Define what stem cells are The types of stem cells and their properties The methods of stem cell culture Medical roles of stem cells

1. STEM CELLS
PRESENTER: ZELEKE ENDALEW (MSc)
MODERATOR: Dr. Solomon Genet( PHD)
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2. Objectives
 Define what stem cells are
 List the types of stem cells and their
properties
 List the methods of stem cell culture
 List the medical roles of stem cells
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3. Outline
 Definition
 Types of stem cells
 Stem cells and organogenesis
 Stem Cell Culture
 Medical roles of stem cells
 Summary
 References
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4. What are Stem Cells?
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 A stem cell is a relatively unspecialized cell
that can reproduce itself indefinitely.
 able to differentiate into any cell of an organism
and have the ability of self-renewal.
 exist both in embryos and adult cells.

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 They have two unique properties that enable
them to do this
 They can divide over and over again to produce
new cells.
 As they divide, they can change into the other
types of cells that make up the body.

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What factors account for the stemness and
cell differentiation capability of ES cells?
 Expression of high levels of active telomerase.
 allows them to escape senescence.
 Expression of specific genes.
 A gene called Oct4, is exclusively expressed in
ES cells.
 Codes for a transcription regulator.
 A core set of transcription regulators defines and
maintains the ES cell state.

7. Types of Stem Cell
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There are three main types of stem cell:
 Embryonic stem cells
 Supply new cells for an embryo as it grows and
develops into a baby.
 Are said to be pluripotent,
 they can change into any cell in the body.

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https://pubmed.ncbi.nlm.nih.gov/16923385/

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Adult stem cells
 Supply new cells as an organism grows and to
replace cells that get damaged.
 Are said to be multipotent
 they can only change into some cells in the body,
not any cell.

10. Adult stem cells…
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 Blood (or 'haematopoietic') stem cells can only
replace the various types of cells in the blood.
 Skin (or 'epithelial') stem cells provide the
different types of cells that make up our skin
and hair.

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Induced pluripotent stem cells (iPS cells)
 Stem cells that scientists make in the
laboratory
 Induced’ means that they are made in the lab
by taking normal adult cells, like skin or blood
cells, and reprogramming them to become
stem cells.
 Just like embryonic stem cells, they are
pluripotent so they can develop into any cell
type

12. Stem cell Types …
 Stem cells are of many types, specialized
for the genesis of different classes of
terminally differentiated cells—
 intestinal stem cells for intestinal epithelium,
 epidermal stem cells for epidermis,
 hematopoietic stem cells for blood
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13.  Tissue Renewal That Does Not Depend on
Stem Cells:
 Insulin Secreting Cells in the Pancreas and
 Hepatocytes in the Liver
 Some types of cells can divide even though
fully differentiated
 allowing for renewal and regeneration without the
use of stem cells.
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14.  insulin-secreting cells (β cells) of the
pancreas
 Sequestered in cell clusters called islets of
Langerhans
 contain no obvious subset of cells specialized
to act as stem cells
 yet fresh β cells are continually generated
within them
 Renewal occurs by simple duplication of the
existing insulin-expressing cells, not by
means of stem cells.
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15. Hepatocyte
 renew by simple duplication of fully differentiated
cells
 normally live for a year or more and renew
themselves through cell division at a very slow rate
 Within a day or so after either sort of damage, a
surge of cell division occurs among the surviving
hepatocytes, quickly replacing the lost tissue.
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16.  If two-thirds of a rat’s liver is removed, for
example, a liver of nearly normal size can
regenerate from the remainder by
hepatocyte proliferation within about two
weeks.
 Both the pancreas and the liver contain
small populations of stem cells that can be
called into play as a backup mechanism for
production of the differentiated cell types in
more extreme circumstances.
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17.  Some Tissues Lack Stem Cells and Are Not
Renewable
 the auditory epithelium and the retinal
epithelium lack stem cells, and their sensory
receptor cells—the sensory hair cells in the
ear, the photoreceptors in the retina—are
irreplaceable.
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18. Fibroblasts
 When a tissue is injured, the fibroblasts nearby
proliferate, migrate into the wound and
 produce large amounts of collagenous matrix that
helps to isolate and repair the damaged tissue.
 are the easiest of cells to grow in culture
 a feature that has made them a favorite subject for
cell biological studies.
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19.  Figure: The family of connective tissue cells.
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20.  Figure : Control of fibroblast differentiation by the physical properties of the extracellular matrix.
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21. Myoblasts
 Precursors of skeletal muscle fibers.
 after a period of proliferation, they stop dividing,
 expression of a muscle-specifc genes required for
terminal differentiation
 Fuse with one another to form multinucleate skeletal
muscle fibers
 Once differentiation and cell fusion have occurred,
the cells do not divide
 The nuclei never again replicate their DNA.
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22.  Some Myoblasts Persist as Quiescent Stem Cells in
the Adult
 able of serving as myoblasts are retained
 small, flattened, and inactive cells lying in close
contact with the mature muscle cell
 The process of muscle repair by means of satellite
cells is, however, limited.
 exhaustion of their regenerative capacity
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23. myoSatellite cells
 Are the stem cells of adult skeletal muscle
 held in reserve in a quiescent state
 available when needed as a self-renewing
source of terminally differentiated cells.
 If the muscle is damaged or stimulated to
grow, these cells are activated to proliferate
 their progeny can fuse to repair the damaged
muscle or to allow muscle growth.
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24. BLOOD CELLS
 Are all generated from a common stem cell, located
in the bone marrow.
 hematopoietic stem cell is thus multipotent
 giving rise to all the types of terminally
differentiated blood cells as well as some other
types of cells, such as the osteoclasts in bone
 have limited life-spans and are produced throughout
life.
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25.  Bone Marrow Contains Multipotent Hematopoietic
Stem Cells, Able to Give Rise to All Classes of Blood
Cells
 The developing blood cells and their precursors,
including the stem cells, are intermingled with one
another
 The stem cells constitutes a tiny fraction of the bone
marrow population
 about 1 cell in 50,000–100,000
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26. Figure: Depiction of hematopoiesis cell lines
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27. Neural Stem Cells
 Stem cells capable of generating new neurons are
hard to find.
 Were thought to be absent many years.
 Can be manipulated in culture and used to
repopulate the central nervous system.
 It is now discovered that neural stem cells that
generate both neurons and glial cells do persist in
certain parts of the adult human brain.
 There is continuing turnover of neurons in the
hippocampus.
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28.  Neural stem cells can be obtained from The
hippocampal region or fetal brains
 Grown in culture, and then grafted back into
other sites in the brain,
 generate neurons appropriate to the new
location.
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29.  Neural stem cells from pluripotent stem
cells can be grafted into an adult brain.
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30.  Embryonic Stem (ES) Cells Can Generate Any Part
of the Body.
 A fertilized egg, or an equivalent cell produced by
nuclear transplantation can generate a whole new
multicellular individual.
 can be taken from an early mouse embryo, at the
blastocyst stage.
 A class of stem cells called embryonic stem cells
through cell culture can be driven from it.
 Originate from the inner cell mass of the early
embryo.
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31.  Figure : Production and pluripotency of ES cells.
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32.  iPS cells also be derived from adult human
cells and from various other differentiated cell
types besides fibroblasts.
 ES and iPS cells can be guided to generate
specific adult cell types and even whole
organs.
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33.  Cells of one specialized type can be forced to
transdifferentiate directly into another.
 Fibroblasts can be made to transdifferentiate
directly into heart muscle cells.
 By forcing expression of combination of
factors: Gata4, Mef2c, and Tbx5
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 Reprogramming focuses on the expression of
oncogenes such as
 octamer-binding transcription factor-4 (Oct4),
 Kruppel-like factor-4 (Klf4)
 c-myelocytomatosis (c-Myc)
 enhanced by a downregulation of genes
promoting genome stability, such as p53.

35. Stem Cells and organogenesis
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 After fertilization, the zygote usually divides
rapidly, or cleaves, to form many smaller cells.
 during this cleavage, the embryo does not
grow.
 a blastula—typically a solid or a hollow fluid-
filled ball of cells is then formed.

36. Stem Cells and
organogenesis…
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 Complex cell rearrangements called
gastrulation
 transform the blastula into a multilayered
structure containing. a rudimentary internal gut.
 Some cells of the blastula remain external,
constituting the ectoderm, which will give rise
to the epidermis and the nervous system.

37. Stem Cells and
organogenesis…
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 A blastocyst is formed after the fusion of sperm
and
ovum fertilization. Its inner wall is lined with
short- lived stem cells, namely, embryonic stem
cells.
 Blastocysts are composed of two distinct cell
types:
 The inner cell mass (ICM), develops into epiblasts
and induces the development of a foetus.
 the trophectoderm (TE).

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 The trophectoderm continues to develop
 forms the extraembryonic support structures
needed for the successful origin of the embryo,
such as the placenta
 As the TE begins to form a specialized support
structure, the ICM cells remain
undifferentiated, fully pluripotent and
proliferative
 allows them to form any cell of the organism

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 Human embryonic stem cells (hESCs) are
derived from the ICM.
 During the process of embryogenesis, cells
form aggregations called germ layers
 Each germ layers eventually give rise to
differentiated cells and tissues of the foetus
and, later on, the adult organism.

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 After hESCs differentiate into one of the germ
layers, they become multipotent stem cells,
 potency limited to only the cells of the germ layer.
 Pluripotent stem cells will then be formed all
over the organism as undifferentiated cells
 Able to proliferate by the formation of the next
generation of stem cells
 differentiation into specialized cells under certain
physiological conditions.

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 Although the derivation of ESCs without
separation from the TE is possible, such a
combination has growth limits.
 proliferating actions are limited, co-culture of
these is usually avoided.

42. Stem cell Culturing
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 Cells are placed in a culture dish filled with
culture medium.
 Passage is inefficient but popular process of
sub-culturing cells to other dishes.

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 Phenotypic pluripotency assays
 Recognizing undifferentiated cells is crucial in
successful stem cell therapy.
 Stem cells appear to have a distinct morphology
 a prominent nucleolus, high nucleus to cytoplasm
ratio
 Cells appear to be flat with defined borders, in
contrast to differentiating colonies.
 appear as loosely located cells with rough
borders.

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 When stem cells differentiate, the methylation
process silences pluripotency genes
 reduces differentiation potential, although other
genes may undergo demethylation to become
expressed.

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hESC derivation and media
 hESCs can be derived using a variety of
methods, from classic culturing to
microsurgery.
 hESC differentiation must be specified to avoid
teratoma formation.
 hESCs spontaneously differentiate into
embryonic
bodies (EBs).

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 EBs can be studied instead of embryos or
animals to predict their effects on early human
development.

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 The essential part of these culturing
procedures is a
separation of inner cell mass to culture future
hESCs.
 Particular attention must be taken in
controlling spontaneous differentiation.
 When the colony reaches the appropriate size,
cells must be separated.

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 Cell passaging is used to form smaller clusters of
cells on a new culture surface.
There are four important passaging procedures.
Enzymatic dissociation
 is a cutting action of enzymes on proteins and
adhesion domains that bind the colony.
 It is crucial to not leave hESCs alone after
passaging.
 Solitary cells are more sensitive and can easily
undergo cell death
 collagenase type IV is an example.

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Manual passage
 Focuses on using cell scratchers.
 The selection of certain cells is not necessary.
 This should be done in the early stages of cell
line derivation

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Trypsin utilization
 allows a healthy, automated hESC passage.
 However, there is a risk of decreasing the
pluripotency and viability of stem cells.

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Ethylene diamine tetraacetic acid (EDTA)
 indirectly suppresses cell-to-cell connections
by chelating divalent cations.
 Their suppression promotes cell dissociation.
 Prevent joining of cadherins (calcium dependent
adhesion) between cells
 Prevent clumping of cells
 Detaching adherent cells for passaging

52. Stem cell Culturing …
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 Stem cells require a mixture of growth factors
and nutrients to differentiate and develop.
 The medium should be changed each day.
 Traditional culture methods used for hESCs
are
 mouse embryonic fibroblasts (MEFs) as a feeder
layer
 bovine serum as a medium.

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 First feeder layer-free culture can be
supplemented with serum replacement,
combined with laminin .
 This causes stable karyotypes of stem cells
and pluripotency lasting for over a year.
 Initial culturing media can be
 serum (e.g. foetal calf serum (FCS)
 artificial replacement such as
 Synthetic serum substitute (SSS),
 Knockout Serum Replacement (KOSR), or
 StemPro.

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 Turning point in stem cell therapy
John B. Gurdon in 1962
 Challenged the dogma that the specialized
cell is irreversibly committed to its fate.
 Successfully cloning frogs by transferring a
nucleus from a frog’s somatic cells into an
oocyte.
 demonstrated that it is possible for a somatic
cell to again acquire pluripotency.

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Davis R.L. in 1987
 showed that reprogramming cells is possible,
and it can even be used to transform cells from
one lineage to another
 enforced expression of genes that were
originally found in myoblasts caused the
conversion of fibroblasts into myoblasts.
 myogenic differentiation 1 (Myod1)

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Shinya Yamanaka and Kazutoshi Takahashi In
2006
 discovered that it is possible to reprogram
multipotent adult stem cells to the pluripotent
state.
 avoided endangering the foetus’ life in the
process.

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 Four transcription factors (Oct-3/4, Sox2,
KLF4, and c-Myc).
 Are mainly expressed in embryonic stem cells
 could induce the fibroblasts to become pluripotent
 This new form of stem cells was named
iPSCs.
 One year later, the experiment also succeeded
with human cells.

59. Medical roles of Stem Cells
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 play a large role in developing restorative
medicine.
 The difference between a stem cell and a
differentiated cell is reflected in the cells’ DNA.

60. Medical roles of Stem cells…
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 Many serious medical conditions, such as birth
defects or cancer, are caused by improper
differentiation or cell division.
 Several stem cell therapies are possible,
among which are treatments for Heart failure,
retinal and macular degeneration, tendon
ruptures, and diabetes type 1

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Hematopoietic stem cell transplantation
 the most popular stem cell therapy.
 the most tissue-specific stem cells
 experimentally studied for more than 50 years.
 HSCs are responsible for the generation of all
functional haematopoietic lineages in blood.

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limitations
 There is a limited number of transplantable
cells
 an efficient way of gathering them has not yet
been found.
 problem with finding a fitting antigen-matched
donor for transplantation.
 Viral contamination or any immunoreactions
can cause a reduction in efficiency

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Stem cells as a target for pharmacological testing
 Stem cells can be used in new drug tests.
 Each experiment on living tissue can be
performed safely on specific differentiated cells fro
pluripotent cells
 If any undesirable effect appears, drug formulas
can be changed until they reach a sufficient level
of effectiveness.

64.  Figure: Use of iPS cells for drug discovery and for analysis and treatment of genetic disease.
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 Stem cells as an alternative for arthroplasty.
Rejuvenation by cell programming.
 using cell reprogramming technology in elder
animals and humans to erase marks of ageing
without removing the epigenetic marks of cell
identity.

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 Cells from aged individuals have
 different transcriptional signatures,
 high levels of oxidative stress,
 dysfunctional mitochondria, and
 shorter telomeres than in young cells

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 There is a hypothesis that when human or
mouse adult somatic cells are reprogrammed
to iPSCs, their epigenetic age is virtually reset
to zero.

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Cell-based therapies
 Stem cells can be induced to become a
specific cell type that is required to repair
damaged or destroyed tissues.

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 It is possible to generate healthy heart muscle
cells and later transplant them to patients with
heart disease.
 it can be possible to induce stem cells to
differentiate into insulin-producing cells

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Fertility diseases
 Scientists showed that it is possible to form
sperm from iPSCs
 Young adults at risk of losing their
spermatogonial stem cells (SSC), mostly
cancer patients can benefit from it

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 Therapy for incurable neurodegenerative
diseases
 Parkinson’s disease, Alzheimer’s disease
(AD), and Huntington disease.

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 Brain tissue from aborted fetuses was used on
patients with Parkinson’s disease
 showed that therapies with pure stem cells are an
important and achievable therapy.

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Stem cell use in dentistry
 There are stem cells in the periodontal
ligament
 capable of differentiating into osteoblasts or
cementoblasts
 their functions were also assessed in neural cells
 Stem cells of the root apical areas are able to
recreate periodontal ligament.

75. Medical role…
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 Adult stem cells are currently used to treat
some conditions, for example:
 Blood stem cells are used to provide a source of
healthy blood cells for people with some blood
conditions:
 Thalassaemia
 cancer patients who have lost their own blood stem
cells during treatment.

76. Medical roles…
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 Age-related macular degeneration (AMD)
 a condition in which cells in the retina of the eye
called retinal pigment epithelium (RPE) cells stop
working.
 stem cells could be used as a new form of
treatment in the future:
 using iPSCs to produce new RPE cells in the lab
that can then be put into a patient’s eye to replace
the damaged cells.

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An illustration showing how stem cells can be used to produce retinal pigment epithelium
(RPE) cells that can
be used to treat patients with age-related macular degeneration (AMD).
Image credit: Genome Research Limited

78. Medical roles
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 Stem cells could be used to generate new organs
for use in transplants:
 damaged organs can be replaced by obtaining
healthy organs from a donor
 donated organs may be 'rejected' by the body as
the immune system sees it as something that is
foreign.

79. Summary
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 A stem cell is a relatively unspecialized cell
that can reproduce itself indefinitely and act
as internal repair systems of the body.
 The three types of Stem cells are ESCs,
Adult stem cells and iPSC
 Stem cells can be isolated and be cultured
under appropriate conditions.
 Stem cells may be one way of generating
new cells that can then be transplanted into
the body to replace those that are damaged
or lost.

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 Differentiated stem cells can be reprogrammed
to give undifferentiated cells capable of
differentiation into different lineage.
 Stem cells when specialized gives the genesis
of different classes of terminally differentiated
cell.
 Stem cells have immense medical role including
the cure for neurodegenerative diseases.

81. References
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 Takahashi, K. and Yamanaka, S. (2006) ‘Induction of Pluripotent
Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by
Defined Factors’, Cell, 126(4), pp. 663–676. doi:
10.1016/j.cell.2006.07.024.
 Albert, B., Johnson, A. and Lewis, J., 2015. Molecular Biology of
The Cell. New York: Garland Science.
 Khan, F. A. et al. (2018) ‘Isolation, Culture, and Functional
Characterization of Human Embryonic Stem Cells: Current Trends
and Challenges’, Stem Cells International. Edited by V. Sorrenti,
2018, p. 1429351. doi: 10.1155/2018/1429351.
 Campbell’s molecular biology of the cell eleventh edition.
 Rahman, M. et al. (2016) ‘STEM CELL AND CANCER STEM CELL:
A Tale of Two Cells’, Progress in Stem Cell, 3(02), p. 97. doi:
10.15419/psc.v3i02.124.
 Li, Y., Xu, C. and Ma, T. (2014) ‘In vitro organogenesis from
pluripotent stem cells’, Organogenesis, 10(2), pp. 159–163. doi:
10.4161/org.28918.