Professional Documents
Culture Documents
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
(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.
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
CaCl2
Calcium Chloride
BDE
Beta diene
LRC
DMEM
PBS
FBS
HSS
Dispase
Henke salt solution
BrdU
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
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)
(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
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.
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
Dispase-detached-keratinocyte-sheets
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
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
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
Biosafety cabinet
III.
IV.
V.
VI.
Centrifuge
Microscope, inverted
Flow cytometre
DMEM Powder
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.
DMEM Powder
NaHCo3
Glucose
Antibiotic-Antimyotic sol.
0.5mg
0.075gm
0.225gm
500l
Method of preparation:
III.
9.55g/100ml
0.20g
0.20g
8.0 g
1.15g
Method of Preparation:
Trypsin 1X sol.
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
Standard
For chromosomal aberration
For staining
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
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,
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
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
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.
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
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
Lavker, R. M. and Sun, T.-T. Epidermal stem cells. J. Invest. Dermatol. (Suppl.)
81, 121S-127S, 1983.
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
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
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