Pluripotent Stem Cells

Question 1

What is pluripotency?

Answer 1

The ability of a cell to give rise to every tissue in the body

Question 2

What are the 3 “germ layers”?

Answer 2

• Ectoderm (skin and neural tissue)
• Endoderm (liver and pancreas)
• Mesoderm (cartilage, muscle blood and bone)

Question 3

What are the two types of pluripotent stem cell?

Answer 3

• Embryonic stem cells - derived from the ICM of the blastocyst (generate an entire embryo)
• Induced pluripotent cells (forced expression in fibroblasts using 4 different genes)

Question 4

How do you derive Embryonic stem cells?

Answer 4

• Isolate the ICM from a blastocyst before it implants
• Grown on a layer of irradiated mouse fibroblast feeder cells (contains all ther factors needed for pluripotency - irradiated so they dont grow)
• Cells are dissociated and re-plated onto a fresh layer of feeder cells to avoid over-growth

Question 5

What are the main stages of the development from an egg to a body?

Answer 5

• migration
• cell division
• differentiation into specific types
• apoptosis at appropriate points (e.g. between digits)

Question 6

What do ectoderm cells differentiate into?

Answer 6

They differentiate to neuroepithelia, which then go to neural stem cells. These are multipotent and can go become astrocytes, oligodendrocytes and neurons.

Question 7

How do cells know when to differentiate?

Answer 7

Cells secrete proteins called growth factors which drive the differentiation

Question 8

What proteins have been found to promote neural differentiation in vitro?

Answer 8

• Bone morphogenic protein 4 (BMP4)

- Fibroblast growth factor (FGF)

Question 9

What can be used to encourage cardiac differentiation?

Answer 9

Wnt inhibits cardiac differentiation, so you can use Wnt inhibitors

Question 10

What are the 4 stages of pancreatic development?

Answer 10

• Gut tube formation (day 9)
• The dorsal and ventral buds then form
• The gut tube then branches to form various organs
• Endocrine progenitors migrate in mesenchyme whey they differentiate to form islets.

Question 11

Give 3 examples of tissue engineering that has been successful

Answer 11

• Biodegradable polymers -> nerve regeneration
• Hydrogels for intervertebral disk regeneration
• Electrospun fibres for artificial tendons
• Inkjet printing of cells for tissue engineering
• Glass windpipes seeded with MSCs from patient

Question 12

Name 3 disadvantages of Embryonic stem cells

Answer 12

• Ethical problems
• Transplant involves immunosuppression
• Immune matching would need 1000s of viable lines to be generated
• Very hard to derive
• Currently only ~300 human ES cell lines worldwide, and very few clinical trials

Question 13

What 4 factors have been found to produce induced pluripotent stem cells?

Answer 13

Sox2
Klf4
Oct4
c-Myc (increases growth)

Question 14

What are the 3 criteria that determine if a cell is pluripotent?

Answer 14

• Form colonies like ES cells
• Express panel of genes associated with pluripotency
• Express Oct4, sox2 and nanog in the nucelus + tra-1-60, ssea4 and e-cadherin on the cell surface.

Question 15

How can we functionally assess their pluripotency?

Answer 15

• If you grow them in 3D, they form mini embryo-like structures with all 3 germ layers, but in a disorganised manner - smooth muscle actin is used as a marker for mesoderm, afp as an endoderm marker and bIII tubulin as an ectoderm marker
• Their ability to form teratomas (tumour-like structures) when implanted under the skin of a mouse
• Introduce Green fluorescent protein into iPS cell, transplant it into a mouse blastocyst and then a pregnant female - get a green mouse.

Question 16

What is one of the main safety aspects of iPS cells?

Answer 16

Many of the genes put into cells to make them iPS cells are oncogenes - can cause cancer as they make cells grow fast

Question 17

Give a method of making iPS

Answer 17

• Electroporation to make holes in the cell membrane allowing DNA to enter
• 8 days later, replate the cells onto mouse embryonic fibroblast feeder
• next day, change medium to pluripotency medium
• Day 18, iPS colonies will start forming

Question 18

What is the three step process of iPS reprogramming?

Answer 18

• initiation (increasing proliferation rate, resisting cell death and shifting metabolism from OxPhos to glycolysis and undergoing Mesenchymal to Epithalial Transition)
• maturation (demethylation of pluripotency gene promoters- allowing RNA transcriptase to access them and cause expression of pluripotency-associated genes)
• stabilisation (X chromosome activation, transgene independence)

Question 19

Give 3 advantages of iPSs

Answer 19

• share most attributes of ES cells (self-renewal and pluripotency)
• generated by patient’s own cells so they wont be rejected
• no ethical issues

Question 20

Name 5 different diseases that have had iPS cell lines produced

Answer 20

• Duchenne muscular dystrophy
• Parkinson’s
• huntingdon’s
• Type 1 diabetes
• Down’s

Question 21

Give 3 disadvantages of iPSs

Answer 21

• Current methods involve inserting genes that would cause cancer
• Current methods are too inefficient to guarantee production of iPS cells for each patient
• may not differentiate as reliably will all cell types

Question 22

How does this process occur?

Answer 22

• WNT inhibition leads to cardiomtyocyte differentiation
• BMP4, WNT ad ACTIVIN give rise to mesoderm
• ACTIVIN gives rise to endoderm