Directed differentiation of human embryonic stem cell‐line HUES9 to dopaminergic neurons in a serum‐free defined culture niche

• DOI: 10.1002/cbin.10012
• Publication Date: 2013-08-06T09:52:41Z
• Authors: Indrani Datta; Kavina Ganapathy; Surendra Mohan Ta...; Ramesh Bhonde
• Keywords: Directed differentiation; Embryoid body; Proliferation Marker

Abstract Although there are several reports on differentiation of human embryonic stem cells to dopaminergic neurons, notable heterogeneity exists in the reported yields tyrosine hydroxylase (TH)‐positive cells. For benchmarking performance and efficiency standards future applications hESC‐derived is thus a dire need well‐defined directed protocols. Pal et al. [Pal 2009 Exp Biol Med (Maywood) 234:1230–3] demonstrated predisposition HUES9 towards ectodermal lineage, but neurons has not yet been reported. Therefore, we report here simple two‐step protocol using suitable ECM serum‐free induction medium for generating from HUES9‐derived embryoid bodies. Flow cytometry analysis neural progenitors obtained after first step gave an enriched yield immune‐positive nestin (99.6 ± 0.1%), musashi12 (98.1 1.5%) Sox2 (95.4 2.6%). Most these also expressed proliferation marker Ki67 (83.8 1.5%), whereas presence undifferentiated cell Oct4 was negligible. In second step, when were exposed midbrain cues sonic hedgehog fibroblast growth factor 8 along with bFGF, differentiated showed upregulation dopaminergic‐related transcription factors Nurr1 Engrailed1. Immunocytochemistry flow that positive mature neuronal Map2ab (96.2 TH (71.9 4.4%). Thus, data demonstrate novel findings nutrient supplements.