Derivation of homogeneous GABAergic neurons from mouse embryonic stem cells Christina Chatzi 1 , Roderick H. Scott, Jin Pu, Bing Lang, Chizu Nakamoto, Colin D. McCaig ⁎, Sanbing Shen ⁎ School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK abstract article info Article history: Received 20 March 2009 Accepted 23 March 2009 Available online 5 April 2009 Keywords: Embryonic stem cells Embryoid body GABAergic neurons Retinoic acid Embryonic stem cells (ESCs) promise an unlimited source of defined cells for cell transplantation therapy, while protocols for derivation of homogeneous populations of desirable cell types are yet to be developed and/or refined. Gamma aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the central nervous system, and disturbed GABAergic signaling is associated with a host of neurological conditions. We developed a simple ES cell differentiation protocol which led to the production of uniform GABAergic neurons in ∼ 2 weeks. The differentiation protocol involved treatment of embryoid bodies (EBs) with high concentrations (10 -5 –10 -4 M) of all-trans-retinoic acid (RA) for 3 days. After plating these EBs on attached dishes in neural supportive medium, 93–96% of the cells became GABA-positive neurons in 7–11 days. These cells also expressed immature neuronal markers with voltage-gated delayed rectifier potassium currents, suggesting that they were immature GABAergic neurons. The technology may have implications for modeling and treatment of GABAergic signaling-related diseases and injuries. © 2009 Elsevier Inc. All rights reserved. Introduction Embryonic stem cells (ESCs) can be maintained as undiffer- entiated cells or differentiated into cell types derived from three germ layers (Smith, 2001), and therefore promise an unlimited source of defined cells for stem cell-based therapies. However, protocols for homogeneous production of particular cell types are yet to be developed and/or refined. Among the differentiating reagents, retinoic acid (RA) remains one of the most effective factors. RA treatment, in conjunction with embryoid body (EB) formation, has been used to derive glia and functional neurons (Bain et al., 1995; Fraichard et al.,1995; Strübing et al., 1995). However, the end-products of the differentiation procedures invariably are heterogeneous. Attempts to overcome the heterogeneity in ES cell differentiation have included the use of a drug-resistance transgene and/or cell sorting capacity for specific lineages of neurons (Lenka et al., 2002; Li et al., 1998), immunopanning to select neuronal or glia progenitors (Mujtaba et al., 1999) and combining growth factors, differential adhesion and proliferation in defined media (Liu et al., 2000; Okabe et al., 1996). Recently, homogeneous neural progenitor cells with radial glial characteristics were reported by combining growth factors with RA-treated EBs, which differentiated towards a relatively homogeneous population of glutamatergic cortical pyramidal cells (Bibel et al., 2004; Liour et al., 2006). Dopaminergic (Kim et al., 2007; Parmar and Li, 2007) and motor (Kim et al., 2006; Miles et al., 2004; Parmar and Li, 2007; Soundararajan et al., 2007; Wichterle et al., 2002) neurons also were derived from ESCs. After transplantation into animal models of Parkinson's disease and spinal injury, they were capable of promoting phenotypic recovery (Kim et al., 2007; McDonald et al., 1999; Wichterle et al., 2002). However, very little progress has been made on differentiation of GABAergic neurons. Gamma aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system. A decrease in GABAergic neurotransmission is associated with many severe neurological disorders including epilepsy, schizophrenia, Huntington's disease, chronic pain, anxiety and other mood disorders (Benes et al., 2007; Brambilla et al., 2003; Malizia et al., 1998). Additionally, cerebral ischemia or stroke is followed by an inhibition of both synthesis and release of GABA in the brain and degeneration of striatal and hippocampal neurons (Brambilla et al., 2003; Green et al., 2000; Saji et al., 1994; Schwartz-Bloom and Sah, 2001). GABAergic drugs (such as benzodiazepines), which facilitate GABA transmission through modulation of GABA A receptors, have been used clinically for years to treat anxiety and seizures. However, their effectiveness depends on GABA availability (Dubnick et al., 1983). Additionally, the supply of GABA depends on local GABAergic neurons, as GABA cannot be transported efficiently from the bloodstream into the brain. Previously, it was reported that up to 23% of GABA-positive neurons were derived from mouse ESCs in 27 days with a protocol involving generation of neural progenitors and subsequent with- drawal of mitogens (Hancock et al., 2000; Westmoreland et al., 2001). Experimental Neurology 217 (2009) 407–416 ⁎ Corresponding authors. Fax: +44 1224 555719. E-mail addresses: cchatzi@burnham.org (C. Chatzi), c.mccaig@abdn.ac.uk (C.D. McCaig), sanbing.shen@abdn.ac.uk (S. Shen). 1 Current address: Burnham Institute for Medical Research, Development & Aging, 10901 North Torrey Pines Rd, La Jolla, CA 92037, USA. Tel.: +1858 646 3100x3660. 0014-4886/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.expneurol.2009.03.032 Contents lists available at ScienceDirect Experimental Neurology journal homepage: www.elsevier.com/locate/yexnr