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Stem cells have the capacity to self-renew and retain their undifferentiated phenotype even after undergoing numerous cycles of cell division or differentiate into one or more type(s) of specialized, function-specific daughter cells (Lajtha, 1979). Based on a nucleotide pulse-chase experiment by Cotsarelis and colleagues (1990), epidermal stem cells were identified by their slow cycling nature which allows them to retain DNA labels for prolonged periods. Although they infrequently proliferate, they have a high proliferative potential and increase their rate of proliferation during development and wound healing to maintain tissue homeostasis. Populations of stem cells are present in continually renewing tissues such as the epidermis, intestine, muscle, brain and the haematopoietic system (Weissman et al, 2001), to provide a source of differentiating cells (Lajtha, 1979). Within these tissues, they often reside in niches which act as a specialized environment that regulates their proliferation and differentiation (Fuchs et al., 2004).
Figure 1. Anatomy of the skin
The skin functions as the primary interface between the body and the external environment. It acts as a protective barrier against several environmental threats such as mechanical injury, ultraviolet radiation, thermal damage, dehydration, chemical insult and infection (Kameda et al., 2002). The skin is composed of the interfollicular epidermis (IFE) interspersed with the pilosebaceous units that contain the hair follicles (HF) and sebaceous glands (SG) (Figure 1; Gambardella and Barrandon, 2003; Fuchs, 2007). The HF is a complex structure composed of several compartments including the bulge and isthmus (Figure 1). The lower two-thirds of the HF periodically undergoes regeneration cycles comprising of an active growth phase (anagen), driven by stem cell populations residing in the HF, followed by destruction (catagen) and finally a quiescent phase (telogen) (Paul, 1998). The IFE consists of four layers of keratinocytes - basal layer, spinous layer, granular layer and stratum corneum (Figure 1). In the IFE, proliferation specifically occurs in the basal layer where progenitor cells are found (Watt, 1998). These cells terminally differentiate and move through the suprabasal layers to the surface of the tissue where they are shed (Watt, 1998). Due to this continual turnover, regular renewal is necessary for new cells to be generated and replace those that are lost in order to maintain epidermal homeostasis. This balance is crucial not only for optimal tissue function, but also to prevent ulceration resulting from excess cell loss and cancer resulting from excess cell production. The requirement for constant cell replenishment in the epidermis has led to extensive research on the mechanisms involved in maintaining its homeostasis.
1.3 Possible Origin of IFE Progenitor Cells
In my project, I will be focusing on the IFE and how it is maintained in steady state. Different studies have suggested several possible origins of IFE. Bulge stem cells have been reported to give rise to multiple lineages of the skin, including the HF, SG and IFE during grafting and wound healing (Oshima et al., 2001; Taylor et al., 2000; Blanpain et al., 2004). Although these studies have made major contributions in understanding stem cell role and potency, they do not reflect the function and behaviour of these cells during normal homeostatic condition. Indeed, genetic lineage tracing experiments have revealed that during skin homeostasis, the contribution of bulge cells is restricted to lineages of the pilosebaceous unit which consist of the HF and SG (Morris et al., 2004; Zhang et al., 2009). Furthermore, genetic labelling studies indicate that the bulge stem cells maintain the HFs but not the IFE in the absence of trauma (Levy et al., 2005). In support of that, a study involving the ablation of bulge cells by transgenic expression of a suicide gene showed that the HFs were completely lost but the IFE remained intact (Ito et al., 2005).
However, a couple of studies have reported populations in the isthmus, the region directly above the follicle bulge, generating progenitor cells that migrate and contribute to the IFE. Jensen et al. (2009) reported that Lrig1+ cells express low levels of Sca1 which is highly expressed in the IFE. In addition to that, Snippert et al. (2010) reported Lgr6+ cells, which reside in the isthmus, contributing to all cell lineages of the skin during development and postnatally. They suggested that Lgr6, a member of the family of orphan G-protein-coupled receptors (GPCR), is a marker for a primitive epidermal stem cell. Besides, these findings provided further evidence in support of the idea that the epidermis contain a hierarchy of stem cells in the epidermis, from stem cell to various progenitors, some of which might be actively cycling while others are normally quiescent but generate a broader array of cell fates.
1.4 Classical Theories on Epidermal Homeostasis
1.4.1 Stem/Transit Amplifying (TA) Cell Hypothesis
Different mechanisms have been described to account for how a single layer of proliferative basal cells generates and maintains a multilayered differentiated epidermis. According to the stem/transit amplifying (TA) cell hypothesis, the proliferative compartment of epidermal keratinocytes is heterogeneous, consisting of the self-renewing stem cell and its progeny, the short-lived TA cell (Figure 2; Potten, 1974). A small population of slow-cycling basal stem cells, which only makes up only 10% of the IFE cells, divides and produces a large number of more rapidly cycling TA cells (Figure 2, Potten, 1974; Jones et al., 1995). TA cells are proliferating keratinocytes that have limited self-renewal capacity and a high probability of withdrawing from the cell cycle and undergoing terminal differentiation (Potten, 1974). As the TA cells terminally differentiate to give rise to post-mitotic keratinocytes, they undergo a decline in expression of surface integrins, leading to detachment from the basement membrane and migration vertically through the suprabasal layers towards the epidermal surface (Fuchs and Horsley, 2008). The difference in proliferative potential indicates a hierarchy in the epidermal cells.
Figure 2. Stem/Transit Amplifying (TA) cell hypothesis. Slow-cycling, self-renewing stem cells (yellow) generate an actively-cycling, short-lived population of TA cells (purple) which undergoes three rounds of cell division before terminally differentiating into post-mitotic cells (blue).
1.4.2 Epidermal Proliferative Unit (EPU) Model
Within the Stem/TA cell hypothesis, it was further proposed that the epidermis is arranged into columns, known as epidermal proliferative units (EPUs; Figure 3; Mackenzie, 1970). This model was supported by the argument that since the differentiated cells can only migrate upwards on differentiation, each column must be maintained by the basal cells beneath it (Mackenzie, 1970). All EPUs are of a constant size with the boundaries that coincide with those of the cornified cells in the stratum corneum (Figure 3). Potten (1974) proposed that a stem cell lay at the centre of each EPU since fewer mitoses were observed in basal cells lying beneath the centre of the columns than those at the periphery. Morris et al. (1985) came to the same conclusion with a study involving DNA labelling with [3H]thymidine which demonstrated the existence of label-retaining cells in the center of the restricted population of EPU. Each EPU contains approximately 10 keratinocytes which are considered to be clonal descendants of the central stem cells (Allen and Potten, 1974). These models predict that the basal layer clone size must become time-independent and clones will eventually reach a constant upper size limit corresponding to an EPU.
Figure 3. Epidermal Proliferative Unit (EPU) model. Epidermis organized into columns comprising of a single centrally located stem cell (yellow) which generates TA cells (purple) that in turn generate post-mitotic cells (blue) that leave the basal layer and migrate vertically upwards as they differentiate into cornified cells (green hexagon) in the stratum corneum.
1.5 Committed Progenitor (CP) Model
In sharp contrast to this traditional model, Clayton and collaborators (2007) introduced a new model, called the committed progenitor (CP) model which suggests that all cycling basal epidermal cells are identical and may undergo unlimited number of divisions. These cells, referred to as CP, maintain normal epidermis with no measurable contribution of stem cells. Jones and Simons (2008) supported this claim by reporting that cycling cells do most of the epidermal renewal work while the stem cells remain dormant; therefore, continual stem cell proliferation is not required.
Clayton et al. (2007) performed a large-scale quantitative in vivo lineage tracing in mouse tail skin epidermis using the Cre-lox system to induce YFP labelling to track the fate of cells. Firstly, by looking at two-cell clones at early time points staining for markers of proliferation or differentiation and looking at cell location, they found that all cycling basal cells are identical and that division of CP has three possible outcomes, resulting in two progenitor cells, two post-mitotic cells or one of each (Figure 4). The fates are adopted at random; however, since the probabilities of the symmetric fates are balanced over the whole CP cell population (Figure 4), equal numbers of cycling and differentiating cells are produced, ensuring tissue homeostasis. Due to this stochastic behaviour, the CP is predicted to give rise to clones with a wide range of sizes.
Secondly, they tracked the sizes of clones over a one-year time course at a single-cell resolution in vivo and found that the average size of persisting clones increased linearly with time. This is in disagreement with the EPU model which suggests that a fixed number of basal layer stem cells regionally maintain a defined number of surrounding TA cells, and the overall clone size should level off at the size of a single EPU rather to continuing to expand. Furthermore, they found that clone size distribution scales with time, which suggests that only one type of cell dividing at one average rate exists in basal epidermis. This is incompatible with the Stem/TA cell hypothesis which suggests a heterogeneous population dividing at different rates.
Figure 4. Committed Progenitor (CP) model. All cycling basal cells (dark blue) stochastically adopt one of the three possible fates following division, generating two progenitor cells (dark blue), two post-mitotic cells (light blue) or one of each type. Post-mitotic cells in the basal layer differentiate into suprabasal cells. The percentages represent the proportion of cells adopting each fate.
However, there were a few limitations to this study. They used a single colour to track the clones. To avoid the overlap between adjacent clones they had to ensure that clones would be far from each other. Consequently, they tailored their system to label only 1 in 600 cells. With this low density staining, there is a higher probability of missing a rare population of stem cells. Moreover, they used the Ah promoter which is a ubiquitous promoter that expresses Cre in both proliferating and differentiating cells in the IFE (Kretzschmar and Watt, 2012). Besides that, the study was only conducted in tail epidermis which may not be applicable to epidermis in other body sites. The ultrastructural organization of epidermis at different body sites is not identical (Allen and Potten, 1976). Dorsal and ear epidermis are arranged in a fixed structure of precise columns with regular alternating interdigitation of cells at the boundaries for all six neighbouring EPUs, predicting vertical movement (Figure 5; Potten, 1974; Allen and Potten, 1974). Ultrastructural studies revealed that the tail skin lacks these regular columns of suprabasal cells and the squames at the skin surface are imbricated, suggesting that basal epidermal cells in the murine tail may migrate at an angle rather than directly upwards (Figure5; Allen and Potten, 1976). In spite of that, Doupe et al. (2010) who used EYFP-marked lineage analysis in murine ear epidermis reached similar conclusions.
Figure 5. Structural variations of epidermis in dorsal and tail skin.
In an incremental change from the CP model which suggests that there is no hierarchy in the epidermal cells, a recent study by Mascre et al. (2012) suggested otherwise. This quantitative analysis of clonal fate data and proliferation dynamics using two different inducible CreER constructs, K14-CreER and involucrin (Inv)-CreER demonstrated the existence of two distinct proliferative cell populations that differed in their proliferative dynamics, gene expression profile and ability to repair epidermis after injury. K14-CreER expression targets all basal layer cells including stem cells which persist through the animal's lifetime and contribute to long-term regeneration and repair of the epidermis. Expression of Inv-CreER, which has been reported to predominantly label suprabasal cell as well as some basal cells (Lapouge et al., 2010), is claimed to be specific to CPs which persist for shorter periods and only provide a short-term contribution to the wound healing. This study demonstrated the existence of slow-cycling stem cells that promote tissue repair and more rapidly cycling progenitors that ensure the daily maintenance of the epidermis. This study however shares similar limitations than the CP model as it does rely on low density staining and may also miss the staining of rare stem cells.
1.6 Lineage Tracing Using Rainbow Technology
Inducible Cre-lox recombination system are commonly used to genetically mark and track the fates of progenitor cells and their progeny. It allows the switching on of gene expression by DNA excision, inversion or interchromosomal recombination (Branda and Dymecki, 2004). One of the major advantages of this technique is the ability to temporally and spatially control the activity of Cre with inducible recombination.
Cre recombinase, which is typically fused to the modified human estrogen receptor (ER), is expressed under the control of a tissue- or cell-specific promoter in one mouse line. This mouse line is crossed with another mouse line in which a reporter is flanked by loxP sequence. In the absence of ligand tamoxifen, the Cre recombinase-ER fusion protein (CreER) is kept inactive in the cytoplasm by heat shock proteins (hsp; Figure 6). Upon treatment, tamoxifen binds to ER causing the receptor to change confirmation, leading to a release from its hsp chaperones (Figure 6). The activated CreER localizes to the nucleus, where Cre can catalyse the recombination the loxP sites and excise the floxed sequence (Figure 6), activating a reporter in cells that express the promoter.
Figure 6. Inducible Cre-lox system. Without ligand binding, the inactive CreER fusion protein is being sequestered by hsp (green) in the cytoplasm. Upon treatment, tamoxifen binds to the ER (yellow), activating Cre (white) which translocate to the nucleus and mediates recombination between lox sites to excise the floxed sequence (pink) and allow expression of a reporter (blue).
It appears that to overcome an important limitation of previous studies, it is required to label all basal keratinocytes to be able to account for the activity of rare stem cells. Multicolour reporter constructs are beings used increasingly for lineage tracing with two or more markers. These constructs make it possible to examine how different cell types contribute to the maintenance and repair of a given tissue. Lox variants have been developed with mutations that prevent them from recombining with the canonical loxP site, while allowing them to recombine specifically with identical lox sequences (Lee and Saito, 1998). Livet and colleagues (2007) exploited the advantages of this to enable combinatorial expression of several reporters in a stochastic manner by alternating the variant lox sites, lox2272, with canonical loxP in the same construct. This configuration forces Cre to choose between two mutually exclusive events; therefore, two recombination events are initially possible, but only one can occur (Livet et al., 2007). Excision between either pair of identical lox site removes one of the other pair, thereby preventing further recombination (Livet et al., 2007).
We will use a transgene designed by Livet et al. (2007), called Brainbow 1.0. This transgene consist of three different reporters - dTomato, mCerulean and EYFP, flanked by two different pairs of lox sites. Since only the first reporter following the promoter is expressed, the cells will express the red fluorescent protein, dTomato in the absence of any Cre induction. When Cre activity is induced, many cells will lose dTomato expression and switch on either mCerulean or YFP expression depending on Cre's choice of excision events. If Cre excises at the lox2272 site, it results in cells expressing mCerulean whereas when Cre excises at the loxP site, it results in cells expressing EYFP. Each cell in these mice has two copies of the transgene; hence, there is a possibility of a cell expressing two different colours. The combination of the primary colours results in cells possibly expressing pink (mCerulean+dTomato), orange (dTomato+YFP) and white (mCerulean+YFP) as well. Altogether, there are six different colours for tracking the cells. These genomic modifications are stable and they are inherited by the progenies of that particular cell, making it easy to follow clone size over time.
Figure 7. Brainbow 1.0 construct.
2. Research Project
2.1 Hypothesis and Aims
Table 1. Contradiction between different models/hypotheses.
According to the Stem/TA cell hypothesis, a heterogeneous population with determined fate is responsible for epidermal homeostasis. They include the stem cell which gives rise to the TA cell that can only undergo a limited number of cell division. The decreasing proliferative potential from stem cells to TA cells suggests a hierarchy in the epidermal cells. On the other hand, the CP model proposed that a homogeneous population with stochastic fate and ability to undergo unlimited number of division maintains normal epidermis. This model reported no involvement of stem cells and no hierarchy in the epidermal cells. In theory, the clones will eventually reach an upper size limit as proposed by the EPU model. However, Clayton et al. (2007) observed an increase in clone size with time. Furthermore, the CP model states that the IFE is not derived from the HF but from the CPs that are already present in the IFE. In contrast, Jensen et al. (2009) and Snippert et al. (2010) observed contributions of a stem cell population in the HF to the IFE. Clearly, observations contradict with the theories proposed; therefore, we are proposing a new model. We hypothesize that there is a population of stem cell in the HF that gives rise to the CP in the IFE (Figure 8B), which stochastically adopts one of the three possible fates following division (Figure 8A).
Figure 8. Hypothesis
2.1.2 Preliminary Data
Our preliminary data clearly shows that the tail epidermis of Skinbow mice consists of clones with different sizes (Figure 9). This characteristic is consistent with CP model as well as our hypothesis, which suggests a wide range of clone sizes correlating with the stochastic behaviour of cycling basal cells.
Figure 9. Cross section of the tail epidermis of Skinbow mice showing the IFE.
Another preliminary data in Figure 10 shows a YFP-expressing population of stem cell in the isthmus, a region in the hair follicle, contributing to the IFE which supports our hypothesis.
Figure 10. Cross section of a Skinbow mouse epidermis.
We have two specific aims to validate this hypothesis.
a) The first aim of the experiment is to evaluate the evolution of clone size over time with high density multicolour staining.
This aim will help determine if the clone size increase described in the CP model holds true in the high density staining model.
b) The second aim of the experiment is to determine if the clones attached to the hair follicles are larger in size.
The aim will more clearly evaluate the possibility that stem cells from the hair follicle participate in the IFE at steady state. It is expected that in such case, clones around hair follicle would have a larger size as they will contain more primitive cells immediately deriving from hair follicle stem cells.
2.2 Experimental Design and Expected Outcomes
Mouse lines, CAGGSBOW and Krt14Cre/ERT2 have been previously generated and imported. CAGGSBOW mice were developed with our close collaborator, Dr. Livet by microinjecting a Brainbow 1.0 cassette under the control of the ubiquitous CAG promoter. Krt14Cre/ERT2 mice are transgenic for the ligand-inducible form of Cre, CreERT2 that is expressed from the Keratin 14 (K14) promoter. [cite ref for these mice: PNAS by Fuchs - jaxmice.com] Mice were maintained within the animal facilities in University of Queensland Centre for Clinical Research (UQCCR) and Herston Medical Research Centre (HMRC). As depicted in Figure 11, CAGGSBOW mice will be crossed with Krt14Cre/ERT2 mice to produce a double transgenic mouse called Skinbow. K14 allows expression of Cre in the basal layer of mouse epidermis and the outer root sheath of hair follicle (Figure 12; Fuchs and Green, 1980). These compartments are the most likely to contain all types of stem cells. Then, the Cre recombinase was activated 20 days after birth (postnatal day 20, P20) by intraperitoneally injecting 100µl of 10mg/ml tamoxifen (dissolved in corn oil) per mice once a day for five consecutive days. At P20, the HF is completely developed and it is ready to enter the first hair cycle. The mice will then be sacrificed at five different time points post induction - 1 week, 3 weeks, 5 weeks, 3 months and 6 months. All procedures are approved by the University of Queensland animal ethics committee under project UQCCR/380/09.
2.2.2 Tissue preparation
The back skin tissue will be dissected at telogen phase for better identification of anatomical locations within hair follicles. For fluorescence-activated cell sorting (FACS) analysis, the subcutaneous tissue and fat will first be removed from the back skin before being digested with 1X trypsin overnight at 4 C to allow separation of epidermis from dermis. The epidermis and dermis will be digested with trypsin and collagenase [how long what temp.], respectively, after which the cell suspensions will be strained and combined.
For confocal microscopy, the tissues will be fixed in 4% paraformaldehyde (PFA) for 2 hours and stored in 20% sucrose. Then, the tissues will be counterstained with DAPI nuclear stain overnight and cleared using FocusClear for 2 days to allow up to 100µm penetration of confocal microscope lasers.
2.2.3 Flow cytometry
Cells will be analysed using a Gallios Flow Cytometer (Beckman Coulter) on the basis of dTomato-, mCerulean- and EYFP-expressing cells after gating out apoptotic cells using a live gate on forward and side scatter (7AAD). This analysis will allow us to quantitatively assess the expression of fluorescent protein in the IFE of the Skinbow mice and determine the respective frequency of the different colours upon recombination.
2.2.4 Confocal microscopy
IFE wholemounts will be flat-mounted on a slide and observed under the Zeiss LSM 710 confocal microscope using 100X magnification. Since the colours in the Skinbow mice are close in spectrum, we find that Multi Channel mode is the most appropriate mode as it allows us to select the most suitable emission range for our fluorochrome. We will use excitation wavelengths of 514nm, 561nm, 458nm and 405nm for EYFP, dTomato, mCerulean and DAPI, respectively. The colours will be separated into three different channels which will be acquired sequentially. The first channel consists of dTomato and DAPI whose wavelengths are far enough apart to avoid overlapping emission, the second channel consists of EYFP and the third channel consists of mCerulean. This multi-acquisition sequence allows also for correction of the level of intensity of the different fluorochrome. Indeed, Cerulean being weak in intensity, it needs stronger excitation signal. Moreover, the pinhole, which determines the slice thickness from which light emission is gathered, will be set to a diameter of ~50µm for EYFP, dTomato and DAPI whereas for mCerulean, which is a relatively weak fluorophore, pinhole diameter will be adjusted to ~100µm to optimize its detection. Three dimensional Z stacks will be acquired containing the entire IFE down to the bulge. Each multichannel Z-stack image will be obtained for analysis using the same settings described.
Figure 11. Experimental outline
Figure 12. Expression of K14 in cells in the basal layer of the IFE and HF.
2.2.5 Data analysis
To address the first aim, the proportion of different clone sizes will be counted at each time point. Each clone consists of cells with similar colour in direct contact with one another. The data collected will be analysed using chi-square for trend to determine if the distributions of clone sizes are significantly different at various time points. There are two expected outcomes from this experiment:
a) If the epidermis consist of clones that remain at constant size corresponding to that of an EPU even at late time points, we can conclude that the epidermal progenitor cells behave as described by the classical theories, Stem/TA cell hypothesis and EPU model (Figure 13A).
b) If there is an increase in the size of clones with time, we can conclude that the epidermal progenitor cells behave as described in the CP model (Figure 13B).
Figure 13. Expected outcomes of experiment addressing Aim 1
This experiment will confirm the mechanisms by which epidermal homeostasis is maintained.
To address the second aim, we will simply compare the sizes of clones that are attached or not attached to a hair follicle. By scrolling through the layers of the Z-stack images, we can locate the HFs and identify clones that are attached to them. We will analyse the data collected using t-test to determine if the average sizes of clones attached are significantly different from that of clones not attached to the hair follicle. There are two expected outcomes of this experiment:
a) If there is no difference in the sizes of clones attached and not attached to a hair follicle, we can conclude that the committed progenitor in the interfollicular epidermis does not derive from stem cells in the hair follicle and that there is no hierarchy of epidermal cells depending on the hair follicle environment (Figure 14A).
b) If the clones that are attached to the hair follicles are significantly larger in size compared to those that are not attached, we can first conclude that a more proliferative cell exists; hence, there is a hierarchy. Since the larger clones are attached to a hair follicle, we can conclude that this proliferative cell originates from the hair follicle and may give rise to the committed progenitor in the interfollicular epidermis. (Figure 14B)
Figure 14. Expected outcomes of experiment addressing Aim 2
With this experiment, we can establish if there is a hierarchy in epidermal cells and if the CP in the IFE originates from a stem cell population in the HF.
The strengths of this study include the use of the novel Rainbow technology which has not been attempted in tracking the fates of epidermal progenitor cells and their progeny. This multicolour analysis and high density staining allows us to label and investigate the effect of rare populations of stem cells that were most probably missed by Clayton et al. (2007). Furthermore, with the use of K14 promoter, we can induce labelling that is unbiased and specific to cells we are examining in this study. Also, we are conducting our experiments on murine back skin which has not been previously examined.
One of the limitations of experiment is the possibility of overlapping of clones. As compared to infinite number of colours, 6-colour labelling tends to underestimate the contribution of smaller clones and overestimate the contribution of larger clones whose larger size is probably due to the merging of clones. In attempts to overcome this problem, a computer simulation has been generated to correct values and will be used in the final analyses. However, this should not affect out conclusion in Aim 1 as the progression or stability in size of clones will also be reflected without this correction.
Another limitation is that once a clone reaches a certain size, it will always be in contact with one or more hair follicle. The probability of being attached to a hair follicle increases linearly with size up to a given size and then is 100%. A preliminary analysis was performed to overcome this problem. This analysis revealed that there is a higher proportion of clones for a given size attached to a hair follicle than expected, suggesting a role of hair follicles in epidermal homeostasis.
Table 2. Timetable
This study will address one of the major paradigms in stem cell biology of the skin. This matter has been highly debated in the past 30 years with theories that have been proposed and changed over time. The experiments proposed here will allow the most comprehensive analysis ever attempted having the potential for durably modifying out understanding of stem cells in the IFE.
Chronic wounds represent a major global burden due to the rapidly growing health care costs. In United Kingdom, the reported cost of wound care in 2006-2007 was £9.89 million (Vowden et al., 2009). In the United States, it is claimed that US$25 billion is spent annually on treating chronic wounds (Sen et al., 2009). These figures urgently demands for medically-effective yet cost-efficient treatment methods.
This study will contribute to developing a better definition of stem cells in epidermal tissue and gaining a better understanding of their potential. This study provides tools for identifying the appropriate cell type for cell therapies and may have important implications in regenerative medicine in particular for skin repair in chronic wounds or severely burnt patients.