Turning A Skin Into A Neuron

TURNING A SKIN CELL INTO A NEURON
This research project concerns the induction of pluripotent stem cells, originally mouse
fibroblasts, the growth and maintenance of Induced Pluripotent Stem Cell (iPS) colonies and
the later differentiation of these cells, demonstrating the possibility to turn a skin cell into a
neuron.
Aquest treball abarca la inducció de cèl·lules pluripotens induïdes (iPS), provinents de

fibroblasts de ratolí, el creixement i manteniment de colònies d’aquestes iPS i la seva
conseqüent diferenciació, tot demostrant la possibilitat de convertir un fibroblast en una
neurona.
Medicine is an evolving field. For thousands of years humans have wondered about
illness, cared for the ill and tried to cure the dying. This process has changed drastically
through time, but the basic principles it treats are the same.
In times like these, the needs of patients have changed, and continue to change.
Medicine adapts to these new needs and demands, producing every year more and more
incredible feats. The barrier between reality and science-fiction is sometime s hard to
distinguish: heart transplants, epigenetics, IVF, genetic engineering…
One of the biggest tasks for scientists now is understanding where illnesses come from,
and why they happen how and when they happen. Sometimes this is easy, but other times
scientists must go back to the moment in which sperm fertilises the ovum in order to fully
understand why a disease developed and, most importantly, to find a cure to this illness.
In recent years, huge medical advances have taken the field into the 21st century and
adapted to the new needs of the patient, but none more, perhaps, than stem cells.
Stem cells are cells that have the remarkable ability to turn into other types of
differentiated adult cells. This ability to turn into other types of cells has seen them adopt a
hugely important function in internal repair processes, turning into healthy specialised cells
when and where necessary. This happens when the stem cells divides. Upon division, the new
stem cells have the ability to stay as stem cells or to immediately differentiate into specialised
cells, depending on what is needed. Examples of differentiated are many, varying from brain
cells (neurons) to muscle cells (fibroblasts).
When these cells specialise, they act just like any other cell of that type, undergoing
any function that a “normal” cell of that type does. Stem cells can be found all around the
body, and behave slightly differently according to where they are. For instance, stem cells in
bone marrow are constantly dividing to replace dying cells. On the other hand, stem cells in
the heart adopt a more passive role, only dividing under very special circumstances and
conditions. Stem cells found in the adult body are abundant, and are called somatic stem cells.
However, there are other types of stem cells which are paramount in the creation of new
individuals. These embryonic cells are found, as the name suggests, in embryos, where a large
variety of specialised cells need to be made. When the embryo is just 3-5 days old, we have
what is called a blastocyst, which is made up of stem cells (mostly). These cells will give rise to
the entire body of the embryo, made up of many types of cells and forming organs, such as the
heart, the brain and sperm. It is the task of Embryonic Stem Cells to do so.
Research in the early 1980s allowed scientists to learn more about Human ESCs
through mice ESCs. This important research led to the derivation stem cells from embryonic
cells, leading to the discovery and the growth of a colony of these embryonic stem cells (ESCs)
in a laboratory. The embryos used in this research were used for in vitro fertilization purposes,
highliting a very important use of stem cells in modern day medicine. This research turned out
to be hugely useful years later, when scientists were able to turn normal adult cells into stem
cells. These were called Induced Pluripotent Stem Cells (iPSCs), which are the basis of my study.
Stem cells are defined by their three most important characteristics: their high
capacity to divide and renew (!), the fact that they are unspecialised (2) and that they can turn
into specialised cells types (3).
(1) The act of replicating many times is called proliferation, and has been a source of
massive research. A question that proliferation posed was why does these cells
replicate when they do, and what leads to this. For instance, researchers were able
to proliferate into embryonic stem cells for over a year without them
differentiating, but were unable to do so with adult stem cells. A better
understanding of these two fundamental questions would enable medicine to use
such information in order to understand and regulate embryonic development, as
well as understanding possible abnormalities in cell division that leads to cancer.
(2) One of the fundamental properties of a stem cell is that it does not have any
tissue-specific structures that allow it to perform specialized functions. For
example, a stem cell cannot work with its neighbours to pump blood through the
body (like a heart muscle cell), and it cannot carry oxygen molecules through the
bloodstream (like a red blood cell). However, unspecialized stem cells can give rise
to specialized cells, including heart muscle cells, blood cells, or nerve cells.
(3) When unspecialized stem cells give rise to specialized cells, the process is called
differentiation. While differentiating, the cell usually goes through several stages,
becoming more specialized at each step. Scientists are just beginning to
understand the signals inside and outside cells that trigger each step of the
differentiation process. The internal signals are controlled by a cell's genes, which
are interspersed across long strands of DNA and carry coded instructions for all
cellular structures and functions. The external signals for cell differentiation
include chemicals secreted by other cells, physical contact with neighbouring cells,
and certain molecules in the microenvironment. The interaction of signals during
differentiation causes the cell's DNA to acquire epigenetic marks that restrict DNA
expression in the cell and can be passed on through cell division.
Another big question posed by researchers is whether the signals that catalyse differentiation
are external, internal or spontaneous, and exactly why stem cells differentiate when they do.
Adult stem cells typically generate the cell types of the tissue in which they reside. For example,
a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of
blood cells. It is generally accepted that a blood-forming cell in the bone marrow—which is
called a hematopoietic stem cell—cannot give rise to the cells of a very different tissue, such as
nerve cells in the brain. Experiments over the last several years have purported to show that
stem cells from one tissue may give rise to cell types of a completely different tissue. This
remains an area of great debate within the research community. This controversy
demonstrates the challenges of studying adult stem cells and suggests that additional research
using adult stem cells is necessary to understand their full potential as future therapies.
As the reader may have picked up, there are various types of stem cells, depending on where
they come from. We, therefore, class these cells as either:
- Embryonic Stem Cells (ESC)

- Adult Stem Cells (ASC/SC)
- Induced Pluripotent Stem Cells (iPS/iPSCs)
Embryonic Stem Cells come from embryos. This fact, from very early on, poses an
ethical complication, which I will elaborate on later. Nowadays, most ESCs have come from in
vitro fertilization processes which have been donated to science. Understanding this and
appreciating the difference between this and deriving from a woman’s fertilised egg in her
own body is paramount for understanding the ethical challenges it faces.
When we grow cells in a laboratory, we speak of a cell culture. Our ESCs come from a fertilised
egg in a preimplantation stage; a very early stage embryo. This extremely primitive and under-
developed egg is then put into a culture dish, submersed in cell medium, which nourishes it.
These cells quickly divide, and spread over the surface of the dish.
Years ago, scientists coated the bottom layer of the dish with mouse embryonic skin
cells, upon which the ESCs stuck to. These mouse cells were called feeder layers, and released
nutrients into the dish so the ESCs grew better and faster. Researchers have now devised ways
to grow embryonic stem cells without mouse feeder cells. This is a significant scientific
advance because of the risk that viruses or other macromolecules in the mouse cells may be
transmitted to the human cells. The process of generating an embryonic stem cell line is
somewhat inefficient, so lines are not produced each time cells from the preimplantation-
stage embryo are placed into a culture dish. However, if the plated cells survive, divide and
multiply enough to crowd the dish, they are removed gently and plated into several fresh
culture dishes. The process of re-plating or sub-culturing the cells is repeated many times and
for many months. Each cycle of sub-culturing the cells is referred to as a passage. Once the cell
line is established, the original cells yield millions of embryonic stem cells. Embryonic stem
cells that have proliferated in cell culture for six or more months without differentiating, are
pluripotent, and appear genetically normal are referred to as an embryonic stem cell line. At
any stage in the process, batches of cells can be frozen and shipped to other laboratories for
further culture and experimentation. At various points during the process of generating
embryonic stem cell lines, scientists test the cells to see whether they exhibit the fundamental
properties that make them embryonic stem cells. This process is called characterization.
Scientists who study human embryonic stem cells have not yet agreed on a standard
battery of tests that measure the cells' fundamental properties. However, laboratories that
grow human embryonic stem cell lines use several kinds of tests, including:
- Growing and sub-culturing the stem cells for many months. This ensures that the
cells are capable of long-term growth and self-renewal. Scientists inspect the
cultures through a microscope to see that the cells look healthy and remain
undifferentiated.
- Using specific techniques to determine the presence of transcription factors that
are typically produced by undifferentiated cells. Two of the most important
transcription factors are Nanog and Oct4. Transcription factors help turn genes on
and off at the right time, which is an important part of the processes of cell
differentiation and embryonic development. In this case, both Oct 4 and Nanog are
associated with maintaining the stem cells in
an undifferentiated state, capable of self-
renewal.
- Using specific techniques to determine the
presence of particular cell surface markers
that are typically produced by
undifferentiated cells.
- Examining the chromosomes under a
microscope. This is a method to assess
whether the chromosomes are damaged or if
the number of chromosomes has changed. It
does not detect genetic mutations in the cells.
- Determining whether the cells can be re-
grown, or sub-cultured, after freezing,
Mice and Stem Cells
thawing, and re-plating.
- Testing whether the human embryonic stem cells are pluripotent by 1) allowing
the cells to differentiate spontaneously in cell culture; 2) manipulating the cells so
they will differentiate to form cells characteristic of the three germ layers; or 3)
injecting the cells into a mouse with a suppressed immune system to test for the
formation of a benign tumour called a teratoma. Since the mouse’s immune
system is suppressed, the injected human stem cells are not rejected by the mouse
immune system and scientists can observe growth and differentiation of the
human stem cells. Teratomas typically contain a mixture of many differentiated or
partly differentiated cell types—an indication that the embryonic stem cells is
capable of differentiating into multiple cell types.
Maintaining cells in the specific conditions needed to grow them and maintain them is
hugely delicate. A small change in temperature, salinity, pressure, concentration or dozens of
other criteria could lead to changes in the cells. As long as the embryonic stem cells in culture
are grown under appropriate conditions, they can remain undifferentiated (unspecialized). But
if cells are allowed to clump together to form embryoid bodies, they begin to differentiate
spontaneously. They can form muscle cells, nerve cells, and many other cell types. Although
spontaneous differentiation is a good indication that a culture of embryonic stem cells is
healthy, the process is uncontrolled and therefore an inefficient strategy to produce cultures
of specific cell types. So, to generate cultures of specific types of differentiated cells—heart
muscle cells, blood cells, or nerve cells, for example—scientists try to control the
differentiation of embryonic stem cells. They change the chemical composition of the culture
medium, alter the surface of the culture dish, or modify the cells by inserting specific genes.
Through years of experimentation, scientists have established some basic protocols or
"recipes" for the directed differentiation of embryonic stem cells into some specific cell types.
The definition of an Adult Stem Cell is unclear, and has opened up a whole field of
research. All our attempts to define ASCs are mere postulations, based on what we think they
may be. Today, we say that an adult stem cell is thought to be an undifferentiated cell, found
among differentiated cells in a tissue or organ. We do know that so-called adult stem cell can
renew themselves and can differentiate to yield some or all of the major specialized cell types
of the tissue or organ. The primary roles of adult stem cells in a living organism are thought to
be to maintain and repair the tissue in which they are found. Scientists also use the term
somatic stem cell instead of adult stem cell, where somatic refers to cells of the body (not the
germ cells, sperm or eggs). Unlike embryonic stem cells, which are defined by their origin (cells
from the preimplantation-stage embryo), the origin of adult stem cells in some mature tissues
is still under investigation.
Research on adult stem cells has generated a great deal of excitement. Scientists have
found adult stem cells in many more tissues than they once thought possible. This finding has
led researchers and clinicians to ask whether adult stem cells could be used for transplants. In
fact, adult hematopoietic, or blood-forming, stem cells from bone marrow have been used in
transplants for more than 40 years. Scientists now have evidence that stem cells exist in the
brain and the heart, two locations where adult stem cells were not at first expected to reside,
due to the apparent limitation of cells in these two important organs, and the lack of recovery
in illness to the organ. If the differentiation of adult stem cells can be controlled in the
laboratory, these cells may become the basis of transplantation-based therapies.
The history of research on adult stem cells began more than 60 years ago. In the 1950s,
researchers discovered that the bone marrow contains at least two kinds of stem cells. One
population, called hematopoietic stem cells, forms all the types of blood cells in the body. A
second population, called bone marrow stromal stem cells (also called mesenchymal stem cells,
or skeletal stem cells by some), were discovered a few years later. These non-hematopoietic
stem cells make up a small proportion of the stromal cell population in the bone marrow and
can generate bone, cartilage, and fat cells that support the formation of blood and fibrous
connective tissue.
In the 1960s, scientists who were studying rats discovered two regions of the brain
that contained dividing cells that ultimately become nerve cells. Despite these reports, most
scientists believed that the adult brain could not generate new nerve cells. It was not until the
1990s that scientists agreed that the adult brain does contain stem cells that are able to
generate the brain's three major cell types—astrocytes and oligodendrocytes, which are non-
neuronal cells, and neurons, or nerve cells.
Adult stem cells have been identified in many organs and tissues, including brain, bone
marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian
epithelium, and testis. They are thought to reside in a specific area of each tissue (called a
"stem cell niche"). In many tissues, current evidence suggests that some types of stem cells are
pericytes, cells that compose the outermost layer of small blood vessels. Stem cells may
remain quiescent (non-dividing) for long periods of time until they are activated by a normal
need for more cells to maintain tissues, or by disease or tissue injury.
Typically, there is a very small number of stem cells in each tissue and, once removed
from the body, their capacity to divide is limited, making generation of large quantities of stem
cells difficult. Scientists in many laboratories are trying to find better ways to grow large
quantities of adult stem cells in cell culture and to manipulate them to generate specific cell
types so they can be used to treat injury or disease. Some examples of potential treatments
include regenerating bone using cells derived from bone marrow stroma, developing insulin-
producing cells for type 1diabetes, and repairing damaged heart muscle following a heart
attack with cardiac muscle cells.
Scientists often use one or more of the following methods to identify adult stem cells:
(1) label the cells in a living tissue with molecular markers and then determine the specialized
cell types they generate; (2) remove the cells from a living animal, label them in cell culture,
and transplant them back into another animal to determine whether the cells replace (or
"repopulate") their tissue of origin.
Importantly, scientists must demonstrate that a single adult stem cell can generate a
line of genetically identical cells that then gives rise to all the appropriate differentiated cell
types of the tissue, an experience I underwent too. To confirm experimentally that a putative
adult stem cell is indeed a stem cell, scientists tend to show either that the cell can give rise to
these genetically identical cells in culture, and/or that a purified population of these candidate
stem cells can repopulate or reform the tissue after transplant into an animal.
As indicated above, scientists have reported that adult stem cells occur in many tissues
and that they enter normal differentiation pathways to form the specialized cell types of the
tissue in which they reside. Normal differentiation pathways of adult stem cells. In a living
animal, adult stem cells are available to divide for a long period, when needed, and can give
rise to mature cell types that have characteristic shapes and specialized structures and
functions of a particular tissue. The following are examples of differentiation pathways of adult
stem cells that have been demonstrated in vitro or in vivo.
- Hematopoietic stem cells give rise to all the types of blood cells: red blood cells, B
lymphocytes, T lymphocytes, natural killer cells, neutrophils, basophils, eosinophils,
monocytes, and macrophages.
- Mesenchymal stem cells have been reported to be present in many tissues. Those
from bone marrow (bone marrow stromal stem cells, skeletal stem cells) give rise
to a variety of cell types: bone cells (osteoblasts and osteocytes), cartilage cells
(chondrocytes), fat cells (adipocytes), and stromal cells that support blood
formation. However, it is not yet clear how similar or dissimilar mesenchymal cells
derived from non-bone marrow sources are to those from bone marrow stroma.
- Neural stem cells in the brain give rise to its three major cell types: nerve cells
(neurons) and two categories of non-neuronal cells—astrocytes and
oligodendrocytes.
- Epithelial stem cells in the lining of the digestive tract occur in deep crypts and
give rise to several cell types: absorptive cells, goblet cells, Paneth cells, and
entero-endocrine cells.
Skin stem cells occur in the basal layer of the epidermis and at the base of hair follicles.
The epidermal stem cells give rise to keratinocytes, which migrate to the surface of the skin
and form a protective layer. The follicular stem cells can give rise to both the hair follicle and to
the epidermis. A number of experiments have reported that certain adult stem cell types can
differentiate into cell types seen in organs or tissues other than those expected from the cells'
predicted lineage (i.e., brain stem cells that differentiate into blood cells or blood-forming cells
that differentiate into cardiac muscle cells, and so forth). This reported phenomenon is called
transdifferentiation. Although isolated instances of transdifferentiation have been observed in
some vertebrate species, whether this phenomenon actually occurs in humans is under debate
by the scientific community. Instead of transdifferentiation, the observed instances may
involve fusion of a donor cell with a recipient cell. Another possibility is that transplanted stem
cells are secreting factors that encourage the recipient's own stem cells to begin the repair
process. Even when transdifferentiation has been detected, only a very small percentage of
cells undergo the process.
In a variation of transdifferentiation experiments, scientists have recently

demonstrated that certain adult cell types can be "reprogrammed" into other cell types in vivo
using a well-controlled process of genetics. This strategy may offer a way to reprogram
available cells into other cell types that have been lost or damaged due to disease. For
example, one recent experiment shows how pancreatic beta cells, the insulin-producing cells
that are lost or damaged in diabetes, could possibly be created by reprogramming other
pancreatic cells. By "re-starting" expression of three critical beta cell genes in differentiated
adult pancreatic exocrine cells, researchers were able to create beta cell-like cells that can
secrete insulin. The reprogrammed cells were similar to beta cells in appearance, size, and
shape; expressed genes characteristic of beta cells; and were able to partially restore blood
sugar regulation in mice whose own beta cells had been chemically destroyed. While not
transdifferentiation by definition, this method for reprogramming adult cells may be used as a
model for directly reprogramming other adult cell types.
In addition to reprogramming cells to become a specific cell type, it is now possible to

reprogram adult somatic cells to become like embryonic stem cells (induced pluripotent stem
cells, iPSCs) through the introduction of embryonic genes. Please refer to iPSCs a few pages on,
where I elaborate on the cell type that gives way to my experiments. Thus, a source of cells can
be generated that are specific to the donor, thereby increasing the chance of compatibility if
such cells were to be used for tissue regeneration. However, like embryonic stem cells,
determination of the methods by which iPSCs can be completely and reproducibly committed
to appropriate cell lineages is still under investigation.
Many important questions about adult stem cells remain to be answered. They
include:
- How many kinds of adult stem cells exist, and in which tissues do they exist?
- How do adult stem cells evolve during development and how are they maintained
in the adult? Are they "leftover" embryonic stem cells, or do they arise in some
other way?
- Why do stem cells remain in an undifferentiated state when all the cells around
them have differentiated? What are the characteristics of their “niche” that
controls their behaviour?
- Do adult stem cells have the capacity to transdifferentiate, and is it possible to
control this process to improve its reliability and efficiency?
- If the beneficial effect of adult stem cell transplantation is a trophic effect, what
are the mechanisms? Is donor cell-recipient cell contact required, secretion of
factors by the donor cell, or both?
- What are the factors that control adult stem cell proliferation and differentiation?
- What are the factors that stimulate stem cells to relocate to sites of injury or
damage, and how can this process be enhanced for better healing?
iPSCs are differentiated adult cells that have been turned into a stem cell very similar
to Embryonic Stem Cells, having been forced to express genes and factors that are crucial to
these. Although these cells meet the defining criteria for pluripotent stem cells, it is not known
if iPSCs and embryonic stem cells differ in clinically significant ways. Mouse iPSCs were first
reported in 2006, and human iPSCs were first reported in late 2007. Mouse iPSCs demonstrate
important characteristics of pluripotent stem cells, including expressing stem cell markers,
forming tumours containing cells from all three germ layers, and being able to contribute to
many different tissues when injected into mouse embryos at a very early stage in development.
Human iPSCs also express stem cell markers and are capable of generating cells characteristic
of all three germ layers, endo, ecto and mesoderm.
Although additional research is needed, iPSCs are already useful tools for drug
development and modelling of diseases, and scientists hope to use them in transplantation
medicine. Viruses are currently used to introduce the reprogramming factors into adult cells,
and this process must be carefully controlled and tested before the technique can lead to
useful treatment for humans. In animal studies, the virus used to introduce the stem cell
factors sometimes causes cancers. Researchers are currently investigating non-viral delivery
strategies. In any case, this breakthrough discovery has created a powerful new way to "de-
differentiate" cells whose developmental fates had been previously assumed to be determined.
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. The iPSC strategy creates pluripotent
stem cells that, together with studies of other types of pluripotent stem cells, will help
researchers learn how to reprogram cells to repair damaged tissues in the human body.
Viral vectors have been used to transfer transcription factors (Oct4, Sox2, c-myc, Klf4,
and nanog) to induce reprogramming of mouse fibroblasts, neural stem cells, neural
progenitor cells, keratinocytes, B lymphocytes and meningeal membrane cells towards
pluripotency. Human fibroblasts, neural cells, blood and keratinocytes have also been
reprogrammed towards pluripotency. In this review we have discussed the use of viral vectors
for reprogramming both animal and human stem cells. Currently, many studies are also
involved in finding alternatives to using viral vectors carrying transcription factors for
reprogramming cells. These include using plasmid transfection, piggyback transposon system
and piggyback transposon system combined with a non viral vector system. Applications of
these techniques have been discussed in detail including its advantages and disadvantages.
Finally, current clinical applications of induced pluripotent stem cells and its limitations have
also been reviewed. Thus, this review is a summary of current research advances in
reprogramming cells into induced pluripotent stem cells.
It is worth noting that I chose viral-vectors in my experimentation, as they show to be

the most efficient, safe and best-known out of all the options, as expressed by Minal Patel and
Shuying Yang in “Advances in Reprogramming Somatic Cells to Induced Pluripotent Stem
Cells”:
“(d) Direct Reprogramming
More recently, somatic cells have been reprogrammed by addition of selective

transcription factors. These factors include Oct4, Sox2, c-myc, Klf4, Fbx15 and nanog. These
factors alone or in combination were directly introduced into somatic cells through viral vectors
or non-viral vector system and performed direct reprogramming of somatic cells into a
pluripotent cell. Successful direct reprogramming depends upon reprogramming of an adult
somatic nucleus to an embryonic state. To achieve complete reprogramming, DNA methylation,
histone modification and chromatin structure need to reprogram to a state which mimics
embryonic development. The mechanisms of direct reprogramming are a complex process and it
is unclear what role each transcription factor plays towards driving a somatic cell to
pluripotency. Though, recent studies have shed light on the role played by transcription factors
to promote pluripotency.
It has been shown that Oct4, Sox2 and Klf4 work together in combination to control a
set of gene expression and repression programs to maintain a pluripotent cell (Loh, et al., 2006,
Kim, et al., 2008). Expression of these factors including c-myc may lead to a sequence of
epigenetic events which influence chromatin modifications and changes in DNA methylation
which lead to a pluripotent cell. Once a somatic cell is introduced with these factors its
phenotype transforms to a partially reprogrammed state (Wernig, et al., 2007, Okita, et al.,
2007). Studies have shown that c-myc proteins may loosen chromatin structure of somatic cells,
thus rendering them a property similar to pluripotent cells (Takahashi & Yamanaka, et al., 2006).
This structure allows for Oct4 and Sox2 to bind to their target genes and the addition of Klf4
assists them to initiate a key set of ES cell genes in somatic cells (Wernig, et al., 2007). Oct4 and
Sox2 then establish an autoregulatory loop which maintains this pluripotent state in somatic
cells (Masui, et al., 2007).
Direct reprogramming is being widely studied because it presents a possibility of

creating a reprogrammed mature nucleus just by introducing a set of known genes. However,
majority of studies have used retroviruses to induce transduction which may result in random
insertion of genes within a genome. More recently, other techniques are being explored to
perform direct reprogramming.”
There are many ways in which human stem cells can be used in research and the clinic.
Studies of human embryonic stem cells will yield information about the complex events that
occur during human development. A primary goal of this work is to identify how
undifferentiated stem cells become the differentiated cells that form the tissues and organs.
Scientists know that turning genes on and off is central to this process. Some of the most
serious medical conditions, such as cancer and birth defects, are due to abnormal cell division
and differentiation. A more complete understanding of the genetic and molecular controls of
these processes may yield information about how such diseases arise and suggest new
strategies for therapy. Predictably controlling cell proliferation and differentiation requires
additional basic research on the molecular and genetic signals that regulate cell division and
specialization. While recent developments with iPS cells suggest some of the specific factors
that may be involved, techniques must be devised to introduce these factors safely into the
cells and control the processes that are induced by these factors.
Human stem cells are currently being used to test new drugs. New medications are
tested for safety on differentiated cells generated from human pluripotent cell lines. Other
kinds of cell lines have a long history of being used in this way. Cancer cell lines, for example,
are used to screen potential anti-tumor drugs. The availability of pluripotent stem cells would
allow drug testing in a wider range of cell types. However, to screen drugs effectively, the
conditions must be identical when comparing different drugs. Therefore, scientists must be
able to precisely control the differentiation of stem cells into the specific cell type on which
drugs will be tested. For some cell types and tissues, current knowledge of the signals
controlling differentiation falls short of being able to mimic these conditions precisely to
generate pure populations of differentiated cells for each drug being tested.
Perhaps the most important potential application of human stem cells is the
generation of cells and tissues that could be used for cell-based therapies. Today, donated
organs and tissues are often used to replace ailing or destroyed tissue, but the need for
transplantable tissues and organs far outweighs the available supply. Stem cells, directed to
differentiate into specific cell types, offer the possibility of a renewable source of replacement
cells and tissues to treat diseases including macular degeneration, spinal cord injury, stroke,
burns, heart disease, diabetes, osteoarthritis, and rheumatoid arthritis.
Heart muscle repair with adult stem cells. This figure is divided into two panels, with each illustrating a
possible means by which adult stem cells could help regenerate damaged heart muscle. On the left, a
mouse heart is being injected with a syringne of green-labelled adult stem cells. Next, a magnifying glass
shows a close-up of the damaged heart muscle cells (greyish-black) next to an area of healthy heart
muscle (pink). Arrows indicate that the adult stem cells are intermingling with the heart muscle fibres. On
the right, a mouse is shown being injected in the tail blood vessels with a syringe of pink human bone
marrow stem cells. The magnifying glass in this panel again shows a close-up of the damaged heart muscle
cells (greyish-black) next to an area of healthy heart muscle (pink). The pink human bone marrow stem
cells intermingle with the heart muscle fibres and the text indicates that they induce new blood vessel
formation in the damaged heart muscle and also cause proliferation of existing heart blood vessels .
For example, it may become possible to generate healthy heart muscle cells in the
laboratory and then transplant those cells into patients with chronic heart disease. Preliminary
research in mice and other animals indicates that bone marrow stromal cells, transplanted into
a damaged heart, can have beneficial effects. Whether these cells can generate heart muscle
cells or stimulate the growth of new blood vessels that repopulate the heart tissue, or help via
some other mechanism is actively under investigation. For example, injected cells may
accomplish repair by secreting growth factors, rather than actually incorporating into the heart.
Promising results from animal studies have served as the basis for a small number of
exploratory studies in humans (for discussion, see call-out box, "Can Stem Cells Mend a Broken
Heart?"). Other recent studies in cell culture systems indicate that it may be possible to direct
the differentiation of embryonic stem cells or adult bone marrow cells into heart muscle cells.
“Can Stem Cells Mend a Broken Heart?: Stem Cells for the Future Treatment of Heart Disease”
“Cardiovascular disease (CVD), which includes hypertension, coronary heart disease, stroke, and
congestive heart failure, has ranked as the number one cause of death in the United States
every year since 1900 except 1918, when the nation struggled with an influenza epidemic.
Nearly 2,600 Americans die of CVD each day, roughly one person every 34 seconds. Given the
aging of the population and the relatively dramatic recent increases in the prevalence of
cardiovascular risk factors such as obesity and type 2 diabetes, CVD will be a significant health
concern well into the 21st century.
Cardiovascular disease can deprive heart tissue of oxygen, thereby killing cardiac muscle cells
(cardiomyocytes). This loss triggers a cascade of detrimental events, including formation of scar
tissue, an overload of blood flow and pressure capacity, the overstretching of viable cardiac cells
attempting to sustain cardiac output, leading to heart failure, and eventual death. Restoring
damaged heart muscle tissue, through repair or regeneration, is therefore a potentially new
strategy to treat heart failure.
The use of embryonic and adult-derived stem cells for cardiac repair is an active area of research.
A number of stem cell types, including embryonic stem (ES) cells, cardiac stem cells that
naturally reside within the heart, myoblasts (muscle stem cells), adult bone marrow-derived
cells including mesenchymal cells (bone marrow-derived cells that give rise to tissues such as
muscle, bone, tendons, ligaments, and adipose tissue), endothelial progenitor cells (cells that
give rise to the endothelium, the interior lining of blood vessels), and umbilical cord blood cells,
have been investigated as possible sources for regenerating damaged heart tissue. All have
been explored in mouse or rat models, and some have been tested in larger animal models, such
as pigs.”
A few small studies have also been carried out in humans, usually in patients who are
undergoing open-heart surgery. Several of these have demonstrated that stem cells that are
injected into the circulation or directly into the injured heart tissue appear to improve cardiac
function and/or induce the formation of new capillaries. The mechanism for this repair
remains controversial, and the stem cells likely regenerate heart tissue through several
pathways. However, the stem cell populations that have been tested in these experiments vary
widely, as do the conditions of their purification and application. Although much more
research is needed to assess the safety and improve the efficacy of this approach, these
preliminary clinical experiments show how stem cells may one day be used to repair damaged
heart tissue, thereby reducing the burden of cardiovascular disease.
In people who suffer from type 1 diabetes, the cells of the pancreas that normally
produce insulin are destroyed by the patient's own immune system. New studies indicate that
it may be possible to direct the differentiation of human embryonic stem cells in cell culture to
form insulin-producing cells that eventually could be used in transplantation therapy for
persons with diabetes.
To realize the promise of novel cell-based therapies for such pervasive and debilitating
diseases, scientists must be able to manipulate stem cells so that they possess the necessary
characteristics for successful differentiation, transplantation, and engraftment. The following is

a list of steps in successful cell-based treatments that scientists will have to learn to control to
bring such treatments to the clinic. To be useful for transplant purposes, stem cells must be
reproducibly made to:
- Proliferate extensively and generate sufficient quantities of cells for making tissue.
- Differentiate into the desired cell type(s).
- Survive in the recipient after transplant.
- Integrate into the surrounding tissue after transplant.
- Function appropriately for the duration of the recipient's life.
- Avoid harming the recipient in any way.
Also, to avoid the problem of immune rejection, scientists are experimenting with
different research strategies to generate tissues that will not be rejected.
To summarize, stem cells offer exciting promise for future therapies, but significant
technical hurdles remain that will only be overcome through years of intensive research.
My practical phase, or experimentation, was undergone at the Barcelona Centre for

Regenerative Medicine of Barcelona (CMRB).
This process was intended to prove my hypothesis that a fibroblast, through the use
of Yamanaka’s Defined Factors, can be converted into a Induced Pluripotent Stem Cell (iPSCs
or IPS) and subsequently specialised into a neuron. Many of the experiments I underwent
were to check the validity of my main experiment, and these validating experiments
constituted the larger part of my time in the laboratory.
The first stage of my experiment was to obtain a sample of fibroblast cells. Due to
ethical a practical reasons, my sample was of mouse fibroblast cells, originally constituting
muscular tissue on the hind right-hand leg of a male mouse.
In this experiment we recurrently work with various different mediums, which are
prepared beforehand in a relatively large quantity. These mediums are:
- bFGF Solution.
- Collagenase t.IV Solution
- MEF
- iPSC Medium.
This sample was then harvested and prepared. These cells had to be made more
resistent to change and movement in order for us to work on them, and we had to prepare a
solution to keep them alive. This solution is called MEF solution. This MEF solution contained a
thick layer of gelatin, which had nutritional importance to our cells. After this, it was necessary
to fix our cells to their enviroment, so we added a different solution (see Step 5 of Day 0) to do
so. Once they were fixed, we removed this medium and replaced it with the MEF solution
which we had been working with before, in order to stabilise their condition, which lasted
approximately a week. This process of preparation is vital, and is called Transduction.
After Transduction, we had to prepare a medium in which we could reprogram our

fibroblasts. This medium is applied to dishes which are named MEF Culture Dishes. It is on
these dishes that we perform reprogramming. 24 hours after putting the MEFs on MEF Culture
Dishes we aspirate the whole medium, leaving our cells fixed to the walls of our wells. We then
insert to each well the iPSC Medium which we have prepared before. This well is where our
fibroblasts will turn into Induced Pluripotent Stem Cells.
At this point we observed our cells on a daily basis under the microscope to appreciate
any changes that might have been happening.
 To maintain sterile culture conditions, carry out all of the procedures using sterile
laboratory practices in a laminar flow hood.
 You can use the CytoTune®-iPS Sendai Reprogramming Kit to reprogram a wide range
of cell types in proliferative and quiescent states. However, the reprogramming
efficiency may vary among different cell types (~0.01%–1%).
 For successful reprogramming, transduce your cells using all four reprogramming
vectors.
Note: For successful reprogramming, all four Yamanaka factors (i.e., Oct4, Sox2, Klf4,
and c-Myc) need to be expressed in your host cell.
 Each CytoTune®-iPS Sendai Reprogramming Kit of four tubes supplies sufficient
reagents to transduce cells in 2 wells of a 6-well plate (5 × 105 cells/well) at an MOI of
3.
 The titer of each CytoTune® Sendai reprogramming vector is lot-dependent. For the
specific titer of your vectors, refer to the Certificate of Analysis (CoA) available on our
website.
 Viral titers can decrease dramatically with each freeze/thaw cycle. Avoid repeated
freezing and thawing of your reprogramming vectors. Viral titer is not guaranteed for
kits that have been refrozen or thawed.
 Prior to starting, ensure that the media are equilibrated to 37°C and appropriately
gassed.
 For positive control, we recommend performing a reprogramming experiment with
human neonatal foreskin fibroblast cells (strain BJ; ATCC no. CRL2522). Note that
experimental conditions may vary among target cells and need to be optimized for
each cell type. The example given in the following protocol does not guarantee the
generation of iPSCs for all cell types.
μ μ
1. To prepare 1 mL of 10 μg/mL bFGF solution, aseptically mix the following components:
Component Volume
bFGF 10 μg
DPBS without Calcium

980 μL
and Magnesium
10% KSR 10 μL
2. Aliquot and store at –20°C for up to 6 months.
1. Add DMEM/F-12 to Collagenase Type IV to make a 10 mg/mL stock solution. Gently

vortex to suspend and filter sterilize the solution. This solution can be aliquoted and
frozen at –20°C until use.
2. Make a working solution of 1 mg/mL Collagenase Type IV in DMEM/F-12. The working
solution can be used for 2 weeks if properly stored at 2–8°C (store in aliquots to avoid
repeated warming).
1. To prepare 100 mL of complete MEF/fibroblast medium, aseptically mix the following

components:
Component Volume
DMEM 89 mL
FBS, ESC-Qualified 10 mL
MEM Non-Essential
Amino Acids Solution, 1 mL
10 mM
2. Complete MEF medium can be stored at 2–8°C for up to 1 week.

1. To prepare 100 mL of complete human iPSC medium, aseptically mix the following
components:
Component Volume
KnockOut™ DMEM/F-12 78 mL
KnockOut™ Serum
20 mL
Replacement
MEM Non-Essential
Amino Acids Solution, 1 mL
10 mM
GlutaMAX™-I
1 mL
Supplement
β-mercaptoethanol,
100 μL
1000X
Penicillin-Streptomycin
1 mL
(optional)
bFGF (10 μg/mL)* 40 μL
2. * Prepare the iPSC medium without bFGF, and then supplement with fresh bFGF when
the medium is used.
3. Complete human iPSC medium can be stored at 2–8°C for up to 4 weeks.
1. Cover the whole surface of each new culture vessel with Attachment Factor (AF)
solution and incubate the vessels for 30 minutes at 37°C or for 1 hour at room
temperature.
2. Using sterile technique in a laminar flow culture hood, completely remove the AF
solution from the culture vessel by aspiration just prior to use. Coated vessels may be
used immediately or stored at room temperature wrapped in Parafilm® sealing film for
up to 24 hours.
Note: It is not necessary to wash the culture surface before adding cells or medium.
1. Remove the cryovial containing inactivated MEFs from the liquid nitrogen storage tank.
2. Briefly roll the vial between hands to remove frost, and swirl it gently in a 37°C water
bath.
3. When only a small ice crystal remains in the vial, remove it from water bath. Spray the
outside of the vial with 70% ethanol before placing it in the cell culture hood.
4. Pipet the thawed cells gently into a 15-mL conical tube.
5. Rinse the cryovial with 1 mL of pre-warmed MEF medium. Transfer the medium to the
same 15-mL tube containing the cells.
6. Add 4 mL of pre-warmed MEF medium dropwise to the cells. Gently mix by pipetting
up and down.
Note: Adding the medium slowly helps the cells to avoid osmotic shock.
7. Centrifuge the cells at 200 × g for 5 minutes.
8. Aspirate the supernatant and resuspend the cell pellet in 5 mL of pre-warmed MEF
medium.
9. Remove 20 μL of the cell suspension and determine the viable cell count using your
method of choice (e.g., Countess® Automated Cell Counter).
1. Centrifuge the remaining cell suspension (step 9, Thawing Gibco® MEFs) at 200 × g for
5 minutes at room temperature.
2. Aspirate the supernatant. Resuspend the cell pellet in MEF medium to a density of 2.5
× 106 cells/mL.
3. Aspirate the gelatin solution from the gelatin coated culture vessel.
4. Add the appropriate amount of MEF medium into each culture vessel (refer to Table 1,
below).
5. Into each of these culture vessels, add the appropriate amount of MEF suspension
(refer to Table 1, below).
Note: The recommended plating density for Gibco® Mouse Embryonic Fibroblasts
(Irradiated) is 2.5 × 104 cells/cm2.
6. Move the culture vessels in several quick back-and-forth and side-to-side motions to
disperse the cells
across the surface of the vessels.
7. Incubate the cells in a 37°C incubator with a humidified atmosphere of 5% CO2.

8. Use the MEF culture vessels within 3–4 days after plating.
Table 1. Amount of Inactivated MEFs Needed
Volume Volume of
Vessel Growth Number
of MEF
size area of MEFs
media suspension
96-
0.32 1.0 ×
well 2 0.1 mL 4 μL
cm /well 104/well
plate
24-
2 5.0 ×
well 2 0.5 mL 20 μL
cm /well 104/well
plate
12-
3.8 1.0 ×
well 1 mL 40 μL
cm2/well 105/well
plate
6-well 9.6 2.5 ×

2 mL 0.1 mL
plate cm2/well 105/well
60-
5.0 ×
mm 19.5 cm2 5 mL 0.2 mL
105
dish
100-
58.95 1.5 ×
mm 10 mL 0.6 mL
cm2 106
dish
25-
6.3 ×
cm2 25 cm2 5 mL 0.25 mL
105
flask
75-
1.9 ×
cm2 75 cm2 15 mL 0.75 mL
106
flask
The following protocol has been optimized for human neonatal foreskin fibroblast cells
(strain BJ; ATCC no. CRL2522). We recommend that you optimize the protocol for your
cell type.
1. 2 days before transduction, plate human neonatal foreskin fibroblast cells into two
wells of a 6-well plate at the appropriate density to achieve 5 × 105 cells per well on
the day of transduction (Day 0).
Note: We recommend about 80–90% confluency on the day of transduction. Because
overconfluency results in decreased transduction efficiency, we recommend replating
your cells to achieve 80–90% confluency if your cells have become overconfluent
during culturing.
2. Culture the cells for two more days, ensuring the cells have fully adhered and
extended.
3. On the day of transduction, warm 2 mL of fibroblast medium in a water bath.

4. Remove one set of CytoTune® Sendai tubes from the –80°C storage. Thaw each tube
one at a time by first immersing the bottom of the tube in a 37°C water bath for 5–10
seconds, and then removing the tube from the water bath and allowing it to thaw at
room temperature. Once thawed, briefly centrifuge the tube and place it immediately
on ice.
5. Add the indicated volumes of each of the four CytoTune® Sendai tubes (3 × 10 6 CIU
each; see the CoA for the appropriate volume) to 2 mL of fibroblast medium, pre-
warmed to 37°C. Ensure that the solution is thoroughly mixed by pipetting the mixture
gently up and down. Complete the next step within 5 minutes.
Aspirate the fibroblast medium from the cells, and add one half of the solution
prepared in Step 5 to each of the two wells. Place the cells in a 37°C, 5% CO2 incubator
and incubate overnight.
7. 24 hours after transduction, replace the medium with fresh fibroblast medium.
Note: Depending on your cell type, you should expect to see some cytotoxicity 24–48
hours post-transduction, which can affect >50% of your cells. This is an indication of
high uptake of the virus. We recommend that you continue culturing your cells and
proceed with the protocol.
8. Culture the cells for 6 more days, changing the spent medium with fresh fibroblast
medium every other day.
Note: Depending on your cell type, you may observe high cell density before Day
5. We do not recommend passaging your cells onto MEF culture dishes before 7 days
post-transduction.
9. One to two days before passaging the transduced fibroblasts onto MEF feeder-cells,
prepare 100-mm MEF culture dishes.
10. Seven days after transduction (Step 6), fibroblast cells are ready to be harvested and
plated on MEF culture dishes. Remove the medium from the fibroblasts, and wash
cells once with DPBS.
11. To remove the cells from the 6-well plate, use 0.5 mL of TrypLE™ Select reagent or
0.05% trypsin/EDTA following the procedure recommended by the manufacturer and
incubate at room temperature. When the cells have rounded up (1–3 minutes later),
add 2 mL of fibroblast medium into each well, and collect the cells in a 15-mL conical
centrifuge tube.
Note: Because the cells can be very sensitive to trypsin at this point, minimize trypsin
exposure time and incubate the cells at room temperature.
12. Centrifuge the cells at 200 × g for 4 minutes, aspirate the medium, and re-suspend the
cells in an appropriate amount of fibroblast medium.
13. Count the cells using the desired method (e.g., Countess® Automated Cell Counter),
and seed the MEF culture dishes with 5 × 104–2 × 105 cells per 100-mm dish and
incubate at 37°C, 5% CO2 incubator overnight.
Note: We recommend plating 5 × 104, 1 × 105, and 2 × 105 cells per 100-mm dish.
Depending on your cell type, you may need to plate most of your cells on the same
plate to ensure sufficient numbers of colonies.
Note: Set aside any remaining cells for RNA extraction to be used as a positive control
in the RT-PCR detection of the SeV genome.
14. 24 hours later, change the medium to iPSC medium, and replace the spent medium
everyday thereafter.
15. Starting on Day 8, observe the plates every other day under a microscope for the
emergence of cell clumps indicative of transformed cells (see Figure 1).
Note: For BJ fibroblasts, we normally observe colony formation on Day 12 post-
transduction. However,
depending on your cell type, you may need to culture for up to 4 weeks before seeing
colonies.
16. Three to four weeks after transduction, colonies should have grown to an appropriate
size for transfer. The day before transferring the colonies, prepare MEF culture plates
using Attachment Factor-coated 12- or 24-well plates.
Note: We typically harvest colonies closer to three weeks to avoid differentiation.
17. When colonies are ready for transfer, perform live staining using Tra1-60 or Tra1-81
for selecting reprogrammed colonies.
18. Manually pick colonies and transfer them onto prepared MEF plates.
By Day 21 post-transduction, the cell colonies on the MEF culture dishes will have
become large and compact, covering the majority of the surface area of the culture
dish. However, only a fraction of these colonies will consist of iPSCs, which exhibit a
hESC-like morphology characterized by a flatter cobblestone-like appearance with
individual cells clearly demarcated from each other in the colonies. Therefore, we
recommend that you perform live staining with Tra1-60 or Tra1-81 antibodies that
recognize undifferentiated hESCs.
Note: Although colonies of “transformed” cells may emerge as early as 7 days after
transduction, most of these colonies will not be correctly “reprogrammed” cells. iPSCs usually
emerge a little later (around day 14 post-transduction), resemble embryonic stem cells in
morphology, and express the cell surface markers Tra1-60 and Tra1-81.
Of the cells induced, we were able to identify three substantial iPS colonies. These are
identified as larger bodies in the cell medium, usually forming rounded shapes, as seen in the
pictures.
All images on the left-side of the page were taken with a 10x lens on the inverted
fluorescence microscope, with phase contrast (otherwise named Nomarski). All pictures on the
right-side were taken with the same microscope, but with a 20x lens. It is worth noting the
darker shade of the colonies in their centre. Initially, we couldn’t explain this, thinking it may
be a necrotic patch of cells, but later realised these cells had differentiated.
On day three one can observe a substantial growth in colony 2. Colonies one and three show a
feeble amount of intact iPS cells, due to the differentiated epicentre of the cell expanding and
differentiating its surroundings. The forecast for colonies one and two is dull and unpromising.
Although colony two does show a substantial amount of necrotic/differentiated cells in its
centre, upon comparison with day two one sees that this area has not grown, unlike the iPS
area which has, substantially.
Our final iPS colony
Seven days after transduction, only one colony has survived, or remained un-differentiated:
colony 2, as forecast.
By this time, colonies one and three had passed the barrier of iPS-to-differentiated cells, being
7/3, and had reached a proportion of 2/8, making them defunct.
Colony two is a relative success. The differentiated area has maintained in size, and the iPS
area has multiplied by a factor of three.
Another good sign is that colony two has changed its initial shape. Although it has maintained
its diagonal orientation, the left-part of the cell has a new vertex, which is a prosperous sign of
iPS capacity.
Day 1 Day 7
Once iPS colonies have been stabilised, samples of these are injected into mice,
forming teratomas. These cells, inside the mice, differentiate into many types of cells,
belonging to each of the three germ layers. This teratoma is then sampled, sliced and used as a
base for IF experimentation.
Immunofluorescence is a key technique in identifying the capacity of an iPS cell, and is

one of various tests researchers or scientists must undergo in order to check the capabilities of
the cells they have just transduced.
Immunofluorescence (IF) is a common laboratory technique, which is based on the use

of specific antibodies which have been chemically conjugated to fluorescent dyes.
These labelled antibodies bind directly or indirectly to cellular antigens (see below).
The technique has a number of different biological applications including evaluation of cells in
suspension, cultured cells, tissue, beads and in microarrays.
The fluorescent dye is subjected to short-wavelength, high energy light, which is

absorbed and emitted as light of a different wavelength. The emitted fluorescence has a lower
energy than the absorbed light, so the wavelength of the emitted light is longer than that of
the excitation light. The fluorescence can be visualized using fluorescence microscopy. The IF
technique allows for a visualization of the presence as well as the distribution of target
molecules in a sample.
Types of immunofluorescence
Primary (direct)
Primary, or direct, immunofluorescence uses a single antibody that is

chemically linked to a fluorophore. The antibody recognizes the target
molecule and binds to it, and the fluorophore it carries can be detected via
microscopy. This technique has several advantages over the secondary (or
indirect) protocol below because of the direct conjugation of the antibody to
the fluorophore. This reduces the number of steps in the staining procedure
making the process faster and can reduce background signal by avoiding some
issues with antibody cross-reactivity or non-specificity. However, since the
number of fluorescent molecules that can be bound to the primary antibody is
limited, direct immunofluorescence is less sensitive than indirect
immunofluorescence.
Secondary (indirect)
Secondary, or indirect, immunofluorescence uses two antibodies; the

unlabelled first (primary) antibody specifically binds the target molecule, and
the secondary antibody, which carries the fluorophore, recognises the primary
antibody and binds to it. Multiple secondary antibodies can bind a single
primary antibody. This provides signal amplification by increasing the number
of fluorophore molecules per antigen. This protocol is more complex and time
consuming than the primary (or direct) protocol above, but it allows more
flexibility because a variety of different secondary antibodies and detection
techniques can be used for a given primary antibody.
For the purposes of my experiment, I used secondary or indirect IF techniques, as it is

more cost effective and allows a more personalised and flexible result.
Above is a table with the antibodies used, as well as their origin, dilution and volume.
One must remember the basic function of an antibody in order to understand the process: an
antibody searches for a protein. Once we know which protein we’re looking for, we can choose
an antibody which will link onto it. Once our antibody has found the protein, we send another
antibody to look for the antibody previously bound on to the protein. The reader must also
remember that antibodies are, themselves, proteins. This secondary antibody is linked to a
fluorophore, which when irradiated which a known and specific wave-length emits a coloured
light.
As mentioned before, for an iPS to work fully it must be able to code for proteins in all
3 primary germ layers in an embryo: endoderm, ectoderm and mesoderm, which roughly
translates to glands, nerves and muscles for the purposes of this experiment.
The ability to give way to proteins for each of these three layers makes the cell
pluripotent, as it can give way to proteins from all parts of a human embryo, a human being.
To do this, knowledge of proteins is needed. Once we identify proteins that belong to

each individual layer, we can choose antibodies to link to these known proteins, and start IF.
In this image the sample was irradiated with a 488nm wavelength which excited the
fluorophore and emitted a green light. The blue colour shows the agent DAPI, which was
inserted to the sample to emit a blue light when genetic material (DNA) was found. The red
light comes from a secondary antigen, highlighting possible Alfa-1-Fetoprotein location.
Mk.1 shows a good result, with a noteworthy amount of the protein being found. A
large amount of the red was in fact the protein we were searching for, and indicated a good
capacity to code this protein. This, in turn, demonstrates the ability to code for endodermic
structure.
fluorophore and emitted a green light. The blue colour shows the agent DAPI, which was
inserted to the sample to emit a blue light when genetic material (DNA) was found. The red
light comes from a secondary antigen, highlighting possible Alfa-1-Fetoprotein location.
This sample shows a classic miss-leading result. Observe that the green patches,
supposed areas of the protein being examined, are greater than the red patches, areas where
it could possibly be. No doubt there was an external influencer to this result, a contaminator
most likely.
This being said, the final image does show areas of green and red at the same point,
making this result valid. This, in turn, demonstrates the ability to code for endodermic
structure.
fluorophore and emitted a red light, showing Alfa-Smooth-Muscle-Actin . The blue colour
shows the agent DAPI, which was inserted to the sample to emit a blue light when genetic
material (DNA) was found. The green light comes from a secondary antigen, highlighting
possible Alfa-Sarcomeric-Actin location.
Here we see a good result. Although an overruling amount of Alfa-Smooth-Muscle-

Actin, there is a small amount of rough-muscle tissue, too, making it able to code for
mesodermic structures.
fluorophore and emitted a red light, showing Alfa-Smooth-Muscle-Actin (smooth muscle) . The
blue colour shows the agent DAPI, which was inserted to the sample to emit a blue light when
genetic material (DNA) was found. The green light comes from a secondary antigen,
highlighting possible Alfa-Sarcomeric-Actin (striated muscle) location.
Here we see a complete dominance of smooth muscle capacity, in contrast with no

striated muscle presence. This is a valid result, but not very strong, as it does not show variety
in its capabilities. Nevertheless, it does show large amounts of one type of muscle, making it
able to code for mesodermic structures.
Here, contrarily, we see a complete dominance of striated muscle capacity, in contrast

with no smooth muscle presence. This is a valid result, but not very strong, as it does not show
variety in its capabilities. Nevertheless, it does show large amounts of one type of muscle,
making it able to code for mesodermic structures.
Here we see a complete dominance of striated muscle capacity, in contrast with no

smooth muscle presence. This is a valid result, but not very strong, as it does not show variety
Here we see a complete dominance of smooth muscle capacity, in contrast with no

striated muscle presence. This is a valid result, but not very strong, as it does not show variety
Here we see a good result, very similar to Mk.I. Although an overruling amount of Alfa-
Smooth-Muscle-Actin, there is a small amount of rough-muscle tissue, too, making it able to
code for mesodermic structures.
This is the best result. The results show solid capabilities to express both types of
protein types, making this sample very strong, and making it able to code very well for
mesodermic structures.
fluorophore and emitted a green light, showing Beta-III-Tubulin-TUJ1 . The blue colour shows
the agent DAPI, which was inserted to the sample to emit a blue light when genetic material
(DNA) was found. The red light comes from a secondary antigen, highlighting GFAP protein.
A perfect result. Very high capacity to express both proteins investigated, making it an
ideal candidate to code for endodermic structures.
Again, a perfect result. Very high capacity to express both proteins investigated, making
it an ideal candidate to code for endodermic structures.
Not as strong a result as the last, but again very strong. Moderately high capacity to
express both proteins investigated, making it a good candidate to code for ectodermic
structures.
An unbalanced result here, coding well for GFAP but not well for the other protein.
Nevertheless, there is a very small amount of green markings in the bottom left image, making
it a good candidate to code for ectodermic structures.
An unbalanced result here, like the last, coding well for GFAP but not well for the other
protein. It does not code for Beta-III-Tubulin-TUJ1, but does for GFAP, meaning in could code
for ectodermic structures, but only certain types.
An unbalanced result here, like the last, coding well for GFAP but not well for the other
protein. It does not code for Beta-III-Tubulin-TUJ1, but does for GFAP, meaning in could code
for ectodermic structures, but only certain types.
The last images, MAP2_Neurofilament Mk.I-IV, were of particular importance. My

hypothesis was: a fibroblast, through the use of Yamanaka’s Defined Factors, can be
converted into a Induced Pluripotent Stem Cell (iPSCs or IPS) and subsequently specialised into
a neuron. The last images showed my results on a specific layer, the layer I was most
interested in, ectoderm, or the layer in charge of neurone production.
After reading my results a few pages ago, my hypothesis have been proven right. It is
possible to turn a fibroblast, through the use of Yamanaka’s Defined Factors, into an
Induced Pluripotent Stem Cell (iPSCs or IPS) and subsequently specialise differentiate it into
a neuron.
This result is significant. This allows us to, theretically, get our own cells and turn the into
others, in particular neurons. Also, we saw we could express mesodermic tissue, which would
enable us to grow our own heart-cells. Many other types of cells, too. This is a huge leap into
the future of Medical Science, and one that has been invested in thoroughly, and with reason.
As I said in the introduction, th e problems medicine faces in the 21st century have changed,
and this technology adresses a key necessity in the public and gives a route to answering many
questions posed about the human body and its illnesses. The origin of disease is now much
closer to being discovered. Strenuous observation of how these iPS cells grow, differentiate or
die, could give way to answering these vital questions.
However, when doing this project, I felt, in more than one ocasion, chilled. The prospect
of this “unnatural” technology is raw and in a human sense brutal. We are now in a position
not only to solve questions, like we have done for many years now, but to re-formulate them
and the processes we are scrutenising. The days in which disease is delt with, endured and
accepted seem to be going or gone.
It is not hard to imagine how this new tool could reformulate life. If your heart was giving
in, we could mould a new heart from your own cells, painlessly and effortlessly. The same
applies to lungs, liver, kidneys. Donations would, in a sense, obsolete, and no longe necessary.
However, there is a good reason to why this hasn’t happened yet. These prospects are
hugely complicated, and not only from a scientifical point of view. Philosophically and ethically,
they are, some would argue, not viable. Inevitably, if these techniques were not heavily
controlled and regulated, we would be facing the prospect of immomrtal human-beings. This,
of course, is something that belongs to the future, one would hope.
This project has not only been curious, captivating and life-changing for me in a scientific
way, but also as a person. It has taught me self-values that I would like to promote, and has
opened my mind to wider prospects and ideas, which have given me many a restless night of
thinking. I would like to take this opportunity to thank the Barcelona Centre of Regenerative
Medicine for this life-moulding experience. I owe a great deal to their generosity, kindness and
willingness to ¡nspire a generation, as they have done for me.
I would also like to thank my tutor, Mª Pilar Isaiz Rubí, PhD, for her kind advice, patience
and untiring support; to Imperial College London in cooperation with the London International
Youth Sience Forum, for having me present my research at their forum; and to anyone who
reads this, thanking for taking time to read about this wonderful experience.
At the end of the week, once our cells had matured further, we were able to differentiate our
colonies into our desired cell-type. I, of course, chose neurons, which presented me with the
most beautiful picture yet and, may I say, the most spine-chilling one, too.
- CMRB Archives and documents

- Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors
by Kazutoshi Takahashi, Koji Tanabe, Mari Ohnuki, Megumi Narita, Tomoko Ichisaka,
Kiichiro Tomoda and Shinya Yamanaka
- Yamanaka factors critically regulate the developmental signaling network in mouse
embryonic stem cells byLiu X1, Huang J, Chen T, Wang Y, Xin S, Li J, Pei G, Kang J.
- Induced pluripotency: history, mechanisms, and applications by Matthias Stadtfeld
and Konrad Hochedlinger
- www.hindawi.com/journals/jir/2014/149316/
- http://www.ncbi.nlm.nih.gov/pubmed/12580000
- http://www.bioline.org.br/pdf?dv08185
- www.eurodiagnostica.com
- http://stemcells.nih.gov
- https://en.wikipedia.org/wiki/Stem_cell
- www.stemcellsinc.com
- http://www.medicalnewstoday.com/info/stem_cell/
- www.stemcells21.com/
- http://www.awaremednetwork.com/tag/mesenchymal-stem-cell/
- http://www.cell.com/cell/abstract/S0092-8674%2807%2901471-
7?_returnURL=http%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS00928
67407014717%3Fshowall%3Dtrue&cc=y=
- http://www.ncbi.nlm.nih.gov/pubmed/19030024
- https://embryology.med.unsw.edu.au/embryology/index.php/Stem_Cells_-_Induced

Turning A Skin Into A Neuron

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Turning A Skin Into A Neuron

Uploaded by

Copyright:

Available Formats

TURNING A SKIN CELL INTO A NEURON

TURNING A SKIN CELL INTO A NEURON

Aquest treball abarca la inducció de cèl·lules pluripotens induïdes (iPS), provinents de

- Embryonic Stem Cells (ESC)

In a variation of transdifferentiation experiments, scientists have recently

In addition to reprogramming cells to become a specific cell type, it is now possible to

It is worth noting that I chose viral-vectors in my experimentation, as they show to be

“(d) Direct Reprogramming

More recently, somatic cells have been reprogrammed by addition of selective

Direct reprogramming is being widely studied because it presents a possibility of

characteristics for successful differentiation, transplantation, and engraftment. The following is

My practical phase, or experimentation, was undergone at the Barcelona Centre for

After Transduction, we had to prepare a medium in which we could reprogram our

1. To prepare 1 mL of 10 μg/mL bFGF solution, aseptically mix the following components:

DPBS without Calcium

2. Aliquot and store at –20°C for up to 6 months.

1. Add DMEM/F-12 to Collagenase Type IV to make a 10 mg/mL stock solution. Gently

1. To prepare 100 mL of complete MEF/fibroblast medium, aseptically mix the following

2. Complete MEF medium can be stored at 2–8°C for up to 1 week.

bFGF (10 μg/mL)* 40 μL

7. Incubate the cells in a 37°C incubator with a humidified atmosphere of 5% CO2.

Table 1. Amount of Inactivated MEFs Needed

6-well 9.6 2.5 ×

3. On the day of transduction, warm 2 mL of fibroblast medium in a water bath.

Our final iPS colony

Immunofluorescence is a key technique in identifying the capacity of an iPS cell, and is

Immunofluorescence (IF) is a common laboratory technique, which is based on the use

The fluorescent dye is subjected to short-wavelength, high energy light, which is

Primary, or direct, immunofluorescence uses a single antibody that is

Secondary, or indirect, immunofluorescence uses two antibodies; the

For the purposes of my experiment, I used secondary or indirect IF techniques, as it is

To do this, knowledge of proteins is needed. Once we identify proteins that belong to

Here we see a good result. Although an overruling amount of Alfa-Smooth-Muscle-

Here we see a complete dominance of smooth muscle capacity, in contrast with no

Here, contrarily, we see a complete dominance of striated muscle capacity, in contrast

Here we see a complete dominance of striated muscle capacity, in contrast with no

Here we see a complete dominance of smooth muscle capacity, in contrast with no

The last images, MAP2_Neurofilament Mk.I-IV, were of particular importance. My

- CMRB Archives and documents

You might also like