lecture 16 Flashcards
Stem cells cont.
What is the model of transcriptional regulatory circuitry in ES cells?
Oct4, Sox2, Nanog
- activated themselves in an auto-regulatory loop
- also activated each other
- lead to the idea that there is a core pluripotency circuitry (in ES cells only)
What is the expanded transcriptional regulatory circuitry in ES cells?
Nanog, Oct4, and Sox2: Activate: - chromatin remodelling - histone acetylation - histone methylation - TGFBeta signalling
- ES cell transcription factors
Repress: factors that induce: - neurogenesis - mesoderm - endoderm - extra-embryonic endoderm
What is Bivalent status?
- provides a mechanism to maintain pluripotency and repress lineage gene expression
- but not permanently silence lineage gene expression or pluripotency genes
- seesaw status
What are the roles of LIF and BMP4?
- ES cells are derived from the inner cell mass
- under appropriate conditions in vitro can proliferate to form all cell types of an embryo
- originally ES cells grown on fibroblast layer with serum added to media
- Smith’s work has shown that LIF can replace fibroblast layer (blocks mesoderm and endoderm differentiation)
- also that BMP4 can replace serum (blocks neural differentiation)
- adding LIF and BMP4 to media suppresses all differentiation
What are signals for maintaining pluripotency and bivalent status?
- cell seesaws between being an ES cell and a progenitor cell
- activating the MAP kinase pathway tips the balance towards becoming a progenitor cell (and lineage commitment)
- high levels of Nanog will swing it back to being an ES cell
- tagged ES cells for Nanog and Oct 4, levels of Nanog turned on and off thus suggesting bivalent status
- balance between two states possible within the same cell, Thus: bivalent status
- the cell is in equilibrium between maintaining pluripotency and becoming committed to a specific lineage
- Oct4 and Sox2 remained the same
- can’t turn off lineage commitment pathway
What is the ground state of pluripotency?
experiment to see if they could keep the cells in pluripotent state/not in an equilibrium of being on and off
- LIF and 2i
- tested inhibitors of:
- FGF — MAP kinase pathway (PD03)
- GSK3 — non-canonical Wnt pathway (CHIR)
in presence of LIF and 2 inhibitors (PD03 and CHIR, known as 2i) differentiation is blocked and >90% of cells are nanog and oct 4 positive
this minimal requirement for self-renewal is known as the ground state of pluripotency
Therefore:
- the ground state is critically dependent on levels of nanog expression
What is nuclear re-programming?
- retroviral vector transfection: Oct4, Sox2, c-myc, Klf-4 (tried about 30 different tfs in different permutations and combinations)
- found that the above four could induce pluripotency in/alter phenotype of fibroblasts
- re-programmed into pluripotent ES cells
- put these cells through the rigid tests
- i.e. induced Pluripotent Stem cells
What do ES cells look like?
- high nucleus to cytoplasm ratio
How do we reprogramme somatic cells into iPS cells?
- somatic cell + oct 4, sox 2, Klf4, c-myc (transgenes)
- reprogramming process: 1 - 4 weeks duration
partially reprogrammed:
- ES cell like appearance
- initiation of MET
- downregulation of somatic gene expression
- retroviral vector expressed
- oct 4 endogenous locus not active
fully reprogrammed:
- activation of endogenous Core Pluripotency Circuitry
- silencing of transgenes
- complete epigenetic resetting
- cytoskeletal remodelling
- chromatin remodelling
- oct 4: initiates nanog expression
- oct 4 probably the most upstream transcription factor in the process of reprogramming of somatic cells (not the same as normal)
- other ‘magic brews’ have been determined but Oct 4 is non-negotiable
- probably most upstream in regulatory circuit
- cannot be substituted in this process
How similar are ES and iPS cells?
Similar:
- morphology, age-related telomeres, surface markers, overall gene expression
Differences:
- takes a long time to make iPS cells and very few reprogrammed
- need human oocytes to derive human ES cells
- some studies have found differences in genetic/gene/protein expression but some studies have not
- differences could be due to reprogramming OR pre-existing genetic and epigenetic 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 versus control iPS cells may not reflect disease but may be due to variation of cell lines
- despite much work over the last 7 years, issue is not resolved
What is our current understanding of stem cells?
- a somatic cell nucleus is not permanently specialised
- can be induced to change its — given correct signals
- so generating any cell type from a ES cell or iPS cells is just a matter of finding the correct signals
What are our current problems with stem cells?
- ethical issues — use of human embryonic cells (iPS eliminates this?)
- tumour cells: not all become specialised and the remaining can become cancerous
- purification techniques are improving
- LIF and 2i treatment has led to new developments
- all ES and iPS cells can now be cultured in ‘ground state’
What are applications of ES and iPS cells?
- availability of new/rare cell populations in large numbers
- derive ES cells from donor blastocyst/or generate iPS cells from patient’s somatic cells cell type
- want to put these cells into a master cell bank
- add signals in culture to create specialised cell required
- transplant into patient
- using own cells means immunocompatible
e. g. rat embryonic stem cell-derived oligodendrocyte progenitors for the treatment of spinal cord injury — was not super effective
e. g. human embryonic stem cell-derived oligodendrocyte progenitors for the treatment of spinal cord injury: Katie Sharify
