Lecture 23- Stem Cells and Regenerative medicine

Question 1

Developmental Genetics

Answer 1

The field that examine the genetic control of processes, whereby a one cell embryo > entire organism

Many of these processes repeated in adult life (can cause disease)

Loss of control of genetic pathways involved in development often involved in disease (cancer)

Stem cell biology involved in both the disease AND the treatment, as well as understanding the processes.

Question 2

‘Model Systems’ that are used for developmental genetics research

Answer 2

-Forms of life other then humans that allow us to do research on stem cells
Organisms at a less complex level then humans, used to do experiments for
a) ethical reasons and
b) they are simpler

eg) yeast> flat worms> mouse

Question 3

Stem cells

Answer 3

‘self-renewing progenitor cells’ that can generate one or more specialised cell types.
Their daughter cells can ‘self-renew’ or differentiate into a more specialised cell

Question 4

Totipotent stem cells

Answer 4

Can differentiate into ALL the cell types in an organism, including the extra-embryonic tissues

Form part of embryo and part of the placenta

Question 5

Pleuripotent stem cells.

Answer 5

Can differentiate into all the cell types and the 3 embryonic germ layers but NOT extra-embryonic tissue

Form the inner part of the embryo

Question 6

Multipotent stem cells

Answer 6

can differentiate into cells of multiple, but a limited number, of lineages (not all germ layers).
Thought to be predisposed by more diverse cells eg) pleuripotented

Question 7

Pleuripotent cells can be

Answer 7

• embryonic stem cells

- adult stem cell: many tissues have resident stem cells. Blood stem cells can travel to various

Question 8

Stem cell ‘plasticity’

Answer 8

The more immature the stem cell, the closer it is to being ‘pleuripotent’, the more cell types it can differentiate into. (more plastic)

Question 9

Genetic Modification

Answer 9

Genetically modify model systems and see how this changes things.

Types of Modification

• Remove genes (gene knockouts)
  eg) study function of an unknown gene by removing it in a mouse
• Change or insert genes (transgenic cells and animals)

Methods:

• Homologous Recombination; swapping over segments of DNA. We can artificially hijack this
• Zinc finger nucleases (ZFNs); guided by enzymes

Question 10

Homologous Recombination

Answer 10

Normal gene is replaced with a gene you’ve generated in the lab.
Make segments of it that are the same as the normal gene. This allows you to ‘swap’ your gene with the gene of interest, inserting it into the DNA. Sometimes when this is done we also insert in other sequences that can be recognised by specific enzymes. Then you’ve inserted sequences in the genome that you can use at will, to swap in/out different genes.
Powerful technique for generating a template to do lots of genetic research

Question 11

Genome-scale CRISPR-Cs9 Knockout screening. GECKO system

Answer 11

Millions of cells cultured
Each cell has a different gene of the genome knocked out using this technology.
Cells treated with drugs, all should be killed BUT if a cell has a particular gene knocked out that’s required for drug killing, then this can teach us what are the genes are involved in drug resistance.

Question 12

History of Stem Cells

Answer 12

Mouse ES cells first described in 1981, which enabled gene knockouts in mice (Martin Evans). Huge acceleration of knowledge.

Human ES cells first described in 1998

1997: Then ‘cloning’, DOlly the sheep progressed. George W. Bush limited funding for a while.
2006: Generated ‘induced pluripotent stem cells’ (iPS) from adult cells by inserting four genes. From adult back to stem cells. Generation of any tissue by regenerated stem cells (not having to use embryonic stem cells) HUGE STEP

2009: Obama lifted George Bushs
2010: Spinal injury treated with human ES cells
2014: First therapeutic cloning- using human ES cells from adult cells (skin cells from diabetic woman reprogrammed into insulin-producing B cells)

Question 13

Feeder Layer

Answer 13

embryonic fibroblasts that are required to grow embryonic stem cells on.
Stem cells don’t exist well by themselves, need very specific environments that keep them undifferentiated.

ES cells grow in colonies on this feeder layer, are also treated with LIF (leukemia inhibitory factor)
Feeder layer + LIF keeps stem cells as pleuripotent as possible,, stopping them differentiating

Question 14

Induced Pluripotent Stem (iPS) cells

Answer 14

adult cells > pluripotent stem cells

Question 15

iPS cells for therapy

Answer 15

-build up banks of cells from patients that can be reprogrammed (from a variety of methods) into a variety of different cell types.
These different cell types can be used to identify drugs for diseases.

These iPS cells can be made cells from patient with disease, then investigate them .

altered iPS cells could replace could replace gentically impaired ones.
iPS biobanks of cells for biomarker in new drugs

Question 16

Spermatogonial stem cell transplantation to preserve male fertility after chemotherapy.

Answer 16

Chemo targets mitotically active cells, eg) sperm and ovarian cells.
The idea of taking a biopsy of testes, preserving it and then re-transplanting these spermatogonial stem cells is very powerful.
These stem cells can also be removed an kept safe in the testes of another animal (they go back into the testes to keep them in the stem cell ‘niche’)

Question 17

stem cells + genetic modifications have multiple uses

Answer 17

balding 
blindnes
cancer 
EVERYTHING
Rapidly developing areas

Question 18

Zinc finger Nucleases

Answer 18

ZFNs use specific enzymes to mediate change, rather then us just hijacking the system. This is a little more effective.