lecture 4: stem cell niches Flashcards
1
Q
What is the bivalent state?
A
- ES cells are in a constant state of flux
- Probably true of promoters in all cells of our body but hard to see in vivo cf in vitro
- need to actively keep genes required for differentiation ON
- continuous, active process
- seesaw effect: can easily repress or activate SCs to self renew or move along a pathway of differentiation
2
Q
What is 2i culture and the ‘ground state’ of pluripotency?
A
- There are genes that encourage growth (allowing it to divide) and genes for viability (maintaining the state of the cell)
- can divide or differentiate depending on the signals in culture
- ES cell secretes a factor called FGF4
- Bind to FGF receptor on the ES cell
- i.e. acting in an autocrine fashion
- stimulates a signalling pathway which is the Mek/Erk parthway which leads to differentiation
- thought that LIF works on this pathway
- tested a lot of inhibitors of pathways and found that PD03 would block the signal transduction pathway activated by binding of FGF therefore blocking differentiation
- did a whole lot of permutations and combinations and discovered they could use CHIR (block a GSK3) therefore enabling the cell to self renew
- so blocking FGF is preventing differentiation, blocking GSK3 (part of wnt signalling) inhibits ability to self renew
- found that just putting those two inhibitors into the culture with no serum, fibroblasts, BMP led to about 90% of cells expressing nanog, with LIF got 95% of cells (still not 100%)
- blocking the differentiation using 2i
- at ground state all cells are stable, expressing nanog
- FGF in negative feedback system
- when FGF is low, high nanog and vice versa (see saw effect)
- fluctuating levels of nanog in conventional culture, bivalent state
- In 2i, all cells express Nanog at high levels - the ‘ground state’ of pluripotency ~ pre-implantation epiblast
- ground state defined as stable, uniform molecular signature, pluripotent stem cells, expressing high levels of Nanog
- culture of ES cells with both CHIR and PDO3 inhibitors is called 2i culture (enhanced by addition of LIF, so called 2i+LIF)
- 2i+LIF culture has enabled ES cell derivation from all strains of mouse and also from rat
- BUT, 2i + LIF culture does not support the derivation or maintainance of human ES cells
3
Q
What is conventional culture?
A
- serum and feeder layers
- BMP and LIF
4
Q
What is the role of nanog?
A
- nanog -/- embryos fail to make epiblast
- nanog -/- ES cells differentiate
- therefore essential for acquisition of pluripotency in vivo and in vitro
5
Q
What are mouse iPS cells?
A
- take ordinary skin cells
- put them into culture - they turn into fibroblast cells
- look a bit stringy, no particular shape
- retroviral vector transfection: Oct4, Sox2, c-myc, Klf-4
- viral expression vectors
- changed shape and function
- beautiful, round, high nuclear to cytoplasm ratio ES cells
- put these cells through the four tests for pluripotency
- iPS cells - induced pluripotent stem cells
- re-programmed into pluripotent-like ES cells + germline chimerism
- many different combinations
- possible to take any cell and reprogramme it back to pluripotency with all four tests
- took 24 tfs that they knew were expressed in ES cells
- put them in viral vectors and transfected 24 into those fibroblast cells
- then took one out and so on to see what was the minimum they could use
- interesting and clever experiment
6
Q
What does reprogramming a cell involve?
A
- sequential activation of pluripotency marker gene expression
- take somatic cell and express viral vectors + Oct5, Sox2, Klf4 and c-myc
- wait 4 - 5 weeks - this is how long it takes to get to iPS cell
- early reprogramming (1-2 weeks):
- retroviral vectors expressed
- gain ES cell morphology and proliferation rate
- (MET)
- if you started with an epithelial cell instead of a mesenchymal cell that would be one less step
- wouldn’t need Klf4
- ES cell markers AP/SSEA1
- loss of somatic gene expression
- no pluripotent endogenous genes expressed (e.g. Oct4, Nanog, Sox2)
- no chimeras
- late reprogramming (3 - 4 weeks):
- retroviral vectors silenced
- de novo expression of methylases/acetylases
- all pluripotent genes expressed
- somatic genes hypermethylated
- germline chimerism
- exogenous Oct4 acts in early reprogramming and Nanog during late reprogramming
- if somatic/reprogramming cell does not express Nanog during late phase, reprogramming does not contine – ‘essential for acquisition of pluripotency’
- in a culture dish of 10 million cells will be lucky if 5% become induced pluripotent cells
7
Q
How similar are human ES and iPS cells?
A
Similarity:
- morphology
- age-related telomeres
- surface markers
- overall gene expression
differences:
- takes a long time to make iPS cells and very few cells are reprogrammed
- need human blastocysts to derive human ES cells
- some studies have found differences in genetic/gene/protein expression
- but some have not (e.g. differences in histone modifications and DNA methylation)
- differences could be due to reprogramming OR pre-existing genetic and epigentic differences within individual parent cells (i.e. normal biological variation)
- but does raise concerns about usefulness for therapeutic applications
- differences in disease specific iPS cells verus control iPS cells may not reflect diseas by may be due to variation of cell lines
- this issue of difference/similarity is not yet resolved
8
Q
What is a stem cell niche?
A
- describes the microenvironment in which stem cells are found and which interacts with stem cells to regulate stem cell fate
- ‘niche’ can be in vivo or in vitro stem cell micro environments
- “Stem-cell populations are established in ‘niches’ – specific anatomic locations that regulate how they participate in tissue generation, maintenance and repair. The niche saves stem cells from depletion, while protecting the host from over-exuberant stem-cell proliferation. It constitutues a basic unit of tissue physiology, integrating signals that mediate the balanced response of stem cells to the needs of organisms. Yet the niche may also induce pathologies by imposing aberrant function on stem cells or other targets. The interplay between stem cells and their niche creates the dynamic system necessary for sustaining tissues, and for the ultimate design of stem-cell therapeutics… The smiple location of stem cells is not sufficient to define a niche. The niche must hae both anatomic and functional dimensions” - David T. Scadden (2006). The stem-cell niche as an entity of action. Nature
9
Q
Show the complexity of the stem cell niche in the context of the adult human stomach
A
- stem cells sit at the base of the glands and the base of the pits
- gives rise to the four other cell types in the gland: pit mucus cell, parietal cell, gland mucus cell, endocrine cell
- next to many other tissues
10
Q
What are some possible factors and interactions regulating a stem cell niche?
A
possible interactions:
- cell-cell interactions
- cell-ECM interactions
- cell-BM or basal lamina interactions
- cell-blood vessel or nerve cell interactions
via
- adhesion molecules, gap junctions, tight junctions etc
- hemi-channels (allow communication between cell and the interstitial fluid environment)
- ECM proteoglycans (can sequester molecules and inhibit signallin or release when needed)
- secreted factors e.g. Growth factors, cytokines, hormones
- physiochemical nature of the environment: - pH, oxygen and carbon dioxide levels, oxidative state, concentration of metabolites/ions/ATP etc
- and i am sure you will learn about other factors/components
11
Q
Why is the niche so important?
A
- components of the niche - basal lamina directed gene expression in the mammary gland
- effect of a normal niche on tumour cells (EC cells and chimera formation)
12
Q
What is the basement membrane?
A
- the epithelial layer (or epithelium) lie on a connective tissue layer (or stroma) containing various cell types, including stromal (or mesenchymal) cells, and a complex ECM composed of proteins including collagens, laminins and proteoglycans
- at the interface between the two tissues (epithelium and stroma) is a basement membrane which is thin but made up of three layers (lamina lucuda, lamina densa, reticular lamina) of specialised ECM composed of proteins including laminin, type IV collagen and entactin
- the BM provides structure and anchoring but also influences cell proliferaton, differentiation, cell metabolism, growth and provides pathways for migration etc
- cells (usually epithelial type) are attached via hemidesmosomes, integrins etc
13
Q
What kinds of stem cell divisions can you have in the niche?
A
- asymmetric: SC and daughter cell
- symmetric
14
Q
What is an example of the importance of the basal lamina?
A
- basal lamina directed gene expression in the mammary gland
- they took mammary gland epithelial cells (epithelial cells in culture will form tight junctions and become a connected layer of cells)
- if you grow these just on the plastic (no substrate etc) these cells will continue to divide and make an epithelial layer
- great if you want to make skin cells for burn victims
- cell cycle genes are on and tissue specific genes are off
- cells divide
- if you grow these cells on a basal lamina (some kind of coating e.g. laminin, collagen etc)
- those cells start to form a ball of cells and express integrins that anchor them to the basal layer
- turning off cell cycle genes and turn on the tissue specific genes
- cells stop dividing and begin differentiation
- tissue specific genes expressed in progressive manner
- basal lamina grown all the way around that ball of cells
- epithelial layer forming more/different tight junctions
- beginnings of formation of lumen
- basal lamina circled all the way around, epithelial cells line a lumen
- i.e. basal lamina has allowed those cells to form a secretory gland
- secretion of milk proteins etc
- simply adding basal layer has allowed flat layers of cells to form a tissue specific, functionally organised gland
- when mammary epithelial cells are cultured on plastic flasks or petri dishes, they will divide and express cell division genes (c-myc, CyclinD1) but mammary gland specific genes for milk proteins lactoferrin, casein and why acidic protein (WAP) - are not expressed
- if the mammary epithelial cells are cultured on a layer of basal lamina coating the plastic glasks or petri dishes, the cells become attached to the basal lamina via integrins forming a colony of cells, stop dividing, switch off c-myc and CyclinD1, and begin to express lactoferrin and p21 (an inhibitor of the cell division)
- eventually the epithelial cells arrange themselves into a secretory gland wrapped in a basal lamina
- during this change in structure, other milk protein genes, casein and WAP, are switched sequentially
- the epithelial cells form a single layer of epithelial cells (epithelium), lining a lumen, held together by tight junctions
- the cells have become similar to a milk secreting gland and begin to secrete milk proteins into the lumen
- there is evidence to suggest that it is the binding of integrins to the basal lamina that stimulates the Ras/MEK/ERK signal transduction pathway leading to changes in specific gene transcription
15
Q
What are embryonic germ (EG) cells and embryonal carcinoma (EC) cells?
A
- EG cells are stem cells derived from primordial germ cells that are pluripotent
- EC cells are stem cells derived from the undifferentiated cells in a teratocarcinoma and are pluripotent
- teratocarcinoma cells are primordial germ cells that are malignant
- EC cells were used before ES cells were developed and led to developments in chimera formation by Stewart and Mintz (1981)
