Sunteți pe pagina 1din 44

A DISSERTATION ON

A method to isolate and culture dermal fibroblast and


epidermal stem cell and arsenic toxicity in balb/c
mouse
SUBMITTED TO THE
DEPARTMENT OF LIFESCIENCES
CSJM UNIVERSITY,KANPUR

IN PARTIAL FULFILMENT
FOR THE
DEGREE OF MASTER OF PHYLOSOPHY
IN LIFESCIENCES

BY
Rashmi Upadhyay
M.PHIL. LIFE SCIENCES(II semester)
Department of LIFE SCIENCES
CSJM University,KANPUR

UNDER THE SUPERVISION OF


Dr. Sushil Kumar
Scientist F & Head

Environmental Carcinogenesis Division


CSIR-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)
Head
Department 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.
5.

OBJECTIVE
MATERIALS & METHODS

6.

7.
8.

RESULTS

DISCUSSION
REFERENCES

Abbreviations Used
Abbreviation
GPM

Full form
Growth Promoting Media

EP SC

Epidermal Stem Cell

CaCl2

Calcium Chloride

BDE

Beta diene

LRC

Label Retaining Cells

DMEM

Dulbeccos Modified Eagle medium

PBS

Phosphate Buffered Saline

FBS

Fetal Bovine Serum


Dpase

HSS

Dispase
Henke salt solution

BrdU

Bromo Deoxy Uridine


Co
Fi

Collagen
Fibronectin

Chelax

Resin

Introduction

Our 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 found
in 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 cells possess 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. Reproducibleisolation 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 labelretaining 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 cells
Stem cells differ from other kinds of cells in the body. All stem cells
regardless 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 cells
regardless 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 Literature
During 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 Tissue
The 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 cells
Significant 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 indepth 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 G 0 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. Reepithelization 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 transitamplifying 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. Immunofluorescence 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 cells
Locating 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 vivo
Further 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, donorderived 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 vitro
For 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]3d 104s24p3. 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 trioxide is the inorganic compound with the formula As2O3. This
commercially important oxide of arsenic is the main precursor to other arsenic
compounds, including organoarsenic 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 in keratin) and eliminated over a period of weeks or
months. The first symptoms of acute arsenic poisoning by ingestion are
digestive problems: vomiting, abdominal pains, diarrhea often accompanied

by bleeding. Sub-lethal doses can lead to convulsions, cardiovascular


problems, inflammation of the liver and kidneys and 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 Setup


1. Equipment
I.

Autoclave Vertical (Macro scientific works, Delhi, India),


II.

Biosafety cabinet
III.

IV.

Incubator, dual chamber

V.
VI.

Centrifuge

Microscope, inverted

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

NaHCo3

Glucose

Antibiotic-Antimyotic sol.

0.5mg
0.075gm
0.225gm
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


Potassium chloride
Sodium Chloride
Anhydrous sodium phosphate dibasic

0.20g
0.20g
8.0 g
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.

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 CaCl 2+1%
antibiotic mixture is mixed to make 50ml GPM.

Colchicine
8 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 selenite

Standard
For a month amount needed

5.6mg/kg body weight


33.6mg dissolved in 23ml miliQ H2O
2. Colchicine

Standard
For chromosomal aberration

4mg/kg body weight


8mg dissolved in 2ml PBS
3. Giemsa

For 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 fibroblast
Skin-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-Medium
Isolate and culture dermal fibroblasts Asphyxiate 2d old BALB/c mice and
soak carcass with BetadineTM solution 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 in PBS-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
25cm2 Nunc 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 CO2 incubator. 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 culture
Use neonatal mouse skin fibroblast at passage 3-5 to generate FibroblastConditioned-Medium Seed fibroblasts (1106cells) in T25cm2 Nunc-flask
containing Fibroblast-Growth-Medium and culture to near-60% confluence at
37C in 5%CO2 Wash adherent cells with PBS once before adding 15ml lowCa2+-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 medium
Prepare Fibroblast-Conditioned Medium (CM2) using Secondary Culture
Rinse primary fibroblast-culture twice using PBS after collecting CM1.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 CO2 incubator. 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 15ml SMEM to 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 cytometry


After 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 shape is
characteristic of an undifferentiated keratinocyte (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

Discussion
In 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.

REFERENCES

Adams, J. C. & Watt, F. M. Fibronectin inhibits the terminal differentiation of

human keratinocytes. Nature 340, 307309, 1989.


Albers, K. M., Greif, F., Setzer, R. W. & Taichman, L. B. Cell cycle withdrawal in

cultured keratinocytes. Differentiation 34, 236-240, 1987.


Alonso, L. & Fuchs, E. Stem cells in the skin: waste not, Wnt not. Genes Dev.

17, 11891200, 2003.


Amoh, Y. et al. Nestin-linked green fluorescent protein transgenic nude mouse

for imaging human tumour angiogenesis. Cancer Res. 65, 53525357, 2005(d).
Arnold, I. & Watt, F. M. c-Myc activation in transgenic mouse epidermis results
in mobilization of stem cells and differentiation of their progeny. Curr. Biol. 11,
558568 2001.
Barrandon, Y. & Green, H. Three clonal types of keratinocyte with different

capacities for multiplication. Proc Bickenbach JR, Chism E: Selection and


extended growth of murine epidermal stem cells in culture. Exp Cell Res

244:184-195, 1998.
Bickenbach JR, McCutecheon J, Mackenzie IC: Rate of loss of tritiated
thymidine label in basal cells in mouse epithelial tissues. Cell Tissue Kinet

19:325-333, 1986.
. Natl Acad. Sci. USA 84, 23022306 1987.
Bickenbach JR: Identification and behavior of label-retaining cells in oral
mucosa and skin. J Dent Res 60:1611-1620, 1981.
Bjornson CRR, Rietze RL, Reynolds BA, Magli MC, Vescovi AL: Turning brain

into blood: a hematopoietic fate adopted by adult neural stem cells in vivo.
Science 283:534-537, 1999.*
Blanpain C, Fuchs E Epidermal stem cells of the skin. Annu Rev Cell Dev Biol

22:339373, 2006.
Blanpain, C. et al. Self-renewal, multipotency, and the existence of two cell

populations within an epithelial stem cell niche. Cell 118, 635648, 2004.
Clayton E, Doupe DP, Klein AM, Winton DJ, Simons BD, Jones PH A single
type of progenitor cell maintains normal epidermis. Nature 446:185189, 2007.
Collins, M.K.L., Perkins, G.R., Rodriguez-Tarduchy, G., etal., Growth Factors as
Survival Factors: Regulation of Apoptosis, BioEssays, vol. 16, pp. 133138,
1993.

Evans, R. D. et al. A tumour-associated 1 integrin mutation that abrogates

epithelial differentiation control. J. Cell Biol. 160, 589596, 2003.


Ferraris, C., Bernard, B. A. & Dhouailly, D. Adult epidermal keratinocytes are
endowed with pilosebaceous forming abilities. Int. J. Dev. Biol. 41, 491498,

1997.
Fuchs E Scratching the surface of skin development. Nature 445:834842,

2007.
Fuchs E, Raghavan S Getting under the skin of epidermal morphogenesis. Nat

Rev Genet 3:199209, 2002.


Gallico GG 3rd, OConnor NE, Compton CC, Kehinde O, Green H Permanent
coverage of large burn wounds with autologous cultured human epithelium. N

Engl J Med 311:448451, 1984.


Gandarillas, A. & Watt, F. M. c-Myc promotes differentiation of human

epidermal stem cells. Genes Dev. 11, 28692882, 1997


Grinnell K. L., Bickenbach J. R., Skin keratinocytes pre-treated with embryonic

stem cell-conditioned medium or BMP4 can be directed to an alternative cell

lineage. Cell Prolif. 40, 685-705, 2007.


Hager B, Bickenbach JR, Fleckman P Longterm. culture of murine epidermal

keratinocytes. J Invest Dermatol 112: 971976, 1999.


Jackson KA, Mi T, Goodell MA: Hematopoietic potential of stem cells isolated

from murine skeletal muscle. Proc Natl Acad Sci 96:14482-14486, 1999.*
Jones PH, Harper S, Watt FM: Stem cell patterning and fate in human

epidermis. Cell 80:83-93, 1995.


Jones PH, Watt FM: Separation of human epidermal stem cells from transit
amplifying cells on the basis of differences in integrin function and

expression. Cell 73:713-724, 1993.


Jones, P. H. Epithelial stem cells. BioEssays 19, 683-690, 1997.
Kleinman HK, Wilkes CM, Martin GR. Interaction of fibronectin with collagen

fibrils. Biochemistry. 20(8):2325-30, 1981. *


Krause DS, Theise ND, Collector MI, et al: Multi-organ, multi-lineage

engraftment by a single bone marrow-derived stem cell. Cell 105:369-377, 2001


Lavker, R. M. & Sun, T. T. Epidermal stem cells: properties, markers, and
location. Proc. Natl Acad. Sci. USA 97, 1347313475, 2000.

Lavker, R. M. and Sun, T.-T. Epidermal stem cells. J. Invest. Dermatol. (Suppl.)
81, 121S-127S, 1983.

Millis AJ, Hoyle M, Field B. Human fibroblast conditioned media contains


growth-promoting activities for low density cells. J Cell Physiol. 93(1):17-24,

1977.
Mols, J.-P. and Watt, F. M. The epidermal stem cell compartment: variation in

expression levels of E-cadherin and catenins within the basal layer of human

epidermis. J. Histochem. Cytochem. 45, 867-874, 1997.


Morasso, M.I. and Tomic-Canic, M. Epidermal stem cells: the cradle of

epidermal determination, differentiation and wound healing. Biol. Cell. 97, 173
183, 2005.
Morrison, S.J., Shah, N.M., and Anderson, D.J., Regulatory mechanisms in

Stem Cell Biology, Cell, vol. 88, pp. 287298, 1997.


PerezLosada, J. & Balmain, A. Stem cell hierarchy in epithelial cancers.
Nature Rev. Cancer 3, 432441, 2003.
Plopper, G.E., McNamee, H.P., Dike, L.E., et al., Convergence of Integrin and

Growth Factor Receptor Signaling Pathways within the Focal Adhesion


Complex, Mol. Biol. Cell., vol. 6, pp. 13491365, 1995.
Rheinwald, J.B. and Green, H., Epidermal growth Factor and the Multiplication

of Cultured Human Epidermal keratinocytes, Nature, vol. 265, pp. 421424,


1977.

Tani H, Morris RJ, Kaur P: Enrichment for murine keratinocyte stem cells
based on cell surface phenotype. Proc Natl Acad Sci 97:10960-10965, 2000.

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.