Stem cells II

Question 1

What do pluripotent cells not give rise to?

Answer 1

Extraembryonic membranes–> trophectoderm etc

Question 2

What can pluripotent cells give rise to?

Answer 2

Inner cell mass (ICM)

Question 3

Why can cells taken out of a human or mouse embryo not make an entire organism?

Answer 3

They are pluripotent not totipotent

Question 4

Difference between toti and pluripotent cells?

Answer 4

Toti can give rise to extraembryonic membranes (trophectoderm) while pluripotent cannot

Question 5

First step of establishing ES cells from blastocyst ICM?

Answer 5

Took pregnant 129/sv mice and ovariectomised them at day 2.5

Question 6

What are 129/sv mice?

Answer 6

Prone to teratocarcinoma

Question 7

What is teratocarcinoma?

Answer 7

Tumours in the testes/ovaries

Question 8

What is ovariectimisation?

Answer 8

Remove ovary after mouse has ovulated

Question 9

What happened to the 129/sv mice after they were ovariectomised?

Answer 9

They were injected with progesterone

Question 10

Why were the 129/sv mice injected with progesterone?

Answer 10

It delays implantation of the egg

Question 11

Effect of delaying implantation on the ICM?

Answer 11

ICM continues to proliferate

Question 12

What happens 4-5 days after the progesterone injection into the 129/sv mice?

Answer 12

Blastocysts are isolated and placed in drops of media on top of feeder fibroblasts

Question 13

What are the feeder fibroblasts used for the 129/sv mouse blastocysts treated with and why?

Answer 13

Mitomycin C–> prevents them from dividing

Question 14

How does Mitomycin C work?

Answer 14

It crosslinks DNA

Question 15

What happens to the blastocyst cells after they have been grown for a bit?

Answer 15

Trypsinized and plated onto fresh feeders

Question 16

What is trypsinization?

Answer 16

Detaching cells from a substrate (culture dish) using trypsin

Question 17

What is trypsin?

Answer 17

A proteolytic enzyme

Question 18

What specific things are needed to make mESCs?

Answer 18

leukemia inhibitory factor, BMP/serum, mitotically inactivated feeder layers

Question 19

Role of LIF?

Answer 19

Activates STAT3

Question 20

What specific things are needed to prevent differentitaion of mESCs in vitro?

Answer 20

BMP/serum, mitotically inactivated feeder layers

Question 21

Derivation efficiency of mESCs in permissive strains?

Answer 21

at best 30%

Question 22

What molecules were inhibited to promote ESC derivation?

Answer 22

MEK/ERK and GSK3 signalling

Question 23

How were MEK/ERK and GSK3 signalling inhibited?

Answer 23

Small molecule inhibitors–> 3i

Question 24

Effect of LIF and BMP on FGF and ERK?

Answer 24

LIF and BMP inhibit FGF and ERK

Question 25

Which TFs need to be activated for pluripotency maintenance?

Answer 25

Oct4, Sox2 and Nanog

Question 26

Which general genes are repressed to maintain ESC pluripotency?

Answer 26

Differentiation specific genes

Question 27

How are differentiation specific genes repressed in ESC pluripotency maintenance?

Answer 27

Keeping the chromating around the genes in a closed conformation, so the gene expression is repressed

Question 28

How is the chromatin of differentiation specific genes kept closed?

Answer 28

Binding of Polycomb proteins

Question 29

What form of chromatin do polycomb proteins keep chromatin in to silence genes?

Answer 29

Heterochromatin

Question 30

Main general components of maintaining ESC pluripotency?

Answer 30

Activate pluripotency genes Inhibit cell differentiation pathways Inhibit cell differentiation genes