L5 Induced Pluripotent Stem Cells

Question 1

Describe Waddington’s epigenetic landscape model and the two discoveries that have challenged it.

Answer 1

Waddington describes differentiation as a process where things will change from a state of less energy to a more stable state. He likens this to a ball rolling down a hill.

However, The method of pluripotent reprogramming (going from differentiated to pluripotent then back to differentiated) and direct conversion (cells going from different differentiated states) challenge this model.

Question 2

What question was John Gurdon trying to answer and how did he do it?

Answer 2

Is the genetic material present in a differentiated cell complete?

He answered this question by taking the nucleus fromm a differentiated intestinal cell and transferring it to a recipient enucleated egg cell. A complete blastocyst was generated. When this blastocyst was implanted into an adult frog, a complete embryo was produced.

Question 3

What is somatic cell nuclear transfer?

Answer 3

This is a form of cloning:

1) You take a biopsy of cells and take out the nucleus.
2) Transfer this nucleus to an oocyte.
3) Create a clone.

Question 4

What did Yamanaka et al (2006) manage to do?

Answer 4

He induced pluripotent stem cells from embryonic and adult fibroblast cultures.

Question 5

What was the construct that Yamanaka et al (2006) made in their experiment?

Answer 5

They identified the gene Fbx15 which is expressed in ES cells in the early embryo, but is not required for maintaining pluripotency and mouse development.

He genetically engineered antibiotic resistance in this gene:

1) Low levels of the antibiotic allow you to detect is the cell carries the gene.
2) High levels of antibiotic allow you to detect which cells have activated this gene.

Question 6

What two things did Yamanaka et al (2006) do with his 24 candidate genes?

Answer 6

1) They introduced each candidate gene separately into the Fbx15 betaGeo (antibiotic resistance) mouse embryonic fibroblasts, but none survived the antibiotic G418.
2) As method 1 didn’t work, they then tried to do the opposite by creating 22 G418 resistant clones with all 24 candidate genes in the genome. Five of these colonies looked very similar to ES colonies and were shown to express pluripotent cell markers. They then withdrew individual factors in order to determine which pool of these candidate genes are necessary to form the G418 resistant colonies. Four factors were required to induce pluripotent stem cells from MEFs (mouse embryonic fibroblasts).

Question 7

What are Thomson and Yamanaka’s factors?

Answer 7

Yamanaka = Oct3/4, Sox2, c-myc, Klf-4.

Thomson = Oct4, Sox2, Nanog, Lin28

Question 8

What did iPS-MEF 10, 4 and 3 do when injected into adult mice?

Answer 8

iPS-MEF 10 and 4 formed teratomas whereas iPS-MEF3 only produced differentiated cells not the various cell types seen in teratomas.

Question 9

What happened when Yamanaka et al (2006)’s experiment was repeated in adult fibroblasts?

Answer 9

Tail-tips (TTFs) were formed. These cells contributed to all three germ layers when they were injected into the blastocyst of another animal.

Question 10

What are the four different ways to reprogramme a cell?

Answer 10

1) Somatic cell nuclear transfer (rapid).
2) Cell fusion with pluripotent stem cell (rapid).
3) Transcription factor expression (slow).
4) Small molecule exposure (slow).

Question 11

What are the three phases of reprogramming a cell?

Answer 11

1) Early (initiation) phase.
2) Intermediate phase.
3) Late (maturation) phase.

Question 12

What are the five reprogramming factor delivery approaches?

Answer 12

1) Viral vectors deliver genes directly into the genome. However, this is a random integration.
2) Non-integrating vectors only go as far as the cytoplasm.
3) Excisable vectors are very difficult to do.
4) Proteins/mRNA are very expensive to make.
5) Small molecules are an up and coming area of research. They are potentially cheap, but the drugs could affect unintended protein targets.

Question 13

What did Hanna et al (2007) want to do and how did they do it?

Answer 13

They wanted to see if they could produce iPSCs from a mutated condition, repair the condition in vitro, produce the corrected cells then replace the cells in vivo.

They managed to do this in mice with sickle-cell anaemia. They fixed the mutation with homologous recombination, formed embryoid bodies, obtained haematopoietic progenitors and then repopulated the mouse bone marrow with the construct.

Question 14

What can you do with patient-derived iPSCs?

Answer 14

Model human disease and screen for new therapies.

Question 15

What are the issues with hES cells and how do human iPS cells solve some of these issues?

Answer 15

Issues with hES cells:

• Genomic instability.
• Needs a continual supply of high-quality embryos.
• Potential for tumour formation.
• Questions regarding functional differentiation.
• Problem of immune rejection.
• Ethically contentious.

Potentially solved issues:

• No need to administer immunosuppressant drugs.
• Can repair genetic defects by homologous recombination.
• Can repeatedly differentiate iPS cells into desired cell types for continued therapy.
• Less ethical issues.

Question 16

What was discovered in 2011 regarding patient specificity?

Answer 16

It was discovered that the reprogramming process changed the cell meaning that the immune response can be induced and you can get tumours. Specifically, the over-expression of some proteins contributes to the immunogenicity (ability to induce an immune response) of iPSCs derived from the same organism.