STEM CELLS - exam Flashcards
Definition of a stem cell
Generate differentiated cells
Regenerate itself
goal of stem cells
During early development, as well as later in life, various types of stem cells give rise to the specialized or differentiated cells that carry out the specific functions of the body such as skin, blood, muscle, and nerve cells.
types of stem cells
- Embryonic stem cells
o Can differentiate into almost any cell type in the body
o Pluripotent - Adult stem cell (stomatic stem cells)
o Can differentiate into a subset of related cell typed
o Multipotent
EMBRYONIC STEM CELLS
what
- Embryonic stem (ES) cells are formed as a normal part of embryonic development. They can be isolated from an early embryo and grown in a dish.
EMBRYONIC STEM CELLS
potential as therapy
o ES cells have the potential to become any type of cell in the body, making them a promising source of cells for treating many diseases.
EMBRYONIC STEM CELLS
special considerations
o Without drugs that suppress the immune system, a patient’s immune system will recognize transplanted cells as foreign and attack them.
EMBRYONIC STEM CELLS
ethical considerations
o When scientists isolate human embryonic stem (hES) cells in the lab, they destroy an embryo. The ethical and legal implications of this have made some reluctant to support research involving hES cells. In recent years, some researchers have focused their efforts on creating stem cells that don’t require the destruction of embryos.
EMBRYONIC STEM CELLS
how to obtain
- sperm and egg join
- embryo develops fro 5-7 days –> blastocyst
- remove inner cell mass
- grow in dish
- change culture conditions to stimulate cells to differentiate into a variety of cell types (skin, skeletal muscle, neural)
- Cultured ES cells can give rise to a 3-dimensional organ
o Able to differentiate both different cell types are different cell structures
EMBRYONIC STEM CELLS
genetic engineering in mammals
- Most widely used strategy
- Harvest blastocysts from mice
o Take out inner cell mass
o Culture it
o Manipulate it (however your want)
o Reintegrate (injected modified cells are incorporated into blastocysts)
Chimeric mouse
EMBRYONIC STEM CELLS
general strategy for gene targeting in mice
gene targeting in ES cells
- ES cell culture - embryonic stem cells are cultivated from mouse pre-implantation embryos (blastocysts)
- construction of targeting vector - the vector contains pieces of DNA that are homologous to the target gene as well as inserted DNA which changes in the target gene and allows for positive-negative selection
- ES cell transfection - the cellular machinery for homologous recombination allows the targeting vector enables the target vector to find and recombine with the target gene
- proliferation of targeted ES cell - selction for presence of neo and absence of HSV-tk enriches targeted ES cells
EMBRYONIC STEM CELLS
general strategy for gene targeting in mice
gene targeting in ES cells
selection
- Negative selection HSV-Tk (The HSVtk-encoded enzyme is able to phosphorylate certain nucleoside analogs (e.g. ganciclovir, an antiherpetic drug), thus converting them to toxic DNA replication inhibitors)
EMBRYONIC STEM CELLS
general strategy for gene targeting in mice
gene targeting in ES cells
happens in the…
minority of cells
positive selection
choose those that have taken up trait
EMBRYONIC STEM CELLS
general strategy for gene targeting in mice
gene targeting in ES cells
works well in…
mice
EMBRYONIC STEM CELLS
general strategy for gene targeting in mice
from gene targted ES cells to targeted mice
- injection of ES cells into blastocysts - the targeted ES cells are injection into blastocysts where they mix and form a mosaic with the cells of the inner cell mass from which the embryo develops
the injected blastocysts are implanted into surrogate mother - brith and breeding of mosaic mice - the mosaic mice breed with normal mice to produce both gene targeted and normal offspring
- Can take chimeric male mice and cross it with a (normal) female mice
EMBRYONIC STEM CELLS
general strategy for gene targeting in mice
from gene targted ES cells to targeted mice
likely to be…
heteozygous
(one from mother one from father)
need to go through another generation to get homozygous
- mendelian genetics
Induced pluripotent stem cells (iPS cells)
what
- New technology in the last decade
- Has changed what we can do with respect to human stem cell biology
Induced pluripotent stem cells (iPS cells)
what maintains the ES cell state
A core set of transcription factors (OSKMs) maintains the ES cell state
iPS CELLS
what maintains the self renewal state
a group of transcription factors
- Master gene regulator proteins
o Drive their own gene expression and the gene expression of eachother
o Often dysregulated in cancers
In this scenario it is a powerful driver of cell proliferation
* which drives self renewal and loosening of protein structures
* Which allows these transcription factors to get into the bits of DNA theyre meant to bind to
This network of transcription factors drives the stem cell state
iPS CELLS
overview
- Promising source of cells for treating diseases
- iPS cells can be made from patients’ own cells, so no risk of rejection
- safety uncertain due to genetic modifications created
o don’t know what will happen in 10-20 years
iPS CELLS
made?
- isolate cells from patient (skin or fibroblasts); grow in a dish
- treat cells with ‘reprogramming’ factors Oct4, Sox2, klf4, Myc
- wait a few weeks
- pluripotent stem cells
- change culture conditions to stimulate cells to differentiate into a variety of cell types (blood cells, gut cells, cardiac muscle cells)
iPS CELLS
Induced pluripotent stem (iPS) cells are created artificially in the lab by “reprogramming” a patient’s own cells. iPS cells can be made from readily available cells including fat, skin, and fibroblasts (cells that produce connective tissue).
iPS CELLS
potential as therapy
Mouse iPS cells can become any cell in the body (or even a whole mouse). Although more analysis is needed, the same appears to be true for human iPS cells, making them a promising source of cells for treating many diseases. Importantly, since iPS cells can be made from a patient’s own cells, there is no danger that their immune system will reject them.
iPS CELLS
special considerations
iPS cells are much less expensive to create than ES cells generated through therapeutic cloning (another type of patient-specific stem cell; see below). However, because the “reprogramming” process introduces genetic modifications, the safety of using iPS cells in patients is uncertain.
iPS CELLS
ethical considerations
Therapy involving iPS cells is subject to the same ethical considerations that apply to all medical procedures.
iPS CELLS
an experimental strategy to select iPS cells
- Need a selection strategy
o This is an example - Degree of uncertainty of how these technologies will last over time
o Will the promoter stay turned on
o Only considered for high risk patient (once confirmed safe become more widely used)
iPS CELLS
an experimental strategy to select iPS cells
Fbx15
a gene (Fbx15) that is present in all cells but is normally expressed only in ES and early embryonic cells (although not required for their survival).
PS CELLS
an experimental strategy to select iPS cells
all info
The experiment makes use of a gene (Fbx15) that is present in all cells but is normally expressed only in ES and early embryonic cells (although not required for their survival).
- A fibroblast cell line is genetically engineered to contain a gene that produces an enzyme that degrades G418 under the control of the Fbx15 regulatory sequence.
- G418 is an aminoglycoside antibiotic that blocks protein synthesis in both bacteria and eukaryotic cells.
- When the OSKM factors are artificially expressed in this cell line, a small proportion of the cells undergo a change of state and activate the Fbx15 regulatory sequence, driving expression of the G418-resistance gene.
- When G418 is added to the culture medium, these are the only cells that survive and proliferate.
- When tested, they turn out to have an iPS character.
Differentiated cells can be produced from cultured iPS (or ES) cells
- These cells can be cultured indefinitely as pluripotent cells when attached as a monolayer to a dish.
- Alternatively they can be detached and allowed to form aggregates called embryoid bodies, which causes the cells to begin to specialize.
- Cells from embryoid bodies, cultured in media with different factors added, can then be driven to differentiate in various ways.
Use of iPS cells for drug discovery and treatment of genetic disease
Two main ways this is being considered to use
1. Cell therapy
o iPS targeting of repair a disease causing mutation
2. Drugs discovery
Use of iPS cells for drug discovery and treatment of genetic disease
drug discovery
iPS cells derived from a patient with a genetic disease can be used for analysis of the disease mechanism and for discovery of therapeutic drugs
Use of iPS cells for drug discovery and treatment of genetic disease
cell therapy
genetic defect might be repaired in the iPS cells, which could then be induced to differentiate in an appropriate way and grafted back into the patient without danger of immune rejection.
ADULT STEM CELLS
- Important for growth, healing and replacing cells lost through wear and tear
- Blood and bone marrow stem cells used to treat disease (cells taken used is cells they are related to)
- But are relatively rare and can only become a subset of related cell types
ADULT STEM CELLS
potential as therapy
o Stem cells from the blood and bone marrow are routinely used as a treatment for blood-related diseases.
o However, under natural circumstances somatic stem cells can become only a subset of related cell types.
o Bone marrow stem cells, for example, differentiate primarily into blood cells.
o This partial differentiation can be an advantage when you want to produce blood cells, but it is a disadvantage if you’re interested in producing an unrelated cell type.
ADULT STEM CELLS
special considerations
o Most types of somatic stem cells are present in low abundance and are difficult to isolate and grow in culture.
o Isolation of some types could cause considerable tissue or organ damage, as in the heart or brain.
o Somatic stem cells can be transplanted from donor to patient, but without drugs that suppress the immune system, a patient’s immune system will recognize transplanted cells as foreign and attack them.
ADULT STEM CELLS
ethical considerations
o Therapy involving somatic stem cells is not controversial; however, it is subject to the same ethical considerations that apply to all medical procedures.
TWO WELL CHARACTERISED ADULT STEM CELL TYPES
- Stem cells in epithelia of small intestine
- Haematopoietic stem cells
Stem cells in epithelia of small intestine
- Small intestine renews itself at a greater rate than any other tissue in the body
- Molecular mechanisms are particularly well understood
Stem cells in epithelia of small intestine
- RENEWAL OF GUT LINING
- The pattern of cell turnover and proliferation in the epithelium that forms the lining of the small intestine.
- Stem cells (red) lie at the crypt base, interspersed among nondividing differentiated cells (Paneth cells).
- ## Progeny of the stem cells move mainly upward from the crypts onto the villi; after a few quick divisions, they cease dividing and differentiate—some of them while still in the crypt, most of them as they emerge from the crypt.
Stem cells in epithelia of small intestine
- RENEWAL OF GUT LINING
Paneth cells
The Paneth cells, like the other nondividing differentiated cells, are continually replaced by progeny of the stem cells, but they migrate downward to the crypt base and survive there for many weeks.
- This amplification component is really important because in adult cells there are not a lot of stem cells
Stem cells in epithelia of small intestine
TYPES OF DIFFERENTIATED CELL
- All of these cells are generated from undifferentiated multipotent stem cells living near the bottoms of the crypts
Stem cells in epithelia of small intestine
TYPES OF DIFFERENTIATED CELL
microvili
- The microvilli on their apical surface provide a 30-fold increase of surface area, -
o not only for the import of nutrients
o but also for the anchorage of enzymes that perform the final stages of extracellular digestion, breaking down small peptides and disaccharides into monomers that can be transported across the cell membrane.
Stem cells in epithelia of small intestine
TYPES OF DIFFERENTIATED CELL
goblet cells
- Goblet cells secrete mucus;
o these are the commonest of the secretory cell types. Paneth cells secrete (along with some growth factors) cryptdins—proteins of the defensin family that kill bacteria.
Stem cells in epithelia of small intestine
TYPES OF DIFFERENTIATED CELL
enteroendocrine cells
- Different subtypes of enteroendocrine cells secrete serotonin and peptide hormones into the gut wall (and thence the blood).
- Cholecystokinin is a hormone released from enteroendocrine cells in response to the presence of nutrients in the gut.
o It binds to receptors on nearby sensory nerve endings, which relay a signal to the brain to stop the feeling of hunger once one has eaten enough.
CHOICE OF TWO DAUGHTERS OF A STEM CELL
remain as a stem cell or be terminally differentiated
A lot of the understanding of this came from the gut system
CHOICE OF TWO DAUGHTERS OF A STEM CELL
how is this choice made?
- Each daughter produced when a stem cell divides can either remain a stem cell or go on to become terminally differentiated.
- In many cases, the daughter that opts for terminal differentiation undergoes additional cell divisions before terminal differentiation is completed; such cells are called transit amplifying cells.
Two theoretical ways for a stem cell to produce daughters with different fates
asymmetric
independent choice
Both of these models have to come up with a means of maintaining a small number of stem cells and generating enough progeny that will go on to differentiate to maintain the tissue
CHOICE OF TWO DAUGHTERS OF A STEM CELL
Asymmetric division
- (A) The asymmetric division strategy gives a clone consisting of precisely one stem cell plus a steadily increasing number of differentiating cells in proportion to the number of cell divisions.
- Asymmetric division hypothesises that you can have something that’s localised within the cell to a certain region which creates the stem cell
- Therefore when the cell divides only one of the daughter cells inherit the determinant (whatever it is) and become a stem cell again
- The other daughter cell will become the committed cell
CHOICE OF TWO DAUGHTERS OF A STEM CELL
independent choice
- (B) The independent-choice strategy is more variable in its outcome.
- With a choice made at random by each daughter and with a 50% probability for each one to remain a stem cell or differentiate, there is, for example, a 25% chance at the first division that both daughters will differentiate, so that the clone eventually goes extinct.
- Or, at this division or later, a preponderance of daughters may chance to retain stem-cell character, creating a clone that persists and increases in size.
- With the help of some mathematics, the probability distribution of clone sizes generated from a single stem cell at any given time can be predicted on this stochastic assumption.
- The observations in the gut and elsewhere fit the stochastic independent-choice strategy, but not the asymmetric-division strategy.
Is each type of differentiated cell produced by a single type of stem cell?
- Each daughter produced when a stem cell divides can either remain a stem cell or go on to become terminally differentiated.
- In many cases, the daughter that opts for terminal differentiation undergoes additional cell divisions before terminal differentiation is completed; such cells are called transit amplifying cells.
do all the progeny in the epithelial (gut) come from a common stem cell?
experimental system used a genetic marker to label descendants of stem cells
have a stem cell specific promoter –> in this context the Lgr5 gene
do all the progeny in the epithelial (gut) come from a common stem cell?
- the lgr5 gene
- The Lgr5 gene is specific for gut stem cells
- Only active in gut epithelial cells
Have a GFP marker - Green fluorescent protein
do all the progeny in the epithelial (gut) come from a common stem cell?
- the CreERT2 gene
- An active recombinases
o Recombinases are enzymes that can cause genetic recombination - This is inactive until you expose a cell that is expressing it to the drug tamoxifen
o This makes it active
do all the progeny in the epithelial (gut) come from a common stem cell?
- Can put in another expression vector that is on a ubiquitous promoter
- So this will be expressed in any cells
o However cannot be expressed because of the blocking sequence - In the presence of inactivated recombinase this blocking sequence can be excised and so it can be expressed
do all the progeny in the epithelial (gut) come from a common stem cell?
- findings
Gut stem cells are multipotent
- Lgr-5-expressing stem cells and their progeny in small intestine
do all the progeny in the epithelial (gut) come from a common stem cell?
- mechanism
- The Lgr5 gene encodes a member of the family of G-protein-linked transmembrane receptors, and it is expressed specifically in stem cells near the crypt base.
- Because the Lgr5 promoter was used to drive expression of CrERT2, treatment with a low dose of tamoxifen resulted in occasional stem cells expressing LacZ.
- These cells and all of their progeny could subsequently be detected with a blue histochemical stain.
- All of the blue cells in these images derive from a single Lgr5-expressing stem cell. After 60 days, the blue progeny of this cell are seen to extend all the way up a villus.
- These progeny can be shown to include all types of differentiated cells, as well as persistent Lgr5-expressing cells at the crypt base.
- This proves that Lgr5-expressing cells are multipotent stem cells.
What keeps the stem cell in its place and maintains its stem cell character?
- Paneth cells create the stem cell niche
What keeps the stem cell in its place and maintains its stem cell character?
- paneth cells and Lgr5
- There is room for about 15 Lgr5-positive stem cells at the base of each crypt
The surrounding Paneth cells generate signals to activate key signalling pathways in stem cells
Stem cells produce paneth cells and the paneth cells job is to regulate stem cells - Cross talk
what maintains proliferation
the Wnt signalling pathway
what inhibits differentiation
the notch signalling pathway
room for dividing stem cells
- When a stem cell divides to create two stem cells there is not room for both, so one is randomly pushed out to begin differentiation
o The niche is regulating cell renewal
o (not the stem cell itself)
Wnt signaling maintains gut stem cell proliferation
- In adenomas, Wnt signalling cannot be turned off as cells move up the crypt and differentiate so they keep dividing
- A lot of our understanding comes from looking at what happens when things go wrong
o E.g. in cancer
Wnt signaling maintains gut stem cell proliferation
mutations in Apc gene
- The specimen is from a patient with an inherited mutation in one of his two copies of the Apc gene.
- A mutation in the other Apc gene copy, occurring in a colon epithelial cell during adult life, has given rise to a clone of cells that behave as though the Wnt signaling pathway is permanently activated.
- As a result, the cells of this clone form an adenoma—an enormous, steadily expanding mass of giant cryptlike structures.
FUNDAMENTAL QUESTION ANSWERS:
how is choice made
Each daughter produced when a stem cell divides can either remain a stem cell or go on to become terminally differentiated. In many cases, the daughter that opts for terminal differentiation undergoes additional cell divisions before terminal differentiation is completed; such cells are called transit-amplifying cells.
- Choice made at random and/or by environment
o Choice is made by creating an environment by the path cells in which there is a finite amount of space that can maintain the stem cells
FUNDAMENTAL QUESTION ANSWERS:
Is each type of differentiated cell produced by a single type of stem cell?
- A single stem cell can give rise to all four differentiated cell types
FUNDAMENTAL QUESTION ANSWERS:
What keeps the stem cell in its place and maintains its stem cell character?
- Paneth cells induce Wnt and Notch signalling in stem cells to maintain their character
o In the case of the gut the paneth cells
HAEOMATOPIETIC STEM CELLS
The multipotent stem cell normally divides…
divides infrequently to generate either more multipotent stem cells, which are self-renewing, or committed progenitor cells, which are limited in the number of times that they can divide before differentiating to form mature blood cells.
HAEOMATOPIETIC STEM CELLS
As they go through their divisions, the progenitors become…
more specialized in the range of cell types that they can give rise to, as indicated by the branching of this cell-lineage diagram.
HAEOMATOPIETIC STEM CELLS
in adult mammals
all of the cells shown develop mainly in the bone marrow—except for T lymphocytes, which as indicated develop in the thymus, and macrophages and osteoclasts, which develop from blood monocytes.
HAEOMATOPIETIC STEM CELLS
some dendritic cells may also derive from…
monocytes
HAEOMATOPIETIC STEM CELLS
complex system?
yes
the most complex stem cell system
HAEOMATOPIETIC STEM CELLS
commintment is a…
stepwise process
HAEOMATOPIETIC STEM CELLS
divisions of committed progenitors amplify…
numbers of certain subsets
how we learnt about stem cells
Rescue of an irradiated mouse by transfusion of bone marrow cells
- Stem cells represents 1:50,000 – 100,000 of the bone marrow population (rare cell)
- Such experiments have discovered the identity of the stem cells
o Found that a single cell can reconstitute the entire blood system
- A similar procedure was used in humans to treat leukaemia by bone marrow transplantation
o Now blood stem cells are used
HAEOMATOPIETIC STEM CELLS
depend on…
contact with stromal cells
HAEOMATOPIETIC STEM CELLS
depend on contact with stromal cells
- When they lose contact with their niche, stem cells tend to lose their stem cell potential
- But dependence on stromal cells is not absolute because functional stem cells can be found free in the circulation
o Knock out faulty blood system
o Chemotherapy
o To created correct niche / conditions
HAEOMATOPIETIC STEM CELLS
contact dependent interaction
The contact-dependent interaction between the Kit receptor and its ligand is one of several signaling mechanisms thought to be involved in hematopoietic stem-cell maintenance.
- The real system is certainly more complex.
Moreover, the dependence of hematopoietic cells on contact with stromal cells cannot be absolute, since small numbers of the functional stem cells can be found free in the circulation. SCF, stem-cell factor.
CANCER STEM CELLS
(cancer cells with stem cell characteristics)
- Can never know for certain if cancer to completely gone
- Common to relapse within 2 years
- 5 years pseudemen for cure (very unlikely to relapse if haven’t relapsed within 5 years)
Cancer stem cells can be responsible for the growth of some tumors
- Theory that cancers are a mixture of transit amplifying cells and some stem cells
- Tumour cells can be experimentally fractionated to identify a stem cell-like population
Cancer stem cells can be responsible for the growth of some tumors
- IMPLICATIONS FOR TREATMENT
- Big implication for treatments
o Radiotherapy or chemotherapy often kills the fast-growing transit-amplifying cells sparing the slow-growing stem cells, which go on to resurrect the disease
o Relapse likely from cancer stem cell left behind after treatment
o Haven’t managed to eradicate it
