Long-term differentiation of neuronal and astroglial cultures derived from noggin-primed human embryonic stem cells
Human embryonic stem cells (hESCs) provide a nearly unlimited source of neural cells for mechanistic and drug-screening studies. The low-density derivation of neural progenitor cells from hESCs primed with the BMP antagonist noggin has opened the way to generate highly homogenous cultures of astrocytes, oligodendrocytes and neurons. Enriched hESC cultures treated with noggin can be used to yield distinct neuronal and glial subtypes under defined conditions. Laminin and fibronectin are important growth permissive substrates that allow adherence of cultures and direct neural cell fates. However, the effect of these substrates on the differentiation of neuronal and glial cultures has not been comprehensively characterized over prolonged periods. Here we show that the stable, long-term differentiation (21-49 days) of neural cultures derived from noggin-primed hESCs (H9) is substrate-dependent. Characterization of neuronal and glial cultures by immunocytochemistry at sequential weekly intervals over 49 days (d) on both laminin and fibronectin substrates revealed superior adherence, higher cell densities (+17.4%) and more stable differentiation on fibronectin substrate between 21d-49d. Our results show optimal conditions for the generation of S100b+GFAP+A2B5+ type 2 astrocytes (≈35%) at 10.5d, GFAP+S100b+Vimentin(-) type 1 astrocytes (≈89%) at 42d (on fibronectin substrate) and MAP2+ Tubulin ßIII+ neuron-restricted progenitors (≈85%) at 7d-14d (laminin substrate). Furthermore, we report a neuronal to glial shift between 21-28d and a glial to neuronal shift between 42-49d on both substrates. The data presented here provides evidence that the most widely used substrate –laminin, is a key limiting factor during long-term neural differentiation. The beneficial effect of fibronectin substrate, in particularly on glial differentiation, may therefore hold great promise to derive more mature cells as a tool for human 'in vitro' models. Taken together, we anticipate that our findings will contribute to the reliability and robustness of methods for the long-term maintenance and differentiation of neuronal and astroglial cultures.