A BRIEF INFORMATION REGARDING STEM CELLS

1. By,
Sukrutha .J

2. Contents
 Definition
 Properties
 Timeline
 Characterization
 Stem cell markers
 Sources and culture
 Stem cell debate
 Ethical issues
 Applications
 Indian scenario
 Future of stem cells
 References

3. Stem cells are undifferentiated biological cells that can differentiate into specialized
cells and can divide (through mitosis) to produce more stem cells.
 They are found in multicellular organisms.
What makes a cell a stem cell?
 There is a classical criteria for a stem cell, it includes
the following basic properties-
1.self-renewal: the ability to go through numerous cycles
of cell division while maintaining the undifferentiated state. Two mechanisms exist to
ensure this property-
Obligatory asymmetric replication
Stochastic differentiation
2.Potency: the capacity to differentiate into specialized cell types.
What are stem cells?

4. Mode of stem cell differentiation-(self renewal)
Obligatory asymmetric division
Stochastic differentiation

5. Potency specifies the differentiation
potential (the potential to differentiate into
different cell types) of the stem cell

6. 1981, Mouse beginnings
Martin Evans of Cardiff University, UK, then at the University of Cambridge, is first
to identify embryonic stem cells– in mice.
1997, Dolly the sheep
Ian Wilmut and his colleagues at the Roslin Institute, Edinburgh unveil Dolly the
sheep, the first artificial animal clone. The process involves fusing a sheep egg with
an udder cell and implanting the resulting hybrids into a surrogate mother sheep.
Researchers speculate that similar hybrids made by fusing human embryonic stem
cells with adult cells from a particular person could be used to create genetically
matched tissue and organs.
1998, Stem cells go human
James Thomson of the University of Wisconsin in Madison and John Gearhart of
Johns Hopkins University in Baltimore, respectively, isolate human embryonic stem
cells and grow them in the lab.
2001, Bush controversy
US president George W. Bush limits federal funding of research on human embryonic
stem cells because a human embryo is destroyed in the process. But Bush does allow
continued research on human embryonic stem cells lines that were created
before the restrictions were announced
THE STEM CELL TIMELINE-

7. 2005, Fraudulent clones
Woo Suk Hwang of Seoul National University in South Korea reports that his team has
used therapeutic cloning – a technique inspired by the one used to create Dolly –
to create human embryonic stem cells genetically matched to specific people.
Later that year, his claims turn out to be false.
2006, Cells reprogrammed
Shinya Yamanaka of Kyoto University in Japan reveals a way of making embryonic-like
cells from adult cells – avoiding the need to destroy an embryo. His team reprograms
ordinary adult cells by inserting four key genes– forming “induced pluripotent stem
cells”.
2007, Nobel prize
Evans shares the Nobel prize for medicine with Mario Capecchi and Oliver Smithies
for work on genetics and embryonic stem cells.
2009, Obama-power
President Barack Obama lifts 2001 restrictions on federal funding for human
embryonic stem cell research.
2010, Spinal injury
A person with spinal injury becomes the first to receive a medical treatment derived
from human embryonic stem cells as part of a trial by Geron of Menlo Park,
California, a pioneering company for human embryonic stem cell therapies.

8. 2012, Blindness treated
Human embryonic stem cells show medical promise in a treatment that eases
blindness.
2012, Another Nobel
Yamanaka wins a Nobel prize for creating induced pluripotent stem cells, which
he shares with John Gurdon of the University of Cambridge.
2013, Therapeutic cloning
Shoukhrat Mitalipov at the Oregon National Primate Research Center in
Beaverton and his colleagues produce human embryonic stem cells from fetal
cells using therapeutic cloning – the breakthrough falsely claimed in 2005.
2014, Pre-embryonic state
Charles Vacanti of Harvard Medical School together with Haruko Obokata at
the Riken Center for Developmental Biology in Kobe, Japan, and colleagues
announced a revolutionary discovery that any cell can potentially be rewound to
a pre-embryonic state – using a simple, 30-minute technique.

9. 2014, Therapeutic cloning – with adult cells
Teams led by Dieter Egli of the New York Stem Cell Foundation and Young
Gie Chung from CHA University in Seoul, South Korea, independently
produce human embryonic stem cells from adult cells, using
therapeutic cloning.
• Egli’s team use skin cells from a woman with diabetes and demonstrate that
the resulting stem cells can be turned into insulin-producing beta cells. In
theory, the cells could be used to replace those lost to the disease.
2014, Human trials
Masayo Takahashi at the same Riken centre is due to select patients for
what promises to be the world’s first trial of a therapy based on induced
pluripotent stem cells, to treat a form of age-related blindness.
⃰NOTE(about the dolly)-
The production of Dolly showed that genes
in the nucleus of such a mature
differentiated somatic cell are still capable of
reverting to an embryonic totipotent state,
creating a cell that can then go on to develop
into any part of an animal.

10. CHARACTERIZATION OF STEM CELLS-
Flow Cytometric Assays
Although MSCs are not easily defined on the basis of
their cell surface antigen, flow cytometry can be useful for
MSC characterization in some cases.
For instance, simple assays of forward and side scatter
can provide information on the proportion of smaller,
rapidly self-renewing (RS)-type cells to slowly replicating
(SR) cells in a population of MSCs. RS cells typically
exhibit a far lower forward and side scatter profile
compared with the larger, and more complex, SR cells.
⃰Mesenchymal stem cells

11. Clonogenic Assays
One of the most important assets of MSCs is their ability to self-renew in culture.
Although the proliferative capacity of MSCs can be evaluated by a number of means,
including labeled nucleotide incorporation, hemocytometer counts, and flow
cytometry, the most widely accepted method is an appraisal of CFU potential.
In the CFU assay, the culture of MSCs is recovered by trypsinization and the cells are
counted by hemocytometer. One hundred cells are then plated into a 15-cm tissue
culture dish containing CCM and allowed to adhere and proliferate under normal
conditions of expansion.
After 3 weeks, the CFU cultures are washed, fixed, and
stained with Crystal Violet.
The colonies are then counted and the plating efficiency determined. Although there
are more complex variations on the CFU assay utilizing single-cell plating in a 96-well
format, the standard CFU assay remains a consistently reliable measure of replication
potential for MSC cultures.
⃰Colony Forming Unit ⃰Complete Culture Medium

12. Osteogenic differentiation assay
MSCs are defined by their potential to differentiate into mineralizing osteoblast-like
cells in vitro.
This is achieved by incubation of a confluent monolayer of MSCs in the presence of
osteogenic medium for 10–21 days, after which time the mineralized monolayer can be
evaluated by Alizarin Red S (ARS) staining.

13. EPIGENETICS IN STEM CELL DIFFERENTIATION
Embryonic stem cells are capable of self-renewing and differentiating to the desired fate
depending on its position within the body.
•Stem cell homeostasis is maintained through epigenetic mechanisms that are highly
dynamic in regulating the chromatin structure as well as specific gene transcription
programs.
•Epigenetics has been used to refer to changes in gene expression, which are heritable
through modifications not affecting the DNA sequence.
•The mammalian epigenome undergoes global remodeling during early stem cell
development that requires commitment of cells to be restricted to the desired lineage.
•There has been multiple evidence suggesting that the maintenance of the lineage
commitment of stem cells are controlled by epigenetic mechanisms such as DNA
methylation, histone modifications and regulation of ATP-dependent remolding
of chromatin structure.
•Based on the histone code hypothesis, distinct covalent histone modifications can lead to
functionally distinct chromatin structures that influence the fate of the cell.

14. STEM CELL MARKERS

15. MARKERS OF ESCs

16. SOURCES OF STEM CELLS
Embryonic stem cells
Adult stem cells
Induced pleuripotent stem cells
Neural crest stem cells
ES Cells
AS Cells
iPS CellsNCS Cells

17. EMBRYONIC STEM CELLS-
(ES) cells are isolated from the inner cell mass of
blastocysts of preimplantation-stage embryos.
These cells require specific signals to differentiate
to the desired cell type; if simply injected directly,
they will differentiate into many different types of
cells, resulting in a tumor derived from this abnormal
pluripotent cell development (a teratoma).
Mouse ES cells and human ES cells were both
originally derived and grown on a layer of mouse
fibroblasts (called “feeder cells”) in the presence of bovine
serum..
With their potential for unlimited expansion and
pluripotency, ES cells are a potential source for
regenerative medicine and tissue replacement after
injury or disease.
ES cell therapy is at an early stage, but human trials
were first carried out in 2010 on spinal injury victims.

18. The use of adult stem cells in research and
therapy is less controversial than ES cells, because
their production does not require the destruction
of an embryo.
ADULT STEM CELLS
Additionally, when adult stem cells are
obtained from the intended recipient (an
autograft) there is no risk of immune
rejection.
Adult stem cell treatments have been
successfully used for many years to treat
leukemia and related bone/blood cancers
through bone marrow transplants.

19. iPSCs are somatic cells that have been genetically reprogrammed to an embryonic stem
cell–like state by being forced to express genes important for maintaining the defining
properties of embryonic stem cells.
Currently, iPS cells are produced by inserting copies of four stem cell-associated genes;
Oct 3/4, Sox 2, Klf4, and c-Myc (or Oct 3/4, Sox 2, Nanog, and LIN28) into
specialized cells using viral vectors. Shinya Yamanaka produced the first iPS cells from
mouse cells in 2006.
INDUCED PLEURIPOTENT STEM CELLS-
Although additional research is needed, iPSCs are already useful tools for drug
development and modeling of diseases, and scientists hope to use them in transplantation
medicine.
In addition, tissues derived from iPSCs will be a nearly identical match to the cell
donor and thus probably avoid rejection by the immune system.
By studying iPSCs and other types of pluripotent stem cells, researchers may learn how to
reprogram cells to repair damaged tissues in the human body.

21. Neural crest cells are a temporary group of cells unique to vertebrates that arise
from the embryonic ectoderm cell layer, and in turn give rise to a diverse cell lineage
-- including melanocytes, craniofacial cartilage and bone, smooth
muscle, peripheral and enteric neurons and glia.
After gastrulation, neural crest cells are specified at the border of the neural plate
and the non-neural ectoderm. During neurulation, the borders of the neural plate,
also known as the neural folds, converge at the dorsal midline to form the neural tube.
Subsequently, neural crest cells from the roof plate of the neural tube undergo
an epithelial to mesenchymal transition, delaminating from the neuroepithelium and
migrating through the periphery where they differentiate into varied cell types.
The emergence of neural crest was important in vertebrate evolution because many
of its structural derivatives are defining features of the vertebrate clade.
NEURAL CREST STEM CELLS-

22. MIGRATION TO DIFFERENT CELL LINEAGES
Neural crest cells originating from different positions along the anterior-posterior axis
develop into various tissues. These regions of neural crest can be divided into four
main functional domains-
Cranial neural crest
Cranial neural crest migrates dorsolaterally to form the craniofacial mesenchyme that
differentiates into various cranial ganglia and craniofacial cartilages and bones.These cells
enter the pharyngeal pouches and arches where they contribute to the thymus, bones of
the middle ear and jaw and the odontoblasts of the tooth primordia.
Trunk neural crest
Trunk neural crest gives rise to two populations of cells. One group of cells fated to
become melanocytes migrates dorsolaterally into the ectoderm towards the ventral
midline. A second group of cells migrates ventrolaterally through the anterior portion of
each sclerotome. The cells that stay in the sclerotome form the dorsal root ganglia,
whereas those that continue more ventrally form the sympathetic ganglia, adrenal
medulla, and the nerves surrounding the aorta.

23. Vagal and sacral neural crest
The vagal and sacral neural crest cells develop
into the ganglia of the enteric nervous system
and the parasympathetic ganglia.
Cardiac neural crest
Cardiac neural crest develops into melanocytes,
cartilage, connective tissue and neurons of some
pharyngeal arches. Also, this domain gives rise to
regions of the heart such as the musculo-
connective tissue of the large arteries, and part of
the septum, which divides the pulmonary
circulation from the aorta.The semilunar valves
of the heart are associated with neural crest cells
according to new research.

24. WHAT IS NEURAL CRECT AND WHAT ARE THE STEM CELLS DERIVED FROM IT-
Abnormalities in neural crest development cause neurocristopathies, which include
conditions such as frontonasal dysplasia, Waardenburg-Shah syndrome, and DiGeorge
syndrome

27. THE CLASSIC STEM CELL DEBATE-

28. ETHICAL ISSUES CONCERNED IN USING STEM CELLS-

29. APPLICATIONS OF STEM CELLS-

30. Among the major hospitals that carry out stem cell transplant are AIIMS and the Army
Hospital in the capital, the Tata Memorial Centre and Jaslok Hospital in Mumbai and
CMC in Tamil Nadu's Vellore town. There are more than 500 centres in the world where
stem cell transplant is done
Stem cell transplant in India costs a fraction of what it does abroad but the country has
very few centres where the procedure can be done and not enough dedicated medical staff
A stem cell transplant can cost up to Rs.1 crore (approx $223,000) abroad, depending on
the type of procedure where as in India it costs Rs.10-20 lakh in private hospitals, while in
government hospitals it is much cheaper - Rs.3-6 lakh - depending on the type of
procedure.
Stem cell transplant has shown 50 percent success in treating certain kinds of cancers and
even more in other major conditions like beta thalassemia, a genetic blood disorder, and
aplastic anaemia, a condition where bone marrow does not produce sufficient new cells to
replenish blood cells. Stem cell transplant has shown 70-80 percent success in treating non-
malignant diseases
STEM CELL TRANSPLANTATION- INDIAN SITUATION

31. FUTURE OF STEM CELLS
•Cloning
•States still cannot decide
•Stem cell research overseas
•The blind might be able to see
•Stem cells for type 1 diabetes
•President Obama supports stem cell research

32. REFERENCES
1.National institutes of health-http://stemcells.nih.gov/info/Pages/Default.aspx
2.Euro stem cells- http://www.eurostemcell.org/factsheet/cord-blood-stem-cells-current-uses-and-future-challenges
3. R&D Systems- https://www.rndsystems.com/resources/articles/stem-cell-markers
4.http://onlinebiologydegree.org/
5.http://edition.cnn.com/2013/07/05/health/stem-cells-fast-facts/
6.http://www.bioon.com/z/stemcellind2015925/images/w4.pdf
7.http://www.explorestemcells.co.uk/pluripotentstemcells.html
8.http://www.ncbi.nlm.nih.gov/pubmed/18370085
9.http://www.stemcellsfreak.com/p/what-are-stem-cells.html
10.http://humrep.oxfordjournals.org/content/18/4/672.full
11.http://www.deccanherald.com/content/125387/stem-cell-transplant-cheap-india.html
Bibliography
13. STEM CELLS: SCIENTIFIC PROGRESS AND FUTURE RESEARCH DIRECTIONS
14. STEM CELLS HANDBOOK STEM CELLS HANDBOOK EDITED BY STEWART SELL, MD EDITED BY STEWART SELL, MD
15. STEM CELL CULURE EDITED BY JENNY MATHER
16. STEM CELL BIOENGINEERING BY BIJU PAREKKADAN, MARTIN YARMUSH
12.https://en.wikipedia.org/wiki/Stem_cell