lecture 3: pluripotency and iPS cells Flashcards
1
Q
How do we define pluripotency?
A
- functionally and molecularly
2
Q
What is potency?
A
- stem cells are categorised by potency, which denotes the potential of the cell to derive other cell types – how many and what cell types
- potency: the range of developmental options available to a cell
- totipotent: ability to form the entire organism (includes placenta/extra embryonic tissues). In a mammal only the zygote and the blastomeres are totipotent. Not demonstrated for any other mammalian stem cell type.
- pluripotent: ability to form all the lineages of the body. example: embryonic stem cells and Embryonic Germ (EG) cells
- cannot form placenta
- multipotent: ability to form multiple cell types from onelineage. e.g. haematopoietic stem cells which form all the blood type cells
- unipotent: ability to form one cell type. e.g. spermatogonia which can only form sperm
3
Q
What is the first test for pluripotent stem cells?
A
- in vitro differentiation
- take away culture conditions and see random differentiation
- differentiate spontaneoulsy in vitro into derivatives of the three germ layers: ectoderm, mesoderm, endoderm
- least stringent test
- the expression for differentiation markers is not a test for functionality:
- any changes in culture conditions can stress the cells or induce differentiation
4
Q
What is the second test for pluripotency?
A
- teratoma formation
- take 200-400 cells, inject under skin, kidney, abdomen of SCID mouse (no functional immune system)
- formation of teratomas when injected into immune-deficient mice
- differentiate spontaneously in vivo into derivatives of the three germ layers; ectoderm, mesoderm, endoderm due to loss of pluripotency and exposure to signals in the new environment that induce differentiation
- does not test for the ability to promote normal development
- should we be using eggs instead of mice since it is a big imposition on the mouse?
5
Q
What is the third test for pluripotency?
A
- germline chimerism
- white mouse donates blastocyst
- ES cells from black mouse
- inject ES cells from black mouse into blastocyst from white mouse
- inject into pseudopregnant mouse
- generate chimera
- when injected into donor blastocyst, ES cells contribute to all tissues of the resulting offspring
- can test for germline competency
- does not test for complete pluripotency i.e. problems caused by epigenetic defects affecting development
- one of the most important tests for pluripotency
6
Q
What is the fourth test for pluripotent stem cells?
A
- tetraploid complementation
- produced by injecting ES cells into a tetraploid (4n) blastocyst
- the tetraploid embryo can only produce the placental tissues
- epiblast are from the cells that you injected and only from the cells that you injected
- most stringent test for pluripotency
- because 4n host cells cannot contribute to somatic lineages, embryo is exclusively composed of the cells formed from the injected cells
- can test for germline competenct
- does not test for the ability for form trophoblast (placental) lineage
7
Q
What are characteristics of mouse embryonic stem cell colonies?
A
- maintain normal karyotype
- pluripotent ES cells express these stem cell markers: e.g. AP (alkaline phosphatase, highly expressed), SSEA (Stage Specific Embryonic Antigen 1, cell surface marker on ES cells), Oct4, Nanog and Sox 2
- conventional ES culture requires: (mitotically inactivated) Fibroblast feeder layer and serum
- serum and feeder layer-free ES culture
- Feeder layer replaced by LIF (leukaemia inhibitory factors) - suppresses mesoderm and endoderm
- serum can be replaced by BMP (bone morphogenic protein) - suppresses neuroectoderm
8
Q
What are characteristics of human ES cells?
A
- do not depend on LIF
- grow as flat, epithelial colonies (mouse ES cells grow as non-epithelial, domed colonies)
- unlike mouse ES cells, cannot be passaged as single cells
- colonies must be mechanically broken into smaller pieces for passaging
- serum- and feeder-free culture requires Activin (activates Nodal signalling pathway) and FGF growth factors
- why are mouse and (currently used) human ES cells so different?
9
Q
How do mouse and human ES cell characteristics compare?
A
- both express Oct 4, Sox 2, nanog
- human:
- different surface markers (e.g. SSEA-4, hTRA-1 proteins)
- do not express LIF receptor, and gp130
- express some trophoblast markers (e.g. esomesodermin)
- express some differentiation markers (Brachyury, Fgf5, AFP< keratin 14)
- difference in cell cycle, cell death pathways, cytokine gene expression
- differences in signalling pathways
- express vimentin
- can form teratomas, but germline chimera not tested
- require FGF and IGF both in vivo and in vitro
- conclusions:
- human culture conditions not optimal? or
- human cells isolated at later stage of development?
10
Q
Compare pluripotent ES cells and EpiSC
A
- ES cells derived from embryonic day 3.5, 4.5
- EpiSC, 4.5, 5.5
- preimplantation epiblast derived ES cells - best stage for deriving pluripotent stem cells
- post-implantation EpiSC are different - features
- ES are rounded
- EpiSC from both post implantation and preimplantation - easier post
11
Q
What is the difference between pluripotent pre-implantation and post-implantation epiblast stem cells?
A
Pre vs Post
- embryonic tissue: late blastocyst (epiblast) vs egg cylinder (epiblast)
- cultured stem cell: ES cells vs Epi Stem Cells (EpiSC)
- chimeras: yes vs no
- teratomas: yes vs yes
- pluripotency factors: oct4, nanog, sox 2, Klf2, vs oct4, nanog, sox2
- differentiation markers: absent vs Fgf5, Brachyury
- response to LIF: self-renewal vs none
- response to FgF/Erk: differentation vs self-renewal
- response to 2i: self-renewal vs differentiation/apoptosis
- both cell lines are pluripotent: able to form cells of the three primary germ layers, express pluripotency factors but are clearly different
- ‘primed epiblast’ represents a more advanced stage of development than the ‘Naive epiblast’
- thus naive pluripotency versus primed pluripotency
12
Q
What are the mouse post-implantation epiblast-derived stem cells (epiSC)?
A
- derived from the epithelial, post-implantation epiblast and maintained under similar conditions to human ES cells (Activin & FGF2; not LIF-dependent)
- pluripotent, but not-germline-competent
- very similar to human ES cells in morphology and gene expression profile: hypothesised to be equivalent
- thus current hES cells are more like mouse EpiSC than ES cells
13
Q
Why is studying mammalian development and understanding the molecular basis of pluripotency so important?
A
For transplantation therapies:
- how cells form in the embryo
- how to maintain SC and form differentiated cells
- when and what type/stage of cells to transplant into patients
- because we don’t understand there are always some stem cells that defy attempts to turn into a differentiated cell
for intellectual interest
14
Q
How do stem cells divide to produce daughters with different fates?
A
- stem cell divides to produce two daughter cells: one goes on to become terminally differentiated cell and one a stem cell (self renewal)
- million dollar question: why is this cell unaffected?
15
Q
What is the transcriptional regulatory circuitry in ES cells?
A
- thought they by knowing the 353 genes controlled by nanog, sox 2 and oct 4 it would all work out and we would understand pluripotency
- if only it were this simple
- this data suggests that Oct4, Sox2, and Nanog function together to regulate a significant proportion of their target genes in ES cells
16
Q
What is a model of transcriptional regulatory circuitry in ES cells?
A
- all three factors regulate the others (and themselves)
- these factors are now known as: core pluripotency regulators
- Oct4, Sox2, and Nanog collaborate to form a regulatory circuitry in ES cells
