l13stemcells

Question 1

what is a pluripotent cell defined as?

Answer 1

a cell which can generate any cell of the body (including germ cells)

Question 2

what is a stem cell?

Answer 2

A cell which can self renew (in response to a particular signal), and can also differentiate to give rise to a variety of cell types (according to differentiation signals)

Question 3

are embryonic stem cells pluripotent?

Answer 3

yes - early embryonic stem cells are pluripotent but begin to lose pluripotentcy/differentiate during gastrulation

Question 4

what are the defining features of pluripotent stem cells?

Answer 4

• expression of pluripotency factors (nanog, sox2, oct4)

- teratocarcinoma formation

Question 5

When does teratocarcinoma formation occur?

Answer 5

when pluripotent stem cells are grafted onto a mouse kidney- tumour forms containing all cell types

Question 6

what are the pluripotency factors?

Answer 6

Nanog, sox2, oct4

-these 3 transcription facotrs in combination define pluripotent SCs

Question 7

at what stage is the inner cell mass pluripotent?

Answer 7

E4.0 - inner cell mass contains PCs surrounded by trophectoderm

Question 8

why are (human) embryos difficult to study?

Answer 8

• small number of cells
• develop in utero
• ethical issues

Question 9

what critical signals maintain cells in a self renewing, undifferentiated state?

Answer 9

FGF2 and TGFb (human)

BMPs (mouse)

Question 10

how are pluripotent cells captured?

Answer 10

• plate inner cell mass cells on layer of feeder cells (support ES cell growth - iradiated stromal cells)
• once divided a couple of times-disaggregate ESCs and replate
• get ES cells to permantly express reporter transgene e.g. GFP(so ES cells can be seen)
• critical signals (TGFb and FGF2) added to maintain in proliferative state

Question 11

what is a chimeric mouse?

Answer 11

A mouse composed of cells with two different genotypes

• an individual derived from 2 different genotypes
  e. g. mouse ESCs can be reintroduced into normal blastocyst and contribute to normal development - can be followed as are GFP labelled

Question 12

how can adult somatic cells be reprogrammed to a pluripotent fate?

Answer 12

• introduce pluripotency factors (sox2,nanog,oct4 and cmyc)
• c-myc-proliferation
• if cultured in right environment with right factors - can resemble pluripotent cells

Question 13

what are the two approaches to get in vitro differentiation?

Answer 13

1) 3D approach

2) 2D/adherent approach

Question 14

whats happens in the 3D approach to get in vitro differentiation (and give advantages and disadvantages)?

Answer 14

• signals removed which keep cell in undifferentiated state
• cells grown in aggregates/embryoid bodies
• embryoid bodies contain many cell types
• advantage - more accurate representation of invivo differentiation (cell-cell interactions)
• disadvantage - difficult to observe and dissect

Question 15

how is Wnt signalling labelled in embryoid bodies?

Answer 15

Axin:Lacz (when Wnt signalling, axin is active-lacz expressed)

• show wnt initiates formaiton of organoids/mini organs
• heart cells from EBs can beat - showing functionality

Question 16

how can it be confirmed that a cell has differentiated into the correct target cell type?

Answer 16

• is expression of the correct markers

- has functionality (e.g. heart cell - has cardiac AP/ can beat)

Question 17

what happens in the 2D/adherent approach to get in vitro differentiation (and give advantages and disadvantages)?

Answer 17

• plate defined number of ESCs on right substrate/ECM
• remove signals which keep ESCs in undifferentiated state
• grow in defined medium with appropriate amounts of a signal

advantage- can test roles of specific signals
disadvantage - loss of cell/cell contact/communication which may occur in vivo

Question 18

what is an early mesodermal marker?

Answer 18

T(bra)

Question 19

what happens in microcephaly?

Answer 19

• small brain
• get seizures - neurological defects
• autosomal recessive (CDKsrap2) gene)

Question 20

what was shown when a skin fibroblast was taken from a microcephaly patient?

Answer 20

• skin fibroblast taken and intorduced to pluripotency factors - becomes pluripotent (iPS cell)
• was shown that patient iPS cells produced smaller cerebral organoids on a dish with less neural progenitors

Question 21

what virus is associated with mucrocephaly?

Answer 21

• Zika virus
• when cells exposed to zika virus cerebral organoids were smaller
• zika virus increased cell death (marked by cas 3 and labelled zika virus)
• death of cells blocked by emricasan - prevented small organoids / microcephaly phenotype

Question 22

what is an embryonic stem cell?

Answer 22

a pluripotent stem cell which is derived from the inner cell mass of a blastocyst at an early stage - pre-implantation

Question 23

give some neural disease modelling problems

Answer 23

• some diseases have multiple genetic causes
• complex phenotypes
• late onsets
• lack of efficient differentiation protocals

Question 24

what has cell replacement been used to try to treat?

Answer 24

parkinsons (tremors, shaking, stiffness)

• lack of dopinergic neurons in substantia nigra
• TH- tyrosine hydroxylase - enzyme derived in dopinergic synthesis
• hES cells derived from MDA neurons
• transplant hES stem cells into a mouse - model of parkinsons- evidence for improved motor function

Question 25

give the challenges of cell replacement

Answer 25

- immune reaction/tumour formation - positional identity - progenitors vs mature differentiated cells - is it replacement or in vivo regeneration