Lecture #32 - Cloning, stem cells and gene therapy

Question 1

1. What’re are totipotent cells?
2. Pluripotent?
3. Prigressive restriction of cell fate….
4. Exception - (2)

Answer 1

1. Embryo begins as a small number of naïve, totipotent cells (total potential)
2. Embryonic stem cells can give rise to all cell types except trophectoderm (pluripotent - many potentials) aka can’t become placenta
3. Progressive restriction of cell fate until terminally differentiated, and can only give rise to same type of cell.
4. Stem and germ cells are exceptions (germ = eggs or sperm) - stay in embryonic state all their life

In this diagram;

• First blob of cells from mitosis. If took one of these cells - would still get normal development (so docs take cells out of this stage to test for ish)
• Second blob - villi projections on outside and uterian enurium or something is the cavity inside and embryo decides what goes into this
• Compaction stage - definite outside membrane
• 2nd to last - celles on the inside and cells on the outside
• Last pic - the cells inside are you (embryonic stem cells - pluripotent) and cells outside are placenta

Question 2

Are embryonic cells differentiated?

Answer 2

No

Question 3

Embryonic cells: first their ____ becomes determined

Answer 3

Their fate

In this diagram;

• Pluripotent cell
• myoD = if switched on - pushes cell to make muscle cell later in life.
• Currently the other muscle specific genes are off because embryo doesn’t need cntractile units like actin and myosin.

Question 4

Then finally, the embryonic cell is _____ ______

Answer 4

Terminally differentiated

• Each cell type controlled by control genes - as embryo experiences different environments, the control genes code for transcription factor (if on)
• Transcription factor = can go back into nucleus and swtich on/off other genes (eg turn on genes for being muscle and off for eg colour of eye). Now cell must become muscle cell.
• Then cell cycle stopped because you’d made your muscle cells - can’t keep dividing bc they’re sealed and differentiated along this path.

Question 5

Why are stem cells different? (3)

Answer 5

• Stem cells can divide without limit
• They are undifferentiated - don’t have any function as per say
• They can divide to give rise to both stem cells and cells which will go on to differentiate into functional tissue cells

Need stem for replacing tissue that gets worn out/damaged

Question 6

A fertilised egg is _______, able to give rise to ______ via cell division

Embryonic stem cells are ______, Adult stem cells such as ____ cells usually give rise to ______.

________ are used to persuade stem cells to develop into different kinds of differentiated cell.

Answer 6

A fertilised egg is totipotent, able to give rise to all cell types via cell division

Embryonic stem cells are pluripotent, Adult stem cells such as bone marrow cells usually give rise to one or a few cell types as shown in diagram.

Different culture conditions are used to persuade stem cells to develop into different kinds of differentiated cell.

• Replace blood cells (which don’t live long) - replaced by stem cells in bone marrow.
• Just needa know the type of environ that you need for them to differentiate

Question 7

Adult stem cells: renewal tissues

1. What are stem cells important for?
2. What’s another name for blood stem cells?
3. What’s another name for bone marrow stem cells?
4. What’s plasticity?

Answer 7

• Stem cells are important for tissues such as blood and skin which need constant renewing.
• Blood stem cells or haematopoietic stem cells are found in the bone marrow and can be used for transplants
• Bone marrow also contains mesenchymal stem cells which can give rise to bone and cartilage cells. These stem cells may also be able to renew other tissues through plasticity.

Plasticity = The ability of stem cells from one adult tissue to generate the differentiated cell types of another tissue. Can be persuaded to make other cell types (even ones it aint meant to be)

• Stem cells can either:*
• 1. Make 2 stem cells (increases stem cells while adult constant)*
• 2. Assymmetrically - 1 stem and 1 intermediate/transit amplifying cell (divide a few more times to give adult cell)*
• 3. Two transit amplifying cells - lose stem cell completely*

Each tissue has stem cells for renewing tissue

Question 8

Umbilical cord stem cells

1. What’s cord blood banking?
2. What’s iPS?

Answer 8

1. Cord blood banking:

• Stem cells from blood isolated from umbilical cord of newborn babies are kept frozen (banked).
• The stem cells are not pluripotent, but are immature blood stem cells (more plastic than mature blood cells)
• Can be used to treat leukaemias and many other blood diseases (when you grow up)

2. Induced pluripotent stem (iPS) cells

• iPS cells are made by reprogramming adult skin cells
• The technique currently involves genetic engineering, but could be used in therapy in future

Go backward to embryonic state rather than extract from embryo

Question 9

What is gene therapy? (6)

Answer 9

• Gene therapy is based on the idea that it may be possible to alter the genetic code of an individual’s cells (bc only needa put one gene in)
• It is proposed as a way of correcting single gene disorders (such as cystic fibrosis).
• A normal allele could be inserted into the cells of the affected tissue.
• Gene therapy is best applied to stem cells, such as bone marrow cells.
• This is because the corrected stem cells will keep dividing and passing on the corrected gene to all progeny.
• One possibility is to use retroviruses (virus with RNA genome) containing the corrected allele to infect cultured bone marrow cells from a patient, then replace the cells.

Will fix exisiting somatic cells if insert it in e.g. skin (skin cells fixed) but once they strip off - back to same old problem so need gene therapy into stem cells (fixes long term)

Question 10

Appreciate

Answer 10

• Take part of retrovirus genome out that it doesn’t need to survive and then replace with your allele of your gene (that you want inserted)
• Retrovirus incorportate their gene into patient’s DNA

Question 11

Two examples of clones?

Answer 11

• Monozygotic twins/quads (blastcysts before split into 4)
• Cuttings - from garden when you cut part of plant and replant it

Question 12

The first cloning experiments in frogs- somatic cell nuclear transfer

Kinda explain so it follows the diagram

So what actually happens to the nucleus?

Answer 12

Early embryo ———> All or most cell types

Differentiated cell ——-> One cell type

Stem cell ———> Some cell types and more stem cells

(all the three have same DNA)

Apart from a few immune system cells, the complete genome is present in all cells, (genomic equivalence) but…

During differentiation, most animal cells generally lose the ability to regenerate the whole organism because the nuclear DNA cannot reset itself to an embryonic state (chromatin changed)

• As nucleus ages - less likely to access sites you needa make embryo with. Histones have changed from undiff to diff (transcription factors e.g. myoD prevented to activate genes)*
• Take a dipole cell nucleus in egg - causes egg to develop into embryo and then organism*
• Need to get nucleus back to embryo state*

Question 13

Random ish from my powerpoint - just read and understand

Answer 13

1. You can go from simple cells to cells that’re arranged in tissues and organs therefore development - involves changes to genomic DNA (in terms of which genes on/off)
2. Therapeutic cloning = solutions fr diseases long term
3. Progressive differentiation = somatic cells. Cells needa perform functions so shut down possibilities and become fully differentiated

Question 15

Dolly - explain how cloned

Answer 15

1997 - Dolly the Sheep

• First mammal to be cloned from an adult cell
• Forced donor mammary cells into G0 ‘resting’ phase of the cell cycle
• Delivered pulses of electric current to the egg
• Nuclear genome from nuclear donor
• Mitochondrial genome from egg donor
• No DNA from surrogate mother
• Dolly died in 2003 at the age of 6 (young for a sheep)
• Dolly was put down after contracting sheep pulmonary adenomatosis and had developed arthritis at an early age
• Dolly’s nuclear chromosomes are from a 5 year old sheep
• Was she 6 or 11 years old?
• The forced Go cells helped cloning somehow
• The electic pulse imitates the act of fertilisation (trigger embryotic development)
• Nuclear genome from nuclear donor - DNA sequencing
• Mitochondrial - 37 genes that should be identical to donor (so we know not twins ad actual clones)
• Only one Dolly worked

Question 16

Can you clone a clone?

Answer 16

Wakayama and colleagues in Japan have added a chemical called Trichostatin to the cloning procedure for SCNT.

This seems to have removed many of the issues, and the mice are now in their 25th generation of cloning, and appear normal.

That chemical modified procedure - helps chromatin fo back to embryonic state. Helped somatic cell transfer thing work successfully

Question 17

Cloning humans

Answer 17

• Many mammalian cloned embryos and fetuses die before birth
• Long term health effects to cloned mammals and their offspring are not yet understood
• Why would we want to clone humans?

• Reproduce remarkable individuals?
– Cloning doesn’t reproduce an individual personality e.g. identical twins are not the same

• Re-create deceased loved people?

– Cloning cannot bring someone back - it creates someone new

Question 18

Therapeutic cloning (6)

Answer 18

• Dolly is an example of reproductive cloning.

• Therapeutic cloning involves the creation of “personalized” embryonic stem cells
• A donor nucleus is transferred into an enucleated zygote as for reproductive cloning
• The embryo is cultured until the blastocyst stage
• Embryonic stem cells are made from the blastocyst and cultured under various conditions to make cell types to order (e.g. make new liver - replace damaged or disceased organs)
• iPS cells from patients with genetic diseases can also be used to make cells or tissues for drug testing- e.g. cystic fibrosis “minilungs” (can grow lungs now tht cells can be pushed back into embryonic state)

Shortage of human eggs so need to rely more on being able to go back

Question 19

Adult stem cell

Answer 19

stem cells found in adults e.g. in bone marrow

Question 20

Differentiation

Answer 20

the process by which cells become adapted for and tied to one function

Question 21

Embryonic stem cell

Answer 21

cell derived from the inner cell mass of a mammalian embryo, which is pluripotent (ES cells).

Question 22

iPS cell:

Answer 22

a pluripotent cell made by manipulating a differentiated cell.

Question 23

Haematopoietic stem cell:

Answer 23

a stem cell in the bone marrow that generates all the mature blood cell types.

Question 24

Mesenchymal stem cell:

Answer 24

second stem cell found in marrow that generates bone and cartilage cells.

Question 25

Parthenogenesis:

Answer 25

where females give birth to genetically identical clones of themselves.

Question 26

Plasticity:

Answer 26

The ability of stem cells from one adult tissue to generate the differentiated cell types of another tissue.

Question 27

Pluripotent:

Answer 27

able to give rise to more than one cell type, but not all cell types.

Question 28

Reproductive cloning

Answer 28

using nuclear transfer to create genetically identical offspring

Question 29

Stem cell:

Answer 29

an undifferentiated cell that can divide to generate either more stem cells or cells that go on to differentiate.

Question 30

Therapeutic cloning

Answer 30

using nuclear transfer to create genetically identical ES cells.

Question 31

Totipotent:

Answer 31

able to give rise to any cell type.