Lecture 8

Question 1

where are ES cells derived from?

Answer 1

the inner cell mass of blastocysts

Question 2

what feature of ES cells means they can grow forever?

Answer 2

they have high telomerase activity therefore there is no chromosome degradation during cell division - indefinite proliferative potential

Question 3

what is the karyotype of ES cells?

Answer 3

stable diploid

Question 4

what are chimeras?

Answer 4

mixture of genetically different tissues formed by processes such as fusion of early embryos, grafting, or mutation.

Question 5

what is the difference between naive and primed stem cells in mice?

Answer 5

naive - don’t express lineage markers - mouse ES cells

primed - express lineage markers - mouse EpiSC

Question 6

describe the process of Human ES cells derivation?

Answer 6

1) form a blastocyst
2) Remove outer trophectoderm layer to get into the inner cell mass
3) Use antibodies to do this by adding complement which triggers cell lysis where antibody is found and inner cell mass cells are left

Question 7

what are 5 ways to characterise pluripotent stem cells?

Answer 7

1) surface markers
2) genetic - look for correct karyotype - 46XX or 46XY
3) epigenetic - correct methylation of genes - imprinting - which genes are paternally and maternally expressed
4) gene expression
5) functional - look at the germ layers or tissues formed in teratomas

Question 8

how can surface markers be used to characterise stem cells?

Answer 8

complementary antibodies specific to the surface markers can be made - cells carry different combinations of marker proteins on their surface

Question 9

how can genetics be used to characterise stem cells?

Answer 9

they should be a diploid karyotype therefore knowing this you can monitor for abnormalities and make sure they are all still karyotypically normal

Question 10

what do changes in methylation of stem cells represent?

Answer 10

they represent which genes are turned on and off
off = unmethylated
on = methylated

Question 11

what are the 3 ways genes can be imprinted (dosage control of certain genes)?

Answer 11

1) maternally
2) paternally
3) biallelically expressed

Question 12

how does gene expression help to characterise pluripotent stem cells?

Answer 12

1) different genes expressed in primed or naive cells
2) Some genes lost through development
3) as the core genes are changed or lost the cells begin to differentiate

Question 13

what needs to be added to a monolayer to form the correct differentiated cells?

Answer 13

the correct growth factors at the correct times

Question 14

apart from a monolayer what is another way to get differentiated cells from pluripotent stem cells?

Answer 14

use of an embryoid body - cells clump together as an aggregate rather than forming a monolayer

Question 15

what is an advantage of using teratomas for differentiation?

Answer 15

they have a 3D structure and extensive differentiation

Question 16

what are human ES cells poor at compared to mice?

Answer 16

single cell re- plating efficiency

Question 17

what are the different types of stem cells that can be isolated from early mammalian embryos?

Answer 17

XEN - extra embryonic 
TS - trophoblast 
mES - mouse embryonic 
hESC - human embryonic 
epiSC - epiblast

Question 18

what does pluripotent mean?

Answer 18

make all 3 germ layers

Question 19

ES cells are clonogenic what does this mean and what cells aren’t in the lab?

Answer 19

whole population can re-derived from a single cell - human ES cells aren’t - could be the conditions we are using

Question 20

are human PSCs primed or naive?

Answer 20

primed