Human embryonic stem cell-derived neuroectodermal spheres revealing neural precursor cell properties유래 신경전구세포의 특성 분석

Abstract

Neural stem/precursor derived from pluripotent human embryonic stem cells (hESCs) has considerable therapeutic potential due to their ability to generate various neural cells which can be used in cell-replacement therapies for neurodegenerative diseases. However, production of neural cells from hESCs remains technically very difficult. Understanding neural-tube like rosette characteristic neural precursor cells from hESCs may provide useful information to increase the efficiency of hESC neural differentiation. Generally, neural rosettes were derived from differentiating hEBs in attached culture system, however this is time-consuming and complicated. Here, we examined if neural rosettes could be formed in suspension culture system by bypassing attachment requirement. First, we tested whether the size of hESC clumps affected the formation of human embryonic bodies (hEBs) and neural differentiation. We confirmed that hEBs derived from 500x500 m square sized hESC clumps were effectively differentiated into neural lineage than those of the other sizes. To induce the rosette formation, regular size hEBs were derived by incubation of hESC clumps (500x500 m) in EB medium for 1 wk in a suspended condition on low attachment culture dish and further incubated for additional 1-2 wks in neuroectodermal sphere (NES)-culture medium. We observed the neural tube-like rosette structure from hEBs after 7-10 days of differentiation. Their identity as a neural precursor cells was assessed by measuring their expressions of neural precursor markers (Vimentin, Nestin, MSI1, MSI2, Prominin-1, Pax6, Sox1, N-cadherin, Otx2, and Tuj1) by RT-PCR and immunofluorescence staining. We also confirmed that neural rosettes could be terminally differentiated into mature neural cell types by additional incubation for 2-6 wks with NES medium without growth factors. Neuronal (Tuj1, MAP2, GABA) and glial (S100 and GFAP) markers were highly expressed after 2-3 and 4 wks of incubation, respectively. Expression of oligodendrocyte markers O1 and CNPase was significantly increased after 5-6 wks of incubation. Our results demonstrate that rosette-forming neural precursor cells could be successfully derived from suspension culture system and that will not only help us understand the neural differentiation process of hESCs but also simplify the derivation process of neural precursors from hESCs.