means conversion of one differentiated cell type into another. It is a subset
of a wider class of cell type transformations called metaplasias. What is a cell type? Tissues Organs
What is a cell type?
Different concentrations of the inducing factor will result in the activation or repression of genes encoding different transcription factors, and therefore the adoption of different developmental pathways. Repeated occurrence of this process enables an early embryo, consisting initially of a simple mass of similar cells, to develop autonomously into an organism with a very complex pattern of structures.
In the hierarchical process of development, cell populations in a particular state of commitment often change into populations with a new state of commitment, for example the change from foregut endoderm to pancreatic bud. This is not transdifferentiation because the cell states in question are not discrete and persistent, they are transient states of commitment that will spontaneously progess along a default pathway even in the absence of any inducing factors. However there are a few examples where visible cell type does change. One is the development of the epithelial lining of the oesophagus. This is formed as a columnar epithelium, like the rest of the gut tube, and later becomes transformed to a squamous epithelium (Fig.2) (Yu, 2005).
Transdetermination is the name given to the transformations between different imaginal discs in Drosophila. It is associated with regeneration but because the discs are not yet differentiated, it also has aspects of an embryonic heterotopia.
other insects with a complete metamorphosis, the Drosophila
embryo hatches as a larva. This
crawls around, feeds and undergoes two moults.
After about a week it becomes a pupa.
Within the pupal case, the body of the larva undergoes
a drastic metamophosis in which most of the larval tissues
autolyse and the adult tissues and structures arise from the
imaginal discs and other reserve nests of cells (Fig.4) (Cohen,
1993). The discs are
established during embryonic life and proliferate during larval
life. But they do not differentiate
visibly until metamorphosis. There are different discs to form
the head; the legs, wings and halteres of the thorax; and the
Imaginal discs can survive in the undifferentiated state if they are transplanted into the abdomen of an adult fly. Here they are nourished by the haemolymph of the host and can grow indefinitely without differentiating. The fragmentation of discs necessary for repeated transplantation means that they are in a state of continuous regeneration. If they are returned to a late larva in which the hormonal conditions signify the start of metamorphosis, then the implants differentiate in concert with the metamorphosis of the host. In most cases the structures formed are appropriate to the original character of the disc, but sometimes they are appropriate to a different disc (Maves and Schubiger, 2003). For example, tissue from a leg disc may produce wing structures. In a classic but rather intricate experiment involving combined clonal labelling and transdetermination, it was shown that transdetermination could sometimes occur in groups of cells as well as in single cells (Gehring, 1967). This indicated that somatic mutation could not be the mechanism as the same mutation would not be expected to occur in a group of cells simultaneously.
Although transdetermination of Drosophila imaginal discs was traditionally considered a rare event, it can be caused to occur at high frequency by cutting the discs at the right places, the so-called “weak points”, and one such place is the dorsal part of the prothoracic leg disc, where a cut will provoke transdetermination to wing with 95% frequency. Such high frequency enables detailed investigation to take place. It turns out that wounding activates the JNK signalling pathway and this represses expression of Polycomb group genes. Polycomb proteins normally repress expression of the wingless gene, hence Wingless protein starts to be produced and its signalling activity upregulates expression of the vestigial gene, which encodes a transcription factor (Maves and Schubiger, 1995; Lee et al., 2005).
This example indicates the central role of a transcription factor, Vestigial. This is normally expressed in the dorsal discs (wing, haltere) but not ventral ones (legs), and serves to distinguish wing from leg character. It also indicates a role for intercellular signalling (Wingless is the Drosophila homologue of the Wnt factors), which explains why transdetermination may occur in many cells simultaneously. It also introduces a role for chromatin organisation. The Polycomb group proteins tend to shut down gene activity in regions of the chromosome to which they bind, by a variety of mechanisms including the promotion of inhibitory histone methylases and the inhibition of chromatin remodelling proteins.
Comparable phenomena to transdetermination are found in other groups of arthropod. For example, the so called serial heteromorphosis is found in the regeneration of appendages in both crustacea and insects. It has long been known that if a claw is amputated from the shrimp Palinurus, it may regenerate not as a claw but as an antenna (Fig.5). In several types of insect, an amputated antenna may be replaced by parts of a leg (Villee, 1942). Serial heteromorphosis has remained uninvestigated for a long time, but has all the hallmarks of a typical metaplasia.
Wolffian lens regeneration
The only example of transdifferentiation that is conceded to exist by sceptics is the regeneration of the lens found in various species of urodele amphibia (newts and salamanders) (Okada, 1991; Tsonis et al., 2004). Ironically this is a case where careful cell lineage studies have not been done, perhaps because the morphological appearance of transdifferentiation is so compelling.
In Wolffian regeneration, after removal of the lens of the eye, the cells of the dorsal iris start to proliferate, undergo depigmentation, and eventually redifferentiate to form a new lens. The two cell types are very different: iris cells are pigmented epithelial cells similar to those of the pigmented retina while the lens is composed of modified keratinocytes containing high concentrations of crystallin proteins that impart the characteristic transparency. This system has been studied extensively in vitro and it was shown that it was possible to obtain development of lentoids from a clonal culture initiated by just a single pigmented iris epithelial cell of a chick embryo (Eguchi and Okada, 1973). Remarkably the in vitro culture system works for all vertebrates including human foetal iris and retina (Yasuda et al., 1978), and not just for the urodeles that will regenerate following lens ablation in vivo. Like the Drosophila discs, this system offers a connection between wounding and the initiation of a regenerative process. However the mechanism seems to be rather different, consisting of enhanced activity of the proteolytic enzyme thrombin, a component of the blood clotting cascade. Removal of the lens causes damage to vessels of the ciliary body, which release prothrombin into the aqueous humour. This becomes sequestered on the dorsal but not ventral iris because of the local presence of a substance called Tissue Factor, and is then processed to thrombin (Imokawa and Brockes, 2003). The thrombin is necessary for the entry of the iris cells into S phase, and it probably works by releasing another factor present in a latent form in serum.
Some metaplasias have a clinical significance because they predispose to development of cancer. In human histopathology it is not unusual to find foci of particular tissues in the wrong place. Examples are the occurrence of bone in the soft connective tissue, or squamous patches in an epithelium that is normally glandular in histology (Willis, 1962). Metaplasias virtually always arise in tissues that have been subjected to chronic trauma, infection or abnormal hormonal stimulation, hence undergoing continuous regeneration. This association with regeneration is a point of resemblance to the arthropod examples. In some cases it is not clear from static preserved pathological specimens whether the ectopic tissue developed in situ, or migrated from elsewhere. Obviously the latter situation is not relevant to our present concerns. But highly unlikely to arise by migration are the glandular metaplasias where patches of one tissue are found embedded within the epithelium of another. These can be found particularly in the gut and in the female reproductive system, perhaps because these two systems consist of a series of organs arranged as a tube, each organ being lined with a histologically different epithelium (Slack, 1985; Slack, 1986). When a patch of metaplasia occurs it is often composed of the tissue type normally derived from a neighbouring region in the embryo. For example intestinal metaplasia of the stomach means the occurrence of patches of intestinal tissue within the gastric mucosa; and intestine and stomach develop from adjacent territories of the endoderm in the early embryo (Fig.6,7). Less obvious is the condition known as cystitis glandularis, where colonic-type tissue arises in the urinary bladder. This is a quite separate organ to the intestine in the adult, but is derived from neighbouring endoderm in the embryo, as the urinary bladder forms from the proximal part of the allantoic evagination of the hindgut.
Some metaplasias have a clinical significance because they predispose to development of cancer. For example the bronchi are lined with columnar epithelium, but smokers often have patches of squamous metaplasia and it is from within these patches that lung cancer usually arises. Adenocarcinoma of the oesophagus usually arises in areas of Barrett’s metaplasia, a condition in which the normally squamous epithelium of the lower oesophagus becomes converted to columnar type, with gastric and intestinal differentiation patterns. In such cases the metaplasia can be regarded as the first step in a multistep progression to cancer.
A number of studies around the year 2000 suggested that bone marrow stem cells are capable of colonising a wide variety of other tissue types when transplanted into irradiated hosts. Some of these were performed with unfractionated marrow, some with enriched or purified haematopoietic or mesenchymal stem cells. The tissues colonized included virtually everything including numerous epithelia, muscle and neurons. This work generated considerable controversy because it suggested a very different model of development from the conventional one. Instead of cell populations undergoing a series of decisions during embryonic development, in each of which their competence is restricted, the idea was that the whole body is continuously being renewed by highly pluripotent cells from the bone marrow (Blau et al., 2001).
The phenomenon became known as “transdifferentiation” although this is unfortunate as the term was previously used, and is used here, to refer to cases of direct transformation between differentiated cell types.
It now appears that some of the results were due to lodgement of cells in these tissue but without actual differentiation, and others were due to cell fusion, whereby the genetic markers from donor cells became incorporated into host cells (Wagers and Weissman, 2004). There may be some genuine reprogramming of marrow-derived cells to various other tissue types, but this certainly only occurs at very low frequency. Because the hosts are nearly always irradiated, and therefore have considerable tissue damage and widespread tissue regeneration all over the body, it is thought that this situation allows favorable circumstances for the occasional reprogramming event. It is not, however, at all likely that reprogramming occurs on a large scale, or that the bone marrow is a repository for cells that can regenerate the rest of the body.
pluripotent stem cells (iPS cells),
which are extremely similar to embryonic stem cells (ESC), are made by introducing certain genes
into normal cells, and then applying selective conditions such
that those few cells that become reprogrammed to an ES-like
state will grow into colonies. They are discussed here because
the cell type transformation is
The original gene set used was Oct4, Sox2, Klf4 and Myc, called “Yamanaka factors” after the originator (Takahashi and Yamanaka, 2006). OCT4 and SOX2 are key transcription factors regulating the pluripotency of ES cells. The function of Klf4 and Myc are less well defined but they are both expressed in ES cells and known as oncogenes that can cause cancer if activated inappropriately. Their effect may be exerted by promoting cell division and/or by making sites in the chromatin more accessible to the OCT4 and SOX2. Delivery of the Yamanaka factors to mouse fibroblasts using retroviral vectors generates about one ES cell colony per 10,000 input cells. This frequency is typically lower for human cells, but it can be increased by including extra genes or by treating with substances, such as the histone deacetylase inhibitor valproic acid, that open chromatin and increase accessibility of transcription factors to the genome. Of the four genes in the Yamanaka set, only Oct4 is really obligatory, the others can be substituted by other genes, usually members of the ES cell pluripotency factor group, such as Nanog, or genes that stimulate cell division. The critical requirement is that overexpression of the genes can upregulate the autocatalytic network of pluripotency factors characteristic of ES cells.
iPS cells can be made from a variety of mammalian species. Mouse iPS cells will integrate into the mouse embryo following injection into blastocysts and are capable of contributing to all tissues including the germ line. This is the “gold standard” test for pluripotency. It is also found possible to make iPS cells from other cell types than fibroblasts. In particular lymphocytes isolated from blood and stimulated to divide by treatment with appropriate growth factors or lectins can serve as the starting material. This is potentially important because blood samples are so easily obtained from individual people. iPS cells have been made from lymphocytes in which gene rearrangement of antibody or T cell receptor genes has already occurred, which shows that it is possible to reprogram even well differentiated cells by the introduction of suitable transcription factors (Hanna et al., 2008).
The genes are delivered using retroviral or lentiviral vectors that insert their DNA into the chromosomes of the cells and enable high level expression of the products. Eventually the viral-encoded genes usually become “silenced”, that is they cease to be active, probably because the chromatin around them becomes impermeable to the transcription machinery. Continued activity of the introduced genes is detrimental, because, being pluripotency factors, they tend to inhibit differentiation of the cells.
There is a set of standard tests that are used to characterize iPS cells and to establish whether a particular new line has a genuinely ES cell-like phenotype or not. These include: an appropriate gene expression pattern, presence of the correct cell surface markers and alkaline phosphatase activity, colony morphology, the removal of DNA methylation from promoters of endogenous pluripotency genes, and formation of embryoid bodies capable of differentiating into derivatives of all three germ layers. Also the cells should have a normal karyotype and viral genes should be fully silenced. One key property of mouse ES cells is the ability to form chimeras when injected into early mouse embryos.
Human iPS cells can be made using similar methods (Takahashi et al., 2007; Yu et al., 2007). For human iPS cells it is not possible for ethical reasons to inject the cells into embryos, so the standard approach is the teratoma assay where the cells are injected into an immunodeficient host animal. Good quality iPS cells should grow to form a teratoma, and this should contain tissues characteristically derived from all three embryonic germ layers: ectoderm, mesoderm and endoderm.
Although good quality of iPS cells closely resemble ES cells, the random elements in their formation mean that it is easy to isolate imperfect cell lines. It is commonly found that iPS cell lines carry some “memory”, probably encoded in DNA methylation, of the cell type that they used to be. This also leads to a subsequent bias in the ease of differentiation in favor of this cell type.
Differentiated cell types tend to be relatively stable and to persist long term, with or without cell division. However it is sometimes possible to reprogram one differentiated cell type to another by overexpression of specific transcription factors which are responsible for the relevant commitment processes in normal development (Zhou and Melton, 2008). The first example of this was the ability of the myogenic factor MyoD to reprogram a variety of tissue culture cell lines to a myogenic phenotype (Weintraub et al., 1989). More recently, several other examples have been described, including the conversion of pancreatic exocrine cells to hepatocytes (Shen et al., 2000), B lymphocytes into macrophages (Xie et al., 2004), pancreatic exocrine cells to endocrine cells (Zhou et al., 2008), fibroblasts to neurons (Vierbuchen et al., 2010) or fibroblasts to cardiomyocytes (Ieda et al., 2010). Between one and three transcription factors need to be overexpressed to achieve these transformations, typically ones that are involved in the normal embryonic development of the cell type in question. Their function is not simply to activate direct target genes, but to shift the cell into a new stable state of gene expression.Barrett’s metaplasia is a condition in which the lining of the lower end of the oesophagus changes from the normal stratified squamous epithelium into a columnar epithelium with some features of stomach and intestinal linings (Falk, 2002). Barrett’s metaplasia predisposes to oesophageal cancer, whose incidence has risen rapidly in the last three decades and carries a poor prognosis. In a typical individual the zone of transformed tissue contains several different populations of cells with different mutations contributing to the abnormal appearance (Leedham et al., 2008). The disease is associated with the reflux of acid and bile from the stomach and duodenum into the oesophagus, but the mechanism of the initiating event is poorly understood at present. There is also controversy about the cell of origin: it may be oesophageal basal cells, or gland cells, or cells from the adjacent region of the stomach (Wang et al., 2011)
In order to achieve a better understanding of what drives the initial changes in Barrett’s metaplasia we are studying the role of certain genes which are important in intestinal development. Recent evidence suggests that Cdx2, a gene normally important for the development of the intestine but not expressed in the oesophagus, may be involved in Barrett’s metaplasia. We are currently investigating the conditions under which expression of Cdx2 in the oesophagus can prompt transdifferentiation from stratified squamous epithelium into intestinal type epithelium. This should increase our understanding of the disease process and may ultimately enable a form of gene therapy to reverse the metaplastic process.
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Cells, tissues and organs
Transdifferentiation in normal development
Transdifferentiation in Drosophila
Transdifferentiation in regeneration
Transdifferentiation in human pathology
The transdifferentiation controversy
Slack and Tosh Bibliography