Introduction To Stem Cells and Disease

“A ROLE OF STEM CELL IN DIFFERENT DISEASE”
A Project Report
Submitted to the PEOPLE’S UNIVERSITY, Bhopal
In partial fulfillment of the requirement for the degree of
Bachelor of Pharmacy
Submitted by Supervised by
FAIZAN KHAN MR. KAILASH SAHU
PU-022171804A
School of Pharmacy and Research
People’s University Bhanpur, Bhopal - 462037
Session JAN-JUNE-2022
A ROLE OF STEM CELL IN DIFFERENT DISEASE
A Project Report (Project report)

Submitted to the PEOPLE’S UNIVERSITY, Bhopal
In partial fulfillment of the requirement for the degree of
Bachelor of Pharmacy
Submitted by Supervised by
FAIZAN KHAN MR. KAILASH SAHU
PU-022171804A
School of Pharmacy and Research
People’s University Bhanpur, Bhopal - 462037
Session:JAN-JUNE-2022
SCHOOL OF PHARMACY AND RESEARCH Page 2

SCHOOL OF PHARMACY AND RESEARCH

(Approved by AICTE and PCI, and Recognized by Govt. of
Madhya Pradesh affiliated to UGC.)
Forwarding Letter
This is to acknowledge that FAIZAN KHAN has completed his/her project
entitled “A ROLE STEM CELL IN DIFFERENT DISEASE” under the
supervision of MR. KAILASH SAHU in partial fulfillment for the degree of
Bachelor of Pharmacy.
The project is forwarded to the examiner for evaluation.
Director/Principal
SCHOOL OF PHARMACY AND RESEARCH

(Approved by AICTE and PCI, and Recognized by Govt. of

Madhya Pradesh affiliated to UGC.)
Certificate
This is to certify that the project entitled “ A ROLE OF STEM CELL IN

DIFFERENT DISEASE ” which is submitted by FAIZAN KHAN, in the
partial fulfillment of the requirement for the award of the Degree of Bachelor of
Pharmacy by the People’s University, Bhopal, is a record of the candidate’s own
work carried out by him/her under my supervision and guidance.
I recommend the project to be forwarded to the examiner for evaluation.
Date: Guide Name

MR . KAILASH SAHU

ACKNOWLEDGEMENT
THE PROJECT REPORT "A ROLE OF STEM CELL IN DIFFERENT DISEASE “ has been
complied involving the hard work of many. Now is the time to express my heartiest gratitude
towards warn.
First and foremost I acknowledge the contributors of many persons who are dedicated to the
research and improvement,
I got an excellent opportunity to carry out my research work under the expert supervision of
Mr.kailash sahu with profound pleasure. I express my guide for his encouraging, untiring help
reliable, demanding and a personification of the word "efficiency".
I wish to place my profound gratitude and regards to respected Principal Prof. (Dr.)
BHASKAR GUPTA I am very much thankful to Mr. KAILASH SAHU for their guidance,
and my friend FARMAN HUSSAIN inspiration during this course of investigation.
Above all my heart left veneration goes to my caring Parents who are an integral part of my life,
for their love, care, patience, inspiration, moral support and trust on me and well wishes they had
bestowed upon me,
My acknowledgement would not finish without giving thanks and praising the god for being with
me before the start of my work helping my all though it and blessing me to make it.
FAIZAN KHAN
PU-022171804A

CONTENTS
SNO. TOPIC PAGE NO
1 INTRODUCTION 7-10
2 HISTORICAL BACKGROUND 11-14
OF STEM CELL RESEARCH
3 DISCUSSION 15-40
4 CONCLUSION 41
5 REFERNCE 42-43

INTRODUCTION
The concept that stem cells may reside in the periodontal tissues was first proposed almost 20
years ago by Melcher, who queried whether the three cell populations of the Periodontium
(cementoblasts, alveolar bone cells and periodontal ligament fibroblasts) were Ultimately
derived from a single population of ancestral cells or stem cells.
Stem cells have the remarkable potential to develop into many different cell types in the Body
during early life and growth. In addition, in many tissues they serve as a sort of Internal repair
system, dividing essentially without limit to replenish other cells as long as The person or animal
is still alive. When a stem cell divides, each new cell has the potential Either to remain a stem
cell or become another type of cell with a more specialized function, Such as a muscle cell, a red
blood cell, or a brain cell.
Stem cell research has been a debatable issue since many years and now it is Regularly a front
page news because of the controversies regarding the derivation of Stem cells from human
embryos. This research has the potential to affect the lives of Millions of people by offering
unprecedented opportunities for developing new medical Therapies for debilitating diseases and
a new way to explore fundamental questions of Biology
Stem cells are distinguished from other cell types by two important characteristics. First, They
are unspecialized cells capable of renewing themselves through cell division, Sometimes after
long periods of inactivity. Second, under certain physiologic or Experimental conditions, they
can be induced to become tissue- or organ-specific cells with Special functions. In some organs,
such as the gut and bone marrow, stem cells regularly
Divide to repair and replace worn out or damaged tissues. In other organs, however, such as The
pancreas and the heart, stem cells only divide under special conditions. Stem cells are important
for living organisms for many reasons. In the 3 to 5 day old Embryo, called a blastocyst, the
inner cells give rise to the entire body of the organism, Including all of the many specialized cell
types and organs such as the heart, lung, skin, Sperm, eggs and other tissues. In some adult
tissues, such as bone marrow, muscle, and Brain, discrete populations of adult stem cells
generate replacements for cells that are lost Through normal wear and tear, injury or disease.
Given their unique regenerative abilities stem cell research has the potential to impact not Just

one disease but numerous ones e.g. Diabetes, Cancer, Cardiovascular disease, Alzheimer’s
disease, Burn victims, Leukemia, tooth regeneration, bone regeneration and Many more but to be
able to harness such therapeutic potential of stem cells, Scientists need To learn how to direct
them to differentiate appropriately, their therapeutic potential and to Ensure that they do not
continue to multiply in an uncontrolled way and not form a tumor. However, much work remains
to be done in the laboratory and the clinic to understand how To use these cells for cell-based
therapies to treat disease, which is also referred to As regenerative or reparative medicine.
Swirling in this cloud of controversy regarding use of embryonic stem cells scientist has
Postulated certain important facts about adult stem cells as they apply to dentistry. Scientists
Theorize that virtually every tissue in the body contains some type of stem cells. From there It is
possible to consider the idea of medical and specifically dental cell based strategies for Tissue
repair. The current focus on self renewing stem cells provides the opportunity to Understand the
process of tissue turnover. This leads us to the cutting edge of stem cell Research as it applies to
dentistry. The focus of stem cell research as it applies to dentistry is On facial reconstruction. Of
the estimated 1.6 million bone grafts performed annually to Regenerate bone lost due to trauma
or disease, about 96,000 relate to the face and mouth. Field of stem cell research is growing
enormously that scientists are able to identify Potential stem cells in unerupted tooth buds, or
dental pulp. These cells provide the prospect Of restoring dental tissues such as dentin,
cementum, and periodontal ligament and Supporting bone. Today, a dental implant relies on the
ability of bone to interface with metal (usually titanium). Tomorrow, dental scientists imagine
the development of an enamel-like Bio-material fabricated in the shape of a patient’s tooth that
needs to be replaced. And Beyond this, it just may be possible to one day recreate a tooth bud in
the laboratory then Transfer it into the patient’s jaw to develop, grow, and erupt on its own.
Individual teeth generally do not last the lifetime of the individuals without requiring at Least
some repair. The need for replacement of teeth and dental tissue repair therapies is Significant.
As the close association between oral health, systemic health and nutrition Becomes more
apparent, the necessity of proper oral health for long term quality of life Becomes more
appreciated.
Conventional dental treatment has relied on symptomatic treatment and prosthetic Restoration
using artificial materials, and clinicians have tended to concentrate on improving Skills rather

than developing new modes of treatment. To accelerate the clinical application Of newly
developed periodontal therapy, the important issues should be addressed while Developing the
periodontal therapeutic techniques. The ideal system for clinical use will be a Simple procedure
that provides one step delivery of the gene/protein of interest with minimal Manipulation. The
development of such a therapeutic approach will enable wider clinical
Application that will benefit the increasing numbers of the aging population suffering from
Periodontitis. Longer & Vacanti defined tissue engineering as “An interdisciplinary field that
applies the Principles of engineering and the life sciences towards the development of biological
Substitutes that restore, maintain or improve tissue function.” Periodontitis is a disease of the
periodontium characterized by irreversible loss of connective Tissue attachment & supporting
alveolar bone. For many decades, periodontists have been Interested in regenerating tissues
destroyed by periodontitis. Periodontal regeneration can be defined as the complete restoration of
the lost tissues to Their original architecture and function by recapitulating the crucial wound
healing events Associated with their development. Conventional open flap debridement falls
short of Regenerating tissues destroyed by the disease, and current regenerative procedures offer
a Limited potential towards attaining complete periodontal restoration. Recently, the isolation Of
adult stem cells from human periodontal ligament has presented new opportunities for Tissue
engineering. Clearly, in order for such therapies to be successful, a thorough Understanding of
stem cells and their role in regenerating periodontal tissues is required. Following disease
control, periodontal regeneration represents the ultimate goal of Periodontal therapy and entails
the reformation of all components of the Periodontium: Gingival connective tissue, periodontal
ligament, cementum and alveolar bone. Periodontal Regeneration aims to restore these lost
tissues to their original form and function by Recapitulating the crucial wound healing events
associated with periodontal development. Hence an understanding of the processes involved in
the development of the Periodontium is Necessary in order to appreciate the cellular and
molecular events that might occur during Periodontal regeneration. Despite recent interest in
tooth regeneration, the view remains that In a clinical setting the fundamental goal is actually the
regeneration of periodontal tissues Particularly periodontal ligament, because of the dimiculty in
regulating the morphogenesis Of crown and regenerating the tooth root

Tissue engineering for periodontal tissue regeneration is based on 3 approaches
1.Protein based approaches: By using Growth and Differentiation factors.
2. Cell based approaches: By harvesting and differentiating stem cells
3. Gene delivery based approaches: By gene encoding of therapeutic proteins.
Regeneration of tissues destroyed by periodontitis has long been a selfless goal of Periodontal
therapy Periodontal regeneration requires consideration of many features that Parallel
periodontal development, including the appropriate progenitor cells, signaling Molecules and
matrix scaffold in an orderly temporal and spatial sequence. It is clear that Current regenerative
procedures are less than ideal but the identification of stem cells in Human dental tissues in
recent years holds promise to the development of novel, more Effective approaches to
periodontal regeneration and reconstructive therapy. One way Forward is to embrace the field of
stem cell based tissue engineering and adopt an Interdisciplinary approach to periodontal
regeneration. In the context of oro-facial tissue Engineering. Populations of stem cells that form
bone, cementum, dentin and even Periodontal ligament have been identified. The future for stem
cell-based periodontal regeneration is very promising. However, as with All new technologies,
questions often arise at a faster rate than answers. The time is now ripe To move from animal
studies to human clinical trials. The plethora of animal studies carried out to date provide an
overwhelming body of Evidence to support the notion that mesenchymal stem cells can be used
for periodontal Regeneration. However, there are critical steps in moving the field towards
human clinical Utility. Issues such as appropriate delivery devices, immunogenicity, autologous
cells vs. Allogenic cells, which tissues provide the most appropriate donor source, control of the
Whole process and cost-effectiveness are all important considerations that should not be
Overlooked. Furthermore, before we can confidently move forward, the next critical phase is The
systematic validation of specific mesenchymal stem cells as reliable sources for Cytotherapeutic
use Stem cell research opens up the new field of ‘cell-based therapies’ and, as such, several
Safety measures have also to be evaluated Finally, the establishment of large-scale preparation
facilities incorporating the stringent Protocols of good manufacturing procedures will be an
absolute necessity.

REVIEW OF LITERATURE
HISTORICAL BACKGROUND OF STEM CELL RESEARCH
1878:- First reported attempts to fertilize mammalian eggs outside the body.
1959:- First report of animals (rabbits) produced through IVF in the United States.
1960: -Studies of terato-carcinomas in the testes of several inbred strains of mice Indicates they
originated from embryonic germ cells. The work establishes Embryonal carcinoma (EC) cells as
a kind of stem cell.”
1968:-Edwards and Bavister fertilize the first human egg in vitro.’’
1970s:-EC cells injected into mouse blastocyst produce chimeric mice. Cultured SC Cells are
explored as models of embryonic development, although their Complement of chromosomes is
abnormal.”
1978: -Louise Brown, the first IVF baby, is born in England.
1980:-Australia’s first IVF baby, Candace Reed, is born in Melbourne. Scientists Discovered
how to obtain or derive embryonic stem cells from mouse Blastocysts by culturing inner cell
masses on feeder layers of mouse Fibroblasts.
1981:- Evans and Kaufman, and Martin derive mouse embryonic stem (ES) cells From the inner
cell mass of blastocysts. They establish culture conditions for Growing pluripotent mouse ES
cells in vitro. The ES cells yield cell lines with Normal, diploid karyotypes and generate
derivatives of all three primary germ Layers as well as primordial germ cells. Injecting the ES
cells into mice Induced the formation of teratomas.
1984 -88:-One of the milestones of mES cell research was the development of methods To
modify the cells genetically by Doetschman et al. ; Thomas and Capecchi 1987. The feeder
layers of mouse fibroblasts could be replaced with Culture medium containing the growth factor
Leukemia Inhibitory Factor (LIF) by Smith, et al. 1988; Williams, et al. 1988. Andrews, et al.
Developed pluripotent, genetically identical (clonal) cells called embryonal Carcinoma (EC)
cells from Tera-2, a cell line of human testicular Teratocarcinoma. Cloned human teratoma cells
exposed to retinoic acid Differentiate into neuron-like cells and other cell types.

1989:-Pera, et al.derive a clonal line of human embryonal carcinoma (EC) cells, Which yields
tissues from all three primary germ layers. The cells are Aneuploid (fewer or greater than the
normal number of chromosomes in the Cell) and their potential to differentiate spontaneously in
vitro is typically Limited. The behavior of human EC cell clones differs from that of mouse ES
Or EC cells.
1992:-The germ cells in fetal mouse gonads can give rise to permanent pluripotent Stem cell
lines in culture, mEG cells. Matsui, et al. ; Resnick, et al
1994:-Bongso, et al. first described isolation and culture of cells of the inner cell Mass of human
blastocysts. Human blastocyst created for reproductive Purposes using IVF and donated by
patients for research, are generated from The 2 pronuclear stages. The inner cell mass of the
blastocyst is maintained in Culture and generates aggregates with trophoblast-like cells at the
periphery And ES like cells in the center. The cells retain a complete set of Chromosomes
(normal karyotype); most cultures retain a stem cell like Morphology, although some inner cell
mass clumps differentiate into Fibroblasts. The cultures are maintained for two passages.
1995-96:- Non human primate ES cells are derived and maintained in vitro, first from The inner
cell mass of rhesus monkeys and then from marmosets blastocyst. The primate ES cells are
diploid and have normal karyotype. They are Pluripotent and differentiate into cells types
derived from all three primary Germ layers. The primate ES cells resemble human EC cells and
indicate that It should be possible to derive and maintain human ES cells in vitro.
1998:- Thomson, et al. derived human ES cells from the inner cell mass of normal Human
blastocysts donated by couples undergoing treatment for infertility. The cells are cultured
through many passages, retain their normal karyotype, Maintain high levels of telomerase
activity, and express a panel of markers Typical of human EC cells non-human primate ES cells.
Gearhart and colleagues derived human embryonic germ (EG) cells from The gonadal ridge and
mesenchyme of 5-9 week fetal tissue that resulted from Elective abortions. They grow EG cells
in vitro for approximately 20 Passages, and the cells maintain normal karyotypes. The cells
spontaneously Form aggregates that differentiate spontaneously, and ultimately contain
Derivatives of all three primary germ layers. The EG cells do not form Teratomas when injected
into immune deficient mice.

2000:-Under appropriate culture conditions, hES cells were shown to be pluripotent By

differentiating into multiple tissue types by Itskovitz-Eldor, et al.; Reubinoff, et al. Scientists in
Singapore and Australia led by Pera,Trounson, and Bongso derive human ES cells from the inner
cell mass of Blastocysts donated by couples undergoing treatment for infertility. The ES Cells
proliferate for extended periods in vitro, maintain normal karyotypes, Differentiate
spontaneously into somatic cell lineages derived from all three Primary germ layers, and form
teratomas when injected into immune Deficient mice.
2001:-As human ES cell lines are shared and new lines are derived, more research Groups report
methods to direct the differentiation of the cells in vitro. Many Of the methods are aimed at
generating human tissues for transplantation Purposes including pancreatic islet cells, neurons
that release dopamine and Cardiac muscle cells.!
2002:-Vojislav Lekovic, et al. compare the clinical effectiveness of 2 regenerative A In humans:

Combination Of Techniques for intrabony defects PRP/BPBM/GTR versus a combination of
PRP/BPBM & showed that both Combinations of PRP/BPBM/ GTR and PRP/BPBM are
effective in the Treatment of intrabony defects present in patients with advanced chronic
Periodontitis. The result also suggest that GTR adds no clinical benefit to PRP/BPBM.17
2003: -Miura, et al. identified the multipotent stem cells (SHED – Stem Cells from 18 Human
Exfoliated Deciduous Teeth).
2004:-Seo, et al. reported multipotent stem cells from human periodontal Ligament.
2005:-Saito, et al. developed bovine cementoblast cells.
2006:-The dental follicle has long been considered as a multipotent tissue because Of its ability
to generate cementum, bone and PDL from the homogeneous Like ectomesenchymal derived
fibrous tissue (Handa, et al. 2002 a, b; Luan, Et al. 2006). Scientists at Wake Forest University
led by Dr. Anthony Atala& Harvard
2007:-University report discovery of a new type of stem cell in amniotic fluid. This May
potentially provide an alternative to embryonic stem cells for use in Research and therapy.

2008 :-The first published study of successful cartilage regeneration in the human Knee using
autologous adult mesenchymal stem cells is published by 20 Clinicians from Regenerative
Sciences.
2010:-First trial of embryonic stem cells in humans. The trial will test the safety of Using
replacement retinal cells known as retinal pigment epithelial derived From human embryonic
stem cells.
2010: Masaki J Honda, et al. Dental follicle stem cells can be isolated and under Defined tissue
culture conditions, and recent characterization of these stem Cells has increased their potential
for use in tissue engineering applications, Including periodontal and bone regeneration.22
2011:-Wolf-Dieter Grimm, et al. in vivo study clearly showed that human adult PDLSCs
transplanted into an athymic rat model were able to regenerate Periodontal tissue elements at
different levels23
2011 : Duan, et al. have implanted induced pluripotent stem cells into a mouse Periodontal
fenestration defect model. In this model, induced pluripotent
2012:-Stem cells were implanted into the fenestration defect with the aid of a silk Fibroin
scaffold in combination with enamel matrix derivative gel24 Fawzyl EL Sayed KM, et al.
investigated the periodontal regenerative Potential of gingival margin-derived multipotent
postnatal stem/progenitor Cells. Periodontal defects were induced at six sites in eight miniature
pigs in The premolar/molar area (4 weeks). Autologous cells isolated from the Gingival Margin
were magnetically sorted using STRO-1 antibodies and Characterized flow cytometrically for the
expression of CD14, CD31, CD34, CD45, CD117 and STRO-1 surface markers.
2013:-Inukai T, et al. evaluated the effect of conditioned medium from cultured Mesenchymal
stem cells (MSC-CM) on periodontal regeneration . In vitro, MSC-CM stimulated migration and
proliferation of dog MSCs (dMSCs) and Dog periodontal ligament cells (dPDLCs). Cytokine
such as insulin like Growth factor, vascular endothelial growth factor, transforming growth
Factor-B1, and hepatocyte growth factor were detected in MSC -CM. In vivo, Cne-wall critical
size, intrabony periodontal defects were surgically created In the mandible of dogs. Dogs with
these defects were divided into three Groups that received MSC-CM, PBS, or no implants.
Absorbable atelo Collage spong (TERUPLUG®) were used as a scaffold material.

STEM CELL BASICS
The word “stem” actually originated from old botanical monographs from the same Terminology
as the stems of plants, where stem cells were demonstrated in the apical root And shoot
meristems that were responsible for the regenerative competence of plants. Hence also the use of
the word “stem” in “meristem.’ The term “stem cell” first appeared in the literature during the
19th century. Like many Other terms in biology the concept of a stem cell has expanded greatly
with identification Of novel sites and functions. A “stem cell” refers to a clonogenic,
undifferentiated cell That is capable of self-renewal and multi-lineage differentiation. In other
words, a stem Cell is capable of propagating and generating additional stem cells, while some of
its Progeny can differentiate and commit to maturation along multiple lineages giving rise to a
range of specialized cell types.
PROPERTIES OF STEM CELLS
Stem cells are different from the other types of cells in the human body. Although they Can be
harvested from various sources, they all share some of the same properties like Stem cells can
divide and renew themselves Stem cells have a special ability to divide and renow themselves for
extended periods of Time. In fact an initial population of stem cells can produce millions of cells
within few Months in a laboratory setting. When the produced cells remain unspecialized for a

Longer duration that indicates the long term self renewal capacity of the cells. Stem cells are
unspecialized The unspecialized nature of stem cells is an important one. It means that stem cells
lack The specific parts that allow them to perform specialized functions in the body. A stem Cell
does not have a specialized function but it has the capacity to differentiate into a Specialized cell
that can carry out these functions. The exact factors that allow stem cells To be unspecialized are
still not known but once they are determined, stem cell culture Will be made possible in the
laboratories with a greater success. Stem Cells Can Give Rise to Specialised Cells The ability of
stem cells to give rise to specialised cells is a crucial one. In this process of Differentiation,
unspecialised stem cells produce specialised cells. It is thought that a Cell’s genes regulate the
internal signals that trigger this process. These genes carry the Specific code, or instructions, for
all the parts and functions of a cell. External signals are Those outside of the cell, which include
chemicals released from other cells, physical Connections with nearby cells and varions other
molecules in the surrounding area
CLASSIFICATION OF STEM CELLS
There are different types of stem cells which are usually considered for their potential Use in research and
medicine. They can be classified on the basis of:
1.The extent to which they can differentiate into different cell types.
2. Source of stem cells(based on their origin)
1. Extent to which they can differentiate:
a) Totipotent cells
b) Pluripotent cells
c) Multipotent cells
d) Oligopotent cells
e) Unipotent cells
Totipotent cells: From Latin totus, meaning entire total because it has the potential to generate all the
cells and tissues that make up an embryo and that support its development in utero. Cells produced by the
first few divisions of the fertilized eggs are totipotents. These cells can differentiate into embryonic and
extra embryonic cell types. These are the most versatile of the stem cell types. When a sperm cell and an

egg cell Unite, they form a one-celled fertilized egg. This cell is totipotent, meaning it has the Potential to
give rise to any and all human cells, such as brain, liver, blood or heart cells Etc. It can even give rise to
an entire functional organism. The first few cell divisions in Embryonic development produce more
totipotent cells. After four days of embryonic cell Division, the cells begin to specialize into pluripotent
stem cells. WIZU egg as it divides into blastomeres which then IPOH The blastocyst from which
embryonic stem cells are isolated.
b) Pluripotent cells: “Plur”- derived from the Latin plures means several or many. Thus, pluripotent cells
have the potential to give rise to any type of cell, unlike totipotent stem cells which cannot give rise to an
entire organism. These are descents of totipotent cells and can differentiate into cells derived from the
three germ layers. On the fourth day ofdevelopment, the embryo forms into two layers, an outer layer
which will become the placenta, and an inner mass which will form the tissues of the developing human
body. These inner cells, though they can form nearly any human tissue, cannot do so without The outer
layer, so are not totipotent, but pluripotent. As these pluripotent stem cells Continue to divide, they begin
to specialize further.
Three types of pluripotent cells have been found
1.Embryonic Stem Cells: These can be isolated from the inner cell mass (ICM) of the
Blastocyst the stage of the embryonic development when implantation occurs. Embryonic germ
cell (EG cells): These can be isolated from the precursor to the Gonads in aborted tissues,
Embryonic Carcinoma cells: These can be isolated from teratocarcinomas, a tumorThat
occasionally occurs in gonad of a fetus. Unlike the other two, they are usually Aneuploid. All

three of these types of pluripotent stem cells can be isolated from the embryonic or Fetal tissue
can be grown in culture but only with special methods to prevent them from Differentiating.
2. Multipotent cells: These are true stem cells but differentiate into a limited number of types
e.g. the bone marrow contains multipotent stem cells that give rise to all the cells of the blood but
not to other types of cells. Multipotent stem cells are found in adult tissues; perhaps most organs
in the body (brain, liver) contain them where they can replace dead or damaged cells. These adult
stem cells may also be cells that when one accumulates sufficient mutations produce a clone of
cancer cells.
3. Oligopotent stem cells: The ability to differentiate into a few cells. Examples include adult
lymphoid or myeloid stem cells.
4. Unipotent stem cell: A unipotent stem cell refers to a cell that can differentiate along only one
lineage. Due to intense interest in totipotent and pluripotent stem cell, unipotent stem cells have
not received the same attention and research. They do, however, have vast potential to treat
health conditions. The word ‘uni’ itself is derived from the Latin word ‘unus’, meaning one.
Found in adult Tissues and in comparison with other types of stem cells, has the lowest
differentiation Potential. Although the unipotent adult stem cells in the body’s tissues will only
give rise To one cell type, they do still have the important property of self renewal that is shared
by All stem cells. Also, despite their differentiation potential being limited, unipotent cells Still
have therapeutic potential to treat injuries and disease.

2. Stem cells can be classified into four broad types based on their origin.
1. Embryonic stem cells (ESC)
2. Stem cells from the fetus
3. Stem cells from the umbilical cord
4. Adult stem cells
Each of these can be grouped into sub types Some believe that adult and fetal stem cells evolved
from embryonic stem cells and theFew stem cells observed in adult organs are the remnants of
original embryonic stem Cells that gave up in the race to differentiate into developing organs or
remained in cell Niches in the organs which are called upon for repair during tissue injury.
Embryonic stem cells Embryonic stem cells (ESCs) were first reported by Damjanov&Solter,
verifying that In certain strains of mice and human germ cells occasionally gave rise to tumors
termed Teratocarcinommas, containing derivates of all three embryonic germ layers. The
Undifferentiated stem cell components of teratocarcinomas were termed embryonal Carcinoma
cells (EC) and provided an important in vitro model to study cellular Differentiation. In
mammals, the fertilized oocyte, zygote, 2 cell, 4 cell, 8 cell and morula resulting from Cleavage
of the early embryo are examples of totipotent cells (ability to form a complete Organism). Proof
that these are indeed totipotent cells comes from the fact that identical Twins can be generated
from splitting of the early embryo in vitro by micromanipulation In domestic animals. However,
strictly speaking, the fertilized oocyte and blastomeres Cannot be termed “stem cells” because
the making of more of them is limited during Early cleavage division. They, thus, cannot self-
renew even though they have the potential to form a complete Organism. The inner cell mass
(ICM) of the 5 to 6 day old human blastocyst is the source Of pluripotent embryonic stem cells
(hESCs). During embryonic development, the ICM Develops into two distinct cell layers, the
epiblast and hypoblast. The hypoblast forms the Yolk sac which later becomes redundant in the
human, and the epiblast differentiates into the three primordial germ layers (ectoderm, mesoderm
and endoderm).
Embryonic endoderm cells are rather restricted in their developmental pathways. A small
Population of multipotent cells, called the definitive endoderm, gives rise to all of the Endoderm

derived organs in the adult. The definitive endoderm is separated from the Pluripotent ICM
during gastrulation immediately after implantation. The definitive Endoderm comprises an
epithelial sheet of approximately 600 cells that cover the ventral Surface of the embryo. This
sheet later forms the fore gut and hind gut. The foregut later forms the lung, liver, stomach and
pancreas, while it’s more posterior Aspects gives rise to the intestines (mid gut) and cloaca. The
hind gut gives rise to the Rectum and large intestine. Knowing what drives these developmental
pathways is Crucial to understanding the factors and events that lead to differentiation of
embryonic Stem cells to desirable tissues such as the pancreas. Pluripotent embryonic stem cells
can Give rise to many cell types in vitro, including cells specific to endodermal tissues.
Advances in the understanding as to how ES cells differentiate should provide answers For re-
programming of stem cells from adult tissues. Properties of an Embryonic Stem Cell? Derived
from the inner cell mass/epiblast of the blastocyst. Capable of undergoing an unlimited number
of symmetrical divisions without Differentiating (long-term self-renewal). Exhibit and maintain
a stable, full (diploid), normal complement of chromosomes (karyotype). Pluripotent ESC can
give rise to differentiated cell types that are derived from all Three primary germ layers of the
embryo (endoderm, mesoderm & ectoderm). Capable of integrating into all fetal tissues during
development. (Mouse ESC Maintained in culture for long periods can still generate any tissue
when they are Reintroduced into an embryo to generate a chimeric animal).
• Capable of colonizing the germ line and giving rise to egg or sperm cells.
• Clonogenic that is a single ESC can give rise to a colony of genetically identical cells Or clones
which have the same properties as the original cell. Expresses the transcription factor Oct-4,
which then activates or inhibits a host of Target genes and maintains ES cells in a proliferative,
non differentiating state. Can be induced to continue proliferating or to differentiate.
Lacks the GI checkpoint in the cell cycle. ESC spends most of their time in the S Phase of the
cell cycle, during which they synthesize DNA. Unlike differentiated Somatic cells, ESC does not
require any external stimulus to initiate DNA Replication Do not show X inactivation. In every
somatic cell of a female mammal, one of the Two X chromosomes becomes permanently
inactivated. X inactivation does not occur In undifferentiated ES cells. Laboratory tests used to
identify embryonic stem cells” The tests are done to check whether the cells exhibit fundamental
properties of Embryonic stem cells The tests include Capability of long term self renewal Cells

are grown and subcultured for Many months and check for undifferentiated symmetrical
divisions. Expression of protein called OCT. 4 are checked which undifferentiated cells
Typically make. OCT. 4 is a transcription factor, meaning that it helps them Genes on and off at
the right time, which is important part of cell Differentiation Examining the chromosomes for
stable diploid nonnal complement of Chromosomes. Testing whether the embryonic stem cells
are pluripotent
Embryonic germ cells
Primordial germ cells or diploid germ cell precursors transiently exist in the embryo Before they
closely associate with somatic cells of the gonads and then become Committed as germ cells.
Human embryonic germ cells (hEGCs) which are also stem Cells, originate from the primordial
germ cells of the gonadal ridge of 5 to 9 week old Fetuses, hEGCs have been successfully
isolated and characterized. These stem cells are Pluripotent and are able to produce cells of all
three germ layers (ectoderm, mesoderm And endoderm). Fetal stem cells Fetal stem cells are
primitive cell types found in the organs of fetuses. Neural creststem Cells, fetal hematopoietic
stem cells and pancreatic islet progenitors have been isolated in Abortuses Fetal neural stem cells
found in the fetal brain were shown to differentiate Into both neurons and glial cells. 35,36 Fetal
blood, placenta and umbilical cord are rich Sources of fetal hematopoietic stem cells. Umbilical
cord stem cells Umbilical cord blood contains circulating stem cells and the cellular contents of
Umbilical cord blood appear to be quite distinct from those of bone marrow and adult Peripheral
blood. The characteristics of hematopoietic stem cells in umbilical cord blood Have recently
been clarified. The frequency of umbilical cord blood hematopoietic stem Cells equals or
exceeds that of bone marrow and they are known to produce large Colonies in vitro, have
different growth factor requirements, have long telomeres and can Be expanded in long term
culture.

ADULT STEM CELLS
Hematopoietie stem cells (bone marrow and peripheral blood): Bone marrow possesses stem
cells that are hematopoietic and mesenchymal in origin. Hematopoiesis is the production and
maintenance of blood stem cells and their Proliferation and differentiation into the cells of
peripheral blood. The hematopoietic Stem cell is derived early in embryogenesis from mesoderm
and becomes deposited in Very specific hematopoietic sites within the embryo.These sites
include the bone marrow, Liver, and yolk sac. Hematopoietic stem cells can be purified using
monoclonal Antibodies and recently common lymphoid progenitor and myeloid erythroid
progenitor Cells have been isolated and characterized. Bone marrow stem cells may be more
Plastic and versatile than expected because they are multipotent and can be differentiated Into
many cell types both in vitro and in vivo. Mesenchymal stem cells (bone marrow stroma) Bone
marrow is a reservoir of pluripotent stem cells for mesenchymal tissue. Hematopoietic and
mesenchymal cells reside in close contact in the post natal bone marrow cavity. Hematopoietic
stem cells in this cavity are functionally and structurally supported in their blood forming
abilities by a complex structure that as a whole forms the bone marrow stromal network. This
stroma is composed of a heterogeneous variety of cell types, which include reticular cells,

adipocytes, osteogenic cells, smooth muscle cells, endothelial cells and macrophages, among
which bone marrow stromal cells are essential element of hematopoietic environment.
Friedenstein and coworkers (1968) isolated these bone marrow stromal cells for the first time
which were similar to fibroblast cells morphologically, but could differentiate in to various cell
lineages.” In a steady state or in response to injury, turnover of stromal tissue and repair occurs
through the participation of a population of stem cells found in the stromal tissue. Apart from
bone marrow stroma, MSCs can also be derived from periosteum, fat and skin. MSCs are
multipotent cells that are capable of differentiating into cartilage, bone, musele, tendon, ligament
and fat.”There is some recent evidence that there is a rare cell within MSC cultures that is
pluripotent and can give rise not only to mesodermal but to endodermal tissues. The authors have
called this a Multipotent Adult Progenitor Cell. Several investigations by various authors have
reported that the presence of FGF has demonstrated the greatest ability to promote the
proliferation of these cells and in particular, the expression of the osteogenic phenotype.
Recently, cell based engineering approach is to treat large bony defects caused by trauma or
disease.


Gut stem cells
The gastrointestinal epithelial lining undergoes continuous and rapid renewal throughout life.
Differentiation programs thus exist in specific regions of the tract. Epithelial renewal is sustained
with populations of multipotent stem cells residing in distinct anatomic sites governed by
niches.” Major challenge is to identify these niches, the properties of these stem cells and the
molecular mechanisms underlining their fate decisions in appropriate developmental pathways.
These answers will provide clues as to why some patients infected with Helicobacter pylori are
at risk in developing gastric adenocarcinoma. Many patients harbor Helicobacter pylori in their
stomachs but only a percentage goes on to develop pathology. Epithelial cell renewal in the
intestine is sustained by multipotent stem cells located in the crypts of Lieberhahn. In the small
intestine, epithelial cells of enterocytic, goblet and enteroendocrine origin differentiate as they
migrate from a crypt up an adjacent villus and leave the intestine once they reach the villus tip.
In the colon, it is different. Epithelial cells migrate from the crypt to a flat surface cuff that
surrounds its opening. The stem cell hierarchy in the gut and the fact that stem cells and their
progeny are located in well defined anatomic units make the gut an ideal in vivo model for stem
cell research
Liver stem cells
Mammals are said to survive surgical removal of at least 75% of the liver by regeneration. The
original tissue can be restored in 2-3 weeks. This is in contrast to most other organs such as the
kidney or pancreas. Recent evidence strongly suggests that different cell types and mechanisms
are responsible for organ reconstitution, depending On the type of liver injury. In the case of the
liver, regeneration must be distinguished by transplantation (repopulation) with donor cells.
Bone and cartilage stem cells Mesenchymal Stem Cells in bone marrow can differentiate into
bone and cartilage under appropriate conditions. However, if bone or cartilage is injured, are
there stem cells inherent in bone or cartilage to participate in the repair process? Bone itself has
been found to have both uncommitted stem cells as well as committed osteoprogenitor cells. In
addition, when bone is fractured, there is exposed marrow and abundant bleeding with hematoma
formation in the marrow space, which results in good repair potential. In vivo, articular cartilage
has a very limited capacity for repair if injured. It is currently not clear whether there is a

committed chondrocyte progenitor cell located within cartilage. In the presence of injury to
cartilage, stem cells do participate in the repair process. The numbers, however, are small and the
regulatory factors are limited. It is postulated that these cells may be derived from surrounding
tissues such as muscle, bone or other non-cartilaginous tissues.”
Epidermal stem cells (skin and hair)
The human skin comprises the outer epidermis and underlying dermis. Hair and sebaceous
glands also make up the epidermis. The most important cell type in the epidermis is the
keratinocyte which is an epithelial cell that divides and is housed in the basal layer of the
epidermis. Once these cells leave the basal layer they undergo terminal differentiation resulting
in a highly specialized cell called a squame which eventually forms either the hair shaft or the
lipid-filled sebocyte that form an outer skin layer between the harsh environment and underlying
living skin cells.
The epidermis houses stem cells at the base of the hair follicle and their self-renewing properties
allow for the re-growth of hair and skin cells that occurs continuously. New keratinocytes are
produced continuously during adult life to replace the squames shed from the outer skin layers
and the hairs that are lost. Stem cells differentiate into an intermediate cell called the “transient
amplifying cell” which gives rise to the more differentiated cell types inclusive of the
keratinocytes and sebocytes. Neuronal stem cells It has been suggested that a continuous
neurogenic turnover occurs in some limited areas of the central nervous system (CNS). Two
neurogenic regions of the adult mammalian CNS are supposed to be involved in this process: the
subventricular zone (SVZ) of the forebrain and the dental gyrus of the hippocampus which are
considered reservoirs of new neural cells. Thus, neural stem cells (NSCs) are known to reside in
these two areas and they consistently generate new neurons.” In vivo, endogenous NSCs seem to
be able. To produce almost exclusively neurons, while a single NSC in vitro is competent to
generate neurons, astrocytes and oligodendrocytes. NSCs are multipotent progenitor cells that
have self-renewal activity. Although it seems clear at present that the bona fide NSC is the
subventricular zone B cells, the search for self-renewing, multipotent NSCs is in progress and
conflicting information is available in the literature. There has been data to suggest that the SVZ
NSC is an ependymal cell, while others have demonstrated that the SVZ astrocyte is the NSC.” It
was also demonstrated that ependymal cells were unipotent giving rise to only glial cells,

whereas SVZ astrocytes were able to produce multipotent neurospheres that yielded both
neurons and glia. The final fate of the NSC is under tight environmental control and a stem cell
niche has been postulated for the adult mammalian brain.
Pancreatic stem cells:
There has been controversy as to whether the pancreas contains true stem cells. It was reported
that the endocrine cells of the rat pancreatic islets of Langerhans, including insulin-producing
beta-cells, turn over every 40-50 days by processes of apoptosis and the proliferation and
differentiation of new islet cells (neogensis) from progenitor epithelial cells located in the
pancreatic ducts. The administration to rats of glucose or glucagon like peptides resulted in the
doubling of the islet cell mass, suggesting that islet progenitor cells may reside within the islet
themselves. The same authors showed that rat and human pancreatic islets contained an
unrecognized population of cells that expressed the neural stem cell-specific marker nestin.
These nestin-positive cells were distinct from ductal epithelium. These nestin positive cells, after
isolation, had an unusually extended proliferative capacity in vitro, could be cloned repeatedly
and appeared to be multipotential. They were able to differentiate in vitro into cells that
expressed liver and exocrine pancreas markers. The authors proposed that these nestin-positive
islet derived progenitor cells were a distinct population of cells that resided within the pancreatic
islets and participated in neogenesis of islet endocrine cells. More recently, however, in an effort
to pin down the source of new b cells, Dor, et al.” designed transgenic mice in which insulin-
producing cells were prompted to produce HPAP that is detected by blue staining. When the
mice were 6-8 weeks old, the HPAP gene was turned on. Once the HPAP gene was tuned on, b
cells were expected to pass on the gene to daughter cells. If the new b cells came from stem cells,
then they should not be labeled by the stain. After 12 months, the percentage of blue cells was
higher than that in 6 week old mice, suggesting that the b cells replicate themselves and that the
pancreas is unlikely to harbor stem cells that produce large numbers of new b cells. Later,
Seaberg et al. exposed pancreatic cells to culture media that encourage growth of neural stem
cells. One out of every 5000 cells quickly multiplied into groups of cells. The authors suggested
that this grouping was characteristic of stem cells. Additionally. The authors demonstrated the
formation of a variety of cell types from these cell groups when the culture medium was changed
to encourage the cell groups t differentiate. The cell milieu comprised neurons and pancreatic

cells inclusive of b cells based on gene. Profiling. The b cells secreted insulin and when sugars
were added to the culture medium, the b cells put out more than twice as much of insulin. The
unequivocal demonstration of the existence of stem cells in the pancreas was, however, not
proven.
Eye stem cells
Stem cells have been identified in the adult mouse eye. Single pigmented ciliary margin cells
were shown to clonally proliferate in vitro to form sphere colonies of cells that can differentiate
into retinal-specific cell types, including rod photo receptors, bipolar neurons and Muller glia.
The adult retinal stem cells were localized to the pigmentary ciliary margin and not to the central
and peripheral retinal pigmented epithelium.
SOURCES OF STEM CELLS IN ORAL TISSUES:
1. Dental pulp stem cells (DPSC’s)

2. Stem cells from Human exfoliated deciduous teeth (SHED)
3. Periodontal stem cells(PDLSCS)
4. Stem cells from apical papilla (SCAP)
5. Dental follicle progenitor cells (DFPCs)
6. Dental epithelial stem cells
7. Cementoblasts like stem cells
Dental pulp stem cells (DPSCS):- Regenerative capacity of human dentin pulp complex is not
well understood but it is known that upon injury reparative dentin is formed as a protective
barrier for the pulp. So we can anticipate that dental pulp contains the dentinogenic progenitors
that are responsible for dentin repair. The dentin-pulp complex displays exquisite regenerative
potential in response to injury. The post natal dental pulp contains a variety of potential
progenitor stem cells which may participate in dental regeneration. A population of multipotent
mesenchymal progenitor cells known as DPSCs with high proliferative potential for self renewal
has been described and may be important for regenerative
Capacity of tissues.
Potential derivation of stem cells during reparative dentinogenesis may be From”

1. Cell rich layer of pulp adjacent to odontoblasts
2. Perivascular cells
3. Fibroblast
4. Recently: Presence of unique population of post natal dental pulp Stem cells. Capable to give
rise to diverse cells phenotypes. Stem cells human exfoliated deciduous teeth (SHED) Exfoliated
deciduous teeth houses living pulp remnants consisting of connective tissue, blood vessel and
odontoblasts. The isolation of post-natal stem cells from an easily accessible source is
indispensable for tissue engineering and clinical applications. Recent findings demonstrated the
isolation of mesenchymal progenitors from the pulp of human deciduous incisors. These cells
were named SHED and exhibited a high plasticity since they could differentiate into neurons,
adipocytes, osteoblasts and odontoblasts. In vivo SHED cells can induce bone or dentin
formation but, in contrast to DPSC failed to produce a dentin-pulp complex. Miura, et al. 2003
found that SHEDs were capable of differentiating into neural cells. Adipocytes, and odontoblasts
when cultured in vitro with specific medium such as DPSC. After 12 single-colony-derived
SHED clones were transplanted into immunocompromised mice, 25% of the clones
demonstrated a potential to differentiate into odontoblast-like cells and to form ectopic dentin-
like tissue. However, SHEDS were unable to regenerate the dentin-pulp-like complex
completely. In addition, transplanted single-colony-derived SHED clones were capable of
inducing murine host cells to differentiate into osteoblasts and osteocytes, and to form a
significant amount of new bone.
Periodontal ligament stem cells (PDLSCS)
Earlier evidence has shown that PDL contains cell populations that can differentiate into either
cementum-forming cells (cementoblasts) or bone-forming cells (osteoblasts). 54.55 The presence
of multiple cell types within PDL suggests that this tissue contains progenitor cells that maintain
tissue homeostasis and regeneration of periodontal tissue. Enzyme digestion treatment of PDL
releases a population of clonogenic cells with characteristics of postnatal stem cells. The
successful isolation and characterization of PDLSCs have led to the identification of tendon
MSCs by the same approaches.

In vitro characterization of PDLSCs-Multilineage differentiation potential: PDLSC’s express the

MSC-associated markers STRO-1, CDs, and scleraxis – a tendon specific transcription factor,
which is expressed at higher levels in PDLSCs than in Derived Marrow mesenchymal stem cells)
and DPSCS. Immunohistochemical staining and Western blot analysis showed that cultured
PDLSCS expressed an array of cementoblastic/osteoblastic markers 57 Similar to the other
dental stem cells described above, PDLSCs exhibit osteogenic, adipogenic, and chondrogenic
characteristics under defined culture conditions. 58,59,60
HOW STEM CELLS WORK
They have capacity of performing three important functions:
1. Plasticity
2. Homing
3. Engraftment
Plasticity:
Potential to change into other cell types. Under special conditions tissue Specific adult stem cells
can generate a whole spectrum of cell types of other tissues, Even crossing germ layers. This
phenomenon is referred to as Transdifferentiation. It can be induced by modifying the growth
medium when stem cells are cultured in vitro or transplanting them to an organ of the body
different from the one they were originally Isolated from. Four explanations for the phenomenon
of plasticity in post natal stem cells have been proposed A There might be persistent stem cells
from embryonic development with broad developmental potentials which ar maintained with in
the adult bone marrow. (Dao & Verfaille.2005) When transplanted into other organs, these cells
are instructed to differentiate in to tissue specific cells under inductive signals from That specific
tissue
b. True precursors of post natal stem cells with embryonic stem cells like properties persist in
adult bone marrow, such as the multipotent adult progenitor cells. (Jiang. Et al. 2002)
c. May be that the nuclei of the transplanted stem cells undergo reprogramming of the existing
genetic information, expressing new genes and proteins that are consistent Stem cells With the
novel lineage and this might be a result of de-differentiation and re Differentiation.

d. When cell fusion accuracy which is a rare phenomenon reported in vitro and in vivo in tissues
where polyploidy is common, such as hepatocytes, skeletal muscles, cardiac muscles and
purkinje cells of the cerebellum (priller, et al. 2001) as a result, the genetic information of both
fused donor and host cells is partially changed.(Terada, et al. 2002)
1. Homing: These cells migrate to site of tissue damage.

2. Engraftment: To unite with other tissues
STEM CELL RESEARCH
Stem cell biology is a promising and emerging field of the life sciences. The potential of hem
cell technology to develop therapy for many untreatable diseases through cellular placement or
tissue engineering is widely recognized. Keeping in view its potential therapeutic applications,
both basic and translational research are being promoted by the department in various
institutions, hospitals and the industry. Till date, more than 55 programmes have been identified
and supported on various aspects of stem cell research all over the world. MARKERS TO
IDENTIFY STEM CELLS 30,31,32 Coating the surface of every cell in the body are specialized
proteins, called receptors that have the capability of selectively binding or adhering to other
“signaling” molecules. There are many different types of receptors that differ in their structure
and affinity for the signaling molecules. Normally, cells use these receptors and the molecules
that bind to them as a way of communicating with other cells and to carry out their proper
functions in the body. These same cell surface receptors are the stem cell markers (Fig.9)
Scientists have taken advantage of the biological uniqueness of stem cell receptors and chemical
properties of certain compounds to tag or “mark” cells. Stem cell markers are given short hand
names based on the molecules that bind to the stem cell surface receptors. For example a cell that
has the receptor stem cell antigen on its surface is identified as Sca-1. In many cases, a
combination of multiple markers is used to identify a particular stem cell type. So now,
identification of stem cells in shorthand is done by a combination of marker names reflecting the
presence (+) or absence (-) of them. For example, a special type of hematopoietic stem cell from
blood and bone marrow called “side population” or “SP” is described as (CD34, c-Kit, Sca-1). A
second method uses stem cell markers and their fluorescent tags to visually assess cells as they
exist in tissues. In this method, a thin slice of tissue is prepared, and the stem cell markers are
tagged by the signaling molecule that has the fluorescent tag attached. The fluorescent tags are

then activated either by special light energy or a chemical reaction. The stem cells will emit a
fluorescent light that can easily be seen
SECOND METHOD
Under the microscope .Fluorescent tag attached to surface STEM CELL CULTURING Cowing
cells in the laboratory (in vitro) is known as cell culture. For this the respective cells have to be
settled on a natural or an artificial extracellular medium. (ECM) Such a material is called a
scaffold. Human embryonic stem cells are isolated by transferring the ar cell mass using a pipette
into a plastic laboratory culture dish that contains a trient broth known as culture medium. The
cells divide and spread over the surface of the dish. The inner surface of the culture dish is
typically coated with mouse embryonic din cells. This coating layer of cells is called a feeder
layer. The reason for having the pouse cells in the bottom of the culture dish is to give the inner
cell mass cells a sticky surface to which they can attach. Also the feeder cells release nutrients
into the culture medium. Recently, scientists have begun to device ways of growing embryonic
stem cells without the mouse feeder cells. This is a significant scientific advancement because of
the risk that viruses or other macromolecules in the mouse cells may be transmitted to the human
cells. Over the course of several days, the cells of the inner cell mass proliferate and begin to
crowd the culture dish. When this occurs, they are removed gently and plated into several fresh
culture dishes. The process of replating the cells is repeated many times and for many months,
and is called subculturing. Each cycle of subculturing the cells is referred to as a passage. After
six months or more, the original 30 cells of the inner cell mass yield millions of embryonic stem
cells. Embryonic stem cells that have proliferated in cell culture for six or more months without
differentiating, are pluripotent, and appear genetically normal are referred to as an embryonic
cell line. Once cell lines are established, or even before that stage, batches of them can be frozen
and shipped to other laboratories for further culture and experimentation. Human pluripotent
embryonic stem (ES) and embryonic germ (EG) cells have been derived from in vitro cultured
ICM cells of blastocysts (after in vitro fertilization) and from primordial germ cells (PGC)
isolated from aborted fetuses, respectively.(Fig.11) Apart from the cells and the scaffold for the
generation of tissues, suitable culture containers are also required. Though a large number of
sterile containers are available, only a very limited selection exists for tissue growth at present.
Various methods to culture stem cells:

1. Petridishes:-In case of the tissue regeneration, a scaffold is created at the bottom of a

culture dish. The cells are pipetted onto it together with the culture medium. (Fig. 12)
Experiments show that cells settle within hours because of a good interaction with the
biomaterial used. However with a longer duration culture, development into a functional
tissue ceases at a certain time because of the contact of the scaffold to the bottom of a
culture dish on one side. In such a static environment of culture dish, formation of
stationary. Layers occur which in turn result in a negative influence on the tissue
differentiation of the scaffold. Every tissue has special demands that require experimental
adjustments. Conventional single-use culture containers like Petri- dishes allow such a
modification of the tissue environment.
2. Spinner Bottles: Improved methods of tissue creation under in vivo conditions can, for
example, be produced in glass containers with relatively high volume in which the culture
medium can be kept in permanent motion by a magnetic stirrer. (Fig 13) In this way the
tissue construct, hanging on a thread, is exposed to a permanent stream of liquid.
Disadvantages of this method is that the culture medium, like in a petridish is not
exchanged continuously and so more products of metabolism accumulate. Moreover a
higher risk of infection is there during opening of the container.
3. Rotating Bioreactor: Another possibility for improving the culture of tissue constructs is
the rotating bioreactor. It is a cylindrical chamber. A hollow space which houses the
developing tissue construct and the culture medium is located in the interior of the
cylinder. A disk-shaped chamber is then fixed to the axis of an engine-module.
Consequently, the chamber along with the tissue construct is subject to discontinuous
micro gravitation.
This culture is also often conducted in a static environment, where no continuous supply of
nutrients takes place. Released metabolites are not continuously removed and can damage the
maturing tissue through their accumulation. In a modified form of rotating bioreactor it is
possible to fill fresh culture medium into the chamber continuously or discontinuously and to
remove the spent medium. Hollow fiber module: All tissues except epithelium and cartilage
require an intact capillary network for supply with nutrients and oxygen. Such a capillary
network for the developing tissues can be stimulated under in vitro conditions by a hollow fiber
module. (Fig 15) With this it is also possible to place a single hollow fiber into a special culture

container. At the same time the ends of the hollow fiber are connected to a thin tube, through
which the medium flows continuously with the help of a peristaltic pump. Pe cells or the
developing tissue settle on the in and outside of the hollow fiber. These ades have proved the
best in the creation of monoclonal antibodies. The hybridomasir it on the outside of the hollow
fiber, while the interior is streamed by new dum. Oxygen and low molecular weight nutrients are
transported continuously which ps through the wall of the hollow fiber by diffusion. Dow fibers
consist of totally different materials like polysulfone, acryl polymers or se acetate. Apart from
synthesis of monoclonal antibodies and cytokines, effective daction of growth hormones and
insulin has been shown in such modules. Perfusion containers: During the cell culture in the
culture dishes, the static comental conditions can have fatal results especially with the growing
tissues, se deep cell layers cannot be supplied with sufficient nutrients and oxygen and maging
metabolites are not adequately removed. Perfusion containers are a solution in shich a constant
environment exists as they are continuously evenly streamed with fresh um. Maturing tissue
constructs should not be put on the bottom of the reactor. If It leads to formation of stationary
layers between the tissue construct and the bottom te reactor. Thus tissue carriers are needed that
fix the construct mechanically, placing the interior of the container and ensuring that the medium
reaches it evenly. Perfusion ares have an advantage that during construct development the
surrounding mironment can be continuously examined for its quality with respective sensors.
Thus is possible to instantly react to changes of the environment and secure even the nelopment
of the construct. Tissue carriers: Cell of maturing tissue requires a suitable ECM as a foundation
for mal development, to which they can, and on which they can multiply and develop. Cernier
systems are preferably used in order to not damage the constructs and to be able
STEM CELL RESEARCH CENTERS IN INDIA
Research institutes:
1. All indin institute of medical sciences - New Delhi
2. CMC-DBT center for stem cell research, Vellore
3. In stem, Bangalore national centre for biological sciences, Bangalore
4. Indian institute of science, Bangalore

5. NIMHANS, Bangalore
6. National institute of immunology, New Delhi
7. National brain research center, New Delhi
8. Drdo-Inmass, New Delhi
9. National centre for cell science, Pune
10. Rajiv gandhi center for biotechnology, Trivandrum
11. Sree Chitra Tirunal institute of medical sciences and technology
12. Indian institute for chemical biology, Kolkata
13. Bose institute, Kolkata
14. Post-graduate institute of medical sciences and research, Chandigarh
15. Sanjay Gandhi post graduate institute, Lucknow
16. Tata institute of fundamental research, Mumbai
17. National institute for research in reproductive health, Mumbai
18. Advanced center for treatment, research and education in cancer (ACTREC), Navi Mumbai
19. Tata memorial center, Mumbai
20. Centre for cellular and molecular biology (CCMB) Hyderabad
21. LV Prasad eye institute, Hyderabad
2. Stem cell research centre, Hyderabad
Private research institute and company:
1. Advanced neuroscience allies pvt. Ltd
2. Stempeutics research pvt. Ltd
3. Reliance life sciences pvt. Ltd.

4. Nichi in regenerative medicine
5. Ansa research foundation
6. Manipal institute of regenerative medicine
7. Amrita institute of medical sciences
POTENTIAL IMPLICATIONS OF STEM CELLS IN DENTISTRY
1. Regeneration of dental hard tissues
i. Dentine regeneration
ii. Cementum regeneration
iii. Enamel formation
iv. Regenerative endodontics
2. Bone regeneration
i. Regenerating of bone from autologous stem cells
ii. Implant associated bone regeneration
iii. Condyle regeneration
3. Periodontal tissue regeneration
4. Stem cells in sinus augmentation
5. Repair of cleft lip & palate defects
6. Regeneration of irradiated salivary glands:
7. Peripheral nerve regeneration
8. Management of oral cancer
PERIODONTAL REGENERATION
periodontal diseases healing by repair results in a tissue that does not completely restore the
architecture or function of the original tissue, whereas healing by regeneration a new tissue that

is identical in both structure and function to the original produces issue. Identification of stem
cells in postnatal dental tissues has presented exciting possibilities for the application of tissue
engineering and cell based therapies in reconstructive dentistry. Recently multipotent stem cell
population, termed PDLSCs have been isolated from the PDL of extracted human third molar
teeth which give rise to adherent clonogenic clusters that resemble fibroblasts and are capable of
developing into adipocytes, osteoblast and cementoblast-like cells in vitro, and demonstrate the
capacity to produce cementum and periodontal ligament like tissues in vivo. PDLSCs express an
array of cementoblast and osteoblast markers as well as the BMSSCs(Bone marrow stromal stem
cells) associated markers, STRO-1 and CD146 antigens, which are also present on dental pulp
stem cells. The similarity between PDLSCs, DPSCs and BMSSCs suggests that PDLSCs
represent another MSCs-like population A potential tissue engineering approach to periodontal
regeneration involves incorporation of progenitor cells and instructive messages in a
prefabricated 3- dimensional construct, which is subsequently implanted into the site of defect.
This Strategy eliminates some of the limitations associated with conventional regenerative
procedures. The direct placement of the growth factors and progenitor cells into the defect site
overcomes the normal lag phase of progenitor cell recruitment to the site. In another study by
Zhenhua Yang, et al. it has been shown that a multilayered human periodontal ligament cell
sheet could reconstruct the physiological architecture of aperiodontal ligament cementum
complex. Human periodontal ligament cells were salated and cultured in dishes coated with a
temperature-responsive polymer to allow all detachment as a cell sheet. In the control group,
human periodontal ligament cells were cultured in Dulbecco's modified Eagle's minimal essential
medium containing 10% fetal bovine serum and 1% antibiotics. After 3 weeks, scanning electron
microscopy was carried out, in addition to staining for alkaline phosphatase activity and for
calcium (using the Von Kossa stain), Human periodontal ligament cells produced mineral-like
nodules and also showed positive staining for alkaline phosphatase, calcium (Von Kossa) and
mRNA expression of type I collagen. By contrast, in the control group only weak alkaline
phosphatase staining was observed, the Von Kossa stain was negative and there was no
mRNAexpression of type I collagen. Six weeks after transplantation with human periodontal
ligament cells cultured in osteodifferentiation medium, most of the dentin surfaces showed a
newly immature cementum-like tissue formation and periodontal ligament with 92 perpendicular
orientation inserted into the newly deposited cementum-like tissue."

TISSUE ENGINEERING
Tissue engineering is a specialized field of science based on principles of cell biology,

developmental biology and biomaterials science to fabricate new tissues to replace lost of
damaged tissues. Successful tissue engineering requires an appropriate extracellular matrix or
carrier construct which contains regulatory signals and responsive progenitor cells. A potential
tissue engineering approach to periodontal regeneration involves incorporation of progenitor
cells and instructive messages in a prefabricated three- dimensional construct, which is
subsequently implanted into the defect site) Prognoos curred in vitro Progetto cheed onto
aSweded implanted to the detect site Region of connective attachment Tissue engineering:
Defined by Langer and Vacanti, "an interdisciplinary field that applies the principles of
engineering and life sciences towards the development of biological substitutes that restore,
maintain, or improve tissue function" MacArthur and Oreffo, defined tissue engineering as
"understanding the principles of tissue growth and applying this to produce functional
replacement tissue for clinical use Principles of tissue engineering"
1. Conductive approaches
2. Inductive approaches
3. Cell transplantation
Conductive approach: This approach makes use of a barrier membrane to excludeconnective

tissue cells that will interfere with the regenerative process, while enabling the desired host cells
to populate the regeneration site. Best examples are dental implants and guided tissue
regeneration(GTR) membrane. GTR membranes are used to regenerate the periodontal tooth
supporting structures & they are used a material barrier to create a protected compartment for
selective wound healing." nductive approaches: This approach uses a biodegradable polymer
scaffold as avehicle to deliver growth factors and genes to the host site. The growth factors or
genes can be released at a controlled rate, based on the breakdown of the polymer. One limitation
of the inductive approach is that the inductive factors for a particular tissue may not be known.
Cell transplantation: Particular cell of interest can be isolated from the tissue and cultured in a
laboratory with required modification like surface receptors alteration or changing gene
expression and transplanted back to patient. This strategy also uses a similar vehicle for delivery
in order to transplant cells and partial tissues to the host site. The cell transplantation strategy

truly reflects the multidisciplinary nature of tissue engineering that requires a clinician, a
bioengineer and a cell biologist. Clinician: The clinician is required to biopsy a small sample of
tissue containing the cells of interest.Cell biologist: Multiplies the cells and maintains their
function. Bioengineer: The manufacturer of the tissue, the bioreactor and the material onto which
the cells will be placed for transplantation. Lastly, the clinician transplants the engineered tissue
polymer scaffold degrades and is remodeled by host and transplanted cells resulting in complete
natural tissue. The technical requirements for successful cell-based tissue engineeringcan be
divided into two main categories": Engineering issues related to maintenance of
GENE AND CELL-BASED THERAPY
The inherent proliferative and pluripotent capabilities of stem cells may offer lifelong
apportunities for treatment of some important human diseases, including periodontitis, by
repairing, replacing or regenerating damaged tissues. Stem cells may act as suitable vehicles for
the delivery of therapeutic genes in gene therapy, and as therapeutic agents per se in cell-based
therapy. Gene therapy is a new approach for the treatment of human diseases. It relies on genetic
engineering, which involves molecular techniques to introduce, suppress or manipulate specific
genes, thereby directing an individual's own cells to produce a therapeutic agent. In the context
of periodontal regeneration, gene therapy seeks to optimize the delivery of agents such as growth
factors to periodontal defects so that the limitations associated with topical application (ex: short
duration of a action) can be overcome. Two major strategies for delivering therapeutic transgenes
into human recipients are: Direct infusion of the gene of interest using viral or non-viral vectors
in vivo; and Introduction of gene into delivery cells (often a stem cell) outside the body ex vivo
followed by transfer of the delivery cells back into the body. The use of both in vivo and ex vivo
gene delivery strategies via adenoviral (Ad) vectors encoding growth promoting molecules such
as platelet-derived growth factor (PDGF) and bone morphogenetic protein (BMP-7) has been
investigated for its potential in periodontal regeneration by Giannobile and colleagues." Recent
findings in rats have revealed sustained transgene expression for up to 10 days at Ad-BMP-7
treated sites, and enhanced bone and cementum regeneration at Ad- BMP-7and Ad-PDGF
treated sites beyond that of control vectors. The introduction of transgenes into dental stem cells
may offer an alternative to conventional methods because stem cells have the potential to a
sustained source of growth factors for regeneration. However, much work is sall needed to

optimize the number of cells that are virally transduced to express specific in order to maximize
the duration and extent of gene expression, and ultimately to determine the success of gene
transfer techniques in periodontal regeneration. Further genes, research is also needed to address
potential risks of viral recombination and immune responses towards viral antigens which could
potentially hinder the progress of genetherapy in treating periodontal diseases.
STEM CELLS AND PERIODONTAL LIGAMENT
periodontal ligament, which is a highly tibrous and venenular tissue, has one of the hest tumover
rates in the body. Many cells are present in the periodontal ligament including cementoblast,
osteoblasts, fibroblasts, myofibroblasts, endothelial cells, nerve alls and epithelial cells. In
addition to these, a smaller population of progenitor cells has een identified by in vivo cell
kinetic studies. These progenitor cell populations within de periodontal ligament appear to be
enriched in locations adjacent to blood vessels and exhibit some of the classical cytological
features of stem cells, including small size, responsiveness to stimulating factors and slow cycle
time. Thus, we have investigated the thesis that within this group there are cells with
characteristics of mesenchymal stem cells capable of sustained renewal and tissue regeneration.
The concept that stem cells may reside in the periodontal tissues was first proposed almost 20
years ago by Melcher. The most compelling evidence that these cells are present within the
periodontal tissues has been provided by the in vivo and histological studies of McCulloch and
coworkers. Recently, multipotent stem cell populations, termed periodontal ligament stem cells
(PDLSCs), have been isolated from the periodontal ligament of extracted human third molar
teeth." Since periodontal regeneration is essentially a re-enactment of the development process
including morphogenesis, cytodifferentiation, extracellular matrix production and mineralization,
Such processes support our concept that some mesenchymal stem cells remain within the
periodontal ligament and are responsible for tissue homeostasis, serving as a source of renewable
progenitor cells generating cementoblasts, osteoblasts and fibroblasts throughout adult life.
Using cloning techniques, a large number of cells or differing phenotype have been isolated from
the periodontal ligament and regenerating periodontal tissue. Theidentification of putative
mesenchymal stem cell populations within the periodontium has stimulated interest in the
potential use of stem cell based therapies to treat the damaged caused by trauma or periodontal
disease.

CONCLUSION
CONCLUSION:
Stem cells derived from all sources hold immense medical promises, Stem cell therapies have
virtually unlimited medical and dental applications. We have moved on from the surgical model
of care to the medical model and are likely to move onto the biological model of care. The need
of the hour is high-quality research coupled with collaboration between basic scientists and the
clinicians. A team effort engaging the expertise of the molecular biologists, immunologists,
biomaterial scientists, cell biologists, matrix biologists, and practicing dental surgeons is crucial
in attaining the desired goal. Stem cell therapy is no longer science fiction. Recent developments
in the technique of stem cell isolation and expansion together with advances in growth factor
biology and biodegradable polymer constructs have set a stage for successful tissue engineering
of tooth/tooth-related tissues. Stem cell therapy has brought in a lot of optimistic hope amongst
researchers, doctors, and not to forget the patients who are the chief beneficiary of this
innovation. Stem cells regenerate hope and not all that is happening in research is hype. While
there are several barriers that need to be broken down before this novel therapy can be translated
from lab to clinics, it is certain that the future is going to be exciting for all of us "Hope is a
prerequisite for any successful scientific innovation".

Reference
Reference:
1. Ariff Bongso, Eng Hin Lee "Stem Cells: Their definition, classification and sources".Stem
Cells from Benchtop to Bedside: World Scientific. ISBN 978-981-256-126-9. 2005:5.
2. Bethesda, MD: National Institutes of Health, U.S. Department of Health and HumanServices.
Stem Cell Basics: Introduction In Stem Cell Information (World Wide Website). 2002
3. Pamela C Yelick, Joseph P Vacanti. Bioengineered teeth from tooth bud cells, Dent ClinNAm
2006, 50:91 - 203.
4. Samuel E Lynch, Robert J Genco, Robert E Marx. Tissue Engineering. Applications in

maxillofacial surgery and Periodontics, 1" ed. Quintessence Publishing 1999, 614.
5. Pihlstrom BL, Michalowicz BS, Johnson NW. Periodontal diseases. Lancet 2005, 366:1809-
1820.
6. Giuseppe Polimeni, Andreas V Xiropaidis, Ulf ME Wikesjo. Biology and principles

ofperiodontal wound healing/regeneration. Periodontol 2000 2006, 41: 30-47.
7. N Hun, S Gronthos, PM Bartold. Stem cells and periodontal regeneration. Australian Dental
Journal 2008, 53: 108-121.
8. Taka Nakahara. A review of new developments in tissue engineerin therapy for periodontitis.
Dent Clin N Am 2006, 50: 265 - 276.
9. Iwata T, Yamato M, Zhang Z, Mukobata S, Washio K, Ando T, et al. Validation of human

periodontal ligament derived cells as a reliable source for cytotherapeutic use. I Clin Periodontol
2010, 37: 1088-1099.
18. DS Mehta, Tarun Kumar AB, TM Jyothi. Stem cells in dentofacial research at the cross
roads. J Indian Soc Periodontol 2005, 9(2): 91-108.

19. Popular issues on human stem cell history - online book, sited on 12/1/2011
20. Centeno CJ, Busse D, Kisiday J, Keohan C, Freeman M, Karli D. Increased knee cartilage
volume in degenerative joint disease using percutaneously implanted, autologous mesenchymal
stem cells. Pain Physician 2008, 11(3): 343-353.
21. "First trial of embryonic stem cells in humans". BBC News, 2010-10-11,
22. Masaki J Honda, Mari Imaizumi, Shuhei Tsuchiya, Christian Morsczeck. Dental folliclestem
cells and tissue engineering. J Oral Sci. 2010, 52(4): 541-552.
23. Wolf Dieter Grimm, Aous Dannan, Sebastian Becher, Georg Gassmann, Wolfgang Amold,
Gabor Varga, et al. The Ability of Human Periodontium-Derived Stem Cells to Regenerate
Periodontal Tissues: A Preliminary In Vivo Investigation. Int J Periodontics Restorative Dent
2011, 31:94-101.
24. Duan X, Tu Q, Zhang J, Ye J, Sommer C, Mostoslavsky G, et al. Application of induced

pluripotent stem (PS) cells in periodontal tissue regeneration. J Cell Physiol 2011, 226; 150–157.
25. Fawzy El-Sayed KM, Paris S, Becker ST, Neuschl M, De Buhr W, Salzer S, et al.Periodontal
regeneration employing gingival margin-derived stem/progenitor cells: an animal study. J Clin
Periodontol 2012, 39(9): 861-870.
26. Inukai T, Katagiri W, Yoshimi R, Osugi M, Kawai T, Hibi H, Ueda M. Novel application of
stem cell-derived factors for periodontal regeneration. Biochem Biophys Res Commun.013,
430(2): 763-768.
27. Smith A. A glossary for stem-cell biology. Nature 2006, 441:1060.
28. Stem cell basics. http://en.wikipedia.org/wiki/stem cells - 2005
29. Hans R Scholer." The Potential of stem cells: An inventory" In Nikolaus Knoepffler, Dagmar
Schipanski, and Stefan Lorenz Sorgner. Human biotechnology as social challenge. Ashgate
Publishing, Ltd. ISBN 978-0-7546-5755-2. 2007:28.
30. Ann A. Kiessling Phd, Scott C Anderson. Human embryonic stem cells - 2nd edn.New York:
Elsievier 2003, 201: 26-28.


Introduction To Stem Cells and Disease

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“A ROLE OF STEM CELL IN DIFFERENT DISEASE”

School of Pharmacy and Research

People’s University Bhanpur, Bhopal - 462037

A Project Report (Project report)

School of Pharmacy and Research

People’s University Bhanpur, Bhopal - 462037

SCHOOL OF PHARMACY AND RESEARCH Page 2

SCHOOL OF PHARMACY AND RESEARCH

SCHOOL OF PHARMACY AND RESEARCH

SCHOOL OF PHARMACY AND RESEARCH Page 3

(Approved by AICTE and PCI, and Recognized by Govt. of

This is to certify that the project entitled “ A ROLE OF STEM CELL IN

Date: Guide Name

SCHOOL OF PHARMACY AND RESEARCH Page 4

and my friend FARMAN HUSSAIN inspiration during this course of investigation.

SCHOOL OF PHARMACY AND RESEARCH Page 5

2 HISTORICAL BACKGROUND 11-14

OF STEM CELL RESEARCH

SCHOOL OF PHARMACY AND RESEARCH Page 6

SCHOOL OF PHARMACY AND RESEARCH Page 7

SCHOOL OF PHARMACY AND RESEARCH Page 8

SCHOOL OF PHARMACY AND RESEARCH Page 9

Tissue engineering for periodontal tissue regeneration is based on 3 approaches

1.Protein based approaches: By using Growth and Differentiation factors.

2. Cell based approaches: By harvesting and differentiating stem cells

3. Gene delivery based approaches: By gene encoding of therapeutic proteins.

SCHOOL OF PHARMACY AND RESEARCH Page 10

HISTORICAL BACKGROUND OF STEM CELL RESEARCH

1968:-Edwards and Bavister fertilize the first human egg in vitro.’’

1978: -Louise Brown, the first IVF baby, is born in England.

SCHOOL OF PHARMACY AND RESEARCH Page 11

SCHOOL OF PHARMACY AND RESEARCH Page 12

2000:-Under appropriate culture conditions, hES cells were shown to be pluripotent By

2002:-Vojislav Lekovic, et al. compare the clinical effectiveness of 2 regenerative A In humans:

2005:-Saito, et al. developed bovine cementoblast cells.

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STEM CELL BASICS

PROPERTIES OF STEM CELLS

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CLASSIFICATION OF STEM CELLS

2. Source of stem cells(based on their origin)

1. Extent to which they can differentiate:

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Three types of pluripotent cells have been found

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1. Embryonic stem cells (ESC)

2. Stem cells from the fetus

3. Stem cells from the umbilical cord

4. Adult stem cells

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Embryonic germ cells

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ADULT STEM CELLS

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Gut stem cells

Liver stem cells

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Epidermal stem cells (skin and hair)