Pluripotent stem cells and differentiation

Question 1

Why is it difficult to study embryos/stem cells in vivo? (3)

Answer 1

• Small cell numbers
• Ethical problems
• Difficult to observe development in utero

Question 2

What are the main pluripotency factors expressed by pluripotent stem cells? (3)

Answer 2

• Nanog
• Oct4
• Sox2

Question 3

What factors are added to mouse embryonic stem cells to keep them in a self-renewing, non-differentiated state? (2)

Answer 3

• Leukaemia inhibitory factor (LIF)
• BMP

Question 4

What factors are added to human embryonic stem cells to keep them in a self-renewing, non-differentiated state? (2)

Answer 4

• FGF2
• TGFβ

Question 5

How do you acquire embryonic stem cells for testing? (2)

Answer 5

• Introduce reprogramming factors to adult cells to induce pluripotency
• Take embryonic stem cells from an embryo

Question 6

What are the 2 main approaches for in vitro cell differentiation?

Answer 6

• 3D
• 2D (adherent)

Question 7

What is the 3D method for in vitro cell differentiation? (2)

Answer 7

• Remove self-renewal signals (i.e. BMP/LIF for mice, FGF2/TGFβ for humans)
• Grow in aggregates (embryoid bodies/organoids)

Question 8

What is the advantage of the 3D method for in vitro cell differentiation?

Answer 8

Accurately mimics embryonic development in vivo

Question 9

What is the disadvantage of the 3D method for in vitro cell differentiation?

Answer 9

Difficult to observe the roles of individual signals

Question 10

What criteria need to be met when performing in vitro cell differentiation? (2)

Answer 10

• Functionality
• Expression of the correct markers in the correct places

Question 11

What is the 2D (adherent) method for in vitro cell differentiation? (3)

Answer 11

• Plate a specific number of cells on a substrate mimicking their natural environment/ECM
• Remove self-renewal signals (i.e. BMP/ILF for mice, FGF2/TGFβ for humans)
• Grow cells in a defined medium containing appropriate signals (FGF, WNT etc.)

Question 12

What are the advantages of the 2D (adherent) method for in vitro cell differentiation? (2)

Answer 12

• Easier to observe and image
• Easier to test the role of specific signals

Question 13

What is the disadvantage of the 2D (adherent) method for in vitro cell differentiation?

Answer 13

Loss of cell interactions that may occur in vivo

Question 14

What are the applications of in vitro cell engineering? (2)

Answer 14

• Disease modelling
• Cell replacement therapy

Question 15

What has cell replacement therapy been used for? (2)

Answer 15

• Transplantation of dopaminergic neurons developed from stem cells in vitro to treat Parkinson’s disease
• Also macular degeneration

Question 16

How do you capture neural stem cells in vitro (multipotent stem cells)? (3)

Answer 16

• Plate cells on laminin in the presence of FGF2 and EGF (self-renewal factors)
• Cells express undifferentiated markers
• Can induce differentiation into glia and neurons by removing FGF2 and EGF

Question 17

What are the main reprogramming factors that convert somatic cells back to pluripotency? (4)

Answer 17

• Oct4
• Nanog
• c-myc
• Klf4