Cell Biology International ISSN 1065-6995 doi: 10.1002/cbin.10012 RESEARCH ARTICLE Directed differentiation of human embryonic stem cell-line HUES9 to dopaminergic neurons in a serum-free defined culture niche Indrani Datta*, Kavina Ganapathy, Surendra Mohan Tattikota and Ramesh Bhonde Manipal Institute of Regenerative Medicine, Constituent Institute of Manipal University, GKVK Post, Bellary Road, Allalasandra, Yelahanka, Bangalore, Karnataka, India Abstract Although there are several reports on differentiation of human embryonic stem cells to dopaminergic neurons, notable heterogeneity exists in the reported yields of tyrosine hydroxylase (TH)-positive cells. For benchmarking performance and efficiency standards in future applications of hESC-derived dopaminergic neurons, there is thus a dire need of well-defined directed differentiation protocols. Pal et al. [Pal et al. 2009 Exp Biol Med (Maywood) 234:1230–3] demonstrated predisposition of HUES9 towards ectodermal lineage, but the directed differentiation of HUES9 to dopaminergic neurons has not yet been reported. Therefore, we report here a simple two-step protocol using suitable ECM and serum-free induction medium for generating dopaminergic cells from HUES9-derived embryoid bodies. Flow cytometry analysis of the neural progenitors obtained after the first step gave an enriched yield of cells immune-positive for nestin (99.6  0.1%), musashi12 (98.1  1.5%) and Sox2 (95.4  2.6%). Most of these cells also expressed the proliferation marker Ki67 (83.8  1.5%), whereas the presence of the undifferentiated stem cell marker Oct4 was negligible. In the second step, when these neural progenitors were exposed to midbrain cues sonic hedgehog and fibroblast growth factor 8 along with bFGF, the differentiated cells showed an upregulation of dopaminergic-related transcription factors Nurr1 and Engrailed1. Immunocytochemistry and flow cytometry analysis showed that these differentiated cells were positive for the mature neuronal marker Map2ab (96.2  1.5%) and dopaminergic neuronal marker TH (71.9  4.4%). Thus, the data demonstrate novel findings of the directed differentiation of HUES9 to dopaminergic neurons using well-defined serum-free nutrient supplements. Keywords: dopaminergic neurons; extracellular matrix; human embryonic stem cells; mid-brain cues; neural progenitors; neuronal differentiation Introduction Human embryonic stem cells (hESCs) are unique since they are essentially derived from the inner cell mass of preimplantation embryos, and are thus capable of self- renewal and differentiation to all three derm layer lineages. Consequently, hESCs are considered a good reserve model for developmental events and for the generation of terminally differentiated cells in therapeutic strategies. One of the primary hurdles in the area of regenerative neuroscience is the availability of embryonic neural progenitors or special- ised neuronal cells of human origin. The generation of neural progenitors and terminally differentiated cells from hESCs thus holds significant promise for providing a cellular model of human neuronal development, and as a potential reserve for cell replacement therapy in neurodegenerative diseases. While in the last few years there have been several reports on differentiation of hESCs to neural progenitors and dopaminergic cell type, the variability in yield obtained by different research groups is noteworthy (reviewed by Correia et al., 2008). This heterogeneity between reports in the yield of the differentiated tyrosine hydroxylase (TH)-positive cells can be attributed to (i) differences in experimental protocols and induction factors employed, and (ii) inherent differences *Corresponding author: e-mail: indrani.datta@manipal.edu Abbreviations: bFGF, basic fibroblast growth factor; BMP, bone morphogenic protein; DAPI, 4 0 ,6 0 -diamidino-2-phenylindole dihydrochloride; EB, embryoid body; ECM, extracellular matrix; FBS, fetal bovine serum; FGF8, fibroblast growth factor 8; HepG2, hepatocellular carcinoma cell line; hESCs, human embryonic stem cells; MEF, mouse embryonic fibroblast; Nurr1, nuclear receptor related 1 protein; Oct4, octamer-binding transcription factor 4; SHH, sonic hedgehog; Sox2, SRY (sex determining region Y)-box 2; SSEA4, stage-specific embryonic antigen 4; TH, tyrosine hydroxylase 54 Cell Biol Int 37 (2013) 54–64 ß 201 International Federation for Cell Biology 2