The mammalian intestine is coated by a single layer of epithelial cells that is renewed around every five days (Laurens, G. et al. 2009). This high cell turnover makes it a fascinating and extensive organ system for the study of cell proliferation and differentiation. The gut is composed of crypts, which contain intestinal stem cells. These cells are multipotent therefore, they can develop into specialised cells (Howell and Wells, 2011). Through LGR5, an intestinal stem cell marker, it is now possible to envisage stem cells and examine their presence in the gut. Furthermore, one can consider differentiation in a much broader context. This understanding has led to the exploitation of in vitro growth of stem cells as a possible therapeutic in the context of IBD and transplantation. Additionally, the knowledge of in vitro organoids has resulted in the prospect of a pharmaceutical treatment for cystic fibrosis. On top of that, theoretically, intestinal stem cells can be used for biomedical purposes in vivo, for example, in single gene disorders the faulty gene can be removed using CRISPR- Cas 9 and homologous recombination occurs, leaving a healthy replaced gene (Baliou, 2018). The identification and application of stem cells has propagated groundbreaking research, which continues to benefit modern medicine.
The physiology of intestinal tissues is extremely complex. Intestinal tissue can renew with the help of stem cells that are multipotent. The gut optimizes it’s function by increasing the surface area for high absorption rates. It does this by forming crypts of tissues that are composed of stem cells, transit amplifying (TA) cells, paneth cells, enterocytes and enteroendocrine cells, goblet cells and myofibroblast cells. The TA cells produced at the crypt base migrate up the crypt axis and undergo apoptosis at the tips of the villi; this is known through exploration of lineage tracing. TA cells are rapidly proliferative and develop to produce the 4 cell types of the gut crypt. There are also Paneth cells which produce antimicrobial peptides as part of the innate mucosal defense.
To aid in food uptake, there are enterocytes which have microvilli on their surface, this increases the surface area. Additionally, gastrointestinal hormones are secreted to regulate glucose levels, food intake and stomach emptying. Goblet cells produce and maintain the protective mucus layer in the small and large intestine, whilst myofibroblast cells support growth and repair of the intestinal crypts (Widmaier et al.2006).
First of all, it is worth considering that up until relatively recently, stem cells were not known to be self-renewing. Originally, +4 cells, which are around four to six cells at the base of the crypt, were thought to be the stem cell region. They are now thought to be quiescent and only activated as a backup.
Researchers looked into stem cells by studying the mechanisms surrounding them like Wnt signalling (Schuijers, 2012). Wnt is a signalling pathway; it facilitates the stem cells to replace crypt cells (Fevr et al. 2007), essentially it is the driving force behind crypt biology. Wnt signalling regulates the processes associated with differentiated cells, and their compartmentalisation along the crypt-villus axis (Flannagan et al. 2018). In the crypt, LGR5 is the target gene of the Wnt signalling pathway. If Wnt signalling is blocked, then there is a fatal loss of intestinal function. Colorectal cancers are mostly initiated by mutations that activate the Wnt signaling pathway.
To further study the expression of LGR5, an allele with lacZ was integrated into one of the LGR5 alleles creating a generation of heterozygous mice (Barker et al. 2009). LacZ works by encoding an enzyme – β-galactosidase. When expressed it converts a substrate xgal into a blue molecule. This allows you to see where LGR5 is expressed. The cells must be permanently marked in order to lineage trace. This is because LGR5 is only expressed in the crypt base, so GFP will not be expressed when LGR5 is not. In this specific mouse model, Cre was expressed on the LGR5 promoter and this particular Cre was conjugated to the oestrogen receptor so that the Cre was only active when tamoxifen was injected into the animal. When Cre was activated it removed a stop codon from Rosa26 lacZ reporter allowing lacZ to be expressed in stem cells and then any other cell thereafter that the stem cell gave rise to. This confirmed, in vivo, that these LGR5 positive cells were self- renewing and that they divided every day because these genetically modified stem cells were long lived.
The next step in intestinal stem cell research was to attempt to culture individual cells. Individual cells were sorted according to GFP expression using flow cytometry (Hi, low and negative). It was found that certain conditions e.g. a Rho-kinase inhibitor to reduce apoptosis, were necessary to allow growth of these single cells. REFERENCE. Additional other components were needed such as, r-spondin which activates the Wnt pathway, its downstream products are essential for crypt-villus axis structure and notch-agonistic peptide which is essential for continued crypt proliferation. With these favourable conditions, Lgr5-GFPhi cells formed large crypt organoids. However, GFPlow cells rarely formed organoids. One of the main findings of this research was a set of culture conditions allowing long-term culture of intestinal stem cell organoids. In addition, it was found that intestinal crypt-villus units are self-organizing and are capable of being independent of non-epithelial cellular niches when separated from the small intestine.
Figure 1 – Images showing organoids grown from sorted single Lgr5+ cells. EGFP+ stem cells are scattered in the organoid (left), with the right image being positive for RFP (Yui et al. 2012).
Inflammatory Bowel Disease or IBD, is the phrase used for chronic inflammatory disorders of the gastrointestinal tract such as Crohn’s Disease (CD) and Ulcerative Colitis (UC). Causes of IBD are not completely known, however, it is thought that a dysregulated immune response to environmental triggers could be a possible cause. One hundred and sixty-three IBD loci have been identified using the genome-wide association study (GWAS). This portrays that host-microbe interactions have framed the genetic composition of inflammatory bowel disease (Jostins et al., 2012). Current treatments for IBD include anti-inflammatory steroids or immunosuppressants or surgery to remove damaged portions of the intestine.
Animal models are used to characterize gut epithelial cells and explore new treatments. Transplantability of cultured organoids can be tested this way (Yui et al. 2012). Colonic mucosal damage was induced by providing immunocompromised Rag2-/- mice with colitis-inducing dextran sulfate sodium (DSS).
The Rag2 gene was used to ensure that the stem donor cells would not be rejected within the mice. DSS damages the epithelium and increases intestinal permeability. Therefore, the mice developed inflammation of the inner lining of the colon as seen in figure 2.
Figure 2 – Image shows the mice colon at six days; the area is surrounded by oedematous mucosa; an inflammation shown in both the normal and highlighted images (EGFP is used). (Yui et al. 2012).
This was illustrated by weight loss, bloody stool, diarrhoea and epithelial injury in the distal colon. The green parts in figure 2 are highlighted by enhanced green fluorescent protein (EGFP) and portray successful engraftment, therefore, suggesting this could have some therapeutic value. On day sixteen, the recipient mice showed varying degrees of recovery from DSS as seen in FIGURE NUMBER. Multiple EGFP+ areas appeared, as well as visible patches in the treated colons. After four weeks, tube like EGFP+ crypts were observed in the distal colon. The engrafted crypts were entirely EGFP+ suggesting that there is the presence of EGFP+ stem cells. The epithelial integrity was tested using tetramethylrhodamine isothiocyanate conjugated dextran (TRITC-dextran). The levels of TRITC in the blood in transplant mice were compared to levels in the blood of control mice. The results of this test show that transplantation was less successful when freshly isolated donor cells were used. It was also suggested that allowing the stem cells to expand in culture will lead to higher chances of a successful transplantation. REFERENCE. Unfortunately, there are still many problems with using stem cells as a treatment for IBD. For example, cells have difficulty engrafting onto an inflamed gut and expanded epithelial cells can develop their own single nucleotide variant (Behjati et al, 2014). Additionally, cells can develop chromosomal trisomy (Wang et al, 2013).
The Cystic Fibrosis Transmembrane Conductor Receptor (CFTR) encodes for an anion channel essential for fluid and electrolyte homeostasis of epithelia (Cheng et al. 1990). In a normal cell, potassium, sodium and chloride ions will diffuse into the cell via the NKCC1 transporter. Sodium and potassium ions leave via the Na, K-ATPase pump but chloride ions leave via the CFTR channel. CFTR channels are activated by forskolin, which makes adenylyl cyclase bind to the G-protein receptor this triggers cyclic AMP which activates protein kinase A. Protein kinase A then phosphorylates the CFTR channel allowing an afflux of Chloride ions. However, mutations in the CFTR gene causes structural abnormalities in the protein stopping the chloride ions leaving the cell.
Cystic fibrosis is a genetic disease that is recessive. It affects the lungs and gut, causing breathing difficulties and an excess of mucus. It is caused by different mutations in the CFTR gene, the most common mutation is a deletion of phenylalanine at position 508 on exon eleven (Tsui, 1992). Cystic fibrosis affects more than ten thousand people in the United Kingdom with sufferers having a life expectancy of approximately forty years. However, CRISPR-Cas9 technology can be used to treat the mutated cells and allow the CFTR channel to operate correctly. Clustered Regularly Interspaced Short Palindromic Repeat or CRISPR is a genome editing technique that is achieved by transfecting a cell with the Cas9 protein along with a specifically designed guide RNA called gRNA (Wang et al. 2016). This directs the cut through hybridization with its matching genome sequence. When the cell repairs that break, errors can occur to generate a gene knockout or additional genetic modifications can be introduced. Cystic fibrosis can be treated using different methods such as: gene therapy requiring the use of CRISPR-Cas9 and pharmacology using specially designed drugs.
At this current time, in vivo gene therapy does not occur in humans. Consequently, pharmacology is critical in treating cystic fibrosis and other genetic disorders, despite that, it is a complicated process finding the correct drug for each individual patient. This is due to the fact that cystic fibrosis is caused by different mutations in the CFTR gene therefore causing different structural abnormalities in the protein. As such, each specific mutation requires a specific drug to treat it and it is hard to tell which drug will work. Furthermore, it requires a substantial amount of time to see if the drug has had the desired effect. Another factor to consider, is that these drugs are extremely expensive, with six months of treatment costing around half a million pounds. However, with the use of organoids, drug screening can aid in the identification of the correct drug with only a fraction of the price. Drug screening is also very time efficient. With the help of the correct drugs, cystic fibrosis patients can be assisted. With the most common mutation of the CFTR gene, the deletion of phenylalanine at position 508, drugs designed to treat this mutation, ‘chaperone’ the CFTR protein from the endoplasmic reticulum up to the membrane, where it should be (shown in figure 3).
Figure 3 – Self-drawn image representing how the drugs used to treat the del508 mutation would work by guiding the CFTR protein.
This allows chloride ions to be successfully transported and there is no problem with fluid transport. Despite the fact, that organoids have made drug screening possible, in vivo application is not possible at this current time. The complication with using drugs is that a vector is often required to reach the target cell and presently there is not a suitable vector. Researchers have tried various methods such as using a virus to break the cells membrane (Lin and Gallay, 2013) and allow the therapeutic agent to reach the target cell. This was unsuccessful as it is hard to predict the host-virus interactions due to the complexity of them. Additionally, there are several disadvantages to using viruses as vectors: there is the possibility of creating a barrier to resistance and compromising the host cell with a virus. Overall, there is still a long way to go to overcome the significant challenges that in vivo drug targeting presents.
In conclusion, intestinal tissue is extremely complex, with various mice models used to demonstrate the science behind stem cells found in the gut. For example, the investigation of the LGR5 marker was a pivotal turning point in intestinal stem cell research, allowing LGR5 positive cells to be discovered as self-renewing.