Pluripotent Stem Cells

Question 1

Define pluripotent stem cells (PSCs).

Answer 1

• Self-renewing cells capable of differentiating into all derivatives of the 3 germ layers - ectoderm, endoderm and mesoderm
• 2 types of PSCs: embryonic stem cells (ESCs) derived from the inner cell mass and induced pluripotent stem cells (iPSCs) generated directly from adult cells via genetic or chemical manipulation

Question 2

How are embryonic stem cells derived?

Answer 2

• Inner cell mass extracted from embryo
• Cells are seeded onto an cellular matrix with collagen
• ESCs grown on chemically treated mouse fibroblast cells usually turn out the best

Question 3

How are embryonic stem cell lines validated (tested)?

Answer 3

ESCs need to be capable of differentiating into any of the 3 embryonic cell lineages - 3 methods to test this:

1. Inject ESCs into blastocyst, transfer into surrogate mother, study resulting cells to confirm they are derived from injected cells
2. Subcutaneously inject ESCs into immunosuppressed mice and investigating resulting teratomas
3. Producing embryoid bodies (clumps of ESCs) in vitro and investigating them for the presence of cells from the 3 lineages

Question 4

What is the basic principle used in targeted differntiation of PSCs?

Answer 4

• Attempt to mimic the in vivo environment that ESCs are exposed to
• ESCs grown in matrigel with specific growth factors
• Can test whether cell produced is the desired target cell by staining for specific markers

Question 5

Describe the problems associated with growing cells in a dish (in vitro).

Answer 5

• Most microenvironment interactions that happen in vivo between cells types are missing
• The 3D spatial arrangement which often regulates cell behaviour is missing - they form a monolayer in vitro
• Cells may differentiate but remain in a very early stage of development (as they are in the early foetus)

Question 6

Outline how 3D cultures are made.

Answer 6

• ESCs grown with spheroids - 3D culture
• Spheroids better simulate a living cell’s environment compared to a 2D model
• By adding different growth factors we can grow organoids which are embedded in matrigel and used for testing
• Organoids resemble in vivo organs

Question 7

Describe the disadvantages of using embryonic stem cells.

Answer 7

• Ethically contentious - embryo is destroyed
• Transplantation requires immune suppression
• Immune matching between stem cells and patients would involve generating more than 1000 viable ESC lines in UK - only 300 in world
• Making cells viable for transplant difficult because mouse fibroblast must be completely removed

Question 8

What are Yamanaka factors?

Answer 8

• 4 genes that are highly expressed in ESCs - Oct3/4, Sox2, Klf4, c-Myc
• Over-expression can induce pluripotency in human somatic cells

Question 9

Name some examples of iPSC reprogramming methods.

Answer 9

• Lentiviral delivery systems - cheap, effective
• Episomal delivery systems
• Sendai-virus
• Proteins and small molecule methods

Question 10

Explain the process of lentiviral reprogramming of fibroblasts.

Answer 10

• Patient’s fibroblasts extracted and grown in gelatine
• Infected with retroviruses carrying Yamanaka factors
• By day 21-28 we can pick colonies to use

Question 11

Descibe the advantages of using iPSCs.

Answer 11

• iPSCs share most of the attributes of ESCs i.e. self-renewal and pluripotency
• No ethical issues
• Generated from patient’s own cells so they are autologous - no immunorejection
• Excellent platform for disease modelling - e.g. iPSCs used to replicate GnRH neurons which are very hard to biopsy

Question 12

Describe the disadvantages of using iPSCs.

Answer 12

• Genomic instability due to reprogramming
• Incomplete epigenetic reprogramming may impede differentiation towards any cell type
• Cells tend to develop chromosomal abnormalities
• Somatic cells naturally accumulate mutations over time - iPSCs derived from elderly patients may contain mutations that render them unsuitable for regenerative medicine