC cells. They also displayed a self-renewal ability while maintaining their differentiation ability. In in ovo transplantation, the converted NC cells migrated along NC cell migration pathways. Therefore, directly converted NC cells can contribute to the progression of NC cell studies.

ORGANISM(S): Mus musculus

PROVIDER: GSE63896 | GEO | 2017/02/10

SECONDARY ACCESSION(S): PRJNA269357

REPOSITORIES: GEO

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Project description:Recent advances in the stem cell biology have revealed that cell type-specific transcription factors could reset the somatic memory and induce direct reprogramming into specific cellular identities. The induction of pluripotency in terminally differentiated cells has been a major achievement in the field of direct reprogramming. Recent studies have shown that fibroblasts could be directly converted into specific cell types, such as neurons, cardiomyocytes, blood progenitor cells, and epiblast stem cells, without first passing through an induced pluripotent stem cell state3-7. However, direct reprogramming of differentiated cells into somatic stem cell types has not been described yet4. Here we show that a combination of neural-specific transcription factors (Sox2, Klf4, c-Myc, Brn4/Pou3f4 or Sox2, Klf4, c-Myc, Brn4, E47/Tcf3) can induce a neural stem cell (NSC) fate on the fibroblasts. Induced neural stem cells (iNSCs) showed morphology, gene expression, epigenetic features, differentiation potential, and functionality similar to wild-type NSCs. Therefore, our data suggest that cell type-specific defined factors can induce specific stem cell identities on somatic cells. Fibroblasts (5 x 104 cells) were infected with retroviruses for two days, and cells were maintained in NSC media: DMEM/F-12 supplemented with N2 or B27 supplements (Gibco-BRL), 10 ng/ml EGF, 10 ng/ml bFGF (both from Invitrogen), 50 ug/ml BSA (Fraction V Gibco-BRL), and 1x penicillin/streptomycin/glutamine (Gibco-BRL). In order to establish stable iNSC lines, were either manually picked mature iNSC clumps or passaged and seeded the whole dishes of cells onto either gelatin- or laminin-coated dishes. RNA samples to be analysed on microarrays were prepared using QIAGEN RNeasy columns with on-column DNA digestion. 500 ng of total RNA per sample was used as input RNA into a linear amplification protocol (Ambion) involving synthesis of T7-linked double-stranded cDNA and 12 h of in-vitro transcription incorporating biotin-labelled nucleotides. Purified and labelled cRNA was hybridised onto MouseRef-8 v2 expression BeadChips (Illumina) for 18 h according to the manufacturer's instructions. After washing, as recommended, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using iScan reader (Illumina) and accompanying software. Samples were hybridised as biological replicates. 9 samples were analyzed. Fibroblast: CF1 Mouse embryonic fibroblasts, 1 biological rep Control NSC: OG2-Rosa Neural Stem Cells, 2 biological rep 4F iNSC (late): 4 factors induced Neural Stem Cells (late passage), 2 biological rep 4F iNSC (early): 4 factors induced Neural Stem Cells (early passage), 2 biological rep 5F iNSC: 5 factors induced Neural Stem Cells, 2 biological rep

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Generation of neural crest cells by direct lineage conversion

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