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.

SUMMARYThe isolation of human induced pluripotent stem cells (iPSCs)1-3 offers a novel strategy for modeling human disease. Recent studies have reported the derivation and differentiation of disease-specific human iPSCs4-7. However, a key challenge in the field is the demonstration of disease-related phenotypes and the ability to model pathogenesis and treatment of disease in iPSCs. Familial dysautonomia (FD) is a rare but fatal peripheral neuropathy caused by a point mutation in IKBKAP8 involved in transcriptional elongation9. The disease is characterized by the depletion of autonomic and sensory neurons. The specificity to the peripheral nervous system and the mechanism of neuron loss in FD are poorly understood due to the lack of an appropriate model system.Here we report the derivation of patient specific FD-iPSCs and the directed differentiation into cells of all three germ layers including peripheral neurons. Gene expression analysis in purified FD-iPSC derived lineages demonstrates tissue specific mis-splicing of IKBKAP in vitro. Patient-specific neural crest precursors express particularly low levels of normal IKBKAP transcript suggesting a mechanism for disease specificity. FD pathogenesis is further characterized by transcriptome analysis and cell based assays revealing marked defects in neurogenic differentiation and migration behavior. Finally, we use FD-iPSCs for validating the potency of candidate drugs in reversing aberrant splicing and ameliorating neuronal differentiation and migration. Our study illustrates the promise of iPSC technology for gaining novel insights into human disease pathogenesis and treatment.

Canonical animal microRNAs (miRNAs) are generated by sequential cleavage of precursor substrates by the Drosha and Dicer RNase III enzymes. Several variant pathways exploit other RNA metabolic activities to generate functional miRNAs. However, all of these pathways culminate in Dicer cleavage, suggesting that this is a unifying feature of miRNA biogenesis. Here, we show that maturation of miR-451, a functional miRNA that is perfectly conserved among vertebrates, is independent of Dicer. Instead, structure-function and knockdown studies indicate that Drosha generates a short pre-mir-451 hairpin that is directly cleaved by Ago2 and followed by resection of its 3′ terminus. We provide stringent evidence for this model by showing that Dicer knockout cells can generate mature miR-451 but not other miRNAs, whereas Ago2 knockout cells reconstituted with wild-type Ago2, but not Slicerdeficient Ago2, can process miR-451. Finally, we show that the mir-451 backbone is amenable to reprogramming, permitting vector-driven expression of diverse functional miRNAs in the absence of Dicer. Beyond the demonstration of an alternative strategy to direct gene silencing, these observations open the way for transgenic rescue of Dicer conditional knockouts.Slicer | gene suppression | miRNA reprogramming

Pluripotent stem cells (PSCSCs) are defined by their potential to generate all cell types of an organism. The standard assay for pluripotency of mouse PSCSCs is cell transmission through the germline, but for human PSCSCs researchers depend on indirect methods such as differentiation into teratomas in immunodeficient mice. Here we report PluriTest, a robust open-access bioinformatic assay of pluripotency in human cells based on their gene expression profiles

Interactions between newly integrated DNA and the host genome limit the reliability and safety of transgene integration for therapeutic cell engineering and other applications. Although targeted gene delivery has made considerable progress, the question of where to insert foreign sequences in the human genome to maximize safety and efficacy has received little attention. In this Opinion article, we discuss 'genomic safe harbours' - chromosomal locations where therapeutic transgenes can integrate and function in a predictable manner without perturbing endogenous gene activity and promoting cancer.