Dipartimento di Biologia e
Biotecnologie Charles Darwin

Several studies have described the production of various neural cell types from human embryonic stem cells (hESCs), but there has been no systematic study of how different regional identities are established. While positional specification along the neural axes has been studied extensively in model organisms, achieving the same knowledge for hESCs requires the use of well-defined differentiation conditions. We have focused on adherent cultures, which reduce complex cellular interactions compared to suspension cultures, and chemically-defined conditions devoid of matrigel and serum replacement. Confirming recent studies, we show that hESCs cultured in these conditions can be efficiently converted into neuroectoderm by Nodal pathway inhibition along with either Bone Morphogenetic Protein (BMP) inhibition or treatment with Fibroblast Growth Factor (FGF). However, in the presence of Nodal inhibition, BMP inhibition allows specification towards anterior neural fates (forebrain/midbrain), while FGF promotes predominantly posterior fates (hindbrain/spinal cord). Upregulation of endogenous Wnt signalling during differentiation also contributes to neuroectoderm posteriorization. We also examined dorsoventral patterning and found that Hedgehog signalling was endogenously activated in our cultures and promoted ventral forebrain fates, which could be repressed by inhibiting the Hedgehog pathway. Based on these results, we describe efficient forebrain specification by inhibition of Nodal, BMP and Wnt pathways and minimization FGF signalling. Notably, these conditions are not permissive for the specification of retinal progenitor fates, which require functional Nodal and BMP pathways. Our results demonstrate that i) the key mechanisms of neural patterning found in model vertebrate species are preserved in adherent, chemically-defined hESC cultures; ii) reveal new insights into the signals regulating retinal progenitor cell fates; and iii) provide an effective route to generate clinically valuable regionally specific neural progenitors in vitro.