Stem Cells

Question 1

How do you derive ES cells from IVF embryos?

Answer 1

1. grow IVF embryos in medium to a blastocyst stage
2. remove outer trophectoderm layer by adding antibody recognising antigen carried on trophectoderm.
3. add complement to induce complement-mediated killing.
4. replate remaining ICM cells into culture conditions and allow them to grow

Question 2

How do you characterise human pluripotent stem cells?

Answer 2

• Use flow cytometry to look at cell surface markers
• Use microarrays, RNA sequencing or qPCR to look at gene expression profiles
• Use in situ staining to see if the 3 germ layers are able to be formed
• Make teratomas
• Clonogenic assay - replate single cells in isolation and see if they form a colony to test ability to undergo unlimited division

Question 3

What is the difference between mouse and human ES cells?

Answer 3

Human ES cells form a flatter, epithelial-like colony and have more heterogeneity
Mouse ES cells form a more rounded colony and are uniform

Question 4

What is the gold standard of non-integrating delivery of reprogramming factors?

Answer 4

transient transfection with mRNA
:) does not integrate so good for clinical use
:( inefficient