Central neuroendocrine cells are a set of specialized neurons located in the hypothalamus. Rather than forming synapses with other neurons, neuroendocrine cells release their product, neurohormones, into the blood circulation to act on their endocrine targets. The neuroendocrine hypothalamus consists of eight neuronal populations, each expressing specific neuropeptides: oxytocin (OXY), vasopressin (AVP), gonadotropin-releasing hormone (GnRH), growth hormone-releasing hormone (GHRH), thyrotropin-releasing hormone (TRH), corticotropin-releasing hormone (CRH), somatostatin (SS), and dopamine (DA). These central neuroendocrine cells are important because they control most vital functions such as growth, reproduction, metabolism and energy balance, and stress responses (Swanson., 1987).
Endocrine disruptors are broadly defined as exogenous substances that interfere with the production, release, transport, metabolism, binding, action, or elimination of natural body hormones, which are responsible for the maintenance of homeostasis and the regulation of developmental processes (Kavlock et al. 1996; Tilson and Kavlock 1997). These disruptors
Include plasticizers, flame retardants, fungicides, pesticides, and pharmaceuticals (Choi et al. 2004; Diamanti-Kandarakis et al.2009).
In vitro models of neuronal development:
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Until recently, in vitro model systems available to study effects of chemicals on nervous system development were mostly carried out on primary cultures and transformed cell lines (Radio and Mundy 2008). Primary cell cultures contain post mitotic neurons and are, therefore, not suitable for the study of early developmental processes such as proliferation and differentiation. In addition, post mitotic neurons do not divide, such that new cultures need to be prepared frequently from freshly dissected tissues. Transformed cell lines such as the human neuroblastoma SH-SY5Y (Perez-Polo et al. 1979) and the rodent pheochromocytoma PC12 (Greene and Tischler 1976) are derived from tumour's, and the extent to which they are true representatives of brain neural cells is questionable (Radio and Mundy 2008). While these models systems proved invaluable in understanding the mechanisms underlying nervous system function, they are significantly limited for use as models of nervous system development (Radio and Mundy 2008; Breieret al. 2010).
Neural Stem Cells as a Model of Neuronal Development
The strength of hypotheses describing mechanisms of chemical action on the developing nervous system depends on the validity of the model system from which they are derived. Therefore, a significant challenge in neurodevelopmental studies has been to derive in vitro models that accurately recapitulate processes important for the development of the nervous system as observed in vivo, such as proliferation, migration, differentiation, and synaptogenesis (Lein et al. 2005; Polleux and Anton 2005).Study of these developmental processes would benefit substantially from use of neural stem cells, which offer significant advantages over other in vitro model systems. Neural stem cells are derived from pluripotent embryonic stem cells or from multipotent adult progenitor cells isolated from brains of multiple species including rodents and humans (Seaberg and van der Kooy 2003). Neural stem cells possess the capacity of self-renewal in culture and the ability to generate the three major cell types of the nervous system: neurons, astrocytes, and oligodendrocytes (Wobus and Boheler 2005). In the laboratory, neural stem cells are cultured as a monolayer or as free-floating neurospheres, and require the presence of only a few basic fibroblast growth factors (Kornblum2007). Removal of the growth factors from the media and/or addition of a signalling molecule promote a differentiation process that results in a mixed population of neurons and glia (Kornblum 2007). Typically, undifferentiated embryonic stem cells, neural stem cells, and differentiated neurons are characterized by their expression of one or more specific phenotypic markers. For example, Oct-4 is a marker of undifferentiated embryonic stem cells, and both microtubule-associated protein-2 (MAP2) and the neuron-specific class III beta-tubulin (Tuj-1) are markers of mature neurons (Kuegleret al. 2010). Neural stem cells, accordingly, provide a readily available source of cells that differentiate into neurons and glial cells that are morphologically and functionally similar to those found in vivo (Bain et al. 1995).
` Although neural stem cells are suitable for modelling neural development, to date only a handful of studies have examined the influence of endocrine disruptors on neural Stem Cell differentiation. In most of these studies, neural stem cells are differentiated in the presence of a chemical disruptor, and the resulting population of neurons and glia are compared to the control. In these studies, neural Stem Cell cultures proved to be a sensitive and promising in vitro model system to study the influence of endocrine disruptors on neuronal development in general. However, a need still exists for a model of neuroendocrine cells that would enable the study of the mechanisms by which endocrine disruptors and drugs directly affect the development and alteration of metabolism in neuroendocrine cells, which is their primary target in the brain.
Stem Cell-Derived Neuroendocrine Cells:
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Stem cells are undifferentiated and self-renewing cells that are present in most adult tissues. Stem cells hold tremendous potential in advancing the treatment of many diseases and disorders that are currently untreatable. Presently, the utilization of stem cells in neural tissue repair or replacement has been limited. However, a continued understanding of stem cell biology and the pathology of neural diseases may lead to future clinical therapies (Korecka et al., 2007). Stem cells, whether embryonic (ESC) or adult (ASC), have other applications, such as providing models to study disease/injury or in drug screening. In particular, the generation of neurons from stem cells affords the unique opportunity to study human neural processes in primary cells. However, customized protocols must be established to generate specific classes of neurotransmitter-producing cells (Scheffler et al., 2006). Embryonic stem cells are pluripotent and they have the capacity to divide into different types of cells in adult tissues and organs. Although a number of technical problems and ethical concerns need to be addressed with embryonic stem cells. Availability of adult stem cells to repair damaged tissue and to provide different cell models, clearly suggests their potential utility in clinical medicine (Gail., 1981)
Mesenchymal Stem cells:
Adult mesenchymal stem cells (MSCs) are a population of multipotent cells found primarily in the bone marrow. The cells are positive for surface markers CD29, CD 44 and CD 105.They have long been known to be capable of osteogenic, adipogenic and chondrogenic differentiation and are currently the subject of a number of trials to assess their potential use in the clinic. Recently, the plasticity of these cells has come under close scrutiny as it has been suggested that they may have a differentiation potential beyond the mesenchymal lineage. Myogenic and in particular cardio myogenic potential has been shown in vitro. MSCs have also been shown to have the ability to form neural cells both in vitro and in vivo, although the molecular mechanisms underlying these apparent Trans differentiation events are yet to be elucidated. Moreover the protocols used in the already-mentioned neuroendocrine differentiation studies are complicated and their efficiency is low (jackson et al., 2007)
To date, reports of neuroendocrine cells derived from stem cells in vitro are limited. Markakis et al. (2004) were the first group to report that neural stem cells isolated from either the hypothalamus or the hippocampus of 7-wk-old rats, and grown as a monolayer, can be directed to differentiate into all 8 types of hypothalamic neuroendocrine cells when treated with retinoic acid and forskolin. Data suggested that the hypothalamus contains neural stem cells that can be harvested, grown as monolayers, and expanded over multiple generations. Differentiation of these neural stem cells produces neuroendocrine cells that secrete all of the neurohormones found in the hypothalamus in vivo (Markakis et al. 2004). A similar approach was used by Salvi et al. (2009), who showed that neural stem cells isolated from the hypothalamus of rat brain at embryonic day 18 and grown as neurospheres are capable of differentiating spontaneously into hypothalamic neurons, including about 20% that were found to express GnRH. Evidence indicated this culture system represents a useful model to study the molecular mechanisms underlying GnRH-induced neuronal cell differentiation. Taken together, the studies just described demonstrate that neural stem cells derived from rat brains differentiate into neuroendocrine cells in vitro (Salvi et al. 2009). Other investigators focused on the potential of the more versatile pluripotent embryonic stem cells to differentiate into neuroendocrine cells in vitro. Ohyama et al. (2005) showed that mouse embryonic stem cells can be directed to differentiate into hypothalamic dopaminergic neurons following treatment with the signalling molecules Shh and Bmp7. However, Wataya et al. (2008) found that mouse embryonic stem cells are able to spontaneously differentiate into hypothalamic neuroendocrine-like progenitor cells when cultured in media free of growth factors, and subsequently into vasopressin expressing neurons that efficiently release the hormone upon stimulation. The protocols used in the already-mentioned neuroendocrine differentiation studies are complicated and their efficiency is low. Moreover, the role of Bmp7 in the neuroendocrine differentiation of embryonic stem cells is still controversial since Wataya et al. (2008) failed to confirm the positive effects of Bmp7 on the hypothalamic differentiation reported earlier by Ohyama et al. (2005). Till now there is no report of in vitro adult stem cell differentiation into neuro endocrine cells. Owing to the importance of neuro endocrine cells in CNS and to test different drugs on these cells, stem cells are the easy sources to provide large population of hypothalamic matures neuronal cells.
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Isolate adult multipotent stem cells (Cd34+ or mesenchymal stem cells).
Grow and trypsinize the cells.
Perform IHC for positive cell detection.
Plate the cells and treat with retinoic acid and forskolin.
Observe the cells under microscope for morphological changes.
Perform RT-PCR and IHC to detect the mature neural cell markers.
Mature neural cell markers:
Mature neuronal markers such as microtubule-associated protein-2 (MAP2; Figure 2) and neuron-specific class III beta-tubulin (Tuj-1) chromogranin A (CgA), a selective marker of neuroendocrine cells (El Majdoubiet al. 1996; Taupenot et al. 2003).
Mesenchymal stem cells represent a population of cells with the potential to contribute to future treatments for a wide range of acute or degenerative diseases. Significant progress has been made to identify the pharmacological and molecular pathways driving MSC differentiation towards mesenchymal derivatives in vitro and preliminary results indicate that MSCs could be used to generate neural derivatives. Much remains to be done in order to evaluate the physiological relevance of these early observations and to unravel the molecular mechanisms governing their differentiation in vivo. Applications currently under investigation for MSC-based therapies include musculoskeletal and cardiac repair, as well as genetic manipulation of MSCs for gene therapy strategies. Directed differentiation of autologous MSCs towards extra-mesenchymal lineages is an exciting and promising area of stem cell biology, with potential for the repair of tissues where resident stem cells are not accessible, such as the brain. MSCs thus represent an interesting and versatile population of adult stem cells demanding further molecular characterization and functional investigation. Future research will define the extent of their potential as an autologous and allogenic stem cell source for clinical application (Jackson et al., 2007).