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- Title
Differentiation of human embryonic stem cells to neural progenitor cells and neurons on human feeder cells.
- Authors
Yuan, D.; Xie, P.; Lu, G. X.; Lin, G.
- Abstract
Introduction: Human embryonic stem cells (human ESC) could provide available tools for neural development research and potential sources for cell replacement therapy. Traditionally, neural induction was initiated through the formation of embryoid bodies (EB) and found that cells underwent a similar process as in-vivo neural development from a primitive neuroepithelia stage, 'rosette', to a more definitive stage, a neural tube-like structure. Recently some researchers reported the direct induction on mouse feeder cells. Our previous study showed that human feeder cells could well maintain the undifferentiated state of human ESC. In this study, we performed neural differentiation either directly on human feeder cells or through the formation of EB to see if they had the same efficiency and underwent similar process of neural development. Materials/Methods: We used two human ESC lines cultured on human feeder cells for comparing the two distinct inducing methods. Neural progenitors obtained on day 10 and day 17 were used for immunocytochemistry. After floating in the neuralinducing medium for one week, differentiating medium was changed for further maturation. Results: After 7-9 days, cells in both groups took on the appearance of 'rosettes'. Immunofluorescence indicated that rosettes were Pax6+, Musashi+, Nestin+ and Sox1-. Cell counting at day 10 indicated that Pax6+ cells in the direct induction group and EB group was 83.1% and 57.5% respectively. The 'rosettes' turned into neural tube-like structures 5-7 days later, which were stained positive for Pax6, Musashi, Nestin and Sox1. Cell counting at day 16 indicated that cells in the direct induction group were 89.9% Pax6+ and 74.5% Sox1+, while cells in EB group were 67.2% and 68.1% respectively. Cells in both groups had similar gene expression profiles, with neural developmentrelated genes up-regulated and pluripotent genes down-regulated. We further induced these two distinct neural progenitors into midbrain dopaminergic neurons by adding FGF8 and SHH into the differentiating medium. Immunofluorescence at day 28 indicated both groups could differentiate into β-tubulin+ neurons, among which tyrosine hydroxylase-positive cells in day 10 and day 16 group was 10% and 3% respectively. Cells derived from day 10 had a higher expression of midbrain dopaminergic neuron marker En1 and Nkx6.1 than those from day 16. Conclusion: We then concluded that human ESC could be induced into neural progenitor cells by both induction strategies, and that the direct differentiation performed on human feeder cells was more approachable for further transplantation therapy.
- Subjects
EMBRYONIC stem cells; CELLULAR therapy
- Publication
Reproductive BioMedicine Online (Reproductive Healthcare Limited), 2008, Vol 16, Issue S2, pS-34
- ISSN
1472-6483
- Publication type
Abstract
- DOI
10.1016/S1472-6483(10)61548-9