Tyrosine hydroxylase expression is unstable in a human immortalized mesencephalic cell line—Studies in vitro and after intracerebral grafting in vivo Gesine Paul, a, ⁎ Nicolaj S. Christophersen, a,b Heather Raymon, c Caroline Kiaer, a Ruben Smith, a and Patrik Brundin a a Neuronal Survival Unit, Department of Experimental Medical Science, Wallenberg Neuroscience Center, 22184-Lund, Sweden b NsGene A/S, Baltorpvej 154, 2750-Ballerup, Denmark c Celgene Corporation, Signal Research Division, San Diego, CA 92121, USA Received 26 July 2006; revised 9 November 2006; accepted 14 November 2006 Available online 10 January 2007 We have studied the stability of the dopaminergic phenotype in a conditionally immortalized human mesencephalic cell line, MESC2.10. Even though MESC2.10 cells exhibit features of dopaminergic neurons in vitro, none of the cells expressed tyrosine hydroxylase (TH) after transplantation into a rat model of Parkinson’s disease. We examined whether this is caused by cell death or loss of transmitter phenotype. Cells were cultured in differentiation medium, then harvested and replated into the same medium where they continued to express TH, whereas replated cells fed medium lacking differentiation factors (dibutyryl cAMP and glial cell line-derived neurotrophic factor) did not. Interestingly, cultures grown in the absence of differentiation factors could regain TH expression once exposed to differentiation medium. Our data suggest that TH expression in vitro is inducible in neurons derived from the MESC2.10 cell line and that the dopaminergic phenotype of these cells in vivo might be unstable. © 2006 Elsevier Inc. All rights reserved. Introduction In Parkinson’ s disease (PD), dopamine (DA) neurons in the substantia nigra gradually degenerate. This leads to reduced striatal levels of DA and progressive motor symptoms. Currently, pharmacological treatment is symptomatic and cannot prevent or retard progression of the disease (Samii et al., 2004). Replacing lost dopaminergic neurons may be an alternative treatment strategy and around 400 PD patients worldwide have been transplanted with immature dopaminergic neurons derived from human embryonic tissue. The transplanted cells can survive, restore DA levels and partially reverse motor deficits (Bjorklund et al., 2003). However, there is a shortage of human embryonic donor tissue that makes alternative cell sources desirable. Such cells should have high proliferative capacity and generate differentiated dopaminergic progeny in a predictable fashion. Several different types of human neural stem cells have been tested in animal experiments (Paul, 2006), but important questions such as the optimal stage of differentiation for grafting, the stability of the transmitter phenotype after changing the environment and the function of the grafted cells in vivo remain before cells can be applied clinically. Immortalized neural stem cells or progenitor cells are useful tools to study such mechanisms controlling differentiation, cell survival and phenotype stability (Frederiksen et al., 1988; Ryder et al., 1990). Cells are arrested at specific stages of development thereby preventing their terminal differentiation (Cepko, 1989; Snyder et al., 1992). Most commonly, immature neural cells are immortalized using a retroviral vector encoding the v-myc protein that enhances proliferation (Ryder et al., 1990; Snyder et al., 1992). V-myc propagated human neural progenitor cell lines include H6, derived from human 15-week-old fetal telencephalon (Flax et al., 1998), and HNSC.100, derived from human 10-week-old embryo- nic forebrain neurospheres (Villa et al., 2000). By constitutively expressing oncogenes such as v-myc, cell lines proliferate indefinitely in the presence of mitogens. In the absence of mitogens, they exit the cell cycle and differentiate (Flax et al., 1998; Kitchens et al., 1994; Villa et al., 2000). As an alternative to developing cell lines by constitutively expressing oncogenes, a tetracycline-controlled gene expression system has been employed (Hoshimaru et al., 1996). Using this method, the first human mesencephalic cell line (MESC2.10) was derived from the ventral mesencephalon (VM) of an 8-week-old human embryo and immortalized with retroviral infection using the LINX v-myc vector. In this system, a tetracycline-controlled transactivator strongly activates transcrip- tion from a minimal CMV promoter, which, in turn, drives v-myc expression in the absence of tetracycline (Lotharius et al., 2002). Under certain culture conditions, MESC2.10 cells differentiate; they exhibit neurites, generate action potentials and express www.elsevier.com/locate/ymcne Mol. Cell. Neurosci. 34 (2007) 390 – 399 ⁎ Corresponding author. Fax: +46 46 2220531. E-mail address: gesine.paul@med.lu.se (G. Paul). Available online on ScienceDirect (www.sciencedirect.com). 1044-7431/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2006.11.010