Cellular differentiation, Stem cells and modern medicine

Question 1

What are the steps of formation of the embryo?

Answer 1

• Embryo begins as a small number of naive, totipotent cells
• Compaction occurs and cells start to make tight attractions towards each other.
• End up with an embryo made up of two layers: inner cells are cut off from the external environment, outer cells are still exposed to it.
• Progressive restriction of cell fate until terminally differentiated, and can only give rise to the same type of cell

Question 2

What form are the cells in at the beginning of the embryo?

Answer 2

They are totipotent (total potential - can go on and make every single cell type in the body)

Question 3

What are embryonic stem cells classified as?

Answer 3

Pluripotent as they can give rise to any type of cell except the trophectoderm (Placenta)

Question 4

Differentiated cells can only give rise to the same type of cell, what are the two exceptions?

Answer 4

Stem cells (don’t differentiate)

Germ cells (egg and sperm)

Question 5

What is a embryonic precursor cell?

Answer 5

A stage in development of a embryonic cell where it could become any type of differentiated cell

e.g. Muscle specific genes would be turned off as they are not needed to be transcribed

Question 6

What is a determined embryonic cell?

Answer 6

The cell has been determined so it must now differentiate into that specific cell type.

e. g. A muscle cell. It may not look different yet, but its path is already determined to become apart of the muscle fibre.
- Certain control genes which code for transcription factors become activated
- The cell now makes MyoD proteins

Question 7

What is a fully differentiated cell?

Answer 7

The cell is terminally differentiated, playing a functional role in the organism.

e. g.
- MyoD travels to the nucleus and activates genes (turns on its own genes on).
- MyoD is now made continuously
- Other genes to do with muscles are now turned on via MyoD
- The cell is now a working muscle cell and part of the muscle fibre

Question 8

What is genomic equivalence?

Answer 8

Differentiated cells contain all the DNA required to build an entire new organism

Question 9

What was found from the tadpole experiment?

Answer 9

1. It is still possible to get from a fully differentiated cell - tells us all the frog DNA is still present in this cell, it’s just shut down and locked away (hard to wake it up)
2. Most of the experiments fails - as it is very hard to make DNA go back in time and get rid of all the transcription factors

Question 10

What are some features of embryonic stem cells?

Answer 10

• Given no signals to differentiate, embryonic cells will stay pluripotent (given the right conditions, can develop into almost any cell)
• These are “Harvested” from the inner cell mass (future embryo) of mammalian cells
• The cells derived from theses ESCs are genetically identical to the embryo donor

Question 11

What are induced pluripotent cells? (iPS)

Answer 11

• Made by “reprogramming” adult skin cells → make them ‘go back in time’
• Can be made from anyone, and are genetically identical to the source skin cells, As they are also pluripotent, they can generate any cell type.
• Can be used in genetic therapy

Question 12

What are some features of adult stem cells?

Answer 12

• Can divide without limit
• Undifferentiated and multipotent
• Divide to give rise to both stem cells and cells that will go onto differentiate into functional tissue cells

Question 13

What are umbilical cord stem cells?

Answer 13

• From blood isolated from the umbilical cord of newborn babies and are kept frozen
• Multipotent, as they are immature blood stem cells.
• They are less restricted than blood stem cells from adults
• Can be used to treat leukaemia and many other blood diseases

Question 14

What is gene therapy?

Answer 14

Based on the idea that it may be possible to alter the genetic code of an individual’s cells. If you put an extra copy of the gene, it may make good protein which will resolve the problem

Question 15

How can gene therapy be used to cure disorders?

Answer 15

• A normal allele could be inserted into the cells of the affected tissue. Descendants of the stem cells will carry the normal allele. (In single gene disorders)

Question 16

What is regenerative medicine?

Answer 16

Based on the idea the pluripotent stem cells can be used to repair or replace damaged organs or tissues

Question 17

How does regenerative medicine work?

Answer 17

• Pluripotent stem cells can be made from skin cells or blood cells from a patient (genetically identical) or matched donors, or from embryos
• Can be encouraged to differentiate into specific cell types such as neurons, or retinal cells
• The differentiated cells can be transplanted into patients

Question 18

What cell class are fertilised eggs?

Answer 18

Totipotent, able to give rise to all cell types via cell division

Question 19

How do we get information about the function of a

gene from its phenotype?

Answer 19

• By studying organisms that are naturally mutant for a particular gene. Where no natural mutants exist we can make our own
• By studying both these types of mutants we can learn how particular mutations lead to phenotypic changes

Question 20

How do we use genetic technique to find out what a gene does?

Answer 20

• Study organisms that are naturally rare for that gene
• Increase the rate of random mutation, select for a phenotype of interest and sequence the genome to identify the mutation (genetic screen)
• Transgenesis
• Deliberately break a particular gene to see what happens (targeted mutation)

Question 21

What model organisms can be used to make mutants and why?

Answer 21

• Mouse (92% of our genes)
• Zebrafish (70% of our genes)
• Drosophila (44% of human genes)
  These can be easily controlled and are easy to genetically manipulate

Question 22

What is transgenesis?

Answer 22

Engineering a multicellular organism by adding in “foreign” DNA

Question 23

How does transgenesis work?

Answer 23

The foreign gene is inserted into the male pronucleus and it is implanted into the ovary of the animal to grow

Question 24

How do we know if a gene variant is pathogenic?

Answer 24

• We can damage, or modify, the gene we are interested in by genetically modifying an organism or cell
• By examining the organism, or its offspring we should be able to work out what the gene normally does

VIA CRISPR

Question 25

How does CRISPR/Cas9 break the DNA?

Answer 25

Decide which gene you wish to mutate, and design a “guide” RNA that only binds to your gene of interest

• Get this into cells of interest in model organism
• Cas9 enters nucleus and finds target sequence in genome that matches guide RNA and makes double stranded break in DNA @ target site

Question 26

What happens to the target gene after it has been broken by Cas9?

Answer 26

• DNA repair enzymes try to patch up the cut
• Often results in errors as there is no template to read from
• Small InDels are created at the target site, the gene is potentially disrupted, or mutated
• If repair template is provided, it is possible to use this to “edit” that DNA sequence at the cut site

Question 27

What is an example of gene therapy for somatic cells?

Answer 27

Cystic fibrosis (defect in CFTR gene)
- Delivering DNA with functional copy of the CFTR gene to lung epithelial cells

• Extra copy makes good proteins, restoring function to some cells

Question 28

What are some ways to correct genetic disease in germline?

Answer 28

• Pre-implantation genetic diagnosis: in families with an identified risk, IVF can be
  used to make embryos from the parent’s eggs and sperm. These embryos can be
  tested before implantation, and only healthy embryos implanted.
• Three parent babies: where the faulty gene is on the mitochondrial DNA, nuclear transfer to a donor egg can be used.
• CRISPR gene edited babies

Question 29

What are haematopeietic stem cells?

Answer 29

Stem cells which give rise to other blood stem cells. Gives rise to myeloid and lymphoid lineages.

Question 30

What are stem cells important for?

Answer 30

Tissues such as blood and skin which need constant renewing

Question 31

What can adult stem cells divide into?

Answer 31

Other stem cells or progenitor cells which develop into fat, bone or white blood cells