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.
What are teratocarcinomas?
Tumours formed by germ cells
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.
Andrews et al
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.
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
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
What substance prevents stem cell regeneration?
Leukaemia inhibitory factor
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)
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
What type of tumours will form if embryonic stem cells are introduced into adult mice?
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
What are the key genes that are required for pluripotency and also a marker of pluripotency?
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:
- JAK - Stat3
- Grb2 - MAPK
- PI(3)K - Akt
Pluripotent stem cells (PSCs) harbor core properties of self-renewal and pluripotency (Hackett and Surani, 2014, Martello and Smith, 2014).
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
Lecture says - LIF and BMP work together to prevent differentiation and to maintain pluripotency
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.
What happens when we remove LIF from ESC's?
Ball like embryoid bodies form which containifferentiating cells (of ectoderm, mesoderm and endoderm lineages)
Before we introduce ES cells back into mice we can genetically manipulate them in culture. How do we introduce mutant DNA into the cells?
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
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
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
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.