stem cell technologies

Question 1

what is a stem cell?

Answer 1

a stem cell is a relatively primitive cell that is capable of:

• self renewal
• make a range of cell types (potency) multi-potent, pluripotent
• convert to a different cell type (differentiation)

these features allow stem cells to build embryos and tissue (development) and repair tissues (regeneration)

Question 2

what are the two main functions of stem cells?

Answer 2

development

regeneration

Question 3

where are human pluripotent stem cells derived from?

Answer 3

from the early embryo

Question 4

are human and mouse embryonic stem cells the same?

Answer 4

no

Question 5

what can be used to define pluripotent stem cells?

Answer 5

surface markers

Question 6

what are the cell surface pluripotent markers in humans?

Answer 6

SSEA1 -ve 
SSEA3 +ve
SSEA4 +ve
TRA-I-60 +ve
GCTM2 +ve
ThyI +ve
MHC +ve

Question 7

what are the cell surface pluripotent markers in mice?

Answer 7

SSEA1 +ve 
SSEA3 -ve
SSEA4 -ve
TRA-I-60 -ve
GCTM2 -ve
ThyI -ve
MHC -ve

Question 8

what are the molecular markers of stem cells in humans ?

Answer 8

OC T3/4 TDGF1 THY1 GABRB3
N ANOG
SOX2 LEFTYA REX1(ZFP42) GDF3
N ANOG DNMT3B

Question 9

what can be used to define human pluripotent stem cells?

Answer 9

many different genes.

Question 10

what can be used to detect genetic changes?

Answer 10

G-banding 
CGH
SKY
aCGH
Sequencing

Question 11

what genetic changes does G banding detect?

what is the detection limit?

Answer 11

aneuploidy
amplifications and deletions
translocations
rearrangements

5-10Mb

Question 12

what does CGH detect?

what is the detection limit?

Answer 12

aneuploidy
amplifications and deletions

2-3MB

Question 13

what genetic changes does SKY detect?

what is the detection limit?

Answer 13

aneuploidy
translocations
rearrangements

1-5Mb

Question 14

what genetic changes does aCGH detect?

what is the detection limit?

Answer 14

aneuploidy
amplifications and deletions
SNPs

1-100kb

Question 15

what genetic changes can sequencing detect?

what is the detection limit?

Answer 15

sequence changes

1bp

Question 16

why do we get genetic change in stem checks?

Answer 16

selective pressures can act at decision points - the ESC will either self renew, apoptosis or differentiation.

self renewal will increase survival of stem cells
apoptosis and differentiation leads to loss of stem cells.

Human ES cells are subject to selective pressures that affect the decision between self renewal, differentiation and death.
this pressure can be relived by alterations in the genome, by gross chromosomal changes, smaller changed (CNV’s) or point mutations.

cells may alter their genome.

Question 17

what assays can be used to test pluripotency?

ways to differentiate pluripotent stem cells

Answer 17

many ways to differentiate pluripotent stem cells each with pros and cons
methods may be selected on whether you need quantitate, ease of getting germ layers and reproducibility

• embryo body (EB)
• spin EB
• spin EB + growth factors
• Monolayer + growth factors etc
• teratoma

Question 18

what are the advantages and disadvantages of the Embryo assay to test pluripotentcy?

Answer 18

advantages - 3D structure

disadvantages - not reproducible, not directed

Question 19

what are the advantages and disadvantages of the spin embryo body assay to test pluripotentcy?

Answer 19

advantages - 3D structure, differentiation is (i) reproducible
disadvantages - not directed (i) needs different size embryo bodies to cover all lineages

Question 20

what are the advantages and disadvantages of the spin embryo plus growth factors assay to test pluripotentcy?

Answer 20

advantages - 3D structures. differentiation is (i) reproducible (ii) directed
disadvantages (i) needs different size EB’s to cover all lineages (ii) many different factors/markers can be used.

Question 21

what are the advantages and disadvantages of the monolayer and growth factors assay to test pluripotentcy?

Answer 21

advantages - differentiation is (i)reproducible and (ii)directed
disadvantages - No 3D structure
Many different methods / markers

Question 22

what are the advantages and disadvantages of the teratoma assay to test pluripotentcy?

Answer 22

advantages - 3D structures, extensive differentiation

disadvantages - Not reproducible Not directed Very expensive Difficult to quantitate

Question 23

what is in vivo differentiation directed by?

Answer 23

exogenous signalling ligands

Question 24

how can pluripotency be induced

Answer 24

viral transduction

OCT4
SOX2
KLF4
C-MYC

virus that have been used are lentivirus and retrovirus

it has been demonstrated in induction of pluripotent stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture conditions.

Question 25

how are real (bone fide) iPS colonies identified?

Answer 25

morphology protein expression - alkaline phosphatase, pluripotency factors (Nanog, Oct4), surface markers eg Tra1-81, SSEA3 gene expression (Oct4, Sox2, Nanog, Rex1, Fgf4, Esg1, Dppa2/4, hTert, Dnmt3b) differentiation (EB diff to three germ layers, teratomas) epigenetic - methylation erasure at pluripotency loci such as Oct4.

Question 26

what cells have iPS been derived from?

Answer 26

``` neural blood vascular cardiac liver pancreas ```

Question 27

what diseased fibroblasts have induced pluripotent cells been derived from?

Answer 27

diseased iPS cells can be used to investigate the nature of the mutant phenotype and probe whether drug candidates could be useful in treatment ``` parkinson's duchene muscular dystrophy gaucheries disease type 1 diabetes leach-Nyhan syndrome ADA-SCID ```

Question 28

induction of pluripotency - what can we use this for?

Answer 28

pluripotent stem cells grow forever could we capture a genotype form 1)a particular human population 2)a cell from a patient with a genetic disease its in drug discovery regenerative medicine drug discovery toxicology disease models

Question 29

several diseases have now been modelled and tested with drugs. what diseases and what are the genes?

Answer 29

``` Long QT type 1 -->KCNQ1 Long QT type 2 -->KCNH2 timothy syndrome --> CACNAIC Leopard syndrome --> PTPNI I, RAFI, SHOC2 schizophrenia DISCI Alzheimer's disease PS1, PS2 early onset Alzheimer's APP ```

Question 30

how can embryonic stem cells be used to treat disease?

Answer 30

by the differentiation to the appropriate cell type not all diseases are suitable for cell replacement you have to be able to make desired cell, transplant it to the correct place and ensure it integrates