human ENS stem cells
2.1 Introduction
The isolation and culture of ENS stem cells from embryonic mice is well established and there are several different techniques available to do this, although all of them take the dissection and dissociation of the embryonic bowel into a single cell suspension as the first step. ENSC are then enriched and grown in culture using either FACS or magnetic beads to select for the stem cell markers p75(Pomeranz et al., 1993), p75 and α4 integrin(Bixby et al., 2002) and RET(Lo and Anderson, 1995), or by culturing in the form of neurospheres with no immunoselection(Almond et al., 2003, Bondurand et al., 2003, Schafer et al., 2003).
These techniques have been modified to allow for the isolation and culture of postnatal rodent ENSC(Kruger et al., 2002, Bondurand et al., 2003, Suarez-‐ Rodriguez and Belkind-‐Gerson, 2004). Although there are obvious differences between mouse and human colon, most obviously in the size of the tissue and the relative maturity of human colon in respect to rat and mouse colon(Karen Walthall, 2005), it seems logical to attempt to adapt these techniques to the isolation and culture of human postnatal ENSC.
2.1.1 Identification of stem cells
As defined in Chapter 1, a stem cell has the capacity to self-‐renew (make more stem cells by cell division) as well as to differentiate into mature, specialized cells. The identification of ENSC as genuine stem cells is problematic.
There are various cellular markers that have been used to identify ENSC, including Sox10, RET, p75 and nestin (see(Young and Newgreen, 2001) for a review). RET, all undifferentiated neural crest cells within the gut wall express p75 and Phox2b as they begin to colonise the hindgut(Young and Newgreen, 2001). RET, p75 and nestin have all been used in previous studies to identify ENSC (Natarajan et al., 1999, Sidebotham et al., 2001, Schafer et al., 2003, Belkind-‐Gerson et al., 2006). The problem with such markers is that they are not necessarily exclusive to ENSC. RET and Phox2b expression is maintained in cells differentiating into a neural lineage and p75 expression is maintained in cells differentiating into a glial lineage (Young et al., 1999). Nestin has been advocated a good marker for ENSC(Belkind-‐ Gerson et al., 2006, Rauch et al., 2006) but although its expression in glial cells in the CNS is very low, it is expressed postnatally in ENS glia and ICC(Vanderwinden et al., 2002). It can therefore be concluded that there is, at the present time, no specific marker for ENSC.
Another method of identifying neural stem cells is utilising their capacity to form neurospheres(Reynolds and Weiss, 1996). This capacity has led to the concept of the neurosphere assay – the assumption that under specific conditions, only such stem cells will form free floating spherical cellular aggregates(Pacey et al., 2006). However, other cell types will also form spherical aggregates in culture and therefore this assay is not specific enough to positively identify ENSC.
A further method of identifying ENSC is by their behaviour: a true stem cell is able to undergo asymmetric division to produce an identical daughter cell and a more differentiated precursor cell which undergoes subsequent differentiation, in this case to either neurons or glia. ENSPC can therefore be demonstrated by identifying a single cell that goes on to produce both neurons glia within the same colony – a demonstration of clonality(Bondurand et al., 2003). Alternatively, it could also be demonstrated that a neurosphere is capable of generating multiple daughter cells when disaggregated. If these daughter cells have characteristics identical to those of the original neurosphere, and can form further neurospheres
Combinations of the above techniques are therefore employed in the identification ENSC during the course of this thesis.
2.1.2 Summary of work done prior to the commencement of
this thesis
Work done by Almond on the isolation and characterisation of embryonic mouse ENSC has been previously described(Almond et al., 2003) and the techniques used in this thesis build on this work. In summary Almond developed a method of producing ENSC from embryonic mouse caeca at harvested at E11.5 and culturing them in the form of neurospheres. These neurospheres were shown to contain differentiated neurons and glia and contained single cells with the capacity to form both neurons and glia. Furthermore it was possible to clonally expand the neurospheres from a single cell, proving the existence of true ENSC within the neurospheres.
2.2 Aims
The aim of this study was to develop a method of isolating postnatal human ENSPC. Initially, competency in growing embryonic mouse neurospheres previously shown to contain ENSPC was achieved. The method for growing mouse ENSC was then adapted to produce human ENSC in the form of neurospheres. ENSC were identified by immunofluorescence for the markers p75 and nestin, the ability to produce both neurons and glia and the ability to self-‐ renew by the generation of secondary and tertiary neurospheres. ENSC were also identified by their ability to produce neurons and glia once transplanted into aganglionic, embryonic mouse hindgut.
The following sections describe groups of experiments; for each group the methods and results will be described, followed by a discussion of significant results and issues related to the experimental design before moving on to the next section.