LUHMES (Lund human mesencephalic) cells are a subclone of the originally generated MESC2.10 cell line (Lotharius et al., 2002). This cell line was obtained by preparation of precursor cells from embryonic ventral mesencephalic tissue and immortalization of these cells with of a LINX v-myc retroviral vector system (Hoshimaru et al., 1996). In this system, a tetracycline-controlled transactivator (tTA) strongly activates transcription of v-myc from a minimal CMV promoter in the absence of tetracycline. This allows the cells to continuously proliferate in a medium containing bFGF (Fig. 4). For initiation of differentiation, the cells are incubated with medium containing non-toxic concentrations of tetracycline, dibutyryl cyclic adenosine 3’,5’-monophosphate (db-cAMP) and glial cell line-derived neurotrophic factor (GDNF). Since tetracycline abolishes the transcription activation by tTA , the production of v-myc is blocked and the cells readily start to transform into post-mitotic neurons (Fig. 4) (Lotharius et al., 2002). In the following sections, more detailed information will be given on mesencephalic cells and the most important medium constituents.
Figure 4. Biphasic state of LUHMES:
growth and differentiation. See text for description. Abbreviations: bFGF: basic fibroblast growth factor, LTR: long terminal repeat, tTA: tetracycline-controlled transactivator (yellow circles), ires: internal ribosomal entry site, neo:
neomycin resistance gene, CMV:
cytomegalovirus-virus promoter, TET:
tetracycline (purple triangles), db:
dibutyryl, GDNF: glial cell line-derived neurotrophic factor. Slightly modified from Lotharius et al., 2002.
7.1 The ventral mesencephalon
The mesencephalon, or midbrain, is considered as part of the brainstem and is associated with multiple brain functions, such as vision, motor control, alertness and reward (Yin et al., 2009). The ventral (= anterior) mesencephalon is organized into different neuronal populations, including dopaminergic neurons and neurons of the red nucleus (RN) (Blaess et al., 2011). Dopaminergic neurons are further divided into anatomically and functionally distinct subclasses (Bjorklund and Dunnett, 2007). The substantia nigra (SN), located in the lateral-ventral midbrain, projects to the striatum and is involved in the regulation of motor behaviors. The ventral tegmental area (VTA), located more medially, projects to corticolimbic targets and is important for motivational states (Blaess et al., 2011). The functional diversity of these different regions becomes apparent in disease states: in Parkinson’s disease, SN neurons, but not VTA neurons, degenerate, resulting in severe motor deficits. In contrast, abnormalities in the corticolimbic system have been implicated in addiction and schizophrenia (Dagher and Robbins, 2009, Smidt and Burbach, 2007).
Dopaminergic and RN neurons have been shown to arise from ventral mesencephalic precursors that express sonic hedgehog (Joksimovic et al., 2009, Ono et al., 2007). Besides this, only little is known about the factors and genes that control the establishment of the distinct neuronal (sub)classes in the developing human brain (Nelander et al., 2009). In the mouse, the process has been subdivided into 3 major phases. First, the induction of a progenitor field within the neuroepithelium that is competent to generate DA precursors takes
place at an early stage of neural development (approximately at embryonic day 8.5) (Yin et al., 2009). Neurogenin 2 and Mash1 are considered as two of the early fate determinants of DA neurons (Ang, 2006). Following a second phase of specification and early differentiation, DA neurons terminally differentiate and acquire their mature phenotype (outgrowth of axons etc.) at relatively late stages of neurodevelopment (Prakash and Wurst, 2006). During this third phase, Nurr1 and Pitx3 were reported to play important roles (Smidt and Burbach, 2007).
7.2 The growth factor for proliferating LUHMES: bFGF
FGFs are polypeptides which play essential roles in a multitude of biological processes during development and adult life. Deregulation of FGF signaling, on the other hand, has been associated with many developmental syndromes, and with human cancer (Wesche et al., 2011). The FGF family consists of 22 members of closely related peptides which contain 150-300 amino acids and have a conserved core with ~30-60% identity (Itoh and Ornitz, 2004). They signal through 4 homologous high-affinity receptors (FGFR1-4) that have an overall structure similar to most receptor tyrosine kinases (Johnson and Williams, 1993). The prototypic FGFs, FGF1 and FGF2 (also named basic FGF = bFGF), were originally isolated from the brain and pituitary as mitogens for cultured fibroblasts (Gospodarowicz, 1975, 1978). They are paracrine factors, which do not possess a signal sequence for secretion, but utilize a non-classical secretion pathway circumventing the ER (Nickel, 2010).
FGFs are crucial during development, where they have been shown to be key molecules in organogenesis. FGF signaling is for example implicated in the formation of the heart, the lungs, the limbs and, most importantly, the nervous system, where it is implicated in neural induction (Dorey and Amaya, 2010, Powers et al., 2000, Turner and Grose, 2010). In cell culture, FGFs stimulate cell proliferation, survival, migration and differentiation (Dailey et al., 2005, Xian et al., 2005). FGF2 in particular has been shown to support the undifferentiated self-renewal of human embryonic stem cells and is routinely used to cultivate such cells in the laboratory (Lanner and Rossant, 2010).
7.3 The growth factor for differentiating LUHMES: GDNF
GDNF is a distant member of the transforming growth factor β superfamily and was originally isolated from a rat glial cell line (Lin et al., 1993). It is expressed throughout the central nervous system during development and in a more region-restricted manner also in the adult brain (Schaar et al., 1993, Stromberg et al., 1993). GDNF is known to be a potent survival factor for midbrain dopamine neurons both in vivo and in vitro (Lin et al., 1993, Pascual et al., 2008). The major source of GDNF to the midbrain is the striatum, from where it is retrogradely transported to the SN and the VTA (Barroso-Chinea et al., 2005, Tomac et al.,
GFRα1
1995). In addition to its dopaminotrophic function, GDNF plays an essential role in the development and survival of motor and sensory neurons and the development of the kidney (Moore et al., 1996, Pichel et al., 1996).
GDNF signals through a receptor tyrosine kinase named RET (rearranged during transfection), which was first discovered as a proto-oncogene (Takahashi, 2001). However, RET can only be activated, if GDNF first binds to a coreceptor, GFRα1 (GDNF-family receptor-α1). GFRα1 and RET are expressed in several brain regions in the developing and adult brain, including the cerebellum, hypothalamus and hippocampus, with particular abundance in the SN and the VTA (Carnicella and Ron, 2009). In the classical model, a GDNF dimer first binds to either monomeric or dimeric GFRα1, and the GDNF-GFRα1 complex then interacts with two RET molecules, thereby inducing their homodimerization and tyrosine autophosphorylation (Fig. 5) (Airaksinen et al., 1999). This leads to the downstream activation of several signaling cascades, such as the mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-kinase (PI3K) and phospholipase Cγ (PLCγ) pathways (Fig.
5) (Manie et al., 2001, Wells and Santoro, 2009).
Figure 5. GFRα1 and RET mediated GDNF signaling pathways. Slightly modified from Carnicella and Ron, 2009. Autophosphorylation of tyrosine residues is indicated by red circles.
See text for explanations.
7.4 Second messenger supply from outside: db-cAMP
Many hormones and growth factors activate transcription by raising the level of cAMP within cells and thereby regulate proliferation, differentiation, survival and plasticity of cells by triggering programs of gene expression (Maruoka et al., 2010). The second messenger cAMP activates protein kinase A (PKA), which phosphorylates and regulates a variety of cellular proteins. One example is the transcription factor CRE binding protein (CREB), which then binds to the cAMP-responsive element (CRE), a consensus sequence found in promoter regions of many target genes (Johannessen et al., 2004). Regarding the brain-specific functions of cAMP, it has been suggested that cAMP-dependent mechanisms contribute to the maturation and maintenance of several catecholaminergic systems, including sympathetic ganglionic neurons and noradrenergic cells in the brainstem (Rydel and Greene, 1988, Sklair-Tavron and Segal, 1993). Furthermore, it has been shown that the
application of db-cAMP (a cell permeant analogue of cAMP) alone is sufficient to promote development and long-term survival of mesencephalic neuronal cultures (Hartikka et al., 1992, Michel and Agid, 1996). Finally, several groups reported that the extracellular supply of db-cAMP significantly potentiates the survival-promoting effects of GDNF on dopaminergic neurons and that CREB plays a crucial role in neurotrophin signaling (Bonni et al., 1995, Engele and Franke, 1996).