Modelling embryonic development and disease using pluripotent stem cells - 1

Question 1

DEFINITION - pluripotent cell

What is an important property of pluripotency?

Answer 1

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

Question 2

DEFINITION - stem cell

Answer 2

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

Question 3

Properties of early embryonic cells? What do they undergo?

Answer 3

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)

Question 4

2 MAIN IDENTIFIABLE FEATURES OF PLURIPOTENT CELLS

Answer 4

• 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

Question 5

Examples of pluripotency factors and where are they found when observed in mice embryos and how was this identified?

Answer 5

Sox2, Nanog, Oct4

Expressed in the inner cell mass of the developing mouse embryo, this was identified by IN SITU HYBRIDISATION

Question 6

Why is pluripotency often hard to study in vivo? Why is in vitro done instead?

Answer 6

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

Question 7

How are E.S.Cs captured and viewed in vivo?

Answer 7

• 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!

Question 8

What is the relevance of feeder cells that allow growth of E.S.Cs in vitro

Answer 8

Feeder cells provide trophic factors such as BMP (in mouse) that are normally found in the niche of pluripotent embryonic stem cell

Question 9

What are the main properties of E.S cells?

Answer 9

• 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

Question 10

How can you test for an E.S cell in mice?

Answer 10

-Mouse ES cells can be reintroduced to normal blastocysts and contribute to normal development of chimeras.

Question 11

How can you reprogram adult somatic cells and how can this be tested?

Answer 11

• 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!

Question 12

How can we recapitulate development using in vitro differentiation?

Answer 12

• Remove cells from self-renewal conditions

- Put cells in environment that signals them to form the germ layers

Question 13

SIMULATED IN VITRO DIFFERENTIATION - REPORTER MICE

The 3D approach - outline

Answer 13

• 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

Question 14

What are embryoid bodies and what is the disadvantage of the 3D approach of in vitro differentiation

Answer 14

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