L4 - In Vitro Stem Cell Models

Question 1

Define pluripotent cell

Answer 1

Potency to be able to generate all of the cell types of an embryo including the germ cells

Question 2

Define multipotent cell

Answer 2

Capable of differentiation into only a few lineages

Question 3

What are the cells called that surround the inner cell mad

Answer 3

trophectoderm

Question 4

Give examples of pluripotent markers expressed by pluripotent cells

Answer 4

Nanog
Oct4
Sox2

Question 5

What are the two hallmarks of pluripotency

Answer 5

Descriptive and functional

Question 6

Descritptive test for pluripoteny

Answer 6

Expression of various pluripotency markers (Nanog, Oct4, Sox2)

Question 7

Functional test for pluripotency

Answer 7

Grafting of pluripotent cells onto a kidney of a host mouse will give rise to a teratoma

Question 8

What cell types are containined in a teratoma

Answer 8

All of the cell types

Question 9

3 types of ectoderm

Answer 9

Surface
Neural
Neural crest

Question 10

What does the ectoderm go on to form

Answer 10

Skin

Nervous system

Question 11

4 types of mesoderm

Answer 11

Axial
APraxail
Intermediate
Lateral plate

Question 12

What does the mesoderm go onto form

Answer 12

Blood
Heart
Muscle
Kidney

Question 13

Whar happens to potency as devlopment proceeds

Answer 13

Becomes restricited

Question 14

What are used to mimich the niche in

MOUSE

HUMAN

Answer 14

Leukaeamia inhibitory facotr (LIF), BMP

FGF2 and TGFB

Question 15

Describe one method of capturing ES cells

Answer 15

Obtain the cells of the inner cell mass and dissociate
Plate on a layer of feeder cells
Once eSCs have divided a few times then disaggregate and replate

Question 16

What factors would be used to re-program an adult somatic cell to a pluripotent fate

Answer 16

Oct4
Sox2
Klf4
C-myc

Question 17

Describe the gene expression in iPS cells

Answer 17

Express the main pluripotency markers (Nanog, Oct4 and Sox2)

No expression of any genes which are indictaive of differentiation

Question 18

Describe some of the characteristics of iPS cells

Answer 18

Single cell able to generate many identical daughters (self-renewal)

Formation of teratoms in permissive environment

When reintroduced into an embryo they contribute to normal development

Question 19

What is a marker expressed by neural stem cells

Answer 19

RC2

Question 20

Describe a method for capturing multipotent stem cells

Answer 20

Dissociate cells

Plate on laminin in the presence of cytokines FGF2 and EGF

Question 21

Describe the 3D method for in vitro differentiation

Answer 21

Remove signals keeping cells in an undifferentiated state

Grow in aggregates (embryoid bodies/organoids)

Cell have functionality and express the right markers at the right time dependent on their fate

Question 22

Advantage of the 3D method of in vitro differentiation

Answer 22

Resembles embryonic development more closely

Question 23

Disadvantage of the 3D method of in vitro differentiation

Answer 23

Diffiuclt to observe/dissect the roles of individual signals

Question 24

Describe the 2D (Adherent) method of in vitro differentiation

Answer 24

Plate defined number of cells on the right substrate
Remove the signals keeping cells from differentiating
Grow in defined medium with appropriate levels of signals

Question 25

Advantages (2) of 2D/Abberate in vitro differentiation

Answer 25

More traceable system for live imaging | Easier to diisect roles of specific signals

Question 26

Disadvantage of 2D/Abberant in vitro differentiation

Answer 26

Don't get same cell-cell interactions which would be seen in vivo

Question 27

What is microcephaly

Answer 27

Neurodevelopmental disorder in which infants are born with an abnormally small brain

Question 28

Why may in vitro differentiation be needed to model microcephaly

Answer 28

Mice models fail to replicate the condition

Question 29

How may microcephaly be modelling in iPS cells

Answer 29

Take a skin biopsy and culture with Oct4, Sox2, Klf4 and myc to get to the iPS cells Formation of a cerebral organoid

Question 30

What can be seen when iPS cells are made to model microcephaly

Answer 30

fewer DCX neurones and fewer nerual progenitors

Question 31

Potential applications of microcephaly models using iPS cells

Answer 31

Small molecule screens to identify suitable drugs

Question 32

What is familiail dysautonomia

Answer 32

Genetic disorder affecting the development and survival of the neruones controlling involuntary actions May also affect the NS in activites related to senses

Question 33

Early symptoms of familial dysautonomia

Answer 33

``` Poor muscle tone Feeding difficulties Poor growth Lack of tears Frequent lung infections Difficulty maintaining body temperature ```

Question 34

Late symptoms of familial dysautonomia

Answer 34

``` Delayed speech and walking Spinal curvature Reduced perception of pain/temp Poor growth UNstable BP ```

Question 35

familial dysautonomia usually caused by a mutation in which gene

Answer 35

IKBKAP

Question 36

Parkinsons affects how many

Answer 36

1:500

Question 37

Symtpoms of parkinsons

Answer 37

``` Resting temor Slowness of movement Rigidity Dementia Anxiety ```

Question 38

Parkinsons characterised by the ...

Answer 38

Loss of mDA neurones of the substantia nigra

Question 39

How can cell replacement be used in parkinsons

Answer 39

Generate h-ES derived mDA neurones Transplant into mouse model Evidence of improvement in motor function

Question 40

What may be used as a marker in Parkinsons treatment

Answer 40

TH Tyrosin hydroxylase - enzyme involved in the synthesis of dopamine

Question 41

Cell replacement has also be used in the treatment of

Answer 41

AMD - replacing RPE cells