Modelling embryonic development and disease using pluripotent stem cells - 1 Flashcards
DEFINITION - pluripotent cell
What is an important property of pluripotency?
Cell with the ability to generate all cell types of the embryo including germ cells.
Pluripotency is transient – this means the pluripotent state of cells is temporary
DEFINITION - stem cell
An undifferentiated cell of a multicellular organism that can give rise to infinitely more cells of the same type. Other cells can arise by differentiation
Properties of early embryonic cells? What do they undergo?
Pluripotent cells that are only confined to very early developmental stages (pluripotent cells are transient)
Gastrulation occurs which initiates generation of ALL CELL TYPES (the germ layers)
2 MAIN IDENTIFIABLE FEATURES OF PLURIPOTENT CELLS
- express Pluripotency factors
- when P.P. Cs are grafted into a location they give rise to teratocarcinomas (TUMOURS CONTAINING ALL CELL TYPES). Non-pluripotent cells will not give rise to these
Examples of pluripotency factors and where are they found when observed in mice embryos and how was this identified?
Sox2, Nanog, Oct4
Expressed in the inner cell mass of the developing mouse embryo, this was identified by IN SITU HYBRIDISATION
Why is pluripotency often hard to study in vivo? Why is in vitro done instead?
Embryos are v small therefore often difficult to study in the uterus.
They are often observed and captured in a petri dish so that they can be seen and so that clinically relevant populations of cells can be reproduced
How are E.S.Cs captured and viewed in vivo?
- The inner cell mass of embryonic stem cells is dissected away and PLATED on a layer of feeder cells.
- once E.S cells have divided, they are disaggregated and replated - E.S cells express GFP so are distinguishable
- Signals from the feeder cells are used to maintain self renewal properties in the cells!
What is the relevance of feeder cells that allow growth of E.S.Cs in vitro
Feeder cells provide trophic factors such as BMP (in mouse) that are normally found in the niche of pluripotent embryonic stem cell
What are the main properties of E.S cells?
- Express pluripotency factors such as Oct4, Nanog and Sox2
- A single E.S cell can generate an identical daughter cell
- No differentiation genes are expressed in these cells
- Can form a teratocarcinoma hen transplanted
How can you test for an E.S cell in mice?
-Mouse ES cells can be reintroduced to normal blastocysts and contribute to normal development of chimeras.
How can you reprogram adult somatic cells and how can this be tested?
- Take differentiated cell (skin biopsy of human donor cells) and inject with reprogramming factors
- Reprogramming factors include cMyc/Oct4/Sox2
- This generates a IPS cell which can self-renew. The pluripotency can again be tested by attempting to induce teratocarcinomas!
How can we recapitulate development using in vitro differentiation?
- Remove cells from self-renewal conditions
- Put cells in environment that signals them to form the germ layers
SIMULATED IN VITRO DIFFERENTIATION - REPORTER MICE
The 3D approach - outline
- Remove signals that keep cells in an “undifferentiated” state (e.g. BMP/LIF for mouse ES cells or FGF2, TGFβ for human ES cells)
- grow in aggregates in the presence or absence of signals
- Cells remember their ability to self organise, they are forced into a ball of cell and cells differentiate
- Allows capitulation of normal embryonic development
What are embryoid bodies and what is the disadvantage of the 3D approach of in vitro differentiation
Embryoid bodies are aggregates of pluripotent cells that are induced to differentiate by a combination of a change in culture medium (removal of factors that support pluripotency) and allowing the cells to interact in three-dimensional structures.
The 3D approach works well but it is difficult to dissect roles of individual signals
