A DISSERTATION ON

A method to isolate and culture dermal fibroblast and epidermal stem cell and arsenic toxicity in balb/c mouse

SUBMITTED TO THEDEPARTMENT OF LIFESCIENCESCSJM UNIVERSITY,KANPUR

IN PARTIAL FULFILMENTFOR THEDEGREE OF MASTER OF PHYLOSOPHYIN LIFESCIENCES

BYRashmi UpadhyayM.PHIL. LIFE SCIENCES(II semester)Department of LIFE SCIENCESCSJM University,KANPUR

UNDER THE SUPERVISION OFDr. Sushil KumarScientist F & HeadEnvironmental Carcinogenesis DivisionCSIR-IITR (Lucknow)

TO WHOM IT MAY CONCERN

This is to certify that Ms.Rashmi Upadhyay, a student of M.Phil. lifesciences (II semester), CSJM University has completed her six months dissertation work entitled A method to isolate and culture dermal fibroblast and epidermal stem cell and arsenic toxicity in balb/c mouse skin.successfully. She has completed this work from Indian Institute of Toxicology and Research (IITR), Lucknow under the guidance of Dr. Sushil Kumar, Scientist and Head Environmental Carcinogenesis division. The dissertation was a compulsory part of her M. Phil. degree.

I wish her good luck and bright future.

(Prof.Nand Lal)HeadDepartment of Lifesciences

ACKNOWLEDGEMENT

I am desperately searching for words to thank Almighty God for making me an instrument to complete this project.

I am also very thankful and express my deepest gratitude to Dr. Sushil Kumar, Scientist F & Head Environmental Carcinogenesis Section IITR, Lucknow. I am greatly indebted to them for their constant encouragement and providence.

I wish to express my deep sense of gratitude to Mr. Shiv Poojan Shukla for their assistance and cooperation during my project work. It was their useful suggestions, which helped me in completing the project work in time.I am thankful also to Mrs.Akanksha,Mr. Shivam Priya, & Mr. Mukesh K. Verma for their consistent support throughout the endeavour.

I would like to give my regard to Director IITR, Mr.B.D. Bhattacharya ji, Mr.Laxmi Kant Shukla and staff of RPBD section.I am also thankful to my friend Ms. Neha Misra for her co-operation and moral boost up.My acknowledgements would remain incomplete if I fail to express my sincere gratitude and love to my family members for being there for me during the course of this dissertation. Without their support and encouragement, this work would not have been possible.

Date: 31stAugust, 2012 Rashmi Upadhyay

CONTENTS

1. ABBREVIATIONS

2. INTRODUCTION

3. REVIEW OF LITERATURE

4. OBJECTIVE

5. MATERIALS & METHODS

6. RESULTS

7. DISCUSSION8. REFERENCES

Abbreviations UsedAbbreviation Full formGPM Growth Promoting MediaEP SC Epidermal Stem CellCaCl2 Calcium Chloride

BDE Beta dieneLRC Label Retaining CellsDMEM Dulbeccos Modified Eagle medium

PBS Phosphate Buffered Saline

FBS Fetal Bovine Serum

Dpase Dispase

HSS Henke salt solutionBrdU Bromo Deoxy UridineCo Collagen

Fi Fibronectin

Chelax Resin

1IntroductionOur body contains over 200 types of cells, each with a specific job: blood cells carry oxygen; muscle cells contract so that you can move; nerve cells transmit chemical signals. The job of a stem cell is to make new cells. It does this by undergoing an amazing process differentiating, or changing into another type of cell. Each time a stem cell divides, one of the new cells might remain a stem cell while the other turns into a heart, blood, brain, or other type of cell. In fact, stem cells are able to divide to replenish themselves and other cells without any apparent limit.Stem cells are the source, or stem, for all of the specialized cells that form our organs and tissues. There are many kinds of stem cells, but two types have made frequent appearances in the news: embryonic stem cells are present in very earlyand very tinyembryos, and produce the first cells of the heart, brain, and other organs. They have the potential to form just about any other cell in the body. Adult stem cells are found in many tissues of developed organisms, and even in embryos after theyve begun to grow. (A newborn babys body contains adult stem cells). Theyre also foundin the placenta and umbilical cord. Adult stem cells can replenish some tissues lost through normal wear and tear or injury. However, adult stem cells are only able to generate a few specific cell types. Adult stem cells in bone marrow, for example, make new blood cells, and adult stem cells in the skin make the cells that replenish layers of the skin.

Right now, researchers are still learning how to generate and grow stem cells. But simply knowing how to culture the cells in the lab isnt enough. Scientists also need to understand and control how stem cells differentiate to become specific cell types. If researchers can decode the signals that govern differentiation, they may be able to take charge of the process, directing a culture of cells to become a specific cell typeheart, neuron, skin, liver, or whatever kind is needed. With that level of control, researchers could pursue a variety of new treatments. They may be able to grow new tissues to replace damaged ones. For example, many scientists are looking at ways of generating heart cells in the hope of replacing cardiac tissue destroyed by a heart attack. But stem cells may have an even bigger impact by giving researchers new ways to study disease.Because embryonic stem cells have the potential to be turned into almost any kind of cell, researchers can culture cells that mimic a variety of diseases. These cultures would provide an almost unlimited supply of cells that could be used to test thousands of drugs. Such rapid screening could make useful drugs available years earlier than they might be otherwise. In addition, simply watching and deciphering the development of stem cells into tissues will give researchers insight into how cells communicate with each other and form the networks used in functions like learning and memory.Skin is largest organ of the body. It is a water proof barrier that protects internal organs against infection, injury and harmful sun-rays. Mature skin mainly comprises of two tissues: dermis and epidermis, the latter largely composed of appendages and specialized epithelial cells called keratinocytes. Epidermis and keratinocytes are continuously regenerated from basal cell layer, which contains stem cells that proliferate and differentiate to their lineage. When exposed to a carcinogen, tumours of lineage progenitor cells get formed. Tumour is an uncontrolled and the abnormal growth of cells that may be benign or malignant.Stem cellspossess a remarkable potential to develop into many different cell types in the body. Serving as a sort of repair system, they can theoretically divide in unlimited fashion to replenish other cells during the life-time of animal. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell. The latter can either remain stem cell or convert into more specialized cell, such as a muscle cell, a red blood cell, or a brain cell. There are two types of stem cells adult stem cells and embryonic stem cell present in the tissue. Adult (or somatic) stem cells are multipotent undifferentiated cell found in a differentiated tissue that can renew itself and differentiate to give rise to all specialized cell types of ectoderm, mesoderm or endoderm from which it originated. Reproducible-isolation and long-term-culture of epidermal stem cells from adult mouse skin is relatively a new area of biomedical research. Continuously renewing skin contains small undifferentiated stem cells capable of self-renewal and maintaining the differentiating cell population. The murine epidermis stem cells have been identified in literature (Bichenbach and Chism 1998) as label-retaining cells (LRCs) that retain label e.g. [3H]-Thymidine or BrdU for long term and grow in low Ca++ medium. It has been suggested that epidermal stem cell adheres to basement membrane through differential expression of specific adhering protein.Scientists primarily work with two kinds of stem cells from animals and humans: embryonic stem cells and adult stem cells. Stem cell, the targets cells in carcinogenesis, will be used for the future prospects purpose. Further, skin may represent an alternatively ideal source for adult stem cells and have therapeutic implications in many fields. Skin stem cells can be used in tissue engineering.Unique properties of stem cellsStem cells differ from other kinds of cells in the body. All stem cellsregardless of their sourcehave three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types. Stem cells differ from other kinds of cells in the body. All stem cellsregardless of their sourcehave three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types. Embryonic stem cells, as their name suggests, are derived from embryos. Specifically, embryonic stem cells are derived from embryos that develop from eggs that have been fertilized in vitroin an in vitro fertilization clinicand then donated for research purposes with informed consent of the donors. They are not derived from eggs fertilized in a woman's body. The embryos from which human embryonic stem cells are derived are typically four or five days old and are a hollow microscopic ball of cells called the blastocyst. The blastocyst includes three structures: the trophoblast, which is the layer of cells that surrounds the blastocyst; the blastocoel, which is the hollow cavity inside the blastocyst; and the inner cell mass, which is a group of approximately 30 cells at one end of the blastocoel.An adult stem cell is an undifferentiated cell found among differentiated cells in a tissue or organ, can renew itself, and can differentiate to yield the major specialized cell types of the tissue or organ. The primary roles of adult stem cells in a living organism are to maintain and repair the tissue in which they are found. Some scientists now use the term somatic stem cell instead of adult stem cell. Unlike embryonic stem cells, which are defined by their origin (the inner cell mass of the blastocyst), the origin of adult stem cells in mature tissues is unknown.Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step. Scientists are just beginning to understand the signals inside and outside cells that trigger each step of the differentiation process. The internal signals are controlled by a cells genes, which are interspersed across long strands of DNA, and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. The interaction of signals during differentiation causes the cells DNA to acquire epigenetic marks that restrict DNA expression in the cell and can be passed on through cell division. Many questions about stem cell differentiation remain. For example, are the internal and external signals for cell differentiation similar for all kinds of stem cells? Can specific sets of signals be identified that promote differentiation into specific cell types? Addressing these questions may lead scientists to find new ways to control stem cell differentiation in the laboratory, thereby growing cells or tissues that can be used for specific purposes such as cell-based therapies or drug screening

Review of LiteratureDuring embryogenesis, a fertilized oocyte gives rise to a multicellular organism whose cells and tissues adopt different characteristics to perform specified functions of each organ in body. As embryos develop, cells differentiate/proliferate enabling tissues and organs to grow. Even after an animal is fully grown, many tissues and organs maintain homeostasis, a process that represents cells dying natural death or by injury. This remarkable feature has ancient origins, dating back to the most primitive animals, such as sponges and hydrozoans. Throughout evolution, nature has exerted considerable fun and fancy in elaborating on this theme. Some amphibians, for instance, can regenerate a limb or tail when severed, and initiate into neurons. Bird brains can readily regenerate. While mammals seem to have lost at least some of this wonderful plasticity, their liver can partially regenerate provided that injury is not too severe. The epidermis and hair in mammalian skin can readily repair when wounded or cut. Epidermis, hair, small intestine and hematopoietic system are examples of adult tissues that are in a state of dynamic flux in nature even in absence of injury. These tissues continually give rise to new cells that are able to transiently divide.The fabulous ability of an embryo to diversify and that of certain adult tissues to regenerate throughout life is a direct result of stem cells; natures gift to multicellular organisms. Stem cells have both (a) the capacity to renew, i.e. to divide and create additional stem cells, and (b) to differentiate along a specified molecular pathway. Embryonic stem cells are nearly totipotent, reserving the elite privileges of choosing (among most if not all) the differentiation pathways that specify the animal. In contrast, stem cells that reside within adult organ or tissue have more restricted options, often able to select a differentiation program from only possible pathways. Adult stem cell in skin is one of the adult tissues which have stem cell counterpart in their compartment (F. M. Watt 2004, Handbook of stem cells).Organization of Skin TissueThe complex process of skin construction (Fuchs et al; 1990) starts around day 9 of mouse embryonic life through a succession of signal exchange between ectoderm and mesoderm. A very structured tissue emerges that is designed to seal and protect animal body against a diverse range of environmental assault (Hardy, M. H. 1992). The barrier function, or sealing of body from external environment, is essential for survival of animal and is fully completed at day 18, i.e. day before mouse is born (Fuchs, E., and Raghavan, S. 2002).Morphogenesis starts around day 13 and occurs in waves until just after the birth. Hair follicles are specified embryologically, and consequently, the maximum number of hair follicles that an animal will have for the rest of its life is determined before birth (Hardy, M. H. 1992) Skin, although composed of dermis and epidermis, also consists of an innermost basal cell layer of mitotically active keratinocytes in epidermis expressing diagnostic keratins K5 and K15 (Fuch, E Green, H 1980). As these cells withdraw from mitotic cell cycle and commit to a program of terminal differentiation, they still remain transcriptionally active and move upward toward skin surface.Epidermal stem cellsSignificant advances have been made in locating and identifying adult stem cell population from skin and hair follicle (Table 3.1.1, Figure 1, 1.3.1.II). Epidermal stem cells in basal layer are unipotent and enable the regeneration and repair of epidermis in adult skin (So and Epastein 2004, Morasso and Tomic-Canic 2005). These unipotent stem cells are derived from multipotent stem cells in hair follicles. The bulge region of hair follicle is strongly suggested to be a niche of multipotent stem cells (Lavker and Sun 2000, Tumbar et al 2004). Subsets of these follicle-derived multipotent stem cells can be activated, which migrate out of the hair follicles to site of a wound to repair damaged epithelium. However, they contribute little to intact epidermis.The epidermis is a stratified squamous epithelium resting on a basement membrane that separates it from the underlying dermis. Basal cells are mitotically active, but they lose this potential when they detach from the basement membrane and migrate toward the skin surface in a process of terminal differentiation. The process of epidermal stratification during embryonic development and in wound healing is a highly regulated process. Furthermore, epidermis is constantly renewed and remodelled with a strict balance between proliferation and differentiation. The capacity for renewal and repair of cutaneous epithelium rests on the presence of epidermal stem cells (ESC) that reside within both the basal layer of the interfollicular epidermis (IFE) and, at least in rodents, the bulge of hair follicle (Ghazizadeh and Taichman 2001; Blanpain and Fuchs 2006; Gambardella and Barrandon 2003; Watt et al. 2006). ESC are slow cycling in vivo, can self renew, and are responsible for long-term maintenance of tissue. They can be activated by wounding or by in vitro culture conditions to proliferate and to regenerate the tissue. Extensive studies over last 30 years have been focused on adult ESC to define their location, to identify specific markers, and to evaluate their potential in vivo and in vitro. The spectacular improvement in our knowledge was well discussed in a recent provocative paper (Clayton et al. 2007) and in-depth reviews (Fuchs 2007; Watt et al. 2006; Gambardella and Barrandon 2003).Epidermis may be subdivided in three subpopulations-(1) Stem cells located in basal layer, cycle at a slow rate and are thought to sojourn in a quiescent state. They can be identified by continuous [3H]-Thymidine labelling (Potten 1974) or as label retaining cells (LRC) (Bickenbach 1981; Potten et al 1982).(2) Stem cells give rise to a subpopulation of transit-amplifying cells that actively proliferate, have a short life span, and produce differentiated mature keratinocytes. This rapidly expanding subpopulation of transit- amplifying cells is produced through a limited number of divisions of stem cells (Potten and Morris, 1988; Morrison et al., 1997; Watt, 1998).(3) Transit-amplifying cells divide several times while in basal layer before giving rise to committed cells that migrate into suprabasal layer and leave the cell cycle to differentiate terminally.There are different opinions about the position of stem cells. Morphological and kinetic data (Potten 1974, 1983) suggest that the mouse dorsal skin is organized as a series of epidermal proliferative units (EPU), each consisting of a group (family) of cells containing proliferative and functioning elements. The proliferative elements are located in basal layer while the differentiated cells are positioned in a column (one cell wide) above proliferative cell group. The basal cells within the EPU amount to 1011, but only 67 are believed to be actively involved in cell proliferation. Of the remainder, one is Langerhans cell with an immunological function, and two or three are believed to be post mitotic cells awaiting their signal to migrate suprabasally into the functional compartment. Each EPU contains a central cluster of 2-4 cells, among which there is a putative stem cell surrounded by transit- amplifying cells (Potten, 1974). When mouse skin is severely damaged by radiation, only 10% of basal keratinocytes are able to form clones of new epidermis, i.e. each EPU probably possesses a stem cell. Stem cells located in EPU centre, cycle at a slower rate than the cells in periphery and are thought to stay predominantly in G0 state. The population of stem cells of a lower level retains certain self-maintenance ability and, if a space becomes available in the stem cell niche, such cells become actual stem cells. Structural and kinetic observations (Lavker and Sun, 1982) demonstrated that two morphologically distinct spatially segregated subpopulations of basal keratinocytes existed in the human and monkey palm epidermis. One cell subpopulation was located in shallow rete ridges and characterized by a cytoplasm filled with tono-filaments and a highly serrated dermal-epidermal junction. These cells could anchor epidermis to dermis. In contrast, basal keratinocytes of another subpopulation located at the tips of deep rete ridges were characterized by a round outline (nonserrated cells), relatively flattened surface facing dermis, a high nucleocytoplasmic ratio, and a primitive cytoplasm. These cells were also heavily melanized versus lightly melanized serrated keratinocytes. The experiments with [3H]-Thymidine labelling showed that these cells were slow cycling and could be stem cells. It was proposed (Lavker and Sun 1982, 1983) that stem cells are more likely to reside on tips of deep rete ridges. Mackenzie and Bickenbach (1985) studied the position of LRC, slow cycling cells, in mouse palatal and lingual epithelia and ear epidermis using autoradiography and histochemistry. They found that LRC were keratinocytes positioned basally and occupied certain sites within epithelial structural units corresponding to those expected for epithelial stem cells. It was also demonstrated that slow cycling epidermal LRC could be good candidates for stem cells (Morris and Potten 1994). LRC, identified by autoradiography in the dorsal epidermis and hair follicles of adult mice 810 weeks after a twice daily injection of [3H]dT (3 to 5-days after birth), were harvested by trypsinization and cultured from low density on feeder layers of irradiated cells. After five days in culture, LRC were found as pairs and clusters having silver grain counts consistent with the number of divisions (Morris and Potten 1994). These data suggest that stem cells (protected by the microenvironment in quiescent state) are highly proliferative when appropriately stimulated. In human hair follicles, LRC (putative stem cells) with relatively undifferentiated ultra-structure were also found, which appear to be responsive to growth stimulation and to have a high proliferative potential. These cells were located in the outer root sheath below midpoint of the follicle (Cotsarelis et al 1990; Rochat et al 1994; Yang et al 1993). Taylor et al (2000) proposed that in upper part of hair follicle there was a major repository of stem cells that, on one hand, could produce several cell types of follicle and, on the other, could migrate to epidermis in neonatal/ adult mice in response to a penetrating wound. Limat et al (1991) demonstrated that keratinocytes of the follicular outer root sheath, like epidermal keratinocytes, could restore in vitro tissue organization very similar to normal epidermis. Re-epithelization of wounds could also occur at the expense of sweat glands, but the resulting epithelium did not resemble surrounding intact epidermis (Miller et al., 1998).Studies of epidermal stem cells are complicated by the absence of safe morphological markers. Therefore different attempts were undertaken to raise monoclonal antibodies that could have selectively recognized antigens of stem cells (Morhenn et al 1985; Samuel et al 1989). Keratinocytes express some receptors of the integrin family, including 21 (receptor for collagen and laminin), 31 (receptor for laminin and epiligrin), 51 (fibronectin receptor), and 64 (a component of hemi desmosomes). Since there is no safe criterion for isolation of stem cells, different authors use several markers. Expression of integrin 1 can be used as one of such markers. This integrin is a subunit of some integrins expressed by cultured keratinocytes. The keratinocyte integrins not only provide tool for adhesion to extracellular matrix proteins, but also are involved in regulation of terminal differentiation, stratification, and migration. Subpopulations of keratinocytes that differ in the level of 1 integrin expression differ also in proliferative potential (Jones and Watt 1993). This study demonstrated that human keratinocytes capable of forming in vitro actively growing colonies could be isolated directly from epidermis and purified on the basis of their adhesive properties. These cells expressed high levels of integrins 21, 31, and 51 and adhered rapidly to Type-IV collagen, fibronectin, and extracellular matrix deposited by keratinocytes in culture. The most adhesive keratinocytes appeared to fulfil the requirements of stem cells. They founded actively growing colonies that contained, in addition to rapidly adhering cells, slowly adhering and involucrin-positive (terminally differentiated) cells. Thus, the relationship between elevated 1 integrin expression, enhanced adhesiveness, and high proliferative potential was found in cultured keratinocytes. The cultures founded by the cells with a high level of 1 integrin expression was capable of forming epidermis after grafting to mice (Jones et al 1995), which demonstrated the presence of stem cells in these cultures. Such differences in level of integrin expression were used to locate stem cells and transit-amplifying cells in vivo and to isolate each subpopulation directly from human epidermis.In in vivo studies by Jones et al (1995), epidermis from three body sites was examined: adult palm, adult scalp, and neonatal foreskin. Immuno-fluorescence staining with antibodies to 2, 3, and 1 subunits revealed a non-random distribution of putative stem cells and transit-amplifying cells: patches of brightly fluorescent cells were interspersed with stretches of basal cells with a low fluorescence. Dispase-detached-keratinocyte-sheets stained for 2 or 3 integrin subunits. It was found that integrin-fluorescent patches of cells were generated both by unselected cell populations and by rapidly adhering cells. Size of the integrin-fluorescent patches was similar irrespective of unselected or rapidly adhering cell types and it was not affected by a 10-fold difference in plating density. Cell patterning in epidermis might be an intrinsic property of keratinocytes. This kind of keratinocyte patterning was described by Malcovati and Tenchini (1991) who found that within one day of culture in absence of growth factors, all single keratinocytes disappeared and different sized aggregates of cohesive colonies formed in absence of cell division. It was proposed that populations of keratinocytes contained a definite and constant proportion of clusterogenic cells and gregarious cells that attached to the clusters. The gap junctional communication (the fastest transfer of the Lucifer Yellow dye) appeared in the stem cell zone (Bjerknes et al 1985) and gap junctional communication compartments had approximately the same size as EPU (Pitts et al 1988). Those regions of epidermis that corresponded to localization of stem cells in vivo expressed higher levels of the 21 and 31 integrins than the transit amplifying cells.Studies of stem cells in adult organisms emphasized the importance of cellular microenvironment or niche. This concept was proposed by Schofield (1978). According to this concept, specific combinations of growth factors, extracellular matrix molecules, and neighbouring cells ensured the conditions necessary for maintenance of the stem cell phenotype and that the progeny of stem cells would differentiate unless there is also a suitable niche to accommodate them (Hall and Watt 1989).Jones and Watt (1993) found that stem cells most rapidly adhered to type IV collagen and, if they failed to attach, they irreversibly lost their proliferative potential and differentiated (Adams and Watt 1989). Plopper et al (1995) reported that, in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton and spatially integrated within the focal adhesion complex at the site of integrin binding. Such positioning within the focal adhesion complex may serve to immediately integrate signals from soluble mitogens with those resulting from cell binding to the extracellular matrix and transmission of mechanical stresses across the plasma membrane. Autocrine and paracrine-acting mitogenic factors synthesized by the keratinocytes, particularly TGF and TGF (Coffey et al 1987; Partridge et al 1989), may contribute to self-maintenance of stem cells and prevention of apoptosis since it was demonstrated that growth factors produced survival signals in cells (Collins et al 1993). In keratinocytes, it was shown that apoptosis was suppressed by signalling through EGF receptors (Rodeck et al 1997). Maas-Szabowski et al (1999, 2000) described a double paracrine mechanism of keratinocyte growth regulation. It was demonstrated that, in a co-culture with keratinocytes, fibroblasts exhibited an enhanced expression of keratinocyte growth factor and interleukin-1 receptor via release of interleukin 1 and 1 by keratinocytes. It was proposed that adhesion to the basement membrane and cell-cell interactions were important factors of stem cell microenvironment. On initial histological evaluation of mammalian skin, there is no obvious morphologically distinct region, or niche, of the basal layer where stem cells might be located. It has been known from the 1970s (Fuchs, E. & Cleveland, D. W. 1998) that epidermis has number of stem cell lineages (see Table 1). It was initially hypothesized that the entire basal layer consisted of stem cells; then later that the Langerhans cells were stem cells. Radiation dosesurvival studies suggested that stem cells might comprise 27% of basal layer cells (Fuchs E & Cleveland DW 1998). One method of retrospectively demonstrating presence of stem cells in epidermal cultures was to label population of cells and then to use them to reconstitute epidermal tissue in vivo. It is understood that 1012% of murine basal layer cells might be stem cells capable of generating a single maturing column of cells. Another method of identifying tissue stem cells makes use of their slow cycling nature. In a pulsechase experiment, all dividing cells of a tissue incorporate nucleotide analogs such as bromodeoxyuridine (BrdU) or [3H]-Thymidine into newly synthesized DNAs. When the label is chased, only those cells that divide rarely and still reside within the tissue over time will retain their label. In oral epithelium, so-called label retaining cells, or LRCs, are located in discrete regions of tongue and palatal papillae (Bickenbach JR & Hamilton E 1974); in murine ear epidermis, LRCs reside in basal layer, near periphery of differentiating cell columns (Potten et al 1974). Therefore, a model of skin epithelial maintenance emerged in the 1980s in which the periodic division of slow-cycling stem cells in basal layer gave rise to transiently amplifying cells that populated most of the basal layer, dividing two or three times and then moving upward while differentiating into mature skin cells. In 1990s, researchers using [3H]-Thymidine and evaluating label retention in murine haired epidermis discovered that the majority of LRCs in skin resided in bulge region of hair follicle, with only a small fraction of LRCs in basal layer of inter-follicular epidermis (Cotsarelis G et al1990; Morris RJ & Potten CS 1994).The hair follicle is an epidermal appendage that consists of an upper, permanent portion, and a lower, cycling portion that produces the hair (Hardy MH 1992; Alonso L & Fuchs E 2003). The outer root sheath (ORS) is contiguous with and biochemically similar to basal layer of epidermis.Functional Characteristics of epidermal stem cellsLocating the putative epidermal stem cells represented a major achievement, allowing scientists to study biochemical and functional characteristics of this important class of cells. Stem cell of other tissue, such as the hematopoietic system, are replete with cell surface markers that identify nearly every cell type starting with stem cells and extending through the most differentiated forms of the progeny types. Specific markers of epidermal stem cells are not yet known. Although these cells can be identified either in vivo by label retention or in vitro by clonogenicity, nevertheless neither method of identification presently allows easy isolation of stem cells for analysis. Therefore, there is a strong need for specific epidermal stem cell markers. One class of markers is the integrin family of trans-membrane receptors, whose members are responsible for attachment of the basal layer of epidermis to its underlying substratum, i.e. basement membrane (Watt, 2002). Basement membrane is rich in extracellular matrix (ECM) proteins (Adams & Watt 1989), many of which constitute the ligands for integrin heterodimers. When cultured human keratinocytes were isolated by fluorescence-activated cell sorting (FACS) on the basis of their surface integrin 1 levels, cells with highest fluorescence displayed a moderately increased colony-forming efficiency in vitro (Jones & Watt 1993). In this study, colony-forming efficiency correlated with the speed of cell adherence to integrin ligands, including type IV collagen (21) and ECM proteins secreted by keratinocytes (Jones & Watt 1993). In a different study, human keratinocytes, (sorted for hemi-desmosomal integrin 6 which partnered with 4 to robustly attach to basement membrane component laminin5), were shown to have higher proliferative potential than those sorted for the focal adhesion integrin 1, (which partnered promiscuously with 2 (type IV collagen), 3 (laminin - 5), 5 (fibronectin), and 9 (tenascin) in keratinocytes (Kaur & Li, 2000).

Epidermal stem cell in vivoFurther evidence for existence of keratinocyte stem cells came from in vivo studies that demonstrated variation in rate of cell cycling of epidermal cells. Experiments in which young mice were labeled with tritiated thymidine or BrdU revealed that a small percentage of basal keratinocytes retained nuclear label after 30d and for up to 240d (Bickenbach 1981; Morris et al 1985; Bickenbach et al 1986; Cotsarelis et al 1990; Bickenbach and Chism 1998). These slowly cycling cells were referred to as label retaining cells (LRC) and were thought to represent the epidermal stem cell subpopulation, which were quiescent or slowly dividing in - vivo. LRC were capable of forming colonies in vitro (Morris and Potten 1994) and were found to be undifferentiated, based on the expression of known differentiation markers viz. cytokeratins and bullous pemphigoid antigen (Mackenzie et al 1989). Presently, label retention is one of the best available indicators of epidermal stem cells. Prior investigations had attempted to characterize epidermal stem cells. Differing expression levels of adhesion molecules were reported (reviewed by Watt, 1998). Mouse keratinocytes rapidly adhering to a variety of substrates had most of the LRC (Bickenbach and Chism 1998). Keratinocytes with high levels of 1 integrin expression had high colony forming efficiency (Jones and Watt, 1993) suggesting that high expression was a characteristic feature of epidermal stem cell The plasticity of stem cells was illustrated by animal transplantation studies in which donor and recipient strains differed. Using this experimental method, it was shown that injected murine neural stem cells (Bjornson et al, 1999) and skeletal muscle stem cells (Gussoni et al, 1999; Jackson et al, 1999) repopulated the hematopoietic compartment in mice that underwent bone marrow ablation. In addition, following bone marrow transplantation, donor-derived cells were found in murine muscle (Ferrari et al 1998; Gussoni et al 1999) in neural cells of brain (Eglitis and Mezey1997; Brazelton et al 2000; Mezey et al 2000) as well as in canine vascular endothelial cells (Shi et al 1998). Bone marrow transplantation studies also demonstrated that purified murine hematopoietic stem cells could differentiate into epithelial cells of the skin, lung, gastrointestinal tract (Krause et al, 2001), as well as the liver (Lagasse et al, 2000; Krause et al, 2001). Kinetic analysis indicated that they were slow-cycling in vivo,consistent with properties expected of epidermal stem cells (Li et al, 1998; Kaur and Li, 2000).Before in vitro study of the epidermal stem cell, their properties in vivo must be considered. Kinetic studies suggest that dividing population of keratinocytes in epidermis is heterogeneous and that not all dividing cells are stem cells (Potten, 1981; Potten et al. 1982). A model has been put forward in which the daughters of stem cells that are committed to terminally differentiate nevertheless may go through a limited number of further divisions before becoming post-mitotic. These cells (called transit amplifying cells) have profound implication for understanding how proliferation in the epidermis is controlled, since stem and transit amplifying cells might respond in different ways to carcinogens or epidermal wounding.In order to study kinetics of organization of epidermis, it would be useful to be able distinguish between stem cells and transit amplifying cells by criteria other than their proliferative capacity. There is some evidence in vivo that the two populations might differ in cell cycle parameters, with stem cell having a longer cycle time and shorter S-phase than transit amplifying cells (Potten et al.1982). It is possible that cells occupy different position, or niches in the basal layer (Schofield 1978; Potten 1981; Lavker & Sun 1983). That no molecular marker of keratinocyte and cultures eventually senesce after several passages might suggest that stem cell do not exit in culture. However, culture grafted onto suitable recipients from normal epidermis who survived for years stabilized the fact that stem cell persisted, at least early in passage culture (Gallico et al 1984). There is strong evidence for proliferative heterogeneity in culture. Subpopulation of keratinocyte that differed in cell cycle time rate of DNA synthesis have been identified (Dover & Potten 1983; Jensen et al 1985b; Albers et al 1986) and withdrawal from the cell cycle has been shown to occur in specific subset of dividing cells (Albers et al 1987). The proliferative potential of individual keratinocytes is inversely correlated with their size (Barrandon & Green 1985) and may be influenced by their neighbours.Epidermal stem cell in vitroFor epidermal stem cell culture there is no isolation procedure developed that yields a pure population of epidermal stem cells. Several enrichment techniques for epidermal stem cell have been described based on cell surface markers, size, or in vitro adhesion (Bickenbach and Chism 1998; Tani et al 2000). High levels of the integrin subunits 6 or 1 or combinations of markers have been used to select cells with high potential of proliferation (Jones and Sharpe 1994; Johnes et al 1995; Li et al 1998). Rapid adhesion of cells to collagen type IV has resulted in enriched populations of epidermal cells that show very high proliferative capacity (Johnes and Watt 1993; Bickenbach and Chism 1998).In conventional culture, cells are normally maintained and grown on non-biological substratum with nutrient medium containing 5-10% serum. The complexity of serum, however, has made it difficult to clarify how it works on cells in culture. Recent findings have made advances on exploration of "growth factors" for mammalian cell-culture in vitro. Cultured cells are known to regulate their own environment by excreting cellular products into culture medium, for example, the factor promoting cell adhesion and spreading in absence of serum, and the other exerting the mitogenic factor in low concentration of serum. (Millis A. J. T. and M Hoyle 1978 and Millis et al 1977). Alternatively the interaction of cells with substratum is also important in the control of growth and growth patterns of the cells, especially collagen coated substratum is known to enhance adhesion, growth, and differentiation of various types of cells (Kleinman et al 1981).Keratinocyte from epidermis, or other stratified squamous epithelia, can be grown in culture in presence of a feeder layer of 3T3 cells and medium supplemented with a range of additives that prolonged the life span and increased the growth rate of culture (Rheinwald & Green 1975, Rheinwald 1980). After isolation from epidermis, cells originating from basal layer attached to culture dish, divided and gave rise to individual colonies of cells. With time, individual colonies expanded and adjacent colonies merged with one another, displacing the feeder cells. At confluence each dish was covered with a continuous stratified sheet of cells, approximately 6 to 8 layers thick. Human keratinocytes could be passaged several times before undergoing senescence, and did not, with rare exceptions transformed spontaneously (Baden et al 1987; Boukamp et al 1988).

Arsenic is a highly poisonous metallic element in the nitrogen family of group Va in the periodic table. Symbol As; aomic number 33; atomic mass 74.9216; melting point ca 817C; sublimation point ca 613C; specific gravity 6.80 or 7.004; 5.73; valence -3, 0, +3, or +5.; electronic config. [Ar]3d104s24p3. It appears in three allotropic forms, yellow, black, and gray. The stable form is a brittle, steel-gray hexagonal solid that oxidizes rapidly in air, and at high temperatures burns to form a white cloud of arsenic trioxide. Arsenic and some arsenic compounds sublime when heated and convert to gaseous form. In modern times, arsenic acquired a reputation as a toxic compound and a poison. Chronic arsenic exposure is a serious public health problem in some parts of the world Intoxication by this heavy metal can result from breathing sawdust, workplace air, or smoke from arsenic- preserved wood, or from ingesting contaminated water, food, or soil Arsenic is present in high concentrations in well water in many parts of the western United States, South America, and Taiwan. In Bangladesh, the health of millions of people has been adversely affected by contamination of the groundwater by naturally occurring arsenic Widespread use of arsenic-containing herbicides and pesticides, its incorporation into feed as a substance to promote the growth of livestock and poultry, and its industrial use have caused the environmental dispersion of this compound. Furthermore, environmental arsenic is concentrated in many species of fish and shellfish. Consequently, the average daily human intake of arsenic is approximately 300 g, virtually all of this ingested with food and water.Arsenic trioxideis theinorganic compoundwith theformulaAs2O3. This commercially importantoxideofarsenicis the main precursor to other arsenic compounds, includingorganoarsenic compounds. Arsenic trioxide is readily absorbed by the digestive system: toxic effects are also well known upon inhalation or upon skin contact. Elimination is rapid at first (half-life of 12 days), by methylation to monomethylarsonic acid and dimethylarsonic acid, and excretion in the urine, but a certain amount (3040% in the case of repeated exposure) is incorporated into the bones, muscles, skin, hair and nails (all tissues rich inkeratin) and eliminated over a period of weeks or months. The first symptoms of acutearsenic poisoningby ingestion are digestive problems: vomiting, abdominal pains, diarrhea often accompanied by bleeding. Sub-lethal doses can lead to convulsions, cardiovascular problems, inflammation of theliverandkidneysand abnormalities in the coagulation of the blood. These are followed by the appearance of characteristic white lines (Mees stripes) on the nails and by hair loss. Lower doses lead to liver and kidney problems and to changes in the pigmentation of the skin. Even dilute solutions of arsenic trioxide are dangerous on contact with the eyes. The poisonous properties are legendary and the subject of an extensive literature Chronic arsenic poisoning is known as arsenicosis.

Objectives:

1. Isolation and culture dermal fibroblast.

2. Preparation of conditioned medium.

3. Isolation and feeder free culture of epidermal stem cell.

4. Demonstrate arsenic toxicity in BALB/c mice

Material and Reagent Setup1. Equipment

I. Autoclave Vertical (Macro scientific works, Delhi, India),II. Biosafety cabinetIII. CentrifugeIV. Incubator, dual chamberV. Microscope, invertedVI. Milli Q-Biocel System (Millipore),VII. Flow cytometre

2. Reagents used in cell Culture

I. DMEM (Dulbeccos Modified Eagle medium)

DMEM Powder 9.5g/lit (approx. 1g/100ml)Other components of media:- Sodium bicarbonate (NaHCO3) 0.15g/100ml Glucose (C6H12O6) 0.35g/100ml Foetal bovine serum (FBS) 10% Antibiotic-Antimyotic sol. 100X i.e. 1ml/100ml

Method of preparation:-

Dissolved above mentioned components in autoclaved milliQ (100ml) water. Took out 10 ml of prepared medium and discard it and to it add 10 ml FBS. Add antibiotic and antimyotic sol. (100X) at rate of 1ml/100ml.The above prepared media is filtered through a 0.22 Millipore filter and the container is sealed with parafilm.

II. Serum Free Media ( SFM)

Composition for 50 ml: DMEM Powder 0.5mg NaHCo3 0.075gm Glucose 0.225gm Antibiotic-Antimyotic sol. 500l

Method of preparation:-

Dissolved above mentioned components in autoclaved milliQ (30ml) water and then make the volume up to 50ml. The above prepared media is filtered through a 0.22 Millipore filter and the container is sealed with parafilm.

III. PBS ( Phosphate Buffer Saline):-

Dulbeccos phosphate buffer saline 9.55g/100ml

Other components of media:- Potassium phosphate monobasic 0.20g Potassium chloride 0.20g Sodium Chloride 8.0 g Anhydrous sodium phosphate dibasic 1.15g

Method of Preparation:- Measured 90% of final required volume of water, to it added powder medium with stirring. (Do not heat the sol.). If required the pH is adjusted by 1N NaoH or 1N HCL. Now volume was adjusted as per requirement.

IV. Trypsin 1X sol.

Dissolved 2.5g of trypsin in 100 of Hanks balanced salt sol. Filtered through membrane filter of 0.22 . This forms 10X sol. And now this is in PBS i.e. 9ml PBS + 1ML 10X Trypsin, to make 1X sol.

V Reagents used in stem cell culture

Keratinocyte SFM media Chelaxed FBS CaCl2 (0.05M) Antibiotic

Method of preparation of GPM (Growth promoting media)

45ml of stem cell media is taken and 5ml chelaxed FBS +0.05mMmM CaCl2+1% antibiotic mixture is mixed to make 50ml GPM.

Colchicine8 mg colchicine is weighed and dissolved in 2ml PBS in laminar chamber filtered by 0.22 Millipore filter.

0.56% KCl,methanol and acetic acid

Dose preparation:

1. Sodium seleniteStandard 5.6mg/kg body weightFor a month amount needed 33.6mg dissolved in 23ml miliQ H2O

2. ColchicineStandard 4mg/kg body weightFor chromosomal aberration 8mg dissolved in 2ml PBS

3. GiemsaFor staining 5% Giemsa is prepared from stock

METHODOLOGY

Method comprised of different steps:

Animals and treatment Balb/c mice

Approval of Animal Ethics Committee:

The objective of Standard Operating Procedure (SOP) is to ensure quality and consistency in review of research proposals and to prevent infliction of unnecessary pain & sufferings before, during and after experiments on animals, to follow the CPCSEA guidelines under the provision of Section 15 of PCA Act 1960 (Ministry of Environment and Forests, Government of India-2008) and the Gazette of India 1998 for experiments on Animals.Functions of Institutional Animal Ethics Committee (IAEC)IAEC should provide independent, competent and timely review of the ethics of proposed studies before the commencement of a study and regularly monitor the ongoing studies. IAEC will review and approve all research proposals involving animal experiments up to physiological level only with a view to assure quality maintenance and welfare of animals used in laboratory studies while conducting biomedical and behavioral research and testing of product.For experiments on higher animals, the IAEC will forward its recommendation to the CPCSEA, New Delhi, for its approval. IAEC will review the proposals before start of the study as well as monitor the research throughout the study and after completion of the study through six monthly reports, final report and visit of the laboratory in the animal house where the experiments are conducted. The committee will also ensure compliance with all regulatory requirements, applicable guidelines and laws.Balb/c mice 4 mice (weight 25gm) has given 85ppm in oral dose for a month with control in normal tap water.

Materials:

Preparation of FBS, chelex-treated FBS is chelex-treated in batch with Chelex-100 resin (BioRad, cat # 1422832) to remove free Ca++. Use 100 g chelex resin per 500 ml FBS. Swell 20 g chelex resin in 400-500 ml distilled water,leave it to swell for overnight then titrate to pH 7.4 with HCl while stirring (pH will take a while to stabilize during titration). Filter through Whatman #1 paper.Scrape resin slurry into 50 ml FBS and stir at room temp for 3 hr. Filter the chelated FBS through 0.45m filter and discard the resin slurry. Filter the chelated FBS through a 0.2m bottle filter to sterilize it. Aliquot sterile, chelated FBS at 50 ml tube and store at 20C.

Trypsinization

Wash with PBS (2ml ) for two times.Add 1 ml trypsin .put it in incubator for 2-3 minutes.Add 10 times media approx 10 ml to the flask .collect in a 15 ml tube and centrifuge at 300g or 1300 rpm for 10 min.Discard the supernatant and suspend the pellet with 1ml media then pour it into the flask.

To prepare collagen-coated dishes:

Coating of dishes or tissue culture flasks with extra cellular matrix proteins like Collagen or Fibronectin is a common procedure in laboratories involved in cell culture collagen (~3 mg/ml) and fibronectin 10 g/ml, then completely cover the surface of a 25cm flask and 6 well plate with 3-5 ml. Leave it for overnight in UV light . Completely aspirate collagen from the flask and 6 well plate before seeding cell.Isolate and culture murine Skin fibroblastSkin-derived Fibroblast Culture Procedure:Mice pups are taken in petriplate Pups are washed with 70% alcohol. After washing with alcohol washed with betadiene Cut the pups and remove the skin. Remove the fat deposited on skin. Wash with PBS Wash with alcohol. Wash with miliQ H2o.The skin biopsy is cut into 3-4 smaller pieces (in petri dish with a sterile knife) Paste pieces of skin onT25 flask. Dried the flask in the sterile environment. Put 2 ml medium in sterile T25 flask (with filter top) Add very carefully 0.5 ml medium Transfer flask to 37 degrees Celsius, 5% CO2 -> leave flask for a minimum of 3 days After a minimum of 3 days, cells start growing around the skin biopsy; these cells are not yet fibroblasts, but then the skin piece is really attached to the bottom of the flask. When these cells are seen carefully add some extra medium (1 ml medium). If cells are not visible, it is better to add only 0.5 ml medium to prevent the skin pieces from starting to float .T25 flask are seen after every 2-3 days. Probably, fibroblasts are then growing out of the initial cell layer.

Isolate and culture epidermal skin stem cell as per stabilized protocol (Poojan & Kumar 2010)

Mice pups are taken in petriplate.Pups are washed with 70% alcohol.After washing with alcohol washed with betadiene .Cut the pups and remove the skin .Remove the fat deposited on skin.Wash with PBS .Wash with alcohol .Wash with miliQ H2O.After washing the skins are spread on the petriplate. 15mg Dispase is weighed and dissolved in 1 ml Henkes salt solution and 9ml miliQ is added.Parafilm is wraped and leave it in freeze for a night . In the mean time cofi coating (collagen and fibronectin ) was done on 6 well plate and 2 25cm2 flask and leave it for a night in UV light .After this remove coating and wash the plate and flask with PBS.Again peel the skin and churn with scissors . In 50ml tube take skin and 10ml media is added after this parafilm is coated and shake it for 30 minutes.Skin is squezed properly with the help of forceps and filtered with membrane.Spin it for 10 min at 1200rpm .Supernatant is discarded and pellet is dissolved in GPM .Now the equal amount is seeded into flask and plate.Fibroblast-Conditioned-MediumIsolate and culture dermal fibroblasts Asphyxiate 2d oldBALB/c mice and soak carcass with BetadineTMsolution using cotton-foil-wrap and with copious amount of 70%ethanol 10min later. Transfer in 100mm sterile petridish to sterile Laminar Hood and perform all activities now onwards in Laminar Flowhood. Cleanse carcass from all sides using cotton-swab soaked with 70%ethanol followed by keeping dipped in 70%ethanol in Laminar-Flow hood. Place one carcass in 100mm sterile petridish and decapitate.Excise aseptically trunk skin in one piece26. Collect all tissue specimens inPBS-antibiotic solution and process as earlier Place dermis side up in 90mm Petridish and scrap carefully the subcutaneous fat using scalpel. Minse tissue into small pieces (< 1mm3) using sterile curved-scissors. Place tissue pieces in 25cm2Nunc culture flask using sterile forceps under laminar flow hood, and allow attaching in petri-dish by their own adhesiveness. Allow to air dry for ca 15min; it helps to attach the tissue. Add 4ml Fibroblast-Growth-Medium (DMEM-Gibco) submerging carefully the tissue pieces without dislodging explants and incubate at 37C in CO2incubator. Replace fresh medium for the first time on 4thday after seeding and thereafter every 3rdday.Watch fibroblasts growing-out of tissue-fragments, sub-culture to enrich the yield. Note migration of fibroblast with spindle cell morphology 2-3d after starting the culture. Check early confluence (60-70%); and trypsinize using trypsin/EDTA to sub-culture.Prepare Fibroblast-Conditioned Medium (CM1) using primary cultureUse neonatal mouse skin fibroblast at passage 3-5 to generate Fibroblast-Conditioned-Medium Seed fibroblasts (1106cells) in T25cm2Nunc-flask containing Fibroblast-Growth-Medium and culture to near-60% confluence at 37C in 5%CO2 Wash adherent cells withPBSonce before adding 15ml low-Ca2+-SMEM to generate Fibroblast-Conditioned-Medium.Culture for 48h and collect conditioned-medium to store-freeze immediately. These cells can either be discarded or reused to produce secondary conditioned mediumPrepare Fibroblast-Conditioned Medium (CM2) using Secondary CultureRinse primary fibroblast-culture twice usingPBSafter collectingCM1.Add 5ml Fibroblast-Growth-Medium to extend culture until 60% confluence.Trypsinize in 6ml 0.25%trypsin/EDTA at 37C for 5-10min transfer cells into a 15ml sterile Tarson tube. Mix 9vol of complete Fibroblast-Growth-Medium; and pellet cells at 300xg 5min 4C.Split 1:3 and seed using Fibroblast-Growth-Medium (DMEM) to incubate in CO2incubator. After incubation for 2-3d, secondary culture of fibroblast grows to 60-80% confluence. Rinse flasks thoroughly twice using Ca2+-free-PBS (to remove all traces of Ca2+).Add 15mlSMEMto each flask (75cm2) and incubate for 48h; collect secondary conditioned medium after incubation. Save conditioned-medium, filter through 0.45m membrane, aliquot; and store-freeze at -20C. Discard the used-fibroblasts.

Chromosomal aberrations

Animal were sacrificed by cervical dislocation.Colchicine has given 4mg/kg BW.Prior to 3 hour of sacrificing.Bone marrow has flushed out.Centrifuged at 2000 rpm for 5 min .Wash with PBS 2X.Pellet was resuspended in 0.56% KCl for 30 min.Centrifuge at 1000 rpm for 5 min.Collect the pellet and fix the pellet in methanol and acetic acid (3:1) Leave it for overnight at -20 degree .Centrifuge at 1000 rpm for 10 min.Remove the supernatant and leave 500microL .Then spread it on slides.Stain with Giemsa 5%.

Micronuclei detection by flow cytometryAfter treatment animal were sacrificed by cervical dislocation. The bone marrow was flushed out from femurs using FBS.Centrifuge the cells at 1000 rpm for 5 minute pellet was washed with PBS by centrifuging at 2000rpm for 5 min. resuspend the pellet in solution (10Mm NaCl,3.4 Mm sodium citrate,25microgram/ml propidum iodide, 0.01mg RNase from bovine pancreas ,and 0.3 microlitre/ml Triton x). After 1 h at room temperature, an equal volume of solution II was added (78.1mM citric acid, 25microgram/ml PI, 0.25M sucrose.).after 15 min, the suspension was filtered through a 53-mm nylon mesh and stored on ice until flow cytometric analysis.The samples were acquired and analysed on flow cell cytometer (Becton-Dickinson LSR II,San Jose,CA,USA)using Cell Quest software).

RESULTS

In the dermal fibroblast culture isolated dermal fibroblast growing in flask after four days of seeding cells showing fibroblast like morphology in the culture Fig 1 A and get confluenced in five to six days after seeding Fig 1 B. conditioned medium was collected after five passage Fig 1C.

As per previous reported protocol by Poojan & Kumar we successfully isolated and culture epidermal stem cell in growth promoting media in different time intervals from day 1-5 cells growing well in low calcium condition as shown in Fig 2A-E cells are fully grown after one week and look like cuboidal in shape and is reported in literature that cuboidal shapeis characteristic of an undifferentiatedkeratinocyte (Jonathan et al 2009).

In the other experiment arsenic exposed mice bone marrow cells were isolated and we check for the chromosomal abrasion and micronuclei formation. The micronuclei formation is less in control and is increased when exposed with 85 ppm sodium arsenite. .as showed in figure 3. Chromosomes are intact when there is no treatment as shown in control when 85ppm sodium arsenite was given the chromosomal breakage were shown. Fig 4

DiscussionIn the skin, epidermal stem cells in the hair follicle contribute in the repair of the epidermis during wound healing and keratinization. When these stem cells are isolated and expanded in culture, they can give rise to hair follicles, sebaceous glands, and epidermis. In this work, we simply isolating dermal fibroblast and epidermal stem cells from the skin of adult mice and In the another experiment we initially demonstrate arsenic toxicity in adult mice bone marrow cells with the chromosome abrasion and micronuclei formation. As previously reported these cells isolated by this method are epidermal stem cell. Thus these cultured cells may be used in specific applications, such as viral manipulation and grafting toxicity application. These techniques should be useful for directly evaluating stem cell function in normal mice and in mice with skin defects. And in case of arsenic toxicity this basic information may be useful to identify the target cell for skin toxicity.

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Van Neste, D., Stagnet, M. J., Viac, J., Lachapelle, J. M. & Thivolet, J. A new way to evaluate the germinative compartment in human epidermis, using peptide. Br. J. Dermatol. 108, 433-439, 1983. Watt FM, Lo Celso C, Silva-Vargas V. Epidermal stem cells: an update. Curr Opin Genet Dev 16:518524, 2006. Watt FM: Epidermal stem cells: markers, patterning and the control of stem cell fate. Philos Trans R Soc Lond B Biol Sci 353:831-837, 1998.Fig 1 Balb/c skin Dermal Fibroblast cells

ABCDay 4Day 5After Five passageFig 2 balb/c epidermal skin stem cells in culture at different time interval

BDECADay oneDay twoDay threeDay fourDay five2

Fig 3. Arsenic toxicity in balb/c bone marrow cells showing micronuclei formation control85ppm sodium arsenite3

Fig 4. chromosomal abberation in balb/c mouse bone marrow cellscontrol85 ppm sodium arsenite