Formation of neurospheres from human embryonal carcinoma stem cells Grace M. Horrocks, a Lyndsey Lauder, a Rebecca Stewart, a and Stefan Przyborski a,b, * a School of Biological and Biomedical Sciences, University of Durham, South Road, Durham DH1 3LE, UK b ReInnervate Limited, Old Shire Hall, Old Elvet, Durham DH1 3HP, UK Received 15 March 2003 Abstract Embryonal carcinoma (EC) stem cells derived from germ cell tumours are valuable tools for the study of embryogenesis and closely resemble embryonic stem cells. When human TERA2.cl.SP12 EC cells are exposed to retinoic acid and grown as adherent monolayers, approximately 10–15% of cells commit toward becoming neurons whilst the remainder of cells produce non-neuronal cell types. Using established protocols it is possible to isolate and purify neurons from these cultures but such a process takes several weeks and the numbers of neurons produced are relatively low. In this study, we describe the development of novel procedures to enhance neuronal productivity with dramatically increased efficiency, which will be of value for research purposes and drug dis- covery programmes. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Embryonal carcinoma; Stem cell; Human; Neurosphere; Neuron; Mass production; Cell culture; Purification The growth of human neurons in vitro provides sci- entists with a valuable tool for basic research, including drug screening, toxicological testing, and the study of nervous system development and disease in man. In general, primary cultures of terminally differentiated neurons have mostly been used as models to study the development and function of neurons in vitro. However, primary cultures are dependent on a supply of the ap- propriate tissue, are often contaminated by other cell types, and possess little capacity for proliferation thus limiting the number of cells available for experimenta- tion from a single source. During the last decade, several groups reported methods on the isolation of mammalian neural progenitor cells from the embryonic nervous system and the expansion of these cells in response to molecules such as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) [1–6]. Under these conditions, neuroprogenitors grow as suspended aggre- gates of cells commonly referred to as neurospheres. Methods for expanding neuroprogenitor populations also include the mechanical dissociation of neurospheres by tissue chopping [5]. Furthermore, withdrawal of growth factors and addition of serum to cultures of neuroprogenitor cells subsequently induces differentia- tion of neurons and glia [1–5]. Accordingly, non-trans- formed human neural cells derived from primary foetal tissue provide a valuable resource for research and have potential therapeutic applications. The use of these expanded primary cells, however, is not as straightforward when it is necessary to consis- tently produce large amounts of identical material for routine screening purposes. Although Svendsen et al. [5] reported that it is possible to scale up these cultures to produce larger quantities of neural cell types, this is not without considerable effort, technical expertise, and ex- pense. Alternatively, transformed human stem cell lines offer the advantage of reproducibly forming particular differentiated cell types in response to certain stimuli. Despite their oncogenic status making them less at- tractive as a source of tissue for therapeutic use, they remain an important tool in research and development. Moreover, their oncogenic nature can make it easier to maintain and rapidly expand these cells in vitro, often accommodating straightforward handling and scale up, and their more restricted ability to differentiate may be a distinct advantage for the production of certain cell types. Several human cell lines are currently available Biochemical and Biophysical Research Communications 304 (2003) 411–416 www.elsevier.com/locate/ybbrc BBRC * Corresponding author. Fax: +44-191-374-2417. E-mail address: stefan.przyborski@durham.ac.uk (S. Przyborski). 0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00611-9