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

Abstract

Current neural induction protocols in human ES cells (hESCs) rely on embryoid body formation, stromal feeder co-culture, or selective survival conditions; each strategy displaying significant 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 for inducing rapid and complete neural conversion of hESCs under adherent culture conditions. Temporal fate analysis reveals a transient FGF5+ epiblast-like stage followed by PAX6+ neural cells competent of rosette formation. Initial cell density determines the ratio of CNS versus neural crest progeny. Directed differentiation of human iPSCs into midbrain dopamine and spinal motoneurons confirm robustness and general applicability of the novel induction protocol. Noggin/SB431542 based neural induction should greatly facilitate the use of hESC and hiPSCs in regenerative medicine and disease modeling and obviate the need for stromal feeder or embryoid body based protocols.

Notch signaling represses p63 expression in the developing surface ectoderm

“…Human embryonic stem cell-derived ectodermal cells were cultured on collagen-coated plates in defined keratinocyte-SFM medium (Invitrogen/Gibco). Neural differentiation was performed according to previously described protocols (Chambers et al, 2009). Briefly, cells were plated as single cells and incubated for 3 days in DMEM/F12 (Gibco), 20% Knockout Serum Replacement (Invitrogen), 0.1 mM β-ME (Sigma), 10 μM SB431542 (Tocris) and 500 ng/ml Noggin (R&D Systems).…”

Translational potential of human brain organoids

“…Prior to the advent of brain organoids, neuroscientists have strived to establish neural differentiation protocols from the hPSCs. Drawing on the insights learned from early embryogenesis, Chambers et al discovered that blocking TGF β /BMP signal pathways in hESCs resulted in highly efficient neural differentiation 23. This led them to identify specific inhibitors of the SMAD proteins (downstream effectors of TGF β /BMP signaling) as potent neural fate inducers, constituting the basis of the so‐called dual‐SMAD inhibition protocol that is widely used for directed neural differentiation today 23, 24.…”

“…Drawing on the insights learned from early embryogenesis, Chambers et al discovered that blocking TGF β /BMP signal pathways in hESCs resulted in highly efficient neural differentiation 23. This led them to identify specific inhibitors of the SMAD proteins (downstream effectors of TGF β /BMP signaling) as potent neural fate inducers, constituting the basis of the so‐called dual‐SMAD inhibition protocol that is widely used for directed neural differentiation today 23, 24. Upon extended differentiation in culture, these neural stem cells gave rise to functionally mature neurons expressing markers of different cortical layers 24.…”

Modeling heterogeneity in the pluripotent state: A promising strategy for improving the efficiency and fidelity of stem cell differentiation

“…Interestingly, there are significant differences in the efficiency of protocols for deriving differentiated cell types. While dual SMAD inhibition is sufficient to induce rapid and complete neural conversion of human ESCs and iPSCs 110, there are limitations to efficiently differentiate PSCs to other cell types, such as cardiomyocytes 111, kidney cells 112, retinal pigment epithelium 113, or hepatocytes 114. In particular, the differentiated cells produced are largely immature, and resemble the fetal stages of development 111, 114, which hampers their ability to engraft and function in coordination with other cells of the tissue.…”