![]() Neural crest could be identified on the periphery of the PAX6 positive tissue (green) based on (g) AP2, (h) HNK1, (i) PAX7, and (j) p75 expression (red). In the absence of factors that confer regional neuronal specificity, the PAX6 + neural tissue (green) expressed (e) OTX2, and (f) BF1, indicating that the tissue defaults to fore-brain specification. (d) Rosettes are formed when PAX6 + tissue is passaged to conditions promoting rosettes (BASF) confirmed by KI67 (green) and luminal phospho-Histone H3 (red) expression, evidence of interkinetic nuclear migration. The PAX6 positive neural tissue (green) expressed rosette markers (red) (a) Nestin, (b) PLZF, (c) ZO1. Neuralization of hESC by dual SMAD inhibition permits a pre-rosette, neural stem cell with dopaminergic and motoneuronal potential. Abbreviations: N, Noggin SB, SB431542 KSR, knock-out serum replacement medium N2, N2 medium. and the p-value was determined using Student’s T-test. (g) A BAC reporter line (HES5-GFP) was used to quantify the percentage of neural induction for the method using MS5 stromal cells (with Noggin) or dual SMAD inhibition (SB431542 and Noggin). (f) Real-Time PCR for neural and neuronal markers during dual SMAD inhibition differentiation towards neurectoderm. (e) Real-Time PCR for PAX6, OTX2, FGF5, OCT4 during dual SMAD inhibition reveals an epi-stem cell intermediate at day 5. (d) Immunoflouresence for OCT4 (red) and PAX6 (green) expression indicates rapid neutralization occurs by day 7. (c) Real-Time PCR for early germ layer markers CDX2, SOX1, SOX17 and Brachyury. Infrequent neural differentiation (< 10% PAX6 + cells) can be observed when the single factors are used. (b) The dual SMAD inhibition greatly improves neural differentiation (PAX6 expression, green) to greater than 80%. (a) Differentiation scheme used for achieving neural induction can be achieved with the combination of SB431542, an ALK inhibitor, and Noggin, a BMP inhibitor. Noggin/SB431542-based neural induction should facilitate the use of hES and hiPS cells in regenerative medicine and disease modeling and obviate the need for protocols based on stromal feeders or embryoid bodies.ĭual SMAD inhibition allows for a highly efficient feeder-free neural induction in adherent cultures in seven days. Directed differentiation of human induced pluripotent stem (hiPS) cells into midbrain dopamine and spinal motoneurons confirms the robustness and general applicability of the induction protocol. Initial cell density determines the ratio of central nervous system and neural crest progeny. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. ![]() Here we report that the synergistic action of two inhibitors of SMAD signaling, Noggin and SB431542, is sufficient to induce rapid and complete neural conversion of >80% of hES cells under adherent culture conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. ![]() Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. ![]()
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