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Apitsada Khlongkhlaeo, Richard L. Carter, Rangsun Parnpai and Anthony W.S. Chan

Abstract

Background

Feeder-free monolayer culture and the suspension culture of embryoid bodies (EB) followed by adherent culture, rosette selection, and expansion are 2 methods for deriving neural progenitor cells (NPCs). Direct comparison of these 2 methods has not yet been reported.

Objectives

To compare the influence of NPC derivation methods on the properties of NPCs derived from rhesus monkey (Macaca mulatta) embryonic stem cells (rhESCs).

Methods

rhESCs were used to derive NPC lines using 2 different methods. EB were produced from a suspension culture of rhESC clumps and rhESCs were cultured in feeder-free monolayers on poly-L-ornithine/laminin coated plates. NPCs were derived by exposure to induction factors. Cell morphology, neural and nonneural lineage markers were evaluated. We measured the expression of nuclear receptor tailless (TLX), which acts as repressor of glial fibrillary acidic protein (GFAP) expression.

Results

NPCs were successful derived using either method, with homogenous populations based on the expression of nestin (>97%) and Pax6 (>99%) as shown by flow cytometry. No significant difference in NPC specific markers or ability to differentiate into neurons in vitro was found between the methods. However, the expression of GFAP was >400-fold higher in cells produced by the feeder-free method. This distinction was consistent with the lower expression of TLX.

Conclusions

NPCs derived by feeder-free and EB methods share similar morphology and properties. The elevated expression of GFAP and reduced expression of TLX in NPCs derived using the feeder-free method may explain their greater heterogeneity and tendency to differentiate toward cells of an astrocyte lineage.