Stem Cell Technology Notes
Stem Cell Technology Notes
Stem Cell
1. Introduction to Stem cells
Introduction
Stem cells have the remarkable potential to develop into many different cell types during early
life and growth.
They serve as an internal repair system, dividing essentially without limit to replenish cells for
an entire lifespan.
When a stem cell divides, each new cell has the potential either to remain a stem cell
(symmetric division) or become a cell with a more specialized function, such as a muscle cell, a
red blood cell, or a brain cell (asymmetric division).
Stem cells are distinguished from other cell types by two important characteristics.
First, they are unspecialized cells capable of renewing themselves through cell division,
sometimes after long periods of inactivity referred to as "quiescence".
Second, under certain physiologic or experimental conditions, they can be induced to become
tissue- or organ-specific cells with specialized functions
In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and
replace worn out or damaged tissues
In other organs, however, such as the pancreas and the heart, stem cells only divide under
special conditions
Stem cells are important for living organisms for many reasons
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In some adult tissues, such as bone marrow, muscle, and brain, discrete populations of adult
stem cells generate replacements for cells that are lost through normal wear and tear, injury, or
disease
Given their unique regenerative abilities, stem cells offer new potentials for treating diseases
such as diabetes, and heart disease
These are the cells in the quiescence, which is a These are the cells in senescence, which is an irreversible
reversible cell cycle arrest state of stable cell cycle arrest
It occurs due to lack of nutrition and growth
It takes place due to ageing and serious DNA damages
factors
It can re-enter into cell cycle It cannot re-enter into cell cycle
Unlike muscle cells, blood cells, or nerve cells, which do not normally replicate themselves,
stem cells may replicate many times, or proliferate
If the cells continue to be unspecialized then the cells are said to be capable of long-term
self-renewal
Scientists are trying to understand two fundamental properties of stem cells that relate to
their long-term self renewal:
Why can embryonic stem cells proliferate for a year or more in the laboratory without
differentiating, but most non-embryonic stem cells cannot?
What are the factors in living organisms that normally regulate stem cell proliferation
and self-renewal?
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 neighbors 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 under specific conditions.
When unspecialized stem cells give rise to specialized cells, the process is called
differentiation
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While differentiating, the cell usually goes through several stages, becoming more
specialized at each step
Scientists are beginning to understand the signals inside and outside cells that trigger each
arm of the differentiation process
The internal signals are controlled by a cell's genes which carry coded instructions for all
cellular structures and functions
The external signals for cell differentiation include chemicals secreted by other cells,
physical contact with neighboring cells, and certain molecules in the microenvironment
Totipotency vs Pluripotency
Stem cells which are capable of differentiating Stem cells which are capable of
into all types of body cells are totipotent and differentiating into any of the three germ
they can support life. They are present in layers are pluripotent but they cannot
embryo and are called as embryonic stem support life. They are formed in bone
cells. They can form all 230 types of cells marrow, they can differentiate into a fix
types in the body. number of cell types.
Totipotent cells are capable of differentiating
Pluripotent stem cells are differentiated into
into more than 200 functionally distinct type of
three germ layers in the embryos.
body cells in humans
The potential of differentiation is relatively
The potential of differentiation is optimal
low
Late embryonic cells are pluripotent in
Early embryonic cells are totipotent in nature.
nature. Embryonic stem cells and iPS are
The zygote and the spore are totipotent.
pluripotent.
Development of pluripotent stem cells
Totipotent stem cells are derived early.
follows that of totipotent cells.
Introduction
Embryonic stem cells (ESCs) are derived mostly from embryos that have been fertilized
in IVF clinics and donated for research purposes with informed consent of the donors
Human embryonic stem cells (hESCs) are generated by transferring cells from a
preimplantation-stage embryo (blastocyst) into a laboratory culture dish with culture
medium
The inner surface of the culture dish is typically coated with mouse embryonic fibroblasts
that have been treated so they will not divide (feeder layer).
The mouse embryonic fibroblasts provide nutrition and adherence to the stem cells
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The feeder layer provides the cells a sticky surface to which they can attach and also
release nutrients into the culture medium.
Recently, researchers have devised ways to grow embryonic stem cells without mouse
feeder cells.
ESC line
ESCs that have proliferated in cell culture for a prolonged period of time without
differentiating, are pluripotent, and have not developed genetic abnormalities are referred
to as an ESC line.
The process of generating an ESC line is somewhat inefficient, so lines are not produced
each time cells from the blastocyst 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 subculturing the cells is repeated many times and for many
months, each cycle of subculturing the cells being referred to as a "passage"
Once the cell line is established, the initial cells could yield millions of ESCs.
At various points during the process of generating ESC lines, scientists test the cells to see
whether they exhibit the fundamental properties of ESCs (characterization)
Laboratories that grow hESC lines use several kinds of tests, including:
Growing and subculturing the stem cells for many months, which 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
Transcription factors, Oct 4 and Nanog are associated with maintaining the stem cells in an
undifferentiated state, capable of self-renewal (used as selection markers)
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Embryonic stem cells - Generation
One can test whether the hESCs are pluripotent by one of the following ways:
Manipulating the cells so they will differentiate to form cells characteristic of the three
germ layers
Injecting the cells into a mouse with a suppressed immune system to test for the
formation of a benign tumor called a teratoma. This is a gold method for the
pluripotency.
Since the mouse's immune system is suppressed, the injected human stem cells are not
rejected by the mouse immune system and scientists can thus observe growth and
differentiation of the human stem cells
As long as the ESCs 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
So, in order to generate cultures of specific types of differentiated cells, such as heart
muscle cells, blood cells, or nerve cells, for example, scientists try to control the
differentiation of ESCs
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They change the chemical composition of the culture medium, alter the surface of the
culture dish, or modify the cells by inserting specific genes.
Strength/Definitiveness
Pluripotency assay Purpose Length of Assay Nature of Assay
of Assay
Verify ESC
colony-like
morphology of
Colony morphology 10 minutes in vitro Low
clustered,
border-definied
colonies.
Stain for
standard
pluripotency
markers such as
Immunohistochemistry 1-4 days in vitro Medium
Oct4, Tra-1-60,
Sox2. Tra-1-81,
Nanog and
SSEA
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Strength/Definitiveness
Pluripotency assay Purpose Length of Assay Nature of Assay
of Assay
Detect and
quantity
expression
4-6 hours (from
levels of
Real-time PCR RNA extraction in vitro Medium-High
selected
to RT-PCR)
pluripotency
genes; limited
by gene number
Test
differentiation
capability of
PSCs to tissues
of all 3 germ
layers in vitro or
in vivo should be
coupled with
relative
quantification
expression of
gene-expression
from the three
germ-layers.
which can be
done by RT-
Embryoid body PCR. Genes
2-3 weeks in vitro or in vivo Medium-High
formation and analysis include Nanog,
Oct4, Sox2, and
KIF4
(pluripotency
markers): Ncam
and NeuroD
(ectodermal
markers),
Runx2, HNF4a,
and Nkx2.5
(mesodermal
markers), and
Sox17 Albumin
Glut2, and
insulin
(endodermal
markers)
A
comprehensive
Microarray measurement of 1-2 days in vitro Medium-High
gene expression
levels.
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Strength/Definitiveness
Pluripotency assay Purpose Length of Assay Nature of Assay
of Assay
Test
differentiation
Teratoma formation capability into all 1-2 months in vivo High
3 germ layers in
vitro
Introduction
An adult stem cell (ASC) is an undifferentiated cell found among differentiated cells in a
tissue or organ that can renew itself and can differentiate to yield some or all of the major
specialized cell types of that tissue or organ
The primary roles of adult stem cells in a living organism are to maintain and repair the
tissue in which they are found
Unlike the origin of ESCs, the origin of adult stem cells in some mature tissues is still under
investigation
Scientists have found ASCs in many more tissues than they once thought possible which
has led researchers and clinicians to think that ASCs could be used for transplants
In fact, hematopoietic or blood-forming stem cells from bone marrow have been used in
transplants for 40 years
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History of adult stem cells and non-hematopoietic stem cells
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), were discovered a few years later.
The non-hematopoietic stem cells make up a small proportion of the stromal cell population
in the bone marrow, and can generate bone, cartilage, fat, cells that support the formation
of blood, and fibrous connective tissue
In the 1990s, scientists discovered that the adult brain contains stem cells that are able to
generate the brain's three major cell types: astrocytes and oligodendrocytes, which are
both non-neuronal cells, and neurons, or nerve cells
Adult stem cells have been identified in many organs and tissues, including brain, bone
marrow, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis.
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 ASCs in each tissue, and once removed from the
body, their capacity to divide is limited
Scientists are trying to find better ways to grow large quantities of ASCs in cell culture to
treat injury or disease
Eg., regenerating bone using bone marrow-derived stromal cells, developing insulin-
producing cells for type 1 diabetes, and repairing damaged heart muscle with cardiac
muscle cells.
Scientists often use one or more of the following methods to identify ASCs:
Label the cells in a living tissue with molecular markers and then determine the
specialized cell types they generate
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 their tissue of origin
(competitive repopulation assay)
Importantly, it must be demonstrated that a single ASC can generate a line of genetically
identical cells that then gives rise to all the appropriate differentiated cell types of the tissue.
To confirm experimentally that a putative ASC 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
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purified population of these candidate stem cells can repopulate or reform the tissue after
transplant into another animal that are immunocompromised.
In a living animal, ASCs are available to divide, when needed, and can give rise to mature
cell types that have characteristic shapes, specialized structures and functions
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 platelets
Mesenchymal stem cells give rise to a variety of cell types: bone cells (osteocytes),
cartilage cells (chondrocytes), fat cells (adipocytes), and other kinds of connective tissue
cells such as those in tendons
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 oligo dendrocytes.
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Transdifferentiation
A number of experiments have reported that certain ASC types can differentiate into cell
types of 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)
This reported phenomenon is called 'transdifferentiation', giving rise to the term 'stem cell
plasticity'. Although reported in some vertebrates, 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
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Nuclear Reprogramming or Dedifferentiation
Scientists have recently demonstrated that certain adult cell types can be "reprogrammed"
into other cell types in vivo using a well-controlled process of genetic modification referred
to as "nuclear reprogramming"
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 could possibly be
created by reprogramming other pancreatic cells
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 ESCs, determination of the methods by which ¡PSCS can be completely and
reproducibly committed to appropriate cell lineages is still under investigation.
Introduction
In human SCNT experiments, these eggs are obtained through consenting donors, many
times utilizing ovarian stimulation
Procedure
They have to remove the nuclei of one cell and then they have to introduce that nuclei to
another cell whose nuclei has been thrown out. So, it is like 1 nucleus and 1 cell
involvement at the end of this procedure.
The technique consists of taking an enucleated oocyte (egg cell without any nucleus- it is
an unfertilized egg cell) and take that nucleus implanting a donor nucleus from a somatic
(body) cell
The nucleus of the donor egg cell is removed and discarded, leaving it 'deprogrammed'
The nucleus of the somatic cell is also removed but is kept, the enucleated somatic cell is
discarded
The somatic cell nucleus is then inserted into the 'empty‘ (enucleated) egg cell, now
reprogrammed by its host egg cell factors
The egg cell, now containing the somatic cell's nucleus, is stimulated with an electric shock,
which will then begin to divide just like an fertilized egg.
The egg is now viable and capable of producing an adult organism containing all the
necessary genetic information from just one parent
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Applications of SCNT
A potential use of stem cells genetically matched to a patient would be to create cell lines
that have genes linked to a patient's particular disease
By doing so, an in vitro model could be created, would be useful for studying that particular
disease, potentially discovering its pathophysiology, and discovering therapies.
For example, if a person with Parkinson's disease donated his or her somatic cells, the
stem cells resulting from SCNT would have genes that contribute to Parkinson's disease
The disease specific stem cell lines could then be studied in order to better understand the
condition
Another application of SCNT is using the patient specific stem cell lines to generate tissues
or even organs for transplant into the specific patient
The resulting cells would be genetically identical to the somatic cell donor, thus avoiding
any complications from immune system rejection
Experiments
Sir John Gurdon carried out SCNT experiments in Xenopus laevis (frog) way back in the
1950s
Sir Ian Wilmut cloned Dolly the Sheep, being the first successful case of reproductive
cloning of a mammal
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Cell synchronization is a process by which cells in a culture at different stages of the cell cycle are
brought to the same phase. There are many ways of cells synchronization - one of them is nutrient
starvation media. Because there is lack of nutrition, the cells don’t grow well. Everything kinda gets slowed
down. But if we keep them for few hours, they all come to that one particular phase where we could safely
remove the nucleus from the cell. So, after synchronization, he took out the nucleus from the donor cell
and then by cell fusion and using electrical impulses to stimulate them for fertilization. Now , somatic cell
nucleus is going to get influenced by the environment of the egg cell. So, this is a fully differentiated cell and
coming in new environment where the new cell is completely undifferentiated. It is an unspecialized egg
cell. Now, it is going to be reprogrammed into gamete cells or embryonic cells. This is the whole point of
doing this process. After this, they start dividing like a normal egg cell. The lamb or the offspring that grew
resemble genetically like that of the one that donated the somatic cell nucleus and not the egg cell.
In Jan 2014, Yoshiki Sasai and colleagues proposed another method of generating ESCs,
referred to as stimulus-triggered acquisition of pluripotency (STAP) by subjecting ordinary
cells to certain types of stress, such as the application of a bacterial toxin, submersion in a
weak acid, or physical trauma
In Jan 2018, Mu-ming Poo and colleagues reported the generation of two genetically
identical long-tailed macaques, Zhong Zhong and Hua Hua using the same technique
However, Induced pluripotent stem cells (iPSCs) have taken over SCNT but it’s not that
SCNT is completely wiped out, there are still some labs that perform this for some reasons
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and there are ethical issues as well for using this technique as well.
Introduction
Induced pluripotent stem cells (iPSCs) are adult cells that have been genetically
reprogrammed to an ESC–like state by being forced to express genes (transcription
factors) and factors important for maintaining the defining properties of ESCs.
It is also a nuclear reprogramming but it is different as in it just involves one cell, that is a
somatic cell. It doesn’t involve any gamete. Since, it doesn’t involve a gamete therefore
ethical issues are far lesser.
So, these are somatic cells are induced with the pluripotency potential. That’s what we call
as induced pluripotency stem cells.
iPSCs are similar to natural pluripotent stem cells, such as ESCs in many aspects, such as
the expression of certain stem cell genes, chromatin methylation patterns, doubling time,
embryoid body formation, teratoma formation, viable chimera formation, and potency and
differentiability, but the full extent of their relation to natural pluripotent stem cells is still
under investigation.
Chimera - If we want to test the pluripotency for take 8 celled embryo, so we pull out 4 cells
from the embryo and 4 cells we introduce to embryo which are actually induced pluripotent
stem cells. If they are truly pluripotent, that chimera would be viable. If you implant it into
uterus, it would grow into a chimeric species or chimeric animal. So, if they don’t support
embryonic development then they aren’t actually pluripotent.
Experiment
iPSCs were first produced in 2006 from mouse fibroblasts and in 2007 from human
fibroblasts by Shinya Yamanaka's team at Kyoto University, Japan, which is an important
advance in stem cell research (Nobel Prize, 2012). He experimented with 10 different
cocktails (It is a mixture of 4-5 different transcription factors), each containing 4 or 5 or 6
transcription factors. Transcription factors are the determinants of the pluripotency.
Normally, adult cells don’t express this pluripotency factor as they are no longer pluripotent.
So, when they differentiate, they lose pluripotency and they express differentiation factors.
Yamanaka decided which ones are the key and let overexpress these factors and introduce
via a viral vectors. So, overexpression of factors. After that, he has different combination of
factors. The one that achieved the highest level of pluripotency and also with reduced
defects or any other technical problem. That became to be known as Yamanaka cocktail.
Yamanaka used genes that (basically code for transcription factors) had been identified as
particularly important in ESCs, and used retroviruses as carriers to transduce mouse
fibroblasts with a selection of four key genes: Oct3/4, Sox2, Klf4 and c-Myc
Once you introduce this cocktail into somatic cells; somatic cells are turned back into ESC
like cells. Because there is an overexpression of the pluripotent factors. Once this is done,
there is some selection, so we are using antibiotics.
Cells were isolated by antibiotic selection of Fbx15+ cells; this iPSC line showed DNA
methylation errors compared to original patterns in ESC lines and failed to produce viable
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chimeras when injected into developing embryos. So, that means the reprogrammed cells
had some defects.
Another two transcription factors which are also popular among the cocktail is LIN28,
NANOG (major determinant of pluripotency), which is another pluripotent factors.
Independent groups at both MIT and UCLA used the same four pluripotent factors, but they
selected a different marker for detection
Instead of Fbx15, they used Nanog for clone selection which is an important gene in ESCs;
this time they observed that these ESCs clones were very similar to actual ESCs. DNA
methylation patterns and production of viable chimeras indicated that Nanog is a major
determinant of cellular pluripotency.
Yamanaka obtained skin cells (adult somatic cells) or dermal fibroblasts and then introduction of Yamanaka
cocktail which are encoded into the viral vectors and these skin fibroblasts undergo reprogramming. And we
get iPSCs similar to ESCs and then they were gene corrected for sickle cell anemia, they are then put back
into the mouse and the mouse recovered.
This is done with the process of viral packaging. These viruses carry our gene of
interest and they are different from vectors. We have plasmids or vectors, we do a
simple method of transduction. We have buffers and we mix them with a certain ratio.
By transduction cells take up these vectors that are introduced, they would actually
produce viruses in the cell culture supernatant. All the packaged viruses would be
floating in the supernatant. We would collect and then further concentrate and would
introduce those concentrated viruses into the cell culture that is going for
reprogramming.
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Mouse iPSCs demonstrate important characteristics of pluripotent stem cells, including
expressing stem cell markers, forming teratomas and being able to contribute to many
different tissues when injected into mouse embryos.
Human iPSCs also express stem cell markers and are capable of generating cells
characteristic of all three germ layers.
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
therapies. This all can be done in-vitro.
Disadvantages
Viruses are used to genetically alter the cells, the expression of cancer-causing genes
"oncogenes" may potentially be triggered
At the same time, retroviruses are capable of causing cancers, they have genotoxicity (In
genetics, genotoxicity describes the property of chemical agents that damages the genetic
information within a cell causing mutations, which may lead to cancer.) because they can
integrate randomly anywhere in the genome and if they go to the upstream of the proto
oncogene, they would turn it to oncogene and this would lead to cancer.
They could also insert in the middle of tumor-suppressing gene. We need them to suppress
tumors but if retroviruses integrate at several points, it would down regulate them. And
again, it would increase the risk of developing cancers.
In animal studies, the virus used to introduce the stem cell factors sometimes causes
cancers
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
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treatments for humans.
Because of all this, researchers are currently investigating non-viral delivery strategies
Depending on the methods used, reprogramming of adult cells to obtain iPSCs may pose
significant risks that could limit their use in humans.
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
In February 2008, scientists announced the discovery of a technique that could remove
oncogenes after the induction of pluripotency, thereby increasing the potential use of iPSCs
in human diseases
In April 2009, it was demonstrated that generation of iPSCs is possible without any genetic
alteration of the adult cell: a repeated treatment of the cells with certain proteins channeled
into the cells via poly-arginine anchors was sufficient to induce pluripotency (piPSCs)
Epigenetic Mechanisms
Introduction
In epigenetics, we talk about the changes that happen at the surface of the genes.
We call them modifications and some types of alterations, some mechanisms that modulate
the DNA expression.
Intake of different drugs or toxins can have impact on epigenetics. Even lifestyle factors
such as exercise changes your epigenetics.
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Nucleosome assembly
Introduction
Epigenetic features of human and mouse stem cells has provided insights into the
unique properties of pluripotent and lineage-restricted stem cells
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These mechanisms are mediated by transcriptional factors and are designated to alter
the gene function without changes to the DNA sequence. They form a gene regulatory
network, all of which work together. Eg. There is a network that would keep stem cells
in their stem cell state. And then there is a transcriptional factor that would help them
differentiate.
Recent studies have revealed that stem cells maintain their status by multiple levels of
molecular events designed to impose flexible but precise control over the expression of
important regulatory genes.
However, emerging evidence suggests that the genome undergoes major epigenetic
alterations during mammalian development and embryonic stem cells (ESC)
differentiation.
Therefore, the molecular mechanisms that provide control of these epigenetic events
are essential components of stem cell biology, indispensable for establishing and
conveying gene expression patterns during cellular differentiation.
These gene expression patterns not only heavily rely on the transcriptional factor
networks, but also on the properties of the chromatin within the cells, which represent
“epigenetic make-up” of the cell
Is genomic DNA of all the cells like of brain cells, liver cells same because there are
different cells?
So, what makes these cells different is because of epigenetics. This means it is this
important that it determines our cells.
So, epigenetics regulate what genes would be expressed depending on what tissue.
There is something called as epigenetic memory. They do remember who they were
and they are fully functional. But they retain the epigenetic memory. So, those cells
which are capable of retaining epigenetic memory are the ones that would undergo
reprogramming. That is why reprogramming efficiency varies from cell type to cell type
depending on what somatic cells we take.
The multipotency of stem cells is reduced over time due to progressive gene silencing
of differentiation genes; genes active in earlier progenitors are gradually silenced at
developmentally later stages, and subsets of cell type-specific genes are turned on.
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epigenetics (specific modifications that take them down to path). For eg. chromatin
remodeling and chromatin modifications, DNA methylation of CpG dinucleotides.
It has been shown that heterochromatic markers, such as HP1 proteins, and
heterochromatic histone modifications change their localization from dispersed and
very dynamic in ESCs to more concentrated distinct loci during cellular differentiation
Epigenetic changes are dynamic. If we look at the ESCs which are pluripotent stem
cells, not many pluripotent genes are to be expressed. It will be more or less that there
will be a defined loci which carry those genes of pluripotency. They are quite dispersed
in ESC stage and as the differentiation begins they become more open as it is needed
for expression. Then more genes become part of the euchromatin. In the ESC stage
i.e. the pluripotent stem cell stage, we have more widespread heterochromatin, only
short sites of euchromatin which are the representation of the pluripotency marker.
When the differentiation begins or we go down the differentiation pathway, we would
have those dynamic changes and we call them as epigenetic landscapes. This means
that differentiation leads to chromatin remodelling just accompanied by the changes in
the Global Neural architecture.
In mammalian cells, both DNA methylation and specific histone modification are
involved in chromatin silencing; DNA methylation (DNA methylation occurs at the
cytosine bases of eukaryotic DNA) and histone modification are interdependent.
Contrary to this, when mouse hematopoietic stem cells (HSC) were exposed in vitro to
soluble factors known to induce neuronal differentiation of NSCs and ESCs, only a low
number of these HSC cells displayed some features of immature neural-like cells.
In in vivo or in vitro neural environments, these HSCs either died or gave rise to cells
with a mixed macrophage/microglial phenotype.
This set of published data argues that there are no simplified algorithms for trans-
differentiation events and main epigenetic principles of these events have not been
discovered yet.
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A number of TFs have been proposed to control stem cell self-renewal and lineage
progression and are also a powerful mechanism for generating cell diversity.
Progression along the lineage from stem cell to differentiated cell is characterized by
striking morphological and functional changes at each stage of the lineage
commitment.
The sequential expression of TFs and other signaling molecules, which elicit cascades
of gene expression, regulate each other and interact with epigenetic control factors to
form large gene regulatory networks.
A large number of auxiliary TFs have been implicated to play a role in ESC biology in
addition to “master regulators”.
Several attempts to create a TF’s gene regulatory network in ESCs in order to predict
regulatory interactions led to the identification of their roles in gene repression or
activation and revealed a surprising role of nuclear receptors.
Another crucial observation was that the most important direct (primary) targets
activated by the core regulatory network were themselves transcriptional regulators,
which in turn, could activate numerous secondary targets
Highly conservative non-coding elements (HCNE) are DNA sequences that are highly
concentrated near genes encoding developmentally important. TFs and have been
shown to partially overlap with regions of ‘bivalent’ chromatin modifications. Bivalent
chromatin refers to regions of chromatin where heterochromatin and euchromatin are
overlapping. Bivalency is important for maintaining pluripotency of ESCs because it
allows them to switch quickly between being pluripotent and differentiating.
Accordingly, these “bivalent domains” would allow differentiation events to shift the
balance of the chromatin modifications in such a way that the silent genes will be
enriched only for meK27H3 and the active genes will bear only euchromatic
modification, meK4H3.
Interestingly, a significant number of ‘bivalent’ domains are enriched with binding sites
for at least one of the three pluripotency associated factors, OCT4, NANOG and SOX2,
which implies that their formation and/or maintenance might be regulated through these
‘master regulators’.
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Chromatin ‘bivalency’ is posing an interesting hypothesis proposed to provide for
molecular mechanisms involved in regulation of ESC differentiation and maintenance of
pluripotency.
This ‘epigenetic memory’ allows cells to maintain their identity, even when exposed to
extracellular environments that would induce formation of other cell fates. For instance,
HSCs (less plastic) have a strong epigenetic memory as they can’t be coalesced to be
something else unlike Neural stem cells (NSCs) which have a weaker epigenetic
memory and that is the reason they are able to transdifferentiate. So, the plasticity is
associated with epigenetics. Greater the plasticity (can be molded or differentiated to
something else), lesser the epigenetic memory and vice versa. So, NSCs being more
plastic and HSCs being less plastic.
This is also important for maintaining stem cells over time and in preventing tumor
formation
Epigenetic response to extrinsic signals occur through the transcriptional factors network
Stem Cell 23
You tube links
http://www.youtube.com/watch?v=eYrQ0EhVCYA
http://www.youtube.com/watch?v=Xjq5eEslJhw
Introduction
The stem cell niche is the in vivo microenvironment where stem cells both reside and
receive stimuli that determine their fate. Niches are very protective and are responsible for
maintaining the stemness.
The closer they are to stem cell niche, they would be more likely to remain stem cells
without differentiation.
The niche is not simply a physical location for stem cells, rather a place where extrinsic
signals interact and integrate to influence stem cell behaviour
These stimuli include cell-to-cell and cell-matrix interactions and signals (molecules) that
activate and/or repress genes and transcription programs
As a direct consequence of this interaction, stem cells are maintained in a dormant state
but they are metabolically active, induced to self-renewal or committed to a more
differentiated state
Stem Cell 24
Ray Schofield first postulated the hypothesis of a specialized stem cell microenvironment in
1978 and proposed that niches have a defined anatomical location and that removal of
stem cells from their niche results in differentiation.
Historical aspect
The first demonstration and characterization of niche components was conducted in the
gonads of invertebrates such as, Drosophila melanogaster and Caenorhabditis elegans
Examination of these systems, characterized in less complex animals, has led to pivotal
insights into understanding the more complex mammalian niche architecture
It appears that the fundamental anatomical components and molecular pathways of the
niche are highly conserved across species, although their respective roles within the niche
may show distinct variations
Therefore, it has been proposed that it is possible to identify common niche components
that are associated with similar functions
The general niche model involves the association between resident stem cells and
heterologous cell types - the niche cells (other cells which are supporting the niche)
The existence of a heterologous cell type is not essential and components of the
extracellular matrix (ECM) may determine the niche for stem cells.
Notably, a niche may retain its key functions and properties, even in the temporary absence
of stem cells (such as following stem cell depletion through radiation treatment) allowing
recruitment and homing of exogenous stem cells. Stem cell homing refers to the ability of
circulating stem cells or exogenously administered stem cells to locate and enter an
environmental niche.
The niches are present at different locations; they reside in their respective organs. So, a
neural stem cell won’t go to a Hematopoietic stem cells in bone marrow. Niches have
certain signals to attract their own stem cells, not any stem cells. So, the basic components
are similar but they are located in such discrete anatomical location that they can’t afford to
attract stem cells from different parts of the body.
Conserved components of the stem cell niche as seen in various species include:
Stromal support cells (Plays an important role in sending messages or signals to stem
cells which are in close proximity), including cell-cell adhesion molecules and secreted
soluble factors, which are found in close proximity to stem cells.
Extracellular matrix (ECM) proteins that act as a stem cell “anchor” and constitute a
mechanical scaffolding unit to transmit stem cell signaling. Anchoring is ability to retain
or pull stem cells closer to niche.
Blood vessels that carry nutritional support and systemic signals to the niche from other
organs and also participate in the recruitment of circulating stem cells from and to the
niche.
Neural inputs that favor the mobilization of stem cells out of their niches and integrate
signals from different organ systems. Neuronal cues appear to be particularly important
in trafficking of HSCs.
Stem Cell 25
Ferraro F, Lo Celso C, Scadden D. Adult stem cells and their niches. Adv Exp Med Biol. 2010;
695:155-68
In drosophila
In Drosophila ovary, germline stem cells (GSCs) located in the germarium are in
physical contact with cap cells and terminal filaments cells. The physical contact with
cap cells is essential for stemness.
During the process of asymmetric division, GSCs that physically contact cap cells
through E-cadherin junctions retain their stem cell properties, whereas those cells that
lose contact with cap cells differentiate into mature follicle cells.
A similar system, driven by polarity cues, also applies to Drosophila testis, where two
sets of stem cells, GSCs and somatic stem cells (SSCs) are associated with hub cells
at the apical tip of the testis.
Daughter cells that detach from the hub initiate a differentiation program to become,
respectively, spermatogonia and somatic cyst cells.
In C. elegans
In C. elegans 225 germ cells are associated with distal tip cells (DTC) and they are
maintained stem cells through signals from these cells.
Several niches have been identified also in many mammalian tissues: hematopoietic
system, skin, intestine, liver, brain and muscle.
In trabecular bone marrow, hematopoietic stem and progenitor cells (HSPCs) reside along
the endosteal surface close to osteoblastic cells and in proximity to the blood vessels..
Since a niche is defined by its functional regulation of stem cells, the mere physical
proximity of stem cells to other components is insufficient to determine if those components
are part of the stem cell niche.
To date, data indicating a regulatory role for osteoblastic cells has several lines of support
but a role for endothelium is less clear.
Stem Cell 26
Skin stem cells
In the skin, hair follicle stem cells are found in the bulge area of the hair follicles.
While the exact components of the skin stem cell niche have not been fully identified yet,
although critical regulatory cues are derived from the dermal papilla.
These stem cells are important in regeneration of hair follicles while scattered stem cells
attached to the basal membrane that separates epidermis from dermis (epidermal stem
cells) are involved in replacement of interfollicular epidermis. With respect to the skin, there
are different type of stem cells. Skin has three types - (1) Epidermis stem cells (2) Hair
follicles stem cells (3) melanocyte stem cells while Bone marrow has two types - HSCs
(Hematopoietic stem cells) and MSCs (mesenchymal stem cells).
Sebaceous glands are maintained by cells at the base of each gland, but their niche is still
largely unknown.
In adult central nervous system (CNS), neural stem cells (NSCs) have been identified in the
lateral subventricular zone (SVZ) and in the subgranular zone (SGZ) of the dentate gyrus
within the hippocampus.
Within these areas NSCs have been shown to express the astrocyte marker glial fibrillary
acidic protein (GFAP). GFAP-positive astrocytes in SVZ and SGZ are able to give rise to
neuroblasts and subsequently mature neurons. Located in close proximity to NSCs,
endothelial cells are considered niche cells in the CNS.
In the gut, intestinal stem cells (ISCs) reside in the bottom part of the intestinal crypt
interdigitated between Paneth cells.
The area surrounding the crypts is particularly rich with enteric neurons and blood vessels.
Satellite cells
In the muscle, stem cells, known as satellite cells, are located along muscle fibre tracts
attached to the plasma membrane that surrounds each muscle fibre bundle.
In this case, the basal lamina may represent the niche for satellite cells
Overview
Stem Cell 27
Cancer Stem Cells (CSCs)
Introduction
Cancer stem cells (CSCs) are very small proportion of tumor cells which through small
symmetric and asymmetric division can maintain their own population by endless self
renewal and give rise to the bulk of tumor by producing fast dividing progenitor cells which
can differentiate into a variety of cancer cell types.
A significant property of cancer stem cells is their ability to spread cancer to other areas of
the body.
They do this by breaking away from the bulk of the tumor and turn the blood stream and
circulating around the body where they can invade other tissues and start the process of
team information from scratch. This study process is called as metastasis and is a major
contributor of cancer related deaths.
2. The occurrence of relapse. If the relapse is occurring then they are cancer stem cells.
3. Metastasis - They most commonly develop when cancer cells break away from the main
tumor and enter the bloodstream or lymphatic system. These systems carry fluids around
Stem Cell 28
the body. This means that the cancer cells can travel far from the original tumor and form
new tumors when they settle and grow in a different part of the body. That metastasis
property is also because of cancer stem cells. Only cancer stem cells have migration
potential.
Emerging evidence has indicated a subpopulation of stem cell like cells within tumors
(hidden inside tumors), known as cancer stem cells (CSCs), which exhibit characteristics of
both stem cells and cancer cells.
In addition to self-renewal and differentiation capacities, CSCs have the ability to seed
tumors when transplanted into an animal host.
CSCs can be distinguished from other cells within the tumour by symmetry of their cell
division and alterations in their gene expression
The first modern evidence for a role of stem cells in cancer came in 1994 when a leukemia-
initiating cell population was identified from an AML patient by transplantation into SCID
mice (Dominique Bonnet & John Dick)
The leukemia-initiating cells (LICs) were enriched on the basis of cell surface marker
expression (CD34+/CD38−).
CD34 is one of the most widely used markers of hematopoietic stem cells (HSCs) and is
involved in the inhibition of HSC differentiation, HSC expansion, signaling transduction and
anti-adhesion. CD38 is a type II glycoprotein that was originally described as a lymphoid
cell surface differentiation marker.
💡 One out of 10,000 cells in your cancer stem cells is sufficient to initiate cancer
This came into existence because of the resistance of cancer stem cells to conventional
therapy.
Current therapies tend to kill fast dividing cells in the bulk of the tumor but they will also
spare the slower dividing cancer stem cell. These may survive and drive regrowth of the
tumor.
This is why we need to develop therapies that kill the cancer stem cells specifically.
Unfortunately, even cancer stem cell specific therapy may not always be a cure as the bulk
of the tumor cells left behind may develop the ability to convert back into cancer stem cells
and so dry tumor reoccurrence.
But if cancer stem cell targeted therapies are integrated with current cancer treatments, all
of the cells within the tumor will be targeted together destroying the bulk tumor cells before
they get the chance to convert back into cancer stem cells.
It is hoped that new strategy will increase the number of cases which can be cured for good
Stem Cell 29
Blue colored cells are cancer differentiated cells. Yellow colored are cancer stem cells.
They have the ability to seed tumors when transplanted into the host cell.
When these cells are introduced into normal animals, they are capable of initiating cancer
into the host out of nowhere.
These truly have the property to give birth to all the progenitors of cancer.
If the cell is not able to perpetuate cancer then it could be just cancer cells and not cancer
stem cells. Normal cancer cells can’t simply grow in other host. They will differentiate and
die afterwards.
If the cells are capable of giving cancer then only they are cancer stem cells i.e. a cell that
can truly initiate cancer are called as CSCs if not otherwise then they are simply cancer
cells.
CSCs are defined by their ability to generate more SCs (self-renewal) and to produce cells
(progenitors) that differentiate
Asymmetric cell division achieves both tasks, as one progeny retains SC identity and the
other undergoes rounds of cell division and subsequent post-mitotic differentiation. There is
no other cell other than stem cell that can execute asymmetric cell division.
Initially, CSCs were believed to represent a small fraction of the total cell population in a
solid tumor, however it has been claimed that as many as 25% of cancer cells may have
the properties of CSCs. It is the reason why cancer is so difficult to eradicate. It doesn’t
mean every tumor would have 25% of CSCs, it is a higher limit.
In 2003, human CSCs were identified in solid tumors, including breast and brain cancer
The subsequent reports identified CSCs in a variety of tumors, including colon, pancreas,
lung, prostate, melanoma, and glioblastoma
Even 100 CSCs were able to form tumors in NOD/SCID mice. The NSG mouse (NOD scid
gamma mouse) is a brand of immunodeficient laboratory mice, developed and marketed
Stem Cell 30
by Jackson Laboratory, which carries the strain NOD
Expression of cell surface markers such as CD44, CD24, CD29, CD90, CD133, epithelial-
specific antigen (ESA), and aldehyde dehydrogenase1 (ALDH1) have been used to isolate
and enrich CSCs from different tumors.
Surface markers could be the indicator for cancer. They are phenotypic as they are present on surface of
cell. All of these markers have been used to identify and enrich cancer stem cells. These expression
markers are specific for the various tissues and also to some extent, they are specific for the tumor subtype.
lin- stands for lineage negative. Lineage refers to some stem cell property or
precursor like early stem cell. All stem cells will lin-.
One theory believes that CSCs arise from normal stem/progenitor cells (Progenitor
cells are the immediate progeny) which obtain the ability to generate tumors when
encountering a specific genetic mutation or environmental alteration (stem cell
model or hierarchical model).
The red ones are the mutated cells. They have got both the property - stem cell property and
tumorigenic properties. They can establish their own tumor. They give rise to the example for
asymmetric division where Cancer stem cells give rise to progenitor (doesn’t acquire the mutation
for cancer) but the other cancer stem cells would cause tumor. This is because the intrinsic
property of cancer stem cells (CSCs).
Stem Cell 31
Introduction
The only difference between normal stem/progenitor cells and CSCs is that CSCs are tumorigenic
properties.
An alternative theory for the origin of CSCs suggests that they arise from
normal somatic cells which acquire stem cell-like characteristics and malignant
behavior through genetic and/or heterotypic alterations (somatic evolution
model or stochastic model).
Normal somatic cell (undergo reprogramming)—> Stem cell —> cancer stem
cells (malignant properties)
Extra info
In contrast, several studies show that tumor cells with an epithelial phenotype
survive in the circulation and form distant metastasis. Only CSCs have the
Stem Cell 32
property of metastasis, not even cancer differentiated cells and only CSC can
initiate tumors at different location. Wherever the CSCs go, they create a new
niche as cancer can’t survive without its niche.
Treatment of CSCs
For selective targeting, studies looked for specific markers and for proteomic and genomic
tumor signatures that distinguish CSCs from other cells.
Some types of cancer cells can survive treatment with salinomycin through autophagy,
where cells use acidic organelles such as lysosomes to degrade and recycle certain types
of proteins. The use of autophagy inhibitors can kill cancer stem cells that survive by
autophagy. So, we generally use salinomycin with autophagy inhibitors for killing cancer
stem cells.
A differentiated tumor cells can also turn into cancer stem cells then we have to go for dual
approach, conventional and cancer stem cell specific therapy.
Teratoma is a property of stem cell, not any stem cell but pluripotent stem cell. Immature
Teratoma is a benign tumor as it is capable of giving rise to multiple different cell type. Only
when it becomes malignant then it’s cancerous or tumorigenic.
Introduction
Stem Cell 33
W —> wingless mutant of drosophila; nt —> Integration gene. Thus, the int/Wingless family
became the Wnt family and int1 became Wnt1. The name Wnt is a portmanteau
of int. [Int1 is highly conserved across multiple species, including humans and Drosophila].
It includes Wnt which is a series of Growth Stimulatory Factors. There are 19 Wnt genes in
mammals. They are incorporated into exosomes.
Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the
noncanonical Wnt/planar cell polarity pathway, and the noncanonical Wnt/calcium pathway.
The canonical Wnt/β-Catenin pathway leads to the regulation of transcription of Wnt target
genes. β-Catenin is regulated primarily by degradation.
They are highly evolutionarily conserved across the animal species from fruit flies
(Drosophila melanogaster) to humans, so they are highly important to study.
Historical aspect
Wnt signaling was first identified for its role in carcinogenesis, then for its function
in embryonic development
These processes are necessary for proper formation of important tissues including bone,
heart and muscle
Its role in embryonic development was discovered when genetic mutations in Wnt pathway
proteins produced abnormal fruit fly embryos.
Wnt signaling also controls tissue regeneration in adult bone marrow, skin and the intestine
Later research found that the genes responsible for these abnormalities also influenced
breast cancer development in mice.
Importance of pathway
The Wnt/β-Catenin pathway regulates stem cell pluripotency and cell fate decisions during
development. This developmental cascade integrates signals from other pathways,
Stem Cell 34
including retinoic acid, FGF (Fibroblast growth factors), TGF-β (transforming growth factor-
beta), and BMP, within different cell types and tissues
The Wnt ligand is a secreted glycoprotein that binds to Frizzled (Fz) receptors (based on
how it appears), leading to the formation of a larger cell surface complex with LRP5/6 (This
is a glycoprotein receptor related protein). Activation of the Wnt receptor complex triggers
displacement of the multifunctional kinase GSK3β from a regulatory APC/Axin/GSK3β
complex (destruction complex). This is called as destruction complex because when this
complex of protein leads to the phosphorylation, ubiquitination and ultimately a proteasome
of degradation of β-Catenin.
Terminologies
In the absence of Wnt ligand (off-state), β-catenin, an integral E-cadherin cell-cell adhesion
adaptor and transcriptional co-regulator, is targeted by co-ordinated phosphorylation by
CK1α and the APC/Axin/GSK3β complex leading to its ubiquitination (It is a three step
process - E1 - Ubiquitin activating enzyme; E2 - Ubiquitin conjugating enzyme; E3 - Ligase
enzyme) and proteasomal degradation through the β-TrCP pathway. The non-functional,
age old proteins gets degraded by proteasomal degradation.
When the pathway is off, destruction complex is active. It phosphorylates one of its member
(here β-catenin) which would lead them to ubiquitination and then eventual degradation in
proteosome. And there won’t be any transcription.
Stem Cell 35
TCF/LEF is a regulator because it functions differently in the presence of Groucho as well as in the
presence of β-catenin. In the absence of signal, the TCF/LEF is under the influence of Groucho which is a
co-repressor and the entire transcription gets halted.
In the presence of Wnt ligand (on-state), it activates the Frizzled. The activation of Frizzled
leads phophorylation of LRP5/6 and when LRP5/6 (lipoprotein receptor-related protein)
is phosphorylated, it induces translocation of destruction complex to the region of the
membrane near the Frizzled and LRP receptors. When the Dvl binds to LRP, the
Dishevelled (Dvl) gets activated by sequential phosphorylation, polyubiquitination, and
polymerization, which displaces GSK3β from APC/Axin. In the activation of Dvl leads to the
inhibition of the destruction complex, thus preventing β-Catenin from phosphorylated and
preventing β-TrCP from incorporating into the destruction complex and preventing it from
ubiquinating β-Catenin.
When the destruction complex is done away with or they are destroyed themselves then β-
catenin level rises up and it is called as Stablized β-catenin. Stablized β-catenin is
translocated to the nucleus via Rac1 and other factors, where it binds to LEF/TCF
(lymphoid enhancer factor/T-cell factor) transcription factors, displacing co-repressors
and recruiting additional co-activators to Wnt target genes. Also, β-catenin co-operates with
several other TFs to regulate specific target genes, c-Myc and cyclin D1. β-Catenin
dislodges the Groucho and it binds to TCF and leads to transcription of Wnt target genes.
Stem Cell 36
Stem Cell 37
What would happen if Point mutation happens in β-catenin?
Because we know that point mutations would lead to aberrant accumulation. Therefore, it
would lead to gain of function in β-catenin. The pathway will be hyperactive as normally the
β-catenin is kept in check when the pathway is off. It is never allowed to accumulate in the
cytosol normally as it would lead to transcription of Wnt targeted genes.
In the given condition, the pathway would be constitutively active i.e. there won’t be any
regulation.
What would happen if mutation happens in any component of destruction complex because
APC mutation have been known in colon cancer? What would be the fate of mutated APC?
In this case, it would lead to loss of function of the component of the destruction complex.
Because when APC is mutated, it is not going to function. It is going to be degraded. And so is
the case with any other protein that is going to be mutated. So, members of the destruction
complex, when they are mutated, they are going to lose the function. They lose the function so
they are unable to ubiquitinate the β-catenin and therefore β-catenin levels would rise up as like
the previous case in which β-catenin gets mutated.
Extra information
Importantly, researchers have found β-catenin point mutations in human tumors that
prevent GSK3β phosphorylation and thus lead to its aberrant accumulation.
https://www.youtube.com/watch?v=iM-6C5ygkIo
Stem Cell 38
Introduction
The noncanonical Wnt/planar cell polarity (PCP) pathway does not involve β-catenin and
uses NRH1, Ryk, PTK7 or ROR2 as co-receptor. The PCP pathway is activated via the
binding of Wnt to Fz and its co-receptor.
The receptor then recruits Dsh, which uses its PDZ and DIX domains to form a complex
with Dishevelled-associated activator of morphogenesis 1 (DAAM1).
If the rearrangement of cytoskeleton won’t happen then cells will not be able to undergo
any shape change like for various cellular process, they need to change the shape.
Introduction
In the noncanonical Wnt/calcium pathway, the activated Frizzled (Fz) receptor directly
interacts with Dsh, which further activates specific Dsh-protein domains (PDZ and DEP).
Unlike the other two pathways, the Fz receptor directly interfaces with a trimeric G-protein,
which leads to the activation of either PLC (Phospholipase C) or cGMP-specific PDE
Stem Cell 39
(phosphodiesterase). If PLC is activated, the plasma membrane
component PIP2 (Phosphatidylinositol 4,5-bisphosphate) is cleaved into DAG
(diacylglycerol) and IP3 (Inositol trisphosphate). When IP3 binds its receptor on the ER,
calcium is released. And increased concentrations of calcium and DAG can
activate Cdc42 through PKC (Protein kinase C).
With respect to protein function, what is the significance of Ca, why should we have enough
store in the cytosol?
After the nascent peptide of protein is formed, folding occurs. For that protein folding, Ca is
very important. There are other enzymes also that are involved in this but for all of these to
work optimally, they need to have sufficient amount of calcium in the cell.
Introduction
Stem Cell 40
The Hedgehog signaling pathway transmits information to embryonic cells required for
proper cell differentiation. Different parts of the embryo have different concentrations of
hedgehog signaling proteins.
The pathway takes its name from its ligand, an intercellular signaling molecule called
Hedgehog (Hh) found in Drosophila. The signaling molecule Hh, in turn, takes its name
from the animal hedgehog, because of the appearance of fruit fly larvae lacking
the Hh gene.
Larvae without Hh, are short and spiny, resembling the hedgehog animal. The molecule
remains important during later stages of embryogenesis and metamorphosis.
The evolutionarily conserved Hedgehog (Hh) pathway is essential for normal embryonic
development and plays critical roles in adult tissue maintenance, renewal and regeneration.
Proper levels of Hh signaling require the regulated production, processing, secretion and
trafficking of Hh ligands: in mammals this includes Sonic (Shh), Indian (Ihh) and Desert
(Dhh).
All Hh ligands are synthesized as precursor proteins that undergo autocatalytic cleavage
and concomitant (same time) cholesterol modification at the C-terminus and palmitoylation
at the N-terminus, resulting in a secreted, dually-lipidated protein (Two different moiety as
one is cholesterol and palmitic acid).
Here, there are two receptors involved - One receptor binds the ligand Hh and after that it is
internalized; it just shuts off and degrade the ligand. The other receptor which is remaining
into cellular which is now activated after this ligand binding, and this one carries the
function. So, this is the signaling receptor.
Pathway
Hh ligands are released from the cell surface through the combined actions of Dispatched
(DISP) and Scube2, and subsequently trafficked over multiple cells through interactions
with the cell surface proteins LRP2 and the Glypican family of heparan sulfate
proteoglycans (GPC1-6).
Hh ligands initiate signaling through binding to the canonical receptor Patched (PTCH1)
and to the co-receptors GAS1, CDON and BOC.
Stem Cell 41
But when Hh binds to PTCH1, it results in derepression of the GPCR-like protein
Smoothened (SMO) i.e. it is not under the inhibiting function of PTCH1, then that results in
SMO accumulation in cilia and phosphorylation of its cytoplasmic tail. [Generally,
Phosphorylation leads to activation of protein function but in some cases it deactivates also
as in case of beta-catenin].
SMO mediates downstream signal transduction that includes dissociation of GLI proteins
(the transcriptional effectors of the Hh pathway) from kinesin-family protein, KIF7, and the
key intracellular Hh pathway regulator SUFU.
GLI proteins (it is similar to beta-catenin in Wnt pathway) also traffic through cilia and in the
absence of Hh signaling are sequestered by SUFU and KIF7, allowing for GLI
phosphorylation by PKA, GSK3β and CK1α, and subsequent processing into transcriptional
repressors (through cleavage of the C-terminus) or targeting for degradation (mediated by
the E3 ubiquitin ligase β-TrCP).
During deactivation
Stem Cell 42
GliFL (Gli full length). In the absence of Hh ligand, GliFL gets cleaved and is no longer functional. But in
presence of Hh ligand, GliFL gets converted to GliA which can induce expression of Hh target genes.
Stem Cell 43
Feedback Mechanism
Stem Cell 44
PTCH1 is an inhibitory receptor while SMO is the signaling receptor. If there are inactivating
mutations in PTCH1 then this is not going to suppress SMO, so SMO would be on even in
the absence of Hh ligand. So, that would lead to constitutively activation.
In addition to vital roles during normal embryonic development and adult tissue
homeostasis, aberrant Hh signaling is responsible for the initiation of a growing number of
cancers including, basal cell carcinoma (skin), medulloblastoma (brain), and
rhabdomyosarcoma (muscle).
More recently, overactive Hh signaling has been implicated in pancreatic, lung, prostate,
ovarian, and breast cancers.
Thus, understanding the mechanisms that control Hh pathway activity will inform the
development of novel therapeutics to treat a growing number of Hh-driven pathologies.
https://www.youtube.com/watch?v=fzVcJ7fWrVg
Introduction
Transforming growth factor-β (TGF-β) superfamily signaling plays a critical role in the
regulation of cell growth, differentiation, and development in various biological systems.
TGF-β and BMP both requires two receptor. Each receptor is dimeric in nature. The ligand
also binds as dimer.
These ligands bind to a type II receptor dimer, which recruits and phosphorylates a type I
receptor dimer. The type I receptor then phosphorylates receptor-regulated SMADs (R-
SMADs).
Pathway
The binding of R-SMAD (equivalent of beta-catenin and Gli protein) to the type I
receptor is mediated by a zinc finger-containing protein, SARA (SMAD anchor for receptor
activation). The R-SMAD further binds the co-SMAD (SMAD4) and the R-SMAD/co-SMAD
complex translocates to the nucleus where they act as transcription factors and regulate
Stem Cell 45
expression of target genes. SMAD proteins activate or repress transcription through
association with various co-activator or co-repressor proteins.
Inhibitory SMADs
The expression of inhibitory SMADs (I-SMADs) 6 and 7 is induced by both TGF-β and BMP
signaling pathways as part of a negative feedback loop.
SMAD 6 and SMAD7 directly interact with the receptor complex and thus inhibit R-SMAD
activation.
The stability of TGF-β family receptors and/or SMADs are regulated by SMURF E3
ubiquitin ligases and USP4/11/15 deubiquitinases.
TGF-β and BMP pathways are modulated by MAPK signaling at a number of levels.
Moreover, in certain contexts, TGF-β signaling can also affect SMAD-independent
pathways, including Erk, SAPK/JNK, and p38 MAPK pathways (non-canonical pathways).
Stem Cell 46
Overview
https://www.youtube.com/watch?v=Z1gSsKxBN8U
https://www.youtube.com/watch?v=BB49lqTAbrM
Stem Cell 47