Lecture 15 - Totipotent, Pluripotent and Multipotent Stem cells Flashcards
What are the two defining criteria of stem cells?
- Self renewal (have the ability to divide for indefinate periods [although adult stem cells have a shorter life])
- Differentiate into different specialised cell types (e.g. blood cells, skin cells, bone cells)
What is the intermediate cell called between a fully differentiated cell and a stem cell?
A progenitor cell
What is a progenitor cell?
A cell that has commited to a certain cell lineage and will further differentiate into a cell of that lineage
How is a population of stem cells maintained?
By asymmetrical division of stem cells to form a differentiated daughter cell that is committed to a particular cell lineage and a stem cell to renew the population
Why is asymmetrical division by stem cells important?
To ensure the pool of stem cells is not depleted
What are the three possible mechanisms that may control the asymmetric division of stem cells?
Segregated cell polarity regulators
Segregated cell fate determinants
Niche Elements
How do Segregated cell polarity regulators control the asymmetric division of stem cells?
-asymmetric localisation of cell polarity regulators (localisation determines the cell that will become a stem cell) initiates cells division e.g. PAR-aPKC complex
How do Segregated cell fate determinants control the asymmetric division of stem cells?
cell fate determinants are segregated to the cytoplasm of one daughter cell, or associated with the membrane, centrosomes or another cellular constituent that is differently distributed to daughter cells
How do Niche elements control the asymmetric division of stem cells?
Stem cells are surrounded by niche microenvironments
- Regulation of the orientation of the mitotic spindle retains only 1 daughter in the SC niche, so that only one daughter has access to the extrinsic signals necessary for maintaining SC identity
- other daughter cell is exposed to signals away from the niche that induces differentiation
When is the blastocyst formed?
From day 5
What is the blastocyst?
-fluid filled ball with an inner cell mass, surrounded by trophectoderm (made of trophoblasts)
What is implantation of the blastoderm guided by?
The trophectoderm
What do the inner cell mass and the trophectoderm develop into?
ICM
-embryo
Trophectoderm
-extra embryonic tissues e.g. placenta
When are the embryonic stem cells totipotent, pluripotent and what does this mean?
Totipotent
-before blastocyst formation
-very brief and early stage
-can develop into entire organism including the placenta
Pluripotent
-after blastocyst formation the cells of the ICM are pluripotent
-can develop into any cell other than the placenta
How does the division of the cells in the embryo occur initially?
The size of the embryo remains the same, cells divide but do not get bigger
What do the markers of the ES cells allow experimentally?
Allow identification and isolation
What are the common markers of the Mouse ES cells and the human ES cells?
Mouse ES cells
-SSEA1
Human ES cells
-SSEA3/4
Shared
- CD133
- Oct-4
- Nanog
- Sox2
How can you experimentally determine pluripotency (i.e. stem cell functionality)
In vivo method - Teratoma assay
Termatoma (cancer tumours that make cells of all three germ layers)
-inject SC and observe the tissues formed
-if pluripotent then will form all 3 germ layers
-this shows differentiation (1 features of SC) but not self renewal
Prove Self renewal
- using markers, remove cells that are identified as stem cells
- implant the extracted cells and if forms a whole new teratoma then the cells can do self renewal
How is pluripotency maintained?
- the prevention of differentiation
- the promotion of proliferation and self renewal
What is pluripotency regulated by?
several signalling pathways and TFs
What are the signalling factors involved in regulating pluripotency and what are their features?
Leukemia inhibiting factor
- members of the interleukin-6 type cytokine family
- LIF prevent differentiation of mouse embryonic stem cells and promote self renewal to maintain pluipotency
What occurs without LIF in mouse ESC?
ESC spontaenously differentiate
What is the pathway through which LIF acts?
- acts through the LIF receptor (LIFRβ) in association with the signalling component gpi30
- STAT3 is activated by LIF (downstream of LIF) and acts as a transcription factor
What proteins are involved in inputting cues via LIF and the LIFRβ into the mouse ESC?
JAK - janus kinase
SHP2 - SHP2-domain-containing protein tyrosine kinase 2
MEK - nitrogen activated protein kinase (MAPK) and extracellular signal related kinase (ERK) protein kinase
What type of cell can an embryonic stem cell be based upon determined by its signalling?
Trophectoderm - totipotent
ES - pluripotent
Primitive ectoderm - differentiation
What is the action of Sox2-Oct4?
- Inhibit trophectoderm
- promote ES and primitive ectoderm
What is the action of Nanog?
- inhibits primitive ectoderm
- promotes ES
What is the action of STAT3?
- inhibits or promote primitive ectoderm
- promotes ES differentiation
What are the three TFs that regulate the pluripotent state of the ESC?
The interaction of:
- Sox2-Oct4
- Nanog
- STAT3
What are the features of Oct4 (aka Oct3)?
- ‘gate keeper’ TF, expressed by pluripotent ESC
- prevents differentiation
- present at the 4 cell stage of embryo
- at blastocyst stage only expressed in the ICM
- down regulated once cells are committed to a lineage
What experiments showed the action of Oct4? And what were the conclusions from these experiments?
Knockout in mice
- inner cell mass fails to develop
- ES cells differentiate inappropriately into trophectoderm
Overexpression
-ESC differentiation into primitive ectoderm and mesoderm
Conclusions
-specific levels of Oct-4 are required to maintain ES cells in a primitive, pluripotent state
What is the structure and action of Oct4?
TF so associates with DNA via 2 DNA binding domains
-binds and regulates down stream genes that code for factors involved in the pluripotency of ESC: FGF4, Rex1, Sox2
What are the two main roles of Oct 4?
Maintain self renewal and pluripotency
Promote differentation
What are the features of Oct4 in maintaining self renewal and pluripotency?
modulates genes that promote:
- permissive chromatin
- DNA repair
- anti-apoptosis
- anti-differentiation
- inactivate pRb (which is cell cycle linked - more of it stops the cell cycle)
What are the features of Oct4 in promoting differentiation?
modulates genes that promote: -repressive chromatin -DNA checkpoint control -apoptosis -active pRB All this facilitates cellular commitment
What are the features of Sox2?
- cooperative with Oct4, forms a complex
- expression pattern similar to Oct-4
- cooperatively bind DNA
What is the action of the Sox2-Oct4 complex?
- regulate Nanog expression
- cooperate with Nanog to control ES cell pluripotency
What is Nanog?
- TF expressed specifically by ESC in the ICM
- slightly later expression than Oct-4
- induces longevity
What is the action of Nanog?
- Acts as a repressor to prevent transcription of genes required for differentiation
- activates genes that are required for ESC self renewal e.g. Rex1, which is also a target for Oct-4
What are the main factors involved in determining ESC pluripotency?
Oct4/Sox2, Nanog and Rex1 have interrelated roles in determining ESC pluripotency, determined by their relative levels
How do Oct4, Nanog and Sox2 prevent and control differentiation down the germ layer lineages?
Promote self renewal via modulating the action of two groups of genes:
- Activate promoters of self-renewal genes (OCT4, SOX2, NANOG)
- Silence promoters of developmental genes (NEUROG1, PAX6, GATA4)
How are the activating and silencing of genes by Oct4, Sox2 and Nanog acheived?
Through epigenetics
- specifically chromatin packaging
- ESC have open chromatin (permissive and transcriptionally active
What is the state of chromatin as differentiation progresses?
Initially ESC have open chromatin (permissive and transcriptionally active
-as differentiation progresses get closed chromatin (transcriptionally repressive)
What are the changes in chromatin for the transcriptional control of differentiation based on?
- stage of the cell
- what genes are needed/not
What does stopping pluripotency require?
Interacting factors
- specifically LIN1,2,3 (lineage transcription factor)
- Sox2/Oct4/Nanog influence is reduced as lineage is commited to
- LIN’s activated so Sox2/Oct4/Nanog cannot reach DNA
- the specific factor determines which lineage the cell goes down
What happens to telomeres with cell division?
In the adult cell, telomeres shortern with every division, meaning there is a limited no of adult cell division
What occurs to cells without telomerase?
Reach quiescene
What are the features of telomerase in ESM and cancers?
- have more telomerase
- lose telomeres but repeating DNA sequence (TTAGGG) lengths are added back on (5-15 times) so that there is no telomere shortening overall
What type of metabolism is present in ESCs/adults?
ESC
-tend to have glycolytic metabolism
Adults
-phosphorylation based metabolism
What are the features of adult stem cells?
-involved in post natal patterning e.g. remodelling and wound repair
-mainly in bone marrow, skin and colon
-multipotent (differentation potential more limited than the ESC)
-can self renew and differentiate in the cell type of the host tissue in which they reside
-
What is the primary function of adult stem cells?
Maintain steady state of activity of a cell and its resident tissue
-may also help replace cells lost through injury or disease
Why might adult stem cells have reduced self renewal ability compared to ES cells?
Reduced telomerase
-not as much risk of teratomas forming if used in therapy compared to ESC
How can you test the properties of adult stem cells?
Destroy the bone marrow of an animal
Inject stem cells and should completely replenish
-observe markers
What are 5 examples of adult stem cells?
- Haematopoietic
- Epidermal (basal layer of skin)
- Mesenchymal (bone marrow stem cells)
- Neural
- Limbal
What are the features of Neural stem cells?
- in the adult brain (as well as progenitor cells)
- capable of generating neurons and glial cells
- many located in the subventricular zone which lines the walls of lateral ventricles
- self renewing
What is the subventricular zone?
- very inaccessible part of the brain in terms of therapy
- the niche environment for neural stem cells
What is the general differentiation process of neural stem cells?
Neural stem cells
Rapidly dividing daughter cells
Migratory precursors (located near vascularture, increasing possibility for transportation)
Migratory precursors travel to other bits of the brain
What is the specific differentiation process of neural stem cells into glia and neurons?
Glia
-NSC differentiate into Glial restricted precursor cells
-then divide into either oligodendrocyte progenitor cells (then oligodendrocytes) or astrocyte
Neurons
-NSC divide into neuron restricted precursors and then to neurons
What are teh features of limbal stem cells?
- in limbus (or eye), an accessible site
- generate the cornea
- high proliferative potential
- migrate out and around cornea to enable function
- surrounded by niche resident supporting cells
- all eventually differentiate
- form transit amplifying cells before fully differentiating
What is the purpose of niche resident supporting cells surrounding limbal stem cells?
anchor cells and expose future daughter cells to the correct signals for self renewal or differentiation
