Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling

Abstract

Current neural induction protocols for human embryonic stem (hES) cells rely on embryoid body formation, stromal feeder co-culture or selective survival conditions. Each strategy has considerable drawbacks, such as poorly defined culture conditions, protracted differentiation and low yield. 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. Temporal fate analysis reveals the appearance of a transient FGF5(+) epiblast-like stage followed by PAX6(+) neural cells competent to form rosettes. Initial cell density determines the ratio of central nervous system and neural crest progeny. 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. 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.

Figure 1

Dual SMAD inhibition allows for a highly efficient feeder-free neural induction in adherent cultures in seven days. (a) Differentiation scheme used for achieving neural induction can be achieved with the combination of SB431542, an ALK inhibitor, and Noggin, a BMP inhibitor. (b) The dual SMAD inhibition greatly improves neural differentiation (PAX6 expression, green) to greater than 80%. Infrequent neural differentiation (< 10% PAX6⁺ cells) can be observed when the single factors are used. (c) Real-Time PCR for early germ layer markers CDX2, SOX1, SOX17 and Brachyury. (d) Immunoflouresence for OCT4 (red) and PAX6 (green) expression indicates rapid neutralization occurs by day 7. (e) Real-Time PCR for PAX6, OTX2, FGF5, OCT4 during dual SMAD inhibition reveals an epi-stem cell intermediate at day 5. (f) Real-Time PCR for neural and neuronal markers during dual SMAD inhibition differentiation towards neurectoderm. (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). All error bars represent S.E.M. and the p-value was determined using Student’s T-test. Abbreviations: N, Noggin; SB, SB431542; KSR, knock-out serum replacement medium; N2, N2 medium. Scale bars: (b) − 200 µm ; (d) − 50 µm.

Figure 2

Neuralization of hESC by dual SMAD inhibition permits a pre-rosette, neural stem cell with dopaminergic and motoneuronal potential. The PAX6 positive neural tissue (green) expressed rosette markers (red) (a) Nestin, (b) PLZF, (c) ZO1. (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. 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. 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). Upon passage, the neural crest cells gave rise to (k) pigmented cells (l) that expressed HMB45 (green), indicating melanosome synthesis. (m) Dopaminergic neuronal patterning was initiated with the addition of super sonic on day 5–9, followed by the addition of BDNF, ascorbic acid, sonic hedgehog, and FGF8 on day 9–12. Dopaminergic cells were maturated on days 12–19 with BDNF, ascorbic acid, GDNF, TGFb3, and cAMP. Motoneuronal patterning was initiated at day 5 with the addition of BDNF, ascorbic acid, sonic hedgehog, and retinoic acid. Cells were passaged on day 11. (n–p) Without passage, tyrosine hydroxylase (TH) positive cells could be observed by day 19. (p) When passaged en bloc on day 12, more mature processes from TH positive cells were observed. For motoneuron induction, nuclear expression of the motoneuron markers (q) ISL1 and (r) HB9 were observed within a total of 19 days of differentiation from hESC. Scale bars: (a,b,c,e,f,g,h,i,j,o,p,q,r) − 100 µm; (c,d) − 50 µm; (k,l,n) − 200 µm.

Figure 3

Induced pluripotent stem cells (IPS) can be differentiated to neural tissue using dual SMAD inhibition and are patternable to dopaminergic neurons and motoneurons. (a–i, ii) Two IPS clones (IPS^C14, IPS^C27) were generated and screened for OCT4 (red) as well as additional pluripotency factors (Tra-1–81, Tra-1–60, SSEA-4 and Nanog, data not shown). (b-i,ii) the two clones were neuralized by dual SMAD inhibition (PAX6 expression, green), and neural crest could be observed by HNK1 staining (c-i,ii). Neural tissue from the IPS clones could be induced to form rosette-NSCs (d-i,ii) based on KI-67 (red) and phospho-histone H3 (green) expression, motoneurons (e-i,ii) based on HB9 expression (green), and dopaminergic neurons (f-i,ii) based on TUJ1 (green) and TH (red) co-expression. Scale bars: 200 µm − (a); 50 µm − (b,c,d,e,f).