Human embryonic stem (ES) cells are derived from the inner cell mass of the preimplantation embryo. Human ES cells have the capacity to differentiate into various types of cells in the body. Human ES cells are indefinite source of cells for cell therapy in various degenerative disorders including neuronal disorders. Directed differentiation of human ES cells is a prerequisite for their clinical application. The objective of this study is to develop the culture condition for the derivation of neural precursor cells from human ES cells. Neural precursor cells were derived from human ES cells in a stepwise culture condition. Neural precursor cells in the form of neural rosette structures developed into neurospheres when cultured in suspension. Suspension culture of neurospheres has been maintained over 4 months. Expressions of nestin, soxl, sox2, pax3 and pax6 transcripts were upregulated during differentiation into neural precursor cells by RT-PCR analysis. In contrast, expression of oct4 was dramatically downregulated in neural precursor cells. Immunocytochemical analyses of neural precursor cells demonstrated expression of nestin and SOX1. When induced to differentiate on an adhesive substrate, neuro-spheres were able to differentiate into three lineages of neural systems, including neurons, astrocytes and oligo-dendrocytes. Transcripts of sox1 and pax6 were downregulated during differentiation of neural precursor cells into neurons. In contrast, expression of map2ab was elevated in the differentiated cells, relative to those in neural precursor cells. Neurons derived from neural precursor cells expressed NCAM, Tuj1, MAP2ab, NeuN and NF200 in immunocytochemical analyses. Presence of astrocytes was confirmed by expression of GFAP immuno-cytochemically. Oligodendrocytes were also observed by positive immuno-reactivities against oligodendrocyte marker O1. Results of this study demonstrate that a stepwise culture condition is developed for the derivation of neural precursor cells from human ES cells.

INTRODUCTION   MATERIALS AND METHODS    Culture of Human ES Cells    Differentiation of Human ES Cells into Neural Precursor Cells    Immunocytochemical Analysis    RT-PCR Analysis   RESULTS    Differentiation of Human ES Cells into Neural Precursor Cells    Characterization of Neural Precursor Cells   DISCUSSION   REFERENCES

• Kim Sehee(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Hong Ji Young(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Joo So Yeon(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Kim Jae Hwan(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Moon Shin Yong(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Yoon Hyun Soo(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Kim Doo Han(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Chung Hyung Min(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)
• Choi Seong-Jun(Cell and Gene Therapy Research Institute, Pochon CHA University College of Medicine)