This document provides an overview of a student project report on stem cell therapy. It includes sections on the introduction, advantages, disadvantages, types of stem cells, sources, isolation and culture, stem cell division, treatment, medical uses, applications to various diseases and conditions. The report was submitted by a pharmacy student, Rishabh Tiwari, to Rajasthan University of Health Sciences, Jaipur, India under the supervision of a faculty member, to fulfill the requirements of a Bachelor of Pharmacy degree.

1. PROJECT REPORT
ON
“STEM CELL THERAPY”
Submitted to:
Rajasthan University of Health Sciences, Jaipur
For The Partial Fulfilment of
Bachelor of Pharmacy
Batch: 2016-2020
SUPERVISED BY: SUBMITTED BY:
Mr. ABHINAV CHAUDHARY RISHABH TIWARI
ASSISTANT PROFESSOR B.Pharm (Final Year)
SKIP, Jaipur Enrollment No.: 2016/2440
SWAMI KESHVANAND INSTITUTE OF PHARMACY,
Ramanagariya, Jagatpura, Jaipur-302017

2. DECLARATION
This is to submit that this written submission in my project entitled “STEM CELL
THERAPY” is a bonafied and genuine work carried out by me under the supervision of
MR. ABHINAV CHAUDHARY (ASSISTANT PROFESSOR), SWAMI
KESHVANAND INSTITUTE OF PHARMACY, JAIPUR. The source of information
derived from the existing literature has been indicated at the appropriate places in the
body of the project.
This project is original and has not been submitted in apart or full for any degree or
diploma of this or any other university.
Date: (RISHABH TIWARI)
Place: Jaipur Enrollment No. 2016/2440
B. Pharm. (Final Year)

3. ACKNOWLEDGEMENT
I would like to convey my gratitude to respected Prof. HEMLATA DULLAR Principle
of Swami Keshvanand Institute of Pharmacy, Jaipur for her continuous guidance
throughout my project.
It would be the greatest pleasure for me now to express my unbound gratitude
indebtedness to my guide MR. ABHINAV CHAUDHARY (ASSISTANT
PROFESSOR) in Swami Keshvanand Institute of Pharmacy, Jaipur for their guidance
and help throughout my project.
I can never think for completion of this project without valuable suggestion and guidance.
I would also like to convey my gratitude to lecturer, Mr. Shri Ram (Associate
professor), Dr. Sangeeta asija (Professor & HOD), Dr. Santosh Kumar Gupta
(Associate Professor), Mr. Rasheed Ahmed (Assistant professor), Mr. Pankaj
pradhan (Assistant professor), Mrs. Divya Sharma (Assistant professor), Mr. Vivek
Singhal (Assistant professor), Mr. Gaurav Bhaduka (Assistant professor), Mr.
Rohitashav Sharma (Lecturer), and Mr. Dharmendra Kumar (Lecturer) and all
faculty member of Swami Keshvanand Institute of Pharmacy, Jaipur for their valuable
suggestion, appreciation and advice to me.
I would also express thanks to Mr. Anil Joshee (Lab technician), Mr. Mahesh Saini
(Lab Assistant), Mr. Vishnu Sharma (Lab Assistant), Mrs. Deepika Kanwar, Mr.
Arjun Lal Meena (Librarian) for helping me throughout the project.
I express my deep appreciation and thanks to my friend for their ebullient encouragement,
useful discussion and grip over the subject which helped me in the way to broaden my
concept towards this project.
(RISHABH TIWARI)

4. Dedicated to
My Family members
who encouraged and flared passion in me to learn more always and my
profession.
“Pharmacist is the founder stone of the medicine”

5. CONTENTS
S. No. Topic Page No.
1. Introduction 1 – 4
2. Advantages 5 – 6
3. Disadvantages 7 - 9
4. Types of stem cells 10 – 15
5. Sources 16
6. Isolation and culture 17 – 24
7. Stem cell division 25
8. Treatment 26
9. Stem cell therapy 27 - 28
10. Medical uses 29
11. Application 30 - 34
12. Disease and conditions 35 - 66
13. Conclusion 67
14. Reference 68 – 72

6. ABSTRACT
Stem cells are a population of undifferentiated cells characterized by the ability to
extensively proliferate (self-renewal), usually arise from a single cell (clonal), and
differentiate into different types of cells and tissue (potent). There are several sources of
stem cells with varying potencies. Pluripotent cells are embryonic stem cells derived from
the inner cell mass of the embryo and induced pluripotent cells are formed following
reprogramming of somatic cells. Pluripotent cells can differentiate into tissue from all 3
germ layers (endoderm, mesoderm, and ectoderm). Multipotent stem cells may
differentiate into tissue derived from a single germ layer such as mesenchymal stem cells
which form adipose tissue, bone, and cartilage. Tissue-resident stem cells are Oligopotent
since they can form terminally differentiated cells of a specific tissue. Stem cells can be
used in cellular therapy to replace damaged cells or to regenerate organs. In addition,
stem cells have expanded our understanding of development as well as the pathogenesis
of disease. Disease-specific cell lines can also be propagated and used in drug
development. Despite the significant advances in stem cell biology, issues such as ethical
controversies with embryonic stem cells, tumor formation, and rejection limit their utility.
However, many of these limitations are being bypassed and this could lead to major
advances in the management of disease. This review is an introduction to the world of
stem cells and discusses their definition, origin, and classification, as well as applications
of these cells in regenerative medicine.
In recent years, stem cell therapy has become a very promising and advanced scientific
research topic. The development of treatment methods has evoked great expectations.
This paper is a review focused on the discovery of different stem cells and the potential
therapies based on these cells. The genesis of stem cells is followed by laboratory steps of
controlled stem cell culturing and derivation. Quality control and teratoma formation
assays are important procedures in assessing the properties of the stem cells tested.
Derivation methods and the utilization of culturing media are crucial to set proper
environmental conditions for controlled differentiation. Among many types of stem tissue
applications, the use of graphene scaffolds and the potential of extracellular vesicle-based
therapies require attention due to their versatility. The review is summarized by
challenges that stem cell therapy must overcome to be accepted worldwide. A wide

7. variety of possibilities makes this cutting edge therapy a turning point in modern
medicine, providing hope for untreatable diseases.
Keywords: stem cell, stem cell therapy

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INTRODUCTION OF STEM CELLS
Stem cells
Stem cells are cells that can differentiate into other types of cells, and can also divide in
self-renewal to produce more of the same type of stem cells.
In mammals, there are two broad types of stem cells: embryonic stem cells, which
are isolated from the inner cell mass of blastocysts in early embryonic development,
and adult stem cells, which are found in various tissues of fully developed mammals.
In adult organisms, stem cells and progenitor cells act as a repair system for the body,
replenishing adult tissues. In a developing embryo, stem cells can differentiate into all the
specialized cells—ectoderm, endoderm and mesoderm (see induced pluripotent stem
cells)—but also maintain the normal turnover of regenerative organs, such as blood, skin,
or intestinal tissues.
There are three known accessible sources of autologous adult stem cells in humans: bone
marrow, adipose tissue, and blood. Stem cells can also be taken from umbilical cord
blood just after birth. Of all stem cell therapy types, autologous harvesting involves the
least risk. [1]
Adult stem cells are frequently used in various medical therapies (e.g., bone
marrow transplantation). Stem cells can now be artificially grown and transformed
(differentiated) into specialized cell types with characteristics consistent with cells of
various tissues such as muscles or nerves. Embryonic cell lines and autologous embryonic
stem cells generated through somatic stem cells generated through somatic cell nuclear
transfer or dedifferentiation have also been proposed as promising candidates for future
therapies. Research into stem cells grew out of findings by Ernest A.
McCulloch and James E. Till at the University of Toronto in the 1960s. [2]

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Stem cell
Fig.1: Transmission electron micrograph of an adult
stem cell displaying typical ultrastructural
characteristics.
Properties
The classical definition of a stem cell requires that it possesses two properties:
 Self-renewal: the ability to go through numerous cycles of cell division while maintaining
the undifferentiated state. [3]
 Potency: the capacity to differentiate into specialized cell types. In the strictest sense, this
requires stem cells to be either totipotent or pluripotent—to be able to give rise to any
mature cell type, although multipotent or unipotent progenitor cells are sometimes
referred to as stem cells. Apart from this it is said that stem cell function is regulated in a
feedback mechanism.[4]
Self-renewal
Two mechanisms ensure that a stem cell population is maintained:
1. Obligatory asymmetric replication: a stem cell divides into one mother cell that is
identical to the original stem cell, and another daughter cell that is differentiated. When a
stem cell self-renews it divides and does not disrupt the undifferentiated state. This self-

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renewal demands control of cell cycle as well as upkeep of multipotency or pluripotency,
which all depends on the stem cell.
2. Stochastic differentiation: when one stem cell develops into two differentiated daughter
cells, another stem cell undergoes mitosis and produces two stem cells identical to the
original. [3]
Potency meaning
Fig.2: Pluripotent, embryonic stem cells originate as inner cell mass (ICM) cells within a
blastocyst. These stem cells can become any tissue in the body, excluding a placenta.
Only cells from an earlier stage of the embryo, known as the morula, are totipotent, able
to become all tissues in the body and the extraembryonic placenta.
Potency specifies the differentiation potential (the potential to differentiate into different
cell types) of the stem cell.
 Totipotent (a.k.a. omnipotent) stem cells can differentiate into embryonic and
extraembryonic cell types. Such cells can construct a complete, viable organism. These
cells are produced from the fusion of an egg and sperm cell. Cells produced by the first
few divisions of the fertilized egg are also totipotent.
 Pluripotent stem cells are the descendants of totipotent cells and can differentiate into
nearly all cells, i.e. cells derived from any of the three germ layers.

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 Multipotent stem cells can differentiate into a number of cell types, but only those of a
closely related family of cells.
 Oligopotent stem cells can differentiate into only a few cell types, such as lymphoid or
myeloid stem cells.
 Unipotent cells can produce only one cell type, their own, but have the property of self-
renewal, which distinguishes them from non-stem cells (e.g. progenitor cells, which
cannot self-renew). [4]

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Advantages of stem cells
1. Adult stem cells have low rejection rates.
Therapies can be developed from adult stem cells that are taken from each patient. These
cells can then be transformed into various therapies that run a low risk of rejection
because the cells are taken from the individual needing treatment. Even when familiar
umbilical cord blood cells are used to develop treatments, the rejection rates are quite
low. This limits the need for immunosuppressant treatments to maintain a positive quality
of life in the future.
2. Some stem cells can be transformed into pluripotent stem cells.
Adult stem cells, through the use of iPS reprogramming factors, have the ability to be
reprogrammed into pluripotent stem cells. Once this occurs, they can be activated into
mesoderm, endoderm, or ectoderm cells. This process allows for the potential benefits
that embryonic stem cell lines could provide for medical treatments without the need to
destroy embryos to collect the cells that are needed.
3. The current treatment options for stem cells are numerous.
The most common use of stem cell therapy currently used is to treat leukemia and
lymphoma patients with bone marrow transplantation. A stem cell therapy called
Prochymal has been conditionally approved in Canada to manage graft vs. host disease in
children who do not respond to steroid treatments. Holoclar is another potential treatment
that can help people with severe limbal deficiencies because of burns to the eye. In the
US, there are 5 hematopoietic cord blood treatments that have been approved by the FDA.
4. This research gives us insights into how human life works.
Stem cell research allows us to understand how the cells in our bodies work. By
understanding these processes better, it becomes possible to understand how an illness or
disease develops. Even if a stem cell therapy isn’t developed from this research, the
understanding obtained can help to create new treatments that can potentially cure what is
damaging our cells. That allows us to extend average life expectancy rates, stop diseases,
and even reduce the costs of medical treatments.

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5. Because stem cells have regenerative properties, the potential is unlimited.
Imagine being able to grow a replacement organ for one that is failing. Or having a
veteran who lost a limb in an attack could have a replacement grown in a lab setting and
then attached so they don’t need a prosthetic – they could have the real thing. The
potential of stem cell research is unlimited, including offering the chance to improve
mental health. Improving insulin production, repairing damaged heart muscle after a heart
attack, repairing torn tendons or ligaments, and even attacking cancers or viruses.
Embryonic stem cells offer a similar potential, along with the possibility of being able to
treat certain genetic disorders or birth defects so more people could go on to live happy
and healthy lives.
6. Embryonic treatments can be developed through stem cell research.
Many issues that afflict the human condition occur during the initial stages of
development for the embryo. Errors in the cell’s coding can lead to potential birth defects
as the embryo transitions into being a fetus. By studying how stem cells begin to
transform into the 200+ different cell types of the human body, medical science has the
potential to learn how defects, genetic errors, and other problems develop and stop that
process before it starts – even if the parents are carriers of current genetic disorders.
7. Stem cell research could reduce pregnancy loss.
Miscarriage is defined as the spontaneous loss of a pregnancy before the 20th week. Up to
20% of known pregnancies end in a miscarriage, those numbers is likely higher because
most miscarriages occur so early on at the embryonic stage of development that women
don’t realize they’ve become pregnant. Stem cell research offers the potential of reducing
this issue so that more pregnancies can be successful with individualized treatments
developed from this work.
8. Stem cells can self-replicate in enormous numbers.
It only takes a few adult stem cells to create potentially trillions of cells that are
specialized to a certain treatment. With ongoing research, even current cell lines,
including embryonic lines, can continue to self-replicate and provide ongoing research
opportunities.
[5]

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Disadvantages of stem cells
1. Embryonic stem cells can have high rejection rates.
Embryonic stem cell therapies have been known to create several future health problems.
Rejection rates are high for these therapies. Research has shown that these therapies
encourage the development of tumours. Some embryonic stem cells do not respond to the
activation sequences as intended.
2. Adult stem cells have a determined cell type.
Without iPS reprogramming, adult stem cells have a determined cell type. This means
they cannot be changed into different cell tissues. This limits the therapies that can be
developed by stem cell research because the cells, in their raw form, can only involve the
same type of tissue from which they were harvested in the first place.
3. Obtaining any form of stem cell is a difficult process.
To collect embryonic stem cells, the embryo must be grown in a culture. Once harvested,
it takes several months for the stem cells to grow enough to the point where they could be
potentially used for the creation of a therapy. Adult stem cells, especially those which are
obtained from a person’s bone marrow, can be extremely painful to obtain for the patient.
Some individuals may not live anywhere near a facility that has the capabilities of
obtaining those cells, which creates another set of logistics which must be solved.
4. Stem cell treatments are an unproven commodity.
The treatments developed from stem cells are experimental at the current phase of
research. There is the potential of having such a treatment work with current research, but
there is a better chance that nothing could happen. The most effective therapy right now is
hematopoietic stem cell transplantation (HSCtx). They are 90% effective and about
50,000 transplants occur per year.
5. Stem cell research is a costly process.
The cost of a single stem cell treatment that has been approved for use in the United
States is typically about $10,000. Some clinics have found ways to reduce this cost by up
to 20%. Outside of the United States, the costs of a single treatment can be as high as
$100,000. The cost of harvesting an embryo for stem cells is up to $2,000 per instance.

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Services rendered to take adult stem cells may not be included in the treatment cost and
could be several thousand dollars. And, because stem cell treatments are experimental in
most instances, health insurance plans and government-provided benefits do not generally
provide access to them.
6. We do not know if there are long-term side effects to worry about.
Tens of thousands of people are receiving stem cell transplants every year, with efficacy
rates improving each year for the dozens of illnesses and diseases that respond positively
to such treatments. What we do not know yet is if there are long-term consequences to
such therapies, even if there are short-term benefits that are being seen. More than 3,500
different research studies are happening right now to determine the effectiveness of stem
cell research and therapies, but the results are still pending.
7. There will always be some limitation to the research possibilities.
The ethics of stem cell research will always place limitations on the medical potential of
this research. Individuals must decide on their own how they will respond to the ethics of
this research. Is it permitted to alter adult stem cells or umbilical embryonic stem cells?
What are the consequences of destroying an embryo to get stem cells, even if that embryo
was going to be discarded? Some people will feel the entire process is unethical and that
has the potential to hold this research back.
8. Research has been held back by factual contradictions.
Some of the research in this field has been discredited because it contains hundreds of
factual contradictions. This includes some of the pioneering work in stem cell research by
Bodo-Eckehard Strauer, who focused on how stem cells could help to treat cardiovascular
conditions. Current research has had to correct these contradictions before proceeding
with future potential benefits.
9. Research opportunities are somewhat limited, especially in the United States.
In 2001, when the US Government took steps to limit the funding and availability of stem
cell research to just 19 lines. The research itself wasn’t banned, but the severe restrictions
placed on having funding for that research made it virtually impossible to conduct for
more than a decade. Some states in the US have or have an interest in additional
restrictions or complete bans on embryonic stem cell research in its current state.

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10. Adults have very few stem cells.
The treatment options that are available for adult stem cells without reprogramming are
few because the number of cells that adults have are very few. Although they reside in
many different areas of the body, they are isolated from tissue samples and their current
source is unknown. Being able to separate them is a time-consuming and costly process
and self-renewal within the body may be slow to occur.
11. Current embryonic stem cell harvesting requires the death of an embryo.
Harvesting embryonic stem cells and germ cells may offer numerous research advantages
compared to adult stem cells, but current methods of harvesting require the death of the
embryo. Embryonic stem cells also have limited self-renewals, measured at 2 years. Germ
cells can double a maximum of up to 80 times. This limits the research potential of any
existing line.
[5]

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Type of stem cells
1. Embryonic
Embryonic stem cells (ESCs) are the cells of the inner cell mass of a blastocyst, formed
prior to implantation in the uterus. In human embryonic development the blastocyst stage
is reached 4–5 days after fertilization, at which time it consists of 50–150 cells. ESCs
are pluripotent and give rise during development to all derivatives of the three germ
layers: ectoderm, endoderm and mesoderm. In other words, they can develop into each of
the more than 200 cell types of the adult body when given sufficient and necessary
stimulation for a specific cell type. They do not contribute to the extraembryonic
membranes or to the placenta.
[6]
During embryonic development the cells of the inner cell mass continuously divide
and become more specialized. For example, a portion of the ectoderm in the dorsal part of
the embryo specializes as 'neurectoderm', which will become the future central nervous
system. Later in development, neurulation causes the neurectoderm to form the neural
tube. At the neural tube stage, the anterior portion undergoes encephalization to generate
or 'pattern' the basic form of the brain. At this stage of development, the principal cell
type of the CNS is considered a neural stem cell.
The neural stem cells self-renew and at some point transition into radial glial
progenitor cells (RGPs). Early-formed RGPs self-renew by symmetrical division to form
a reservoir group of progenitor cells. These cells transition to a neurogenic state and start
to divide asymmetrically to produce a large diversity of many different neuron types, each
with unique gene expression, morphological, and functional characteristics. The process
of generating neurons from radial glial cells is called neurogenesis. The radial glial cell
has a distinctive bipolar morphology with highly elongated processes spanning the
thickness of the neural tube wall. It shares some glial characteristics, most notably the
expression of glial fibrillary acidic protein (GFAP). The radial glial cell is the primary
neural stem cell of the developing vertebrate CNS, and its cell body resides in
the ventricular zone, adjacent to the developing ventricular system. Neural stem cells are
committed to the neuronal lineages (neurons, astrocytes, and oligodendrocytes), and thus
their potency is restricted.
Nearly all research to date has made use of mouse embryonic stem cells (mES) or
human embryonic stem cells (hES) derived from the early inner cell mass. Both have the
essential stem cell characteristics, yet they require very different environments in order to

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maintain an undifferentiated state. Mouse ES cells are grown on a layer of gelatine as
an extracellular matrix (for support) and require the presence of leukemia inhibitory
factor (LIF) in serum media. A drug cocktail containing inhibitors to GSK3B and
the MAPK/ERK pathway, called 2i, has also been shown to maintain pluripotency in
stem cell culture. Human ESCs are grown on a feeder layer of mouse
embryonic fibroblasts and require the presence of basic fibroblast growth factor (bFGF or
FGF-2). Without optimal culture conditions or genetic manipulation, embryonic stem
cells will rapidly differentiate.
A human embryonic stem cell is also defined by the expression of several
transcription factors and cell surface proteins. The transcription factors Oct-4, Nanog,
and Sox2 form the core regulatory network that ensures the suppression of genes that lead
to differentiation and the maintenance of pluripotency. The cell surface antigens most
commonly used to identify hES cells are the glycolipids stage specific embryonic antigen
3 and 4, and the keratan sulfate antigens Tra-1-60 and Tra-1-81. The molecular definition
of a stem cell includes many more proteins and continues to be a topic of research.
By using human embryonic stem cells to produce specialized cells like nerve cells or
heart cells in the lab, scientists can gain access to adult human cells without taking tissue
from patients. They can then study these specialized adult cells in detail to try to discern
complications of diseases, or to study cell reactions to proposed new drugs.
Because of their combined abilities of unlimited expansion and pluripotency,
embryonic stem cells remain a theoretically potential source for regenerative
medicine and tissue replacement after injury or disease, however, there are currently no
approved treatments using ES cells. The first human trial was approved by the US Food
and Drug Administration in January 2009. However, the human trial was not initiated
until October 13, 2010 in Atlanta for spinal cord injury research. On November 14, 2011
the company conducting the trial (Geron Corporation) announced that it will discontinue
further development of its stem cell programs. Differentiating ES cells into usable cells
while avoiding transplant rejection are just a few of the hurdles that embryonic stem cell
researchers still face.[8]
Embryonic stem cells, being pluripotent, require specific signals
for correct differentiation — if injected directly into another body, ES cells will
differentiate into many different types of cells, causing a teratoma. Ethical considerations
regarding the use of unborn human tissue are another reason for the lack of approved
treatments using embryonic stem cells. Many nations currently have moratoria or

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limitations on either human ES cell research or the production of new human ES cell
lines. [7]
Fig.3: Mouse embryonic stem cells with fluorescent marker
Fig.4: Human embryonic stem cell colony on mouse embryonic fibroblast feeder layer.
2. Fetal
The primitive stem cells located in the organs of fetuses are referred to as fetal stem
cells. There are two types of fetal stem cells:
1. Fetal proper stem cells come from the tissue of the fetus proper, and are generally
obtained after an abortion. These stem cells are not immortal but have a high level of
division and are multipotent.
2. Extraembryonic fetal stem cells come from extraembryonic membranes, and are
generally not distinguished from adult stem cells. These stem cells are acquired after
birth, they are not immortal but have a high level of cell division, and are pluripotent.
3. Adult
Adult stem cells, also called somatic (from Greek σωματικóς, "of the body") stem cells,
are stem cells which maintain and repair the tissue in which they are found. They can be
found in children, as well as adults.
There are three known accessible sources of autologous adult stem cells in humans:
1. Bone marrow, which requires extraction by harvesting, that is, drilling into bone
(typically the femur or iliac crest).
2. Adipose tissue (fat cells), which requires extraction by liposuction.

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3. Blood, which requires extraction through apheresis, wherein blood is drawn from the
donor (similar to a blood donation), and passed through a machine that extracts the stem
cells and returns other portions of the blood to the donor.
Stem cells can also be taken from umbilical cord blood just after birth. Of all stem
cell types, autologous harvesting involves the least risk. By definition, autologous cells
are obtained from one's own body, just as one may bank his or her own blood for elective
surgical procedures.
Pluripotent adult stem cells are rare and generally small in number, but they can be found
in umbilical cord blood and other tissues. Bone marrow is a rich source of adult stem
cells, which have been used in treating several conditions including liver
cirrhosis, chronic limb ischemia and endstage heart failure. The quantity of bone marrow
stem cells declines with age and is greater in males than females during reproductive
years. Much adult stem cell research to date has aimed to characterize their potency and
self-renewal capabilities. DNA damage accumulates with age in both stem cells and the
cells that comprise the stem cell environment. This accumulation is considered to be
responsible, at least in part, for increasing stem cell dysfunction with aging (see DNA
damage theory of aging).
Most adult stem cells are lineage-restricted (multipotent) and are generally referred
to by their tissue origin (mesenchymal stem cell, adipose-derived stem cell, endothelial
stem cell, dental pulp stem cell, etc.). Muse cells (multi-lineage differentiating stress
enduring cells) are a recently discovered pluripotent stem cell type found in multiple adult
tissues, including adipose, dermal fibroblasts, and bone marrow. While rare, muse cells
are identifiable by their expression of SSEA-3, a marker for undifferentiated stem cells,
and general mesenchymal stem cells markers such as CD105. When subjected to single
cell suspension culture, the cells will generate clusters that are similar to embryoid bodies
in morphology as well as gene expression, including canonical pluripotency
markers Oct4, Sox2, and Nanog.
Adult stem cell treatments have been successfully used for many years to treat
leukemia and related bone/blood cancers through bone marrow transplants. Adult stem
cells are also used in veterinary medicine to treat tendon and ligament injuries in horses.
The use of adult stem cells in research and therapy is not as controversial as the use
of embryonic stem cells, because the production of adult stem cells does not require the
destruction of an embryo. Additionally, in instances where adult stem cells are obtained
from the intended recipient (an autograft), the risk of rejection is essentially non-existent.

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Consequently, more US government funding is being provided for adult stem cell
research.
With the increasing demand of human adult stem cells for both research and
clinical purposes (typically 1–5 million cells per kg of body weight are required per
treatment) it becomes of utmost importance to bridge the gap between the need to expand
the cells in vitro and the capability of harnessing the factors underlying replicative
senescence. Adult stem cells are known to have a limited lifespan in vitro and to enter
replicative senescence almost undetectably upon starting in vitro culturing. [9]
4. Amniotic
Multipotent stem cells are also found in amniotic fluid. These stem cells are very active,
expand extensively without feeders and are not tumorigenic. Amniotic stem cells are
multipotent and can differentiate in cells of adipogenic, osteogenic, myogenic,
endothelial, hepatic and also neuronal lines. Amniotic stem cells are a topic of active
research.
Use of stem cells from amniotic fluid overcomes the ethical objections to using
human embryos as a source of cells. Roman Catholic teaching forbids the use of
embryonic stem cells in experimentation; accordingly, the Vatican newspaper
"Osservatore Romano" called amniotic stem cells "the future of medicine".
It is possible to collect amniotic stem cells for donors or for autologous use: the
first US amniotic stem cells bank was opened in 2009 in Medford, MA, by Biocell Center
Corporation and collaborates with various hospitals and universities all over the world. [10]
5. Induced pluripotent
Adult stem cells have limitations with their potency; unlike embryonic stem cells (ESCs),
they are not able to differentiate into cells from all three germ layers. As such, they are
deemed multipotent.
However, reprogramming allows for the creation of pluripotent cells, induced
pluripotent stem cells (iPSCs), from adult cells. These are not adult stem cells, but adult
cells (e.g. epithelial cells) reprogrammed to give rise to cells with pluripotent capabilities.
Using genetic reprogramming with protein transcription factors, pluripotent stem cells
with ESC-like capabilities have been derived. The first demonstration of induced
pluripotent stem cells was conducted by Shinya Yamanaka and his colleagues at Kyoto
University. They used the transcription factors Oct3/4, Sox2, c-Myc, and Klf4 to

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reprogram mouse fibroblast cells into pluripotent cells. Subsequent work used these
factors to induce pluripotency in human fibroblast cells. Junying Yu, James Thomson,
and their colleagues at the University of Wisconsin–Madison used a different set of
factors, Oct4, Sox2, Nanog and Lin28, and carried out their experiments using cells from
human foreskin. However, they were able to replicate Yamanaka's finding that inducing
pluripotency in human cells was possible.
Induced pluripotent stem cells differ from embryonic stem cells. They share
many similar properties, such as pluripotency and differentiation potential, the expression
of pluripotencygenes, epigenetic patterns, embryoid body and teratoma formation, and
viable chimera formation, but there are many differences within these properties. The
chromatin of iPSCs appears to be more "closed" or methylated than that of
ESCs. Similarly, the gene expression pattern between ESCs and iPSCs, or even iPSCs
sourced from different origins. There are thus questions about the "completeness"
of reprogramming and the somatic memory of induced pluripotent stem cells. Despite
this, inducing adult cells to be pluripotent appears to be viable.
As a result of the success of these experiments, Ian Wilmut, who helped create the
first cloned animal Dolly the Sheep, has announced that he will abandon somatic cell
nuclear transfer as an avenue of research.
Furthermore, induced pluripotent stem cells provide several therapeutic advantages.
Like ESCs, they are pluripotent. They thus have great differentiation potential;
theoretically, they could produce any cell within the human body (if reprogramming to
pluripotency was "complete"). Moreover, unlike ESCs, they potentially could allow
doctors to create a pluripotent stem cell line for each individual patient. Frozen blood
samples can be used as a valuable source of induced pluripotent stem cells. Patient
specific stem cells allow for the screening for side effects before drug treatment, as well
as the reduced risk of transplantation rejection. Despite their current limited use
therapeutically, iPSCs hold create potential for future use in medical treatment and
research. [11]

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Sources for stem cells
Most stem cells intended for regenerative therapy are generally isolated either from the
patient's bone marrow or from adipose tissue. Mesenchymal stem cells can differentiate
into the cells that make up bone, cartilage, tendons, and ligaments, as well as muscle,
neural and other progenitor tissues; they have been the main type of stem cells studied in
the treatment of diseases affecting these tissues. The number of stem cells transplanted
into damaged tissue may alter efficacy of treatment. Accordingly, stem cells derived from
bone marrow aspirates, for instance, are cultured in specialized laboratories for expansion
to millions of cells. Although adipose-derived tissue also requires processing prior to use,
the culturing methodology for adipose-derived stem cells is not as extensive as that for
bone marrow-derived cells.While it is thought that bone-marrow derived stem cells are
preferred for bone, cartilage, ligament, and tendon repair, others believe that the less
challenging collection techniques and the multi-cellular microenvironment already
present in adipose-derived stem cell fractions make the latter the preferred source for
autologous transplantation.
New sources of mesenchymal stem cells are being researched, including stem cells
present in the skin and dermis which are of interest because of the ease at which they can
be harvested with minimal risk to the animal. Hematopoetic stem cells have also been
discovered to be travelling in the blood stream and possess equal differentiating ability as
other mesenchymal stem cells, again with a very non-invasive harvesting technique.
There has been more recent interest in the use of extra embryonic mesenchymal
stem cells. Research is underway to examine the differentiating capabilities of stem cells
found in the umbilical cord, yolk sac and placenta of different animals. These stem cells
are thought to have more differentiating ability than their adult counterparts, including the
ability to more readily form tissues of endodermal and ectodermal origin.

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Isolation of stem cells
The isolation, generation, and maintenance of stem cells pose several challenges due to
the propensity of stem cells to differentiate and for variations such as chromosomal and
epigenetic changes to occur in these cells during culture. Protocols are continuously
evolving and vary for different types of stem cells.
[12]
Isolation of Embryonic Stem Cell (ESC) Line
The majority of ESC lines are mouse, not human. The basic protocol for generation of
ESCs is similar for all species.
ESCs are pluripotent stem cells generated from early-stage embryos. A fertilized
embryo is required for the generation of ESCs. Typically, cells are harvested from the
blastocyst 4–5 days post-fertilization. The outer cell layer of the blastocyst, called the
trophoblast, contains a fluid-filled cavity, the blastocoele, and an inner cell mass of 10–20
cells. The inner cell mass, which is also called the embryoblast, is removed for culture.
Occasionally, cells may be obtained from the stage before the formation of the blastocyst,
the morula. [14]
The cells from the inner mass are placed in culture, and those that are viable are
expanded. Generation of ESCs is inefficient; many cells do not adapt to cell culture and
do not survive.
Human, mouse, rat, and other ESC lines are available through commercial vendors. [13]

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Fig.5: Isolation and cultivation of embryonic stem cells
Somatic Stem Cells
Somatic (adult) stem cells are found in most major organs and tissues, and are currently
being isolated from many tissues in the body. The methods of isolation and culture are
dependent on the source and lineage. Many isolation and purification protocols
involve flow cytometry and cell sorting. Positive and negative sorting for cell surface
markers can quickly generate enriched populations. [15]
The number of cells that can be isolated varies greatly depending upon the cell and
tissue type. For example, cardiac stem cells are quite rare, while hematopoietic stem cells

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(HSCs) occur in high enough numbers that they are routinely isolated and used in bone
marrow transplantation. HSCs for bone marrow transplantation are collected either
directly from bone marrow or by apheresis, the removal of white blood cells from
peripheral blood. Prior to apheresis, the donor is injected with granulocyte-colony
stimulating factor to mobilize stem cells from the bone marrow.
Mesenchymal stem cells (MSCs) were originally isolated from bone marrow stroma
but were subsequently found to be present in most tissues of the body including cord
blood, adipose tissue, skin, and periodontal ligaments, which attach teeth to the jaw.
Adipose tissue is becoming an important source of MSCs because adipocytes are easily
accessible and present in relatively high numbers in the body. It has been estimated that 1
g of adipose tissue yields 5,000 MSCs, whereas bone marrow aspirate contains 100–1,000
MSCs per ml (Strem et al. 2005).
Fig.6: Sources of somatic stem cells in the human body.
Induced Pluripotent Stem Cells (iPSCs)
Induced pluripotent stem cells (iPSCs) are somatic cells that have been reprogrammed to
become pluripotent. Theoretically, any somatic cell could be reprogrammed. Practically,

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it has been found that some are relatively straightforward and others are more technically
challenging.
iPSCs were first generated by the introduction of the transcription factors Oct3/4,
Sox2, Klf4, and c-Myc in cells maintained in culture conditions used for ESC (Takahashi
and Yamanaka 2006). Various combinations of these transcription factors have since been
used by other investigators.
Most iPSCs have been generated using retroviral and lentiviral vectors to introduce
transcription factors into stem cells. However, there are concerns with using these viral
vectors. Previous studies of retroviral infection of embryonic cells had suggested that
retroviruses are silenced in these cells. Silencing is an epigenetic process that suppresses
transcription. However, it was demonstrated that silencing was often incomplete and viral
genes could still be expressed. A major consideration for using retroviruses to generate
iPSCs is that the viruses integrate into the host DNA. Depending on the integration site,
integration can have deleterious effects on the cells, altering gene expression and
increasing the risk of tumor formation. Adenoviruses (Stadfield et al. 2008) and Sendai
virus, an RNA virus (Seki et al. 2012), have been used as alternative vectors to transduce
transcription factors because they do not integrate into the genomic DNA of the cell.
Viral-mediated introduction of transcription factors is very inefficient. A more
efficient method was found to be the combination of lentiviruses and micro RNAs
(miRNAs) to reprogram cells (Anokye-Danso et al. 2012). These small RNAs bind to
mRNA and either inhibit translation or cause degradation of transcripts. miRNA clusters,
including miR-290-295 and miR-302-367, have been shown to enhance reprogramming
of somatic cells into iPSCs.
Another approach was the use of miRNA mimics to enhance viral-mediated
transduction of transcription factors. miRNA mimics are double-stranded modified RNAs
that mimic mature miRNAs (fully processed cellular miRNAs). miRNA mimics do not
require a vector; they can be transfected directly into cells. The combined use of
transcription factors and miRNA mimics produces more homogeneous iPSC clones
(Judson et al. 2009). An advantage of using miRNA mimics with transcription factors is
that the transcription factor c-myc, an oncogene, is not required.
Several chemical compounds that modulate enzymes controlling epigenetic
modifications have been evaluated for increasing the efficiency of transduction by
transcription factors. DNA methyltransferase and histone deacetylase (HDAC) inhibitors
were shown to potentiate the efficacy of transduction. The HDAC inhibitor valproic acid

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was the most effective, increasing reprogramming efficiency by 100-fold (Huangfu et al.
2008). As with the miRNA mimics, the use of valproic acid eliminates the need to
transduce with the oncogene c-myc.
A number of other inhibitors of kinases, such as the glycogen synthase kinase-3
(GSK3) inhibitor CHIR99021 and the MEK inhibitor PD0325901, and other enzymes that
are in pathways involved in pluripotency have also been shown to enhance the efficiency
of reprogramming (Zhang et al. 2012). [16][17][18]
Plasmid expression vectors have been used to introduce transcription factors (Okita et
al. 2008), but the efficiency is low, and occasionally expression plasmids can integrate
into genomic DNA.
Somatic cell nuclear transfer (SCNT) is the transfer of an isolated somatic nucleus into
an enucleated egg. The egg is stimulated to divide, usually by an electric shock, though
caffeine has recently been used (Tachibana et al. 2013). The somatic nuclei are
reprogramed by the egg, and the resulting daughter cells become pluripotent stem cells.
In animals, SCNT is routinely used to study stem cells. Recently, SCNT was used to
transfer nuclei from skin fibroblasts to enucleated human eggs to generate pluripotent
stem cell lines (Tachibana et al. 2013). For animal cloning, after a number of cell
divisions, the egg can be implanted into a surrogate, as was done with Dolly, the first
cloned sheep (Wilmut et al. 1997).
Culture of Stem Cells
The culture conditions and types of media used for stem cell culture depend on the type of
stem cell. There are a wide range of protocols and products available for both maintaining
stem cells in an undifferentiated state and for differentiating them into different lineages
and cell types. [19]
Feeder Cell Layers
Originally, all ESC cultures were maintained on feeder cell layers. Inactivated mouse
embryonic fibroblasts (MEFs) were used to provide factors and a substrate that allowed
ESCs to grow and divide. There are several problems associated with MEFs, including
the potential for the introduction of mouse-derived infectious agents, undesirable protein
transfer, and lot-to-lot variation among feeder cells.

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MEFs can either be purchased or freshly generated in the lab. Fibroblasts from ~14–15-
day-old fetal mice are cultured and expanded for 3–4 days. To be used as a feeder layer,
MEFs must be mitotically inactivated, either by irradiation with UV light or incubation
with mitomycin C. Stocks of MEF cells can be frozen either before or after inactivation.
[20]
Fig.7: Preparation of mitotically inactivated mouse embryonic fibroblast (MEF) cells for
use as feeder cells
A consideration in the choice of culture system is that stem cells have been shown to
acquire xenoantigens from culture products derived from other animal species. This was
first shown in hESCs cultured with mouse feeder cells and animal-derived serum. These
stem cells incorporated non-human sialic acid, against which many humans already have
circulating antibodies (Martin et al. 2005). In addition to feeder cells, animal-derived

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products used for culture include serum and other matrices. The potential for stem cells to
cause an immunogenic response can restrict their clinical use.
Feeder-free culture
There are two types of feeder cell-free media: defined media and conditioned media.
These media will support the growth of stem cells and contain factors that inhibit
differentiation. The components of the media and the supplements vary depending on the
type of stem cell and the animal. [21]
Defined medium is a serum-free medium that has been supplemented with recombinant
growth factors and other molecules that are required to support the growth and
pluripotency of stem cells.
Media formulated for stem cell culture often contain factors such as leukemia inhibitory
factor and bone morphogenetic protein to prevent differentiation. Rho-associated coiled-
coil containing protein kinase (ROCK) inhibitors such as Y-27632 and thiazovivin have
been demonstrated to increase the viability of newly isolated stem cells (Chen et al.
2010). For the culture of hESCs, basic fibroblast growth factor (bFGF or FGF2) is usually
present in the media but not for most other stem cell media. Some defined media for
human cell culture may contain bovine serum albumin (BSA) and is therefore not
completely free of animal protein. Additionally, other culture matrices (described below)
are often derived from animal cells.
Long-term culture in serum-free media has been shown to cause epigenetic changes in
cultures as the cells adapt. For instance, it was shown that hESCs start to express CD30, a
marker for certain types of malignancies, when grown in media with knockout serum
replacement but not in media with fetal calf serum (Chung et al. 2010). Epigenetic
changes with different culture conditions have been shown to include methylation,
histone modification, and in female ESCs, X-chromosome inactivation (McKewn et al.
2013).
Cells in culture secrete factors into the media that support cell growth. After cells have
grown and divided for a period of time, the media are removed. This conditioned
media can then be used as a supplement to fresh media. Although there is still concern
about the presence of viruses when using conditioned media, it is much less than when
using cross-species feeder cells. An advantage of conditioned media is that they contain
more factors than defined media. Variability between batches of conditioned media is

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common, so new lots should be tested. Human foreskin fibroblasts are the most common
cell type used to make conditioned media for human cells.
Stem cells not growing on a feeder layer need a matrix for attachment and growth.
A culturematrix containsextracellularmatrix(ECM) proteins and polysaccharidessuchas
vitronectin and proteoglycans. There are many matrices available with varying
combinations of proteins and carbohydrates. Different types and sources of stems cells
require different matrices, and different matrix components can either maintain
pluripotency or help drive differentiation. Cell matrix products containing only human
ECM are available.
Testing of Stem Cells
Frequent testing of stem cultures is necessary due the propensity of cultured stem cells to
undergo genotypic and phenotypic changes or mosaicism. Initially, when newly isolated
cells are at a sufficient density, they are usually screened for pluripotency, appropriate
gene expression, and a normal karyotype. Thereafter, the stability of the stem cells is
confirmed at regular intervals and after frozen stocks are thawed and plated. Additionally,
particularly in lines that use feeder layers, cultures must be checked for contamination.
Differentiation media
There are many formulations for media for directed differentiation of stem cells. Usually,
there is a reduction in growth factors and an addition of other factors. Frequently,
differentiation medium is combined with a change of the culture matrix to help promote
the desired path of differentiation. [22]

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Stem cell division (Lineage)
To ensure self-renewal, stem cells undergo two types of cell division. Symmetric division
gives rise to two identical daughter cells both endowed with stem cell properties.
Asymmetric division, on the other hand, produces only one stem cell and a progenitor
cell with limited self-renewal potential. Progenitors can go through several rounds of cell
division before terminally differentiating into a mature cell. It is possible that the
molecular distinction between symmetric and asymmetric divisions lies in differential
segregation of cell membrane proteins (such as receptors) between the daughter cells.
An alternative theory is that stem cells remain undifferentiated due to environmental cues
in their particular niche. Stem cells differentiate when they leave that niche or no longer
receive those signals. Studies in Drosophila germarium have identified the
signals decapentaplegic and adherens junctions that prevent germarium stem cells from
differentiating. [23]

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Treatment
Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone
marrow transplant is a form of stem cell therapy that has been used for many years
without controversy. [24][25]
Advantages
Stem cell treatments may lower symptoms of the disease or condition that is being
treated. The lowering of symptoms may allow patients to reduce the drug intake of the
disease or condition. Stem cell treatment may also provide knowledge for society to
further stem cell understanding and future treatments. [26]
Disadvantages
Stem cell treatments may require immunosuppression because of a requirement for
radiation before the transplant to remove the person's previous cells, or because the
patient's immune system may target the stem cells. One approach to avoid the second
possibility is to use stem cells from the same patient who is being treated.
Pluripotency in certain stem cells could also make it difficult to obtain a specific cell type.
It is also difficult to obtain the exact cell type needed, because not all cells in a population
differentiate uniformly. Undifferentiated cells can create tissues other than desired types.
Some stem cells form tumours after transplantation; pluripotency is linked to tumour
formation especially in embryonic stem cells, fetal proper stem cells, and induced
pluripotent stem cells. Fetal proper stem cells form tumours despite multipotency.[27]

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Stem cell therapy
Stem-cell therapy is the use of stem cells to treat or prevent a disease or condition.
Bone marrow transplant is the most widely used stem-cell therapy, but some therapies
derived from umbilical cord blood are also in use. Research is underway to develop
various sources for stem cells, as well as to apply stem-cell treatments
for neurodegenerative diseases[29]
and conditions such as diabetes and heart disease,
among others.
Stem-cell therapy has become controversial following developments such as the ability of
scientists to isolate and culture embryonic stem cells, to create stem cells using somatic
cell nuclear transfer and their use of techniques to create induced pluripotent stem cells.
This controversy is often related to abortion politics and to human cloning. Additionally,
efforts to market treatments based on transplant of stored umbilical cord blood have been
controversial.
[28]
Everybody is born different, some are born perfectly healthy and remain healthy
for the rest of their lives, some are born with certain neuromuscular disorders, while some
may develop degenerative disorders. Stem Cell Therapy (SCT) is the treatment of
various disorders, non-serious to life threatening, by using stem cells. These stem cells
can be procured from a lot of different sources and used to potentially treat more than 80
disorders, including neuromuscular and degenerative disorders.
Hematopoietic disorders (eg leukaemia, thallassemia, aplastic anemia, MDS, sickle
cell anemia, storage disorders etc.) affect the bone marrow and manifest with various
systemic complications. Stem cells from a donor (either from cord blood or bone marrow)
are known to reconstitute the defective bone marrow and permanently overcome the
disorder.
Degenerative disorders arise from degeneration or wear and tear of bone, cartilage,
muscle, fat or any other tissue, cell or organ. This could occur due to a variety of reasons,
but it's normally the process known as aging, or 'getting old' that is the biggest cause. The
disorders have a slow and insidious onset but once contracted, can be long-standing, pain-
staking and lifelong. These disorders can affect any organ of the body. The common
degenerative disorders are diabetes, osteoarthritis, stroke, chronic renal failure, congestive
cardiac failure, myocardial infarction, Alzheimer's disease, Parkinson's disease etc.
Stem cells have the ability to build every tissue in the human body, hence have great
potential for future therapeutic uses in tissue regeneration and repair. In order for cells to

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fall under the definition of “stem cells,” they must display two essential characteristics.
First, stem cells must have the ability of unlimited self-renewal to produce progeny
exactly the same as the originating cell. This trait is also true of cancer cells that divide in
an uncontrolled manner whereas stem cell division is highly regulated. Therefore, it is
important to note the additional requirement for stem cells; they must be able to give rise
to a specialized cell type that becomes part of the healthy animal.
The general designation, “stem cell” encompasses many distinct cell types.
Commonly, the modifiers, “embryonic,” and “adult” are used to distinguish stem cells by
the developmental stage of the animal from which they come, but these terms are
becoming insufficient as new research has discovered how to turn fully differentiated
adult cells back into embryonic stem cells and, conversely, adult stem cells, more
correctly termed “somatic” stem cells meaning “from the body”, are found in the fetus,
placenta, umbilical cord blood and infants. Therefore, this review will sort stem cells into
two categories based on their biologic properties - pluripotent stem cells and multipotent
stem cells. Their sources, characteristics, differentiation and therapeutic applications are
discussed.
Pluripotent stem cells are so named because they have the ability to differentiate
into all cell types in the body. In natural development, pluripotent stem cells are only
present for a very short period of time in the embryo before differentiating into the more
specialized multipotent stem cells that eventually give rise to the specialized tissues of the
body . These more limited multipotent stem cells come in several subtypes: some can
become only cells of a particular germ line (endoderm, mesoderm, ectoderm) and others,
only cells of a particular tissue. In other words, pluripotent cells can eventually become
any cell of the body by differentiating into multipotent stem cells that themselves go
through a series of divisions into even more restricted specialized cells.

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Medical uses
For over 30 years, bone marrow has been used to treat people with cancer with conditions
such as leukaemia and lymphoma; this is the only form of stem cell therapy that is widely
practiced. During chemotherapy, most growing cells are killed by the cytotoxic agents.
These agents, however, cannot discriminate between the leukaemia or neoplastic cells,
and the hematopoietic stem cells within the bone marrow. It is this side effect of
conventional chemotherapy strategies that the stem-cell transplant attempts to reverse; a
donor's healthy bone marrow reintroduces functional stem cells to replace the cells lost in
the host's body during treatment. The transplanted cells also generate an immune response
that helps to kill off the cancer cells; this process can go too far, however, leading to graft
vs. host disease, the most serious side effect of this treatment. [30]
Another stem-cell therapy called Prochymal was conditionally approved in Canada
in 2012 for the management of acute graft-vs.-host disease in children who are
unresponsive to steroids. It is an allogenic stem therapy based on mesenchymal stem
cells (MSCs) derived from the bone marrow of adult donors. MSCs are purified from the
marrow, cultured and packaged, with up to 10,000 doses derived from a single donor. The
doses are stored frozen until needed.
The FDA has approved five hematopoietic stem-cell products derived from
umbilical cord blood, for the treatment of blood and immunological diseases.
In 2014, the European Medicines Agency recommended approval of limbal stem
cells for people with severe limbal stem cell deficiency due to burns in the eye.

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Applications
Neurodegeneration
Research has been conducted on the effects of stem cells on animal models of brain
degeneration, such as in Parkinson's, Amyotrophic lateral sclerosis, and Alzheimer's
disease. There have been preliminary studies related to multiple sclerosis.
Healthy adult brains contain neural stem cells which divide to maintain general
stem-cell numbers, or become progenitor cells. In healthy adult laboratory animals,
progenitor cells migrate within the brain and function primarily to maintain neuron
populations for olfaction (the sense of smell). Pharmacological activation
of endogenous neural stem cells has been reported to induce neuroprotection and
behavioural recovery in adult rat models of neurological disorder.
Brain and spinal cord injury
Stroke and traumatic brain injury lead to cell death, characterized by a loss of neurons
and oligodendrocytes within the brain. Clinical and animal studies have been conducted
into the use of stem cells in cases of spinal cord injury.
Heart
Stem cells are studied in people with severe heart disease. The work by Bodo-Eckehard
Strauer was discredited by identifying hundreds of factual contradictions. Among several
clinical trials reporting that adult stem cell therapy is safe and effective, actual evidence
of benefit has been reported from only a few studies.Some preliminary clinical trials
achieved only modest improvements in heart function following use of bone marrow stem
cell therapy.
Stem-cell therapy for treatment of myocardial infarction usually makes use of
autologous bone marrow stem cells, but other types of adult stem cells may be used, such
as adipose-derived stem cells.
Possible mechanisms of recovery include:
 Generation of heart muscle cells
 Stimulating growth of new blood vessels to repopulate damaged heart tissue
 Secretion of growth factor

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Criticisms
In 2013, studies of autologous bone marrow stem cells on ventricular function were found
to contain "hundreds" of discrepancies. Critics report that of 48 reports there seemed to be
just five underlying trials, and that in many cases whether they were randomized or
merely observational accepter-versus-rejecter, was contradictory between reports of the
same trial. One pair of reports of identical baseline characteristics and final results, was
presented in two publications as, respectively, a 578 patient randomized trial and as a 391
subject observational study. Other reports required (impossible) negative standard
deviations in subsets of people, or contained fractional subjects, negative NYHA classes.
Overall there were many more people published as having receiving stem cells in trials,
than the number of stem cells processed in the hospital's laboratory during that time. A
university investigation, closed in 2012 without reporting, was reopened in July 2013.
In 2014, a meta-analysis on stem cell therapy using bone marrow stem cells for
heart disease revealed discrepancies in published clinical trial reports, whereby studies
with a higher number of discrepancies showed an increase in effect sizes.Another meta-
analysis based on the intra-subject data of 12 randomized trials was unable to find any
significant benefits of stem cell therapy on primary endpoints, such as major adverse
events or increase in heart function measures, concluding there was no benefit.
The TIME trial, which used a randomized, double blind, placebo-controlled trial design,
concluded that "bone marrow mononuclear cells administration did not improve recovery
of LV function over 2 years" in people who had a myocardial infarction. Accordingly, the
BOOST-2 trial conducted in 10 medical centers in Germany and Norway reported that the
trial result "does not support the use of nucleated BMCs in patients with STEMI and
moderately reduced LVEF". Furthermore, the trial also did not meet any other secondary
MRI endpoints, leading to a conclusion that intracoronary bone marrow stem cell therapy
does not offer a functional or clinical benefit.
Blood-cell formation
The specificity of the human immune-cell repertoire is what allows the human body to
defend itself from rapidly adapting antigens. However, the immune system is vulnerable
to degradation upon the pathogenesis of disease, and because of the critical role that it
plays in overall defense, its degradation is often fatal to the organism as a whole. Diseases
of hematopoietic cells are diagnosed and classified via a subspecialty of pathology known
as hematopathology. The specificity of the immune cells is what allows recognition of

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foreign antigens, causing further challenges in the treatment of immune disease. Identical
matches between donor and recipient must be made for successful transplantation
treatments, but matches are uncommon, even between first-degree relatives. Research
using both hematopoietic adult stem cells and embryonic stem cells has provided insight
into the possible mechanisms and methods of treatment for many of these ailments.
Fully mature human red blood cells may be generated ex vivo by hematopoietic
stem cells (HSCs), which are precursors of red blood cells. In this process, HSCs are
grown together with stromal cells, creating an environment that mimics the conditions of
bone marrow, the natural site of red-blood-cell growth. Erythropoietin, a growth factor, is
added, coaxing the stem cells to complete terminal differentiation into red blood
cells. Further research into this technique should have potential benefits to gene therapy,
blood transfusion, and topical medicine.
Regrowing teeth
In 2004, scientists at King's College London discovered a way to cultivate a complete
tooth in mice and were able to grow bioengineered teeth stand-alone in the laboratory.
Researchers are confident that the tooth regeneration technology can be used to grow live
teeth in people.
In theory, stem cells taken from the patient could be coaxed in the lab turning into a
tooth bud which, when implanted in the gums, will give rise to a new tooth, and would be
expected to be grown in a time over three weeks. It will fuse with the jawbone and release
chemicals that encourage nerves and blood vessels to connect with it. The process is
similar to what happens when humans grow their original adult teeth. Many challenges
remain, however, before stem cells could be a choice for the replacement of missing teeth
in the future.
Cochlear hair cell regrowth
Heller has reported success in re-growing cochlea hair cells with the use of embryonic
stem cells.
Blindness and vision impairment
Since 2003, researchers have successfully transplanted corneal stem cells into damaged
eyes to restore vision. "Sheets of retinal cells used by the team are harvested from aborted
fetuses, which some people find objectionable." When these sheets are transplanted over

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the damaged cornea, the stem cells stimulate renewed repair, eventually restore vision.
The latest such development was in June 2005, when researchers at the Queen Victoria
Hospital of Sussex, England were able to restore the sight of forty people using the same
technique. The group, led by Sheraz Daya, was able to successfully use adult stem cells
obtained from the patient, a relative, or even a cadaver. Further rounds of trials are
ongoing.
Pancreatic beta cells
People with Type 1 diabetes lose the function of insulin-producing beta cells within the
pancreas. In recent experiments, scientists have been able to coax embryonic stem cell to
turn into beta cells in the lab. In theory if the beta cell is transplanted successfully, they
will be able to replace malfunctioning ones in a diabetic patient.
Fig.8: Mesenchymal stem cells being injected into knee of patient
Orthopaedics
Use of mesenchymal stem cells (MSCs) derived from adult stem cells is under
preliminary research for potential orthopaedic applications in bone and muscle
trauma, cartilage repair, osteoarthritis, intervertebral disc surgery, rotator cuff surgery,
and musculoskeletal disorders, among others. Other areas of orthopaedic research for uses
of MSCs include tissue engineering and regenerative medicine.
Wound healing
Stem cells can also be used to stimulate the growth of human tissues. In an adult,
wounded tissue is most often replaced by scar tissue, which is characterized in the skin by
disorganized collagen structure, loss of hair follicles and irregular vascular structure. In
the case of wounded fetal tissue, however, wounded tissue is replaced with normal tissue
through the activity of stem cells.A possible method for tissue regeneration in adults is to
place adult stem cell "seeds" inside a tissue bed "soil" in a wound bed and allow the stem
cells to stimulate differentiation in the tissue bed cells. This method elicits a regenerative

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response more similar to fetal wound-healing than adult scar tissue
formation. Researchers are still investigating different aspects of the "soil" tissue that are
conducive to regeneration. Because of the general healing capabilities of stem cells, they
have gained interest for the treatment of cutaneous wounds, such as in skin cancer.
Infertility
Culture of human embryonic stem cells in mitotically inactivated porcine ovarian
fibroblasts (POF) causes differentiation into germ cells (precursor cells of oocytes and
spermatozoa), as evidenced by gene expression analysis.
Human embryonic stem cells have been stimulated to form Spermatozoon-like
cells, yet still slightly damaged or malformed. It could potentially treat azoospermia.
In 2012, oogonial stem cells were isolated from adult mouse and human ovaries and
demonstrated to be capable of forming mature oocytes. These cells have the potential to
treat infertility.
HIV/AIDS
Destruction of the immune system by the HIV is driven by the loss of CD4+ T cells in the
peripheral blood and lymphoid tissues. Viral entry into CD4+ cells is mediated by the
interaction with a cellular chemokine receptor, the most common of which
are CCR5 and CXCR4. Because subsequent viral replication requires cellular gene
expression processes, activated CD4+ cells are the primary targets of productive HIV
infection. Recently scientists have been investigating an alternative approach to treating
HIV-1/AIDS, based on the creation of a disease-resistant immune system through
transplantation of autologous, gene-modified (HIV-1-resistant) hematopoietic stem and
progenitor cells (GM-HSPC).
[32]

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Diseases and conditions where stem cell treatment is being investigated
include:
 Diabetes
 Rheumatoid arthritis
 Parkinson's disease
 Alzheimer's disease
 Osteoarthritis
 Stroke and traumatic brain injury repair
 Learning disability due to congenital disorder
 Spinal cord injury repair
 Heart infarction
 Anti-cancer treatments
 Baldness reversal
 Replace missing teeth
 Repair hearing
 Restore vision and repair damage to the cornea
 Amyotrophic lateral sclerosis
 Crohn's disease
 Wound healing
 Male infertility due to absence of spermatogonial stem cells. In recent studies, scientists
have found a way to solve this problem by reprogramming a cell and turning it into a
spermatozoon. Other studies have proven the restoration of spermatogenesis by
introducing human iPSC cells in mice testicles. This could mean the end of azoospermia.
 Female infertility: oocytes made from embryonic stem cells. Recently, scientists have
found the ovarian stem cells, a rare type of cells (0,014%) found in the ovary. It is not
clear their existence yet, but the impact it could have are limitless. It could be used as a
treatment not only for infertility, but also for premature ovarian insufficiency.
Research is underway to develop various sources for stem cells, and to apply stem cell
treatments for neurodegenerative diseases and conditions, diabetes, heart disease, and
other conditions. Research is also underway in generating organoids using stem cells,
which would allow for further understanding of human development, organogenesis, and
modeling of human diseases.

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In more recent years, with the ability of scientists to isolate and culture embryonic stem
cells, and with scientists' growing ability to create stem cells using somatic cell nuclear
transfer and techniques to create induced pluripotent stem cells, controversy has crept in,
both related to abortion politics and to human cloning.
Hepatotoxicity and drug-induced liver injury account for a substantial number of failures
of new drugs in development and market withdrawal, highlighting the need for screening
assays such as stem cell-derived hepatocyte-like cells that are capable of detecting
toxicity early in the drug development process.
[33]
Fig.9: Diseases and conditions where stem cell treatment is being investigated.
Stem cell therapy for treatment of diabetes
Introduction
Diabetes is a disease in which our blood glucose levels are high. Mostly glucose comes
from the foods that we eat. Hormone that is involved in glucose transport into our cells to
get energy is insulin. In diabetes, our body does not able to make insulin it is called
diabetes type 1. In the Type 2 body is not able to use glucose. A number of new
techniques have been used in the past to treat he diabetes in the past these include
improved insulin delivery and glucose monitoring systems, whole pancreatic and islet cell
transplantation, and new methods for ß-cell generation either from pancreatic ducts or
stem cells, or through genetic engineering. Patients with diabetes have been characterized
by relative lack of insulin producing pancreatic β-cells; as a result they are unable to

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establish normal blood glucose level. Islet transplantation has been effective therapy for
producing sustained insulin level in the patients. Due to the lack of donor for the islets
transplantation, this technique has been widely used. Human embryonic stem cells
(hESCs) are has been a good alternative source of this treatment and numerous groups of
cells have been used to differentiate in to insulin producing pancreatic Beta cells.
Knowledge of pancreas development is based on Model organisms but we have not been
fully recognized the pattern of pancreas development. A strategy has been used in which
the Human embryonic progenitor cells have been differentiated in vivo. Human fetal
islet-like cell clusters successfully matured into glucose producing cells in mice,
suggesting that a similar approach may be feasible for hESC derived cells. The diabetics
can be cured by the re-plenishment of beta cells by the trans-plantation of Islets. Tran-
planted Islets can be collect from the two to three donor’s pancreatic donors with
exceeding ten thousand islets equivalent (IEQ)/kg. One of the limits of the islets
transplantation is the storage of the donor’s organs. By the use of stem cell technology
this issues is address well. Stem cells have the capacity of self-renewal and the potential
of differentiating into various cell types. The generations of insulin-producing cells from
the human embryonic stem cells (ESC) and induced pluripotent stem cells (iPS) have
challenges till now. Lumelsky and Assady found that beta cells can be produced by the
application of different physiological condition from the islets structure in-vitro from
ESC. Cells produced by this method have many issues like low insulin production and
lack of response to glucose. They can also cause the diseases like risk of cancer,
controversial ethical issues and functional deficiency. Cells produced from iPS have such
problems. Human adult stem cells can also be used for the production of insulin
producing cells. Expanded mesenchymal stromal cells from human umbilical cord and
placenta, and differentiated them into functional islets in vitro reported that islet-like cell
aggregates derived from stem cells in human adipose tissue ameliorated experimental
diabetes in mice. This is insufficient for the clinical applicable. It is shown that stem cells
are present in the pancreatic duct and islets, that have the ability to differentiate into the
pancreatic exocrine and endocrine with number of pancreatic stem cells increase upon the
destructive immune response. So, that pancreatic stem cells are used for the formation of
functional endocrine cells in vitro condition. Pancreatic stem cells differentiating into
endocrine cells have pancreatic duodenal homeobox- 1(PDX-1) and neurogenin 3. From
the work of Bonner-Weir et al. showed that human pancreatic duct cells expanded and
differentia into glucose responsive islet tissue in vitro given ITS (insulin, transferrin,

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selenium), nicotinamide and keratinocytes growth factor. Ramiya et al. isolated murine
pancreatic ductal epithelial cells into culture and induced them into functional islets
containing alpha, beta and delta cells. From these results, it is shows that some changes in
the mRNA for the islets cells that differentia into some markers, response to glucose in
vitro and reversed insulin de-pendent diabetics into the mice. Pancreatic cells isolate from
the adult pancreas show low proliferative than the fetal pancreatic cells in vitro. Human
fetal pancreatic cells also have the ability to differentiate into the insulin producing cells
in vitro. Human fetal cells not have this ability but also have to correct high blood glucose
efficiency in diabetic animals.
[34]
The limitation of the conventional treatments
In many current cases, diabetic complications are not controlled by the drugs because
they do not provide sufficient control on the blood glucose level. Whole pancreas
transplantation was an effective treatment but it had some serious issues like surgery and
long term immunosuppression. The failure of many conventional processes was a sad
situation not only for the patient and relatives but also for the whole society. The cost of
the treatment was very high due to the increase in the number of the patients of diabetes.
So, the development in the treatment of the diabetes was very important for the patient
and society also.
Islet cell transplantation
This treatment for the diabetes was effective one but the limitation for this treatment was
that the donor cells were not easily available or shortage of them. So, for type 1 diabetes
allogenic transplantation had been explored. Extraction of the islet cells from the donor
pancreas and cells were injected into the portal veins of the liver. This procedure repeated
two to three times and patient hospitalized for two to three months. This type of treatment
improved the diabetic patient condition, if successful. But the limitation for this type of
treatment was that the people who were already immunosuppressed for the other type of
treatment like kidney transplantation were not suitable for this transplantation of islet
cells. It was also possible that the immunosuppression itself the cause of the inhibition of
proper functioning of islet cells or it also induced peripheral insulin resistance. As a
result, only 10% of the patients had been seen insulin independent on the International
Islet Cell Transplantation Registry aіer the transplantation. Promising results had recently

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been rumored from transplantation of huge amounts of island cells from body pancreases
that were not HLA matched into seven patients with diabetes type 1 or had multiple
hypoglycemic episodes or uncontrolled polygenic disorder despite compliance with the
prescribed hypoglycemic agent treatment. All the patients showed standardization of
glycated heamoglobin concentration and lasting independence from the insulin injection
at a median of eleven months follow up. The islet cells were pure i.e. they are free from
the foreign proteins, and this, combined with a glucocortcoid free from the
immunosupressive regimen, with success prevented rejection. Notably, each host versus
graі and immune rejection reactions were apparently avoided. This was a tiny
uncontrolled study, however, and its encouraging results ha to be compelled to be
confirmed in larger irregular controlled trials. Even if any studies make sure the
effectiveness of this approach, the requirement to get 2 to 4 donor pancreases for every
patient and also the uncertainty concerning long term side effects from
immunosuppression probably to limit its application to patients with terribly poorly
controlled diabetic disorder.
Alternative sources of islet cells
Due to the shortage of the donor of the islet cells there was the search for alternative
sources. Several sources are suggested:
• From pigs
• Induction from human pancreatic duct cells
• Fetal pancreatic stem cells
• Induction of insulin producing B cells and each one has its own benefits and downsides.
Xenogeneic islet cells
Porcine islet cells are instructed as a virtually unlimited offer of insulin hormone
manufacturing cells for transplantation. However, the medical specialty barrier to
xenogeneic graft is well bigger than the barrier to human grafts. The development of the
transgenic pigs was a great approach because due to these techniques we get the
humanized pigs that have the more characteristics like the human cells. Xeno antigens
were not present in such type of transgenic pigs but not required for their survival and the
technology would possibly even permit production of pigs one by one matched for
recipients HLA sort. The problem for this type of grafting is the risk of the retrovirus of
the porcine which aіer this made the human their host. Retroviruses lead to permanent

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infection, and there were reports that porcine endogenous retroviruses from porcine cell
lines and lymphocytes would infect human cells in vitro. So, the US Food and Drug
Administration concerned about this fact and stopped the trial with the porcine xenograft
until the already transplanted people had the infection or not. Although 10 Swedish
patients were transplanted with these porcine endogenous cells but not acquired any sort
of infection. Another type of research performed that was recent one in this a transgenic
mouse is transplanted with the endogenous porcine islet and showed infection in almost
all cells but this mouse was diabetic and highly immunodeficient. Expansion and
transdifferentiation of the duct cells of pancreas whereas the character of the pancreatic
duct stem cells continues to be uncertain, recent advances during this space prompted a
high level meeting sponsored by the National institute of Health on stem cells and
pancreatic duct gland development. It had been reported by the Peck et al. that pancreatic
ductal epithelial cells that are isolated from adult non-obese diabetic mice can be grown
in long term cultures and induced to produce functioning islets. These in vitro generated
islets were capable of lowering blood sugar concentrations to close traditionally when
transferred in the diabetic non-obese mice. In the three-month duration of the study mice
remained norm glycemic. Human cells of pancreatic duct were also developed and
produced in vitro but they did not show any proper result when transplanted inside the
body. This promising line of analysis was being pursued by many laboratories. Not solely
would the use of adult donor ductal cells avoid the disputation of the fetal cells however
there were fewer biological issues related to certain alpha cells from duct cells than from,
as an example, embryonic stem cells.
The use of a cell precursor and fetal pancreatic stem cells
Few years ago, vast improvements have made in empathetic fetal endocrine growth.
These gives significant guide further efforts produce islet cells in vitro. The identification
of endocrine predecessor cells in developing pancreas and regulation of differentiation by
definite cellular pathway raises stirring probability that modulation cellular signaling can
used in vitro to grow and distinguish endocrine precursor cells, taken either from
embryonic pancreas from aborted fetuses or using pancreatic duct cells. Once molecular
facts are solved culture conditions can developed to supply unlimited number of
allogeneic a cell for trans-plantation. In 2013 the fetal pancreatic cell was used to produce
active insulin producing cells that was a excellent work by the biotechnologists. In this
work, they took the fetal pancreatic progenitor cells from the aborted embryos and they

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firstly isolated them by identification of the pancreatic progenitor cells by using different
markers e.g. PDX1 and NGN3. Then they provided them the media and performed
culturing. Some islets like structures were formed and they started to produce insulin
producing cells. Then they checked the function of those cells. By observing the results
these cells showed the high efficiency than the normal cells of the body. So, these fetal
cells have the best proliferation and differentiation ability than any kind of other cells.
Embryonic stem cells
Stem cells are powerful biological units have utilized for decades in numerous features of
biology. The mammalian body contains 200 different cell types, which all derive from
fertilized egg cell. The fertilized human egg distributes and rise the primary embryo, at
blastula stage, comprises cluster of totipotent cell from clonal embryonic stem cells
derived. Such ESC proliferated in-definitely in vitro and can induced to differentiate into
numerous different lineages in vitro, containing cardiomyocytes and neural cells, but
differentiation into endodermal cell types has not described. the stem cells follow
appropriate develop-mental pathway in order become insulin producing cells. Soria et al.
by using embryonic stem cells transfected with insulin promoter, resulting insulin
producing cells from mouse ESC which permitted them selectively make insulin
producing cells. Nevertheless, this procedure gives rise proliferating cells, and potentially
malignant cells, rather than matures, post mitotic cells. However, this trial shows that
embryonic stem cells differentiate along pancreatic endocrine path. These embryonic
stem cells are being used to produce functional Beta cells and transplanted to patient for
the treatment of diabetes as shown in the Figure.
Fig.10: Formation of beta cells from IPS or ES cells and then transplantation to
patient.

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Induced pluripotent cells
IPS have high replicative capacity and pluripotency, these cells can be differentiated in to
the insulin producing cells. These cells are highly similar to ES cells that have high
differentiation ability. These are also able to maintain the normal telomere length. These
cells can also differentiate in to the three germ layers that can also aggregate to form
embryoid body. These three germ layers ectoderm, mesoderm and endoderm can be
differentiated in to the different kinds of cells. So, we can also make the pancreatic beta
cells form these cells that can be used for the treatment of diabetes. This process is also
shown in the Figure 1.
Therapeutic cloning
The transfer nucleus of somatic cell from breast tissue into a donor oocyte from which
nucleus has re-moved is used to clone mammalian species. The oocyte is re-placed by
nucleus transfers genetic info of donor. This method used to clone Dolly sheep.
Blastocysts can establish in vitro from oocytes and ESC that genetically identical to
donor. The produced cells from embryonic stem cells established have organized supply
of oocytes to produce therapy for diabetes. Then these cells (ESs) are collected at the
embryonic stage. This development will avoid need of therapeutic cloning as shown in
Figure.
Fig.11: Oocyte is injected with the donor nucleus and developed
in to blastocyst stage in vitro and then embryonic stem cells are
collected at blastocyst stage.

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Mesenchymal stem cell therapy
Stem Cell therapy provide handsome alternative to islet cell transplantation in type 2
diabetic patients. Mesenchymal stem cell therapy is best among autologous adult stem
cells. Mesenchymal stem cells are less pluripotent than embryonic stem cells it renders
the effciency of MSCs to be differentiated into insulin secreting stem cells. Moreover,
MSCs can be isolated different sources like umbilical cord, bone marrow and pancreatic
stroma. MSCs can be obtained from the patient for autologous transplant. This of course
can also be the case for ESCs if reproductive cloning techniques are followed; however,
autologous MSCs from diabetic patients are still remarkably different from ESCs,
because of prolonged exposure to hyperglycemia. Studies in transgenic mice showed that
stem cells engineered to produce insulin did much more efficiently in hyperglycemic
environment. MSCs are niche cells. Their traditional role in the bone marrow is the
formation of the stroma and facilitation of growth, differentiation, and engraftment of
HSCs.
Islets derived from human fetal pancreatic progenitor cells
From 10 to 12 weeks post conception pancreas is composed of many tubes like structures
that are confined within loose mesenchymal stroma. These tubes like structures are
composed epithelial cells that are CD133 positive but insulin negative that indicate
progenitor cells. After digestion with XI collagenase, the mesenchymal tissue was
destroyed and islet-like structures were harvested. The progenitor containing clusters
adhered after 24 hours and the progenitor cells began expanding. These cells exhibited
monolayer growth and proliferated quickly in medium containing bFGF, EGF and LIF,
and confluent cells were epithelial-like.
Conclusion
By using these different kinds of stem cell technologies, we can make the insulin
producing cells that will be helpful in the cure of diabetes that is worldwide disease. Out
of all the stem cells fetal pancreatic cells are the best-known stem cells that have high
efficiency than any other stem cells. Human fetal pancreatic stem cells have excellent
capacity for proliferation; these may be induced to differentiate into insulin-producing
cells resulting in the formation of islet-like structures in vitro. these are capable of
secreting insulin and help to reduce hyperglycemia aіer transplantation in diabetic
animals and resulted islets might become a potential source for islets transplantation in

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treatment for diabetes. In future, the time is near when there will be the fully cure of
diabetes that is seem to be the cause of the most deaths now.
Stem cell therapy for treatment of Parkinson’s disease
Introduction
Parkinson’s disease (PD) is a progressive neurodegenerative disorder resulting from the
loss of dopamine-producing neurons (DAn) in the substantia nigra pars compacta (SNpc).
In addition, PD patients present with the deposition of α-synuclein-positive protein
aggregates called Lewy bodies and neuro-inffammation in various brain regions, further
contributing to the progression of the disease. The loss of SNpc DAn triggers the
recognizable primary motor symptoms, including tremor, rigidity and bradykinesia.
However, the pathology of PD is now known to extend beyond the nigrostriatal
dopaminergic pathway itself, leading to a number of secondary motor and non-motor
symptoms that can be just as debilitating. Although the precise etiology of PD is still
unknown, a variety of pathogenic mechanisms have been proposed. These may include
the loss of trophic support, excessive release of oxygen free radicals, a dysfunctional
mechanism of protein degradation; abnormal kinase activity and impairment of
mitochondrial function.A variety of treatment options are available to help manage motor
symptoms. These include medications in the form of the dopamine precursor levodopa
(L-dopa), dopaminergic agonists, or inhibitors of dopamine breakdown (catechol-O-
methyl transferase and monoamine oxidase inhibitors) or surgical procedures such as
deep brain stimulation (DBS) . With time, however, these treatments cease to be
effective, and some of them are known to develop unpleasant side effects, such as
dyskinesias. Most importantly, these treatments are not a cure. They are not reparative of
basal ganglia circuitry, nor capable of stopping the disease from progressing. For this
reason, alternative treatment options are currently being investigated, among them is
particularly interesting the cell replacement therapy (CRT). Here we summarize general
approaches for experimental and clinical applications of stem cell therapy, discussing the
common issues, different strategies and how they are being developed as a possible
treatment options for Parkinson’s disease. [35]
Transplants of human fetal ventral mesencephalic tissue
Transplants of human fetal ventral mesencephalic (hfVM) tissue have been developed in
the clinic for more than 30 years for PD treatment. these grafts contain immature

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midbrain DAn and their progenitors, which are generally transplanted into the striatum
(the target region of nigral DAn) where they are expected to release and replenish
dopamine levels.Preclinical studies performed in the 1970s and 1980s in animal models
of PD demonstrated that DAn obtained from the fetal midbrain were able to survive
transplantation in animal models of PD, integrate into host tissue, release dopamine and
improve motor function. With this background, several groups were able to conduct open
label clinical trials in PD patients, providing proof of principle that hfVM grafts can be an
effcient and safe treatment option for PD. However, two double-blind, placebo-controlled
clinical trials failed to meet their primary endpoint, and the overall results obtained from
all trials were fairly inconsistent, both between and within trials. For this reason, several
limiting challenges are still being faced in turning fetal VM grafts into a comparative
treatment option for PD. the first challenge is to avoid further inconsistencies in the
results, which can be achieved by establishing better standardization procedures and
improved trial design. The second challenge is the probability of the host developing an
immune reaction, since all the transplants are allogenic. The third, and arguably the
biggest, is the ethical concerns with using fetal tissue, and the diffculty in obtaining
enough tissue for a successful transplant. To help overcome these challenges, the
European consortium TRANSEURO (www.transeuro.org.uk), a multicenter clinical trial
that is currently working to analyze the feasibility and effectiveness of transplantation of
human fetal cell suspensions in PD patients, in hopes of providing more consistent results
for a better understanding of the potential therapeutic benefit (Table 1).Interestingly, in a
recently published study it has shown that grafts of hfVM can survive for at least 24 years
inside the denervated putamen of a Parkinson’s patient, with no signs of inflammation.
the patient had clinically improved during the first decade post-implantation, although
eventually this improvement began decreasing, indicating that the transplant was no
longer functional. The histopathological analysis showed that approximately 12% of the
neurons of the graft were positive for α-synuclein, reflecting the transfer of the host brain
pathology to the implanted neurons.
Human stem cell-based therapies for PD treatment
Due to the above limitations of using fetal tissue, important research efforts are currently
underway to find alternative types of cells for transplantation in PD. Several sources have
been explored, and the most promising so far have been stem cells. Stem cells are
undifferentiated cells that have the ability to differentiate to more specialized cell types.

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Because of these properties, they are currently considered the best option for developing a
uniform source of DAn to be used for cell replacement therapy.
In recent years it has made enormous progress in this field, which has made it
possible to obtain human dopaminergic precursors from different types of stem cells such
as: human Embryonic Stem Cells (hESCs), human induced Pluripotent Stem Cells
(hiPSCs), human Neural Stem Cells (hNSCs) from fetal or adult brains, and even human
Mesenchymal Stem Cells (hMSCs) by transdifferentiation (Figure 1). Basically, stem
cells can be classified as pluripotent, when they retain the ability to differentiate to all cell
types, or multipotent, when they are more specialized and give rise to a specific cell line.
In general, in the research field it is assumed that in order for the stem cells to become a
clinically competitive treatment option, these cells need to be equivalent to those of hfVM
tissue. And aier grafting they must be able to survive, re-innervate the striatum and
integrate into the neural circuitry of the host. They also have to significantly improve
motor symptoms, cause no side effects and meet a number of safety requirements, such as
eliminating the risk of tumor formation, immune response and the development of
dyskinesias.
Human multipotent stem cells
Human neural stem cells
Human Neural Stem Cells (hNSCs) are multipotent stem cells with the ability to generate
all neural cells of the CNS. they can be obtained from fetal, neonatal and adult brains or
from the directed differentiation of pluripotent stem cells. In theory, the human VM
neural precursors are considered the ideal candidates for cell therapies in PD, but as
mentioned above, their use is very limited. Furthermore, they present poor growth
potential, unstable phenotypes (especially upon repeat passage), and survive poorly in the
brain aier grafting.
Different techniques have been developed to optimize the expansion of these cells,
including the formation of neurospheres in the presence of growth factors such as basic
Fibroblast Growth Factor (bFGF) and Epidermal Growth Factor (EGF) or the
transduction with immortalizing genes such as v-Myc, c-Myc or TERT. Furthermore, in a
different approach an effcient method involving the addition of Wnt5a showed a 6-fold
increase in the amount of midbrain DAn obtained, as compared to the starting VM
preparation.

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However, currently there are several clinical trials involving the use of hNSCs of
different origin. the company Celavie Bioscences, LLC, is conducting a clinical trial
consisting in the intraputaminal injection in PD patients of undifferentiated human fetal
stem cells (OK99 cell line) obtained from a fetal brain tissue and growth in a bioreactor.
This is a phase 1, open-label and safety study which aims to analyze the potential effcacy
of this type of grafting for PD treatment (Table 1).
Table1: Human stem cells used currently in clinical trials for treatment of
Parkinson’s disease.