lecture 16

Question 1

What is the model of transcriptional regulatory circuitry in ES cells?

Answer 1

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)

Question 2

What is the expanded transcriptional regulatory circuitry in ES cells?

Answer 2

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

Question 3

What is Bivalent status?

Answer 3

• provides a mechanism to maintain pluripotency and repress lineage gene expression
• but not permanently silence lineage gene expression or pluripotency genes
• seesaw status

Question 4

What are the roles of LIF and BMP4?

Answer 4

• 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

Question 5

What are signals for maintaining pluripotency and bivalent status?

Answer 5

• 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

Question 6

What is the ground state of pluripotency?

Answer 6

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

Question 7

What is nuclear re-programming?

Answer 7

• 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

Question 8

What do ES cells look like?

Answer 8

• high nucleus to cytoplasm ratio

Question 9

How do we reprogramme somatic cells into iPS cells?

Answer 9

• 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

Question 10

How similar are ES and iPS cells?

Answer 10

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

Question 11

What is our current understanding of stem cells?

Answer 11

• 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

Question 12

What are our current problems with stem cells?

Answer 12

• 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’

Question 13

What are applications of ES and iPS cells?

Answer 13

• 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