Differentiation of neurospheres from the enteric nervous system

Abstract

The enteric nervous system (ENS) derives from neural crest cells, which migrate from the neural tube into the developing gut. The neuronal and glial precursor cells migrate mainly from the oral towards the anal end of the gastrointestinal tract. So far, knowledge about the multipotent influences upon the ENS development, especially its neurotrophic support, derives mainly from knock-out models. The in vitro technique of isolating enteric neuronal precursor cells allows to study the effects of various factors upon their appropriate development in more detail. We therefore adapted the method of growing neurospheres, which are agglomerates of neuronal precursor cells and differentiated neurones and glial cells, from the central nervous system (CNS) for the ENS. The gut of NMRI mice at E12 were dissected, mildly dissociated and plated in 25-cm(2) culture flasks. The cultures were maintained in N1 supplemented DMEM/F12 medium with the appropriate neurotrophin cocktails (bFGF, GDNF, Neurturin, CNTF). After several days in culture most of the cells die, while the surviving cells form clusters from which domes, and later spheres arise. The spheres could be harvested and processed for further experiments. First investigations revealed, that the amount of precursor cells was much less in enteric neurospheres as seen in corresponding cultures from the CNS. We found about 43% HNK-1-NCAM+ in enteric and approximately 90% Nestin-+ cells in midbrain neurospheres. Differentiation studies of the enteric neurospheres showed that especially ciliary neurotrophic factor (CNTF) increased the number of enteric neurones (PGP positive), while the amount of HNK-1 precursor cells decreased under the influence of all tested neurotrophins but GDNF. The culture of the freshly dissociated enteric neurospheres in a three-dimensional matrix yielded a secondary network which allows to investigate the pattern formation of the ENS. The generation of enteric neurospheres and the following differentiation and 3D culture in vitro can increase our knowledge of the amount and time point of neurotrophic as well as the ECM-protein influence upon the appropriate development of the ENS.

The Chemokine Stromal Cell-Derived Factor-1 Regulates the Migration of Sensory Neuron Progenitors

“…7a). These neurospheres had an appearance similar to those prepared using neural cells from the CNS (Tran et al, 2004) or the peripheral enteric nervous system (Schafer et al, 2003).…”

ABC transporters, neural stem cells and neurogenesis – a different perspective

“…Furthermore, some adult astrocyte-like GFAP + cells are actually NSCs, which are probably derived from an embryonic glial lineage [46]. Therefore, the 3D neurosphere organization could give some indication of whether a neurosphere is generated by a true stem cell or by a non-stem cell population when the neurospheres are characterized by staining with a stem cell marker like nestin or musashi-1 [47,48]. The DNAbinding dye Hoechst-33342 has been used as a method to identify potential stem cells in a host of tissues, including the bone marrow, heart, lung, muscle, eye and pancreas [9].…”

Divergent fate and origin of neurosphere-like bodies from different layers of the gut

“…They represent a multipotent stem cell population that can differentiate into neurons, glial cells, and myofibroblasts and colonize aganglionic gut explants (1, 2, 4, 16, 20, 25-27, 30, 31). Initially isolated from embryonic gut tissue (2,27,31), they have subsequently been identified in adult guts of rodents and humans (1,14,16,21,25,26,30,33). Recently, Laranjeira et al (18) utilized genetic fate mapping of Sox10, a neural crest cell marker, to demonstrate that in the postnatal gut Sox10-derived cells can act as neuroprogenitor cells and regenerate neurons in response to injury.…”