Embryonic Stem Cells

Question 1

Define pluripotency

Answer 1

Pluripotency – the ability of a cell to contribute to any tissue in the body

In the spectrum of cell potency, totipotency represents the cell with the greatest differentiation potential, being able to differentiate into any embryonic cell, as well as extraembryonic cells. In contrast, pluripotent cells can only differentiate into embryonic cells.

Question 2

What are teratocarcinomas?

Answer 2

Tumours formed by germ cells

Question 3

What are EC cells?

Answer 3

Embryonal carcinoma (EC) cells are the stem cells of teratocarcinomas, and the malignant counterparts of embryonic stem (ES) cells derived from the inner cell mass of blastocyst-stage embryos, whether human or mouse.

https://www.ncbi.nlm.nih.gov/pubmed/16246161

Andrews et al

Question 4

What evidence suggests that EC cells are pluripotent?

Answer 4

In culture, EC cells can be maintaned as EC cells or differentiated into other types of cells.

If EC cells are innoculated into nude mice then differentiated and undifferentiated cells are formed

If they are injected into blastocysts, chimeric mice are born.

Question 5

How are embryonic stem cells gathered?

Answer 5

Embryonic stem cells are pluripotent stem cells derived from the inner cell mass (ICM) of the blastocyst of preimplantation embryos, at which time they consist of 50-150 cells

Derived from dissociating the blastocyst and culturing the ICM

Question 6

How is the trophectoderm distinguished from the ICM?

Answer 6

Trophectoderm expresses CDX2 - can be stained red

ICM expresses OCT4 - can be stained green

Nanog is also a marker of pluripotency

SSEA1 is also a marker for pluripotent embryonic stem cells

Question 7

What substance prevents stem cell regeneration?

Answer 7

Leukaemia inhibitory factor

Question 8

What are the three main characteristics of embryonic stem cells?

Answer 8

They are derived from pre-implantation or peri-implantation embryo

They can self-renew (i.e they can divide to make more copies of themselves without differentiating)

Pluripotency - they can give rise to cells in all thre germ layers (ectoderm, mesoderm and endoderm)

Question 9

What do the three germ layers eventually form?

Answer 9

Ectoderm - brain, spinal cord, nerves, eyes, ears nose

Mesoderm - Muscles, blood, connective tissue and the heart

Endoderm - Gut, stomach, pancreas, liver, lungs, germ cells

Question 10

What type of tumours will form if embryonic stem cells are introduced into adult mice?

Answer 10

Teratomas

Question 11

How do we achieve chimeric mice with ES cells that randomly contribute to all tissues?

Answer 11

ES cells are injected into the host ICM of blastocyst, planted into the uterus of a female

Question 12

What are the key genes that are required for pluripotency and also a marker of pluripotency?

Answer 12

OCT4

Nanog

Question 13

How does LIF maintain pluripotency?

Answer 13

LIf is responsible for the downstream expression of genes such as OCT4 and Nanog which are essential for maintaining pluripotency. It does this through activation of several pathways:

1. JAK - Stat3
2. Grb2 - MAPK
3. PI(3)K - Akt

Question 14

Generic reference

Answer 14

Pluripotent stem cells (PSCs) harbor core properties of self-renewal and pluripotency (Hackett and Surani, 2014, Martello and Smith, 2014).

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719190/

Question 15

What is the role of BMP?

Answer 15

Bone morphogenetic proteins (BMPs) have been shown to sustain self-renewal of naive mESCs together with LIF

Ying et al., 2003

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4719190/

Lecture says - LIF and BMP work together to prevent differentiation and to maintain pluripotency

Question 16

How are human embryonic stem cells maintained in contrast to mouse ESC?

Answer 16

Human ESC’s are more mature and are described as primed for differentiation.

These cells are LIF independant and are usually kept in the presence of fibroblast growth factor 2 and activin A which stabilise this primed pluripotent state.

Question 17

What happens when we remove LIF from ESC’s?

Answer 17

Ball like embryoid bodies form which containifferentiating cells (of ectoderm, mesoderm and endoderm lineages)

Question 18

Before we introduce ES cells back into mice we can genetically manipulate them in culture. How do we introduce mutant DNA into the cells?

Answer 18

Electroporation

Question 19

What is the process of electroporation?

Answer 19

Cells are bathed in a solution containing DNA

An electric shock is passed through them - electroporation (tiny lesions are made in the cell membrane - DNA enters before or during repair)

We then select the cells that have taken up the DNA

We then analyse the survivors after selection and make clones of successfully transfected cells.

These cells are then injected into ICM host of blastocyst

Then implanted into the uterus of the pseudopregnant female

Question 20

Define pseudopregnant female?

Answer 20

The recipient female is mated with a vasectomised male

Over the next 2 days, her uterine wall swells and vascularises, ready for implantation of blastocysts

Question 21

How to we tell which mice are chimeric and which mice aren’t when the female gives birth to her litter?

Answer 21

Often the host blastocyst and the donor ES cells will come from mouse strains with different coat colours - so you can tell which mice are chimeric

Question 22

How do we achieve a genetically modified mouse from a chimeric mouse?

Answer 22

When the genetically modified cells were implanted into the blastocyst - our hope is that some of these modified embryonic stem cells became germ cells (derived from the endoderm)

We have to breed the chimeric mouse with the wild type mice. Hopefully there will be some chimeric mice with the modification in the germ cells (sperm cells) this will then produce offspring with that genetic modification.