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Flashcards in Embryonic Stem Cells Deck (23)
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1

Define pluripotency

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.

2

What are teratocarcinomas?

Tumours formed by germ cells

3

What are EC cells?

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

4

What evidence suggests that EC cells are pluripotent?

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. 

5

How are embryonic stem cells gathered?

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

6

How is the trophectoderm distinguished from the ICM?

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 

7

What substance prevents stem cell regeneration?

Leukaemia inhibitory factor

8

What are the three main characteristics of embryonic stem cells?

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)

9

What do the three germ layers eventually form?

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

Mesoderm - Muscles, blood, connective tissue and the heart

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

10

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

Teratomas

11

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

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

12

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

OCT4

Nanog

13

How does LIF maintain pluripotency?

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

14

Generic reference

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/

15

What is the role of BMP?

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

16

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

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. 

17

What happens when we remove LIF from ESC's?

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

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?

Electroporation 

19

What is the process of electroporation?

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

20

Define pseudopregnant female?

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

21

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

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

22

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

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. 

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