1
Q
Stem Cell Characteristics
A
- ability to self-renew
2. ability to differentiate
2
Q
Potency
A
- number of cell types they can provide
- totipotent: generate full embryo and extraembryonic tissues
- pluripotent
- multipotent: adult stem cells used to regenerate and renew some tissues
3
Q
Embryonic Stem Cells
A
- pluripotent cells making all 3 germ layers
- taken from blastocyst (ICM specifically)
- self-renew indefinitely in culture
- can be stably maintained and expanded in vivo
- useful study tools
4
Q
3 germ layers
A
- cell and tissue layers with different fates
1. ectoderm: epidermis, neuron, pigment cells
2. endoderm: lung, thyroid, pancreatic
3. mesoderm: cardiac/skeletal muscle, blood, etc
5
Q
ES cells generation
A
- isolated ICM put on fibroblast feeder cells
- dissociated cells
- replated cells form ES cell cultures
6
Q
Ethical Issues
A
- involved destruction of a preimplantation embryo
- limited research due to legal restrictions
7
Q
Reprogramming
A
- John Gurdon showed that somatic cell nuclear transfer could form stem cells
- took the nucleus of an oocyte and replaced it with the nucleus of a gut cell and a complete frog could develop
- oocyte contents take the donor nucleus back to the undifferentiated state
- genome has all info to convert cell into organism
8
Q
OSKM Factors
A
- 4 core factors found to reprogram adult cells
- Oct3/4, Sox2, c-Myc, and Klf4 into fibroblasts
- resulting cells behaved like ES
- induced pluripotent stem cells
9
Q
Induced Pluripotent SC
A
- specific set of TF bind to many targets in genome (control elements)
- induces pluripotent genes and represses differentiated genes
- look and divide like ES cells
- relatively inefficient process
- need to pass set of pluripotency tests
- not all ES genes expressed
10
Q
Metastable Equilibrium
A
- TF keep a cell pluripotent but stoichastically signal pathways will increase/decrease a specific TF and cause differentiation
11
Q
Teratoma Test
A
- inject iPS cells into mice and could form teratomas with mixture of cell types from each 3 germ layers
- shows that iPS resemble the pathway of ES cells
12
Q
Tests of Pluripotency for iPS cells
A
- form embryoid bodies in vitro
- demethylation of pluripotency genes
- use GFP to trace iPSC contribution to an embryo
- most key is to see if the iPSC will make an entire embryo
13
Q
Molecular Biology of Pluripotency
A
- ICM expresses Oct4/Sox2 targeting other pluripotency genes
- c-Myc stimulates division and renewal
- Klf4 promotes self-renewal
- large nucleus and small cytoplasm
- OSKM target many genes
- hypothesis that these factors can fluctuate and trigger differentiation into one germ layer or another
14
Q
Lineage Tracing
A
- highlight full progeny of given cell population or cell via genetic tagging
- label the mother cell to get a family tree and determine adult cell origin
15
Q
re-Lox system
A
- one mouse strain with Cre recombinase under control of tissue specific promoter
- another strain with a ubiquitous promoter controlling LoxP and reporter protein
- in tissues expressing Cre, LoxP is recognised and expressed (?)
16
Q
Uses of iPSCs
A
- example in neuroscience: a ALS patient took skin cells and reprogrammed them into iPSCs and redifferentiated into neurons
- from this they found a rare dominant allele of a superoxide dismutase associated with her disease
- new bank of therapy testing cells
- small molecule screening against cells like neurons can identify molecules as candidates/eliminate them earlier
17
Q
Advantages of iPSC
A
- no immune rejection
- no ethical issues
- personalised medicine
18
Q
Waddington’s Epigenetic Landscape
A
- challenged by the idea of direct conversion in which a tissue specific cell directly converts into a related tissue specific cell or into a cell type of another germ layer
- less risk of tumor formation (if the epigenetics of a normal cell isn’t fully reprogrammed it will be biased to form a specific cell type)
19
Q
Multipotent Cells
A
- tissue specific stem cells
- undifferentiated cells in a differentiated tissue or organ
- can self-renew and produce offspring that can differentiate into the cell types of that tissue
- in the embryo they generate enough cells for each tissue but in the adult they are needed for repair and replacement (stay in G0)
- important research area for regenerative medicine but hard to take stem cells and get 100% purity of a tissue
20
Q
Stem Cell Niche
A
- environment of the stem cells
- contains signals maintaining it as a stem cell from matrix, support cells, other SC, physical forces, neurotransmitters
- daughter cells secrete signals to the mother for ‘counting’
- contains group of cells in specific location specialised for maintenance of stem cells
21
Q
Niche Characteristics
A
- physical anchor (stuck in place to remain SC)
- generates factors regulating SC number and fate (spatiotemporal context)
- can support asymmetric division of SCs
22
Q
Gut Stem Cells
A
- Crypt contains SC and paneth cells
- population of transamplifying cells
- nondividing differentiated cells at the villi top
- niche size limit pushes out some daughter cells to differentiate
- two or more SC populations
- Lgr5+ stem cells at crypt base are active an replace epithelium daily
- Lgr4+ stem cells are quiescent reserve cells
23
Q
Gut Stem Cells and Cancer
A
- gut SC niche may include microbiome
- mice fed normal vs high diet with higher fat increasing the rate of tumor proliferation
- PPAR gamma receptor activated promoting the B catenin pathway and cauisng more SC and more transamplifying cells
24
Q
Gut Organoids
A
- biomedical application
- single cell can form an organoid (isolate crypt)
- resource for research, drug screening, disease diagnosis
25
Skeletal muscle stem cells
- between muscle fibers and basal lamina
- usually quiescent (satellite cell)
- damage or injury activates SC division
26
Satellite Stem Cell Niche
- complex: adhesions (cadherins), Notch signals, matrix feedback, stromal cells, neuronal cells, blood vessels
- PAX7 is a marker of stem cells in muscle
27
Satellite cells
- long-lived
- expressed drug resistance channels and resistors of oxidative stress (low metabolic rate)
- primed for activation: contain releasable repressors of myogenic genes
- fuse to damaged myofibers and form new ones before terminally differentiated
28
Satellite Cell Signals
- each state involves specific genes involved in this state
- from quiescence to activation MYOD is a key gene activated
- expansion and differentiation
29
Quiescent Muscle Cellls
- about 500 genes involved includes cell cycle blockers (hold in G0) and myogenic inhibitors (stop differentiation)
- eg. FOX03 expressed in satellite cells in G0. If deleted it causes incorrect self-renewal and spontaneous differentiation increases
- FOX03 regulates Notch signaling and repressed MYOD
30
Duchenne Muscular Dystrophy
- muscle disease of dystrophin
- previous view that dystrophin was disregulated in differentiated cells
- but satellitle cells have loads of dystrophin and this is actually a SC associated disease
- normally, asymmetry of DMD in 1/2 of a cell allows self-renewal after division
- in muscular dystrophy the asymmetry is gone causing division to mess up
- due to mitotic errors: there is a lack of committed muscle progenitors and impaired myofiber repair
31
Ageing and Stem Cells
- old stem cell niches behave badly
- ageing disrupts muscle stem cell quiescence
- satellite cells have bipotentiality and can make muscle or fat
- with age they make fat more which stops powerful contraction
- aged fibers express more Fgf2 and this drives satellite cells out of quiescence and depletes them
32
BrdU+ Label
- labels cells in S phase
| - older cells have more division with more cells going out of G0 more often
33
Parabiosis
- rejuvenation of stem cell niche by sharing circulation
- old mice become healthier and fixed SC defects
- potentially assoicated with Wnt/B cadherin
- research showed young blood reverses age related impairments in cognitive function and synaptic plasticity in mice
34
Neural Stem Cells
- self-renewing, multipotent cells that firstly generate the radial glial progenitor cells that generate the neurons and glia of the nervous system of all animals during embryonic development
- neural cells come from the ectoderm
35
Dorsal Ectoderm
- forms the neural tube as the neural plate folds in
- epidermis on either side brought together
- subdivide rostral-caudal and dorsal-ventral
36
Neural Plate
- mesoderm induces a neural plate
| - BMP4 signal in the ectoderm is inhibited to get the head formation
37
Neurulation
- once the neural plate is formed
- Neurulation refers to the folding process in vertebrate embryos, which includes the transformation of the neural plate into the neural tube
38
Neural Tube
- reverse neural inhibition and avoid border/epithelial differentiation
- specify neural progenitors
- notochord at bottom signals the midline of the tube
39
Notochord & Floor plate
- signalling centers
- floor plate (ventral) secretes hedgehog
- roof plate (dorsal) secretes Wnt
- transplanted notochord causes growth of an ectopic floor plate/motor neurons
40
Dorso-ventral spinal cord patterning
- gradient of retinoic acid and FgF making cells differentiate
- groups express cadherin to differentiate and adhere together
- gene expression/TF affected by signals
41
Neural Tube Defects
- regions of neural tube don't close
- eg. spinal bidufa
- neural tube unprotected
-
42
Organoids and Microchephaly
- thin cerebral cortex with not enough neurons
- used iPSCs via NSCs to prove this was visible
- organoids showed less cells were made
43
Neural Cell Types
- stem cells of the nervous system generate neurons and oligodendrocytes and astrocytes in the embryo and CNS
- multipotent stem cells
- in the embryo you have neuroepithelial cells - then redial glial cells supporting neural cell movement
- in the adult oyu get neuroblasts, oligodendryocytes, B cells, etc.
44
Brain and Spinal Cord Development
- start with forebrain, midbrain, hindbrain, spinal column
- adult brain contains the spinal cord, medulla, cerebellum, midbrain, telencephalon
- IsO boundary between hindbrain and midbrain is a signal center : specific TF expressed based on the boundary placement
- moving the boundary affects structure of brain
- patterns expanding neural progenitors
45
Dopaminergic Neurons
- midbrain only in a small section
- need the right temporal combination of Wnt and hedgehog to differentiate
- early development involves proliferation of neuroepithelial cells, migration down radial glia, and differentiation
46
Outline CNS Development
1. part of embryo designated to future CNS (ectoderm)
2. cells divide (pluripotent)
3. regionalisation and signal centers
4. restriction of cell mixing via adhesion molecules
5. neurons generated and migrate to final positions
6. NSCs produce glia and more neurons
7. connect and generate synapses
8. cull excess
9. myelination
10. remodel
47
Outline Skin Development`
1. from ectoderm
2. form multipotent epidermal stem cells (Wnt promotes BMP and inhibits FGF)
3. keratin markers form
4. SCs divide and cells leave the basement membrane migrate upwards and differentiate
5. form stratified epithelium
6. develop skin appendages
7. maintain skin homeostasis
48
General Structure of Skin
*see diagram
49
Skin Wound Healing
- inflammation and recruitment of immune cells
- cell proliferation
- dampen inflammation and clear dead cells
- remodelling and scarring
- or regeneration of epidermis
- if embryos are damaged there is no scarring (interesting in research)
50
Gut Structure
- stem cells at crypt base interspersed among nondividing cells (Paneth cells)
- their progeny move upwards and after a few division stop dividing and differentiate
- Many cell types are generated from the stem cells
- Four types of differentiated cells
o Absorptive cells: have microvilli + digestive enzymes
o Goblet cells: secrete mucus
o Paneth cells: secrete defensins
o Enteroendocrine cells: secrete serotonin and peptide hormones
51
Cre Lox Tracing
- Crypt Base stem cells are multipotent
o Cre-Lox lineage tracing showed this
o Heritable marker activated in a individual cell and identify itsprogeny
o A modern method for tracking cell lineage uses transgenic animals containing two transgenes, which together drive expression of a readily detected and heritable marker protein in a small subset of stem cells.
o The first transgene (top) carries two adjacent protein-coding sequences, GFP and CreERT2, both expressed under the control of the Lgr promoter that is active only in stem cells and not in their differentiated progeny.
o The CreERT2 gene encodes a chimeric form of the Cre recombinase called CreERT, which consists of Cre recombinase linked to the estrogen receptor protein; this enzyme becomes active as a recombinase only when it binds the artificial estrogen analog tamoxifen.
o The second transgene (bottom) carries a marker gene, LacZ, under the control of a promoter that is active in all cells. The LacZ gene encodes β-galactosidase, an enzyme that can be detected histochemically in tissues However, LacZ expression in the transgene shown here is prevented by a blocking sequence (red) that is flanked by LoxP sites (pink; see Figure 5-66). When tamoxifen is provided, CreERT becomes active—leading to a recombination event that removes the blocking DNA sequence (and leaves one LoxP site behind). As a result, the LacZ marker is expressed. Because this change is heritable, the marker continues to be expressed in all cells descended from those in which a recombination event has occurred.
o With a low dose of the inducer molecule tamoxifen, it is possible to activate the marker at random in just a few widely spaced cells, which, in the course of time, give rise to widely separated and easily distinguished clones of progeny
52
Wnt and Gut Stem Cells
o Hereditary colorectal cancer patients often get small precancerous tumors in the lining of the gut
Caused by intestinal crypt cells having mutations in the Apc gene coding for a protein preventing Wnt overactivation
Losing the gene mimic continual Wnt signaling: suggest the Wnt protein keeps crypt cells proliferating and cessation makes them stop as they leave the crypt
53
Notch and Gut Cells
- Notch signaling controls gut cell diversification and maintains SC state
o Wnt signaling leads to expression of Notch and Delta in the cells of the crypt, and Delta-Notch signaling in the crypt mediates lateral inhibition between adjacent cells. Cells expressing higher levels of Delta eventually activate Notch in their neighbors, adopt a secretory fate, and stop dividing; their neighbors, with activated Notch, are prevented from differentiating and keep on dividing
54
Special Cases
Pancreas and Liver
- Special case: tissue renewal not dependent on SC
- Fully differentiated cells dividing allowing for regeneration
- Insulin secreting pancreatic cells sequestered in islets of Langerhans
o Fresh B cells constantly generated
o Lineage tracing shows this is due to duplication of insulin expressing cells
- Hepatocytes
o Performs livers metabolic functions
o Can perform large divisions to replace lost tissue
55
Reprogramming
- Xenopus experiment showed that a cell nucleus is capable of generating any cell type
- Cytoplasmic factors reprogram the nucleus
o First, the reprogramming in such experiments is not perfect. When the transplanted nucleus is taken from a gut cell, for example, a gene that is normally specific to the gut is found to be expressed persistently, even in the muscle cells of the final animal. Second, the experiment succeeds in only a limited proportion of cases, and this success rate becomes lower and lower, the more mature the animal from which the transplanted nucleus is taken: very large numbers of transplantations must be done to score a single success if the nucleus comes from a differentiated cell of an adult frog.
- In a typical differentiated cell there are mechanisms maintaining the pattern of gene expression cytoplasmic factors cannot easily override, ie. self-perpetuating modifications of chromatin
- The egg contains factors resetting chromatin and wiping out modifications
56
Reprogramming TF
o The process begins with a Myc-induced cell proliferation and loosening of chromatin structure that promotes the binding of the other three master regulators to many hundreds of different sites in the genome. At a large proportion of these sites, Oct4, Sox2, and Klf4 all bind in concert. The binding sites include the endogenous Oct4, Sox2, and Klf4 genes themselves, which eventually creates the types of positive feedback loops just described that makes expression of these genes self-sustaining (see Figure 22-41). But self-induction of Oct4, Sox2, and Klf4 is only a small part of the transformation that occurs. The three core factors activate some target genes and repress others, producing a cascade of effects that reorganize the gene control system globally and at every level, changing the patterns of histone modification, DNA methylation, and chromatin compaction, as well as the expression of innumerable proteins and noncoding RNAs. By the end of this complex process, the resulting iPS cell is no longer dependent on the artificially generated factors that triggered the change: it has settled into a stable, self-sustaining state of coordinated gene expression, making its own Oct4, Sox2, Klf4, and Myc (and all the other essential ingredients of a pluripotent stem cell) from its own endogenous copies of the genes
57
Timothy Syndrome
o By using SC to replace tissues degenerative diseases can be cured
o Avoid immune rejection
o Generate large cell populations for disease and drug mechanisms
o Where a disease has a genetic cause, we can derive iPS cells from sufferers and use these cells to produce the specific cell types that malfunction, to investigate how the malfunction occurs, and to screen for drugs that might help to put it right. Timothy syndrome provides an example. In this rare genetic condition, there is a severe, life-threatening disorder in the rhythm of the heart beat (as well as several other abnormalities), as a result of a mutation in a specific type of Ca2+ channel. To study the underlying pathology, researchers took skin fibroblasts from patients with the disorder, generated iPS cells from the fibroblasts, and drove the iPS cells to differentiate into heart muscle cells. These cells, when compared with heart muscle cells prepared similarly from normal control individuals, showed irregular contractions and abnormal patterns of Ca2+ influx and electrical activity that could be characterized in detail. From this finding, it is a small step to development of an in vitro assay for drugs that might correct the misbehavior of the heart muscle cells.
o Huge advance on the slow costly traditional methods of clinical trials
ICB
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