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MSc - embryo exam > stem cell technologies > Flashcards

stem cell technologies Flashcards

1
Q

what is a stem cell?

A

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)

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2
Q

what are the two main functions of stem cells?

A

development

regeneration

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3
Q

where are human pluripotent stem cells derived from?

A

from the early embryo

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4
Q

are human and mouse embryonic stem cells the same?

A

no

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5
Q

what can be used to define pluripotent stem cells?

A

surface markers

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6
Q

what are the cell surface pluripotent markers in humans?

A 
SSEA1 -ve 
SSEA3 +ve
SSEA4 +ve
TRA-I-60 +ve
GCTM2 +ve
ThyI +ve
MHC +ve
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7
Q

what are the cell surface pluripotent markers in mice?

A 
SSEA1 +ve 
SSEA3 -ve
SSEA4 -ve
TRA-I-60 -ve
GCTM2 -ve
ThyI -ve
MHC -ve
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8
Q

what are the molecular markers of stem cells in humans ?

A

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

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9
Q

what can be used to define human pluripotent stem cells?

A

many different genes.

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10
Q

what can be used to detect genetic changes?

A 
G-banding 
CGH
SKY
aCGH
Sequencing
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11
Q

what genetic changes does G banding detect?

what is the detection limit?

A

aneuploidy
amplifications and deletions
translocations
rearrangements

5-10Mb

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12
Q

what does CGH detect?

what is the detection limit?

A

aneuploidy
amplifications and deletions

2-3MB

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13
Q

what genetic changes does SKY detect?

what is the detection limit?

A

aneuploidy
translocations
rearrangements

1-5Mb

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14
Q

what genetic changes does aCGH detect?

what is the detection limit?

A

aneuploidy
amplifications and deletions
SNPs

1-100kb

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15
Q

what genetic changes can sequencing detect?

what is the detection limit?

A

sequence changes

1bp

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16
Q

why do we get genetic change in stem checks?

A

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.

17
Q

what assays can be used to test pluripotency?

ways to differentiate pluripotent stem cells

A

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
18
Q

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

A

advantages - 3D structure

disadvantages - not reproducible, not directed

19
Q

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

A

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

20
Q

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

A

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.

21
Q

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

A

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

22
Q

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

A

advantages - 3D structures, extensive differentiation

disadvantages - Not reproducible Not directed Very expensive Difficult to quantitate

23
Q

what is in vivo differentiation directed by?

A

exogenous signalling ligands

24
Q

how can pluripotency be induced

A

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.

25
Q

how are real (bone fide) iPS colonies identified?

A

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.

26
Q

what cells have iPS been derived from?

A 
neural 
blood 
vascular 
cardiac
liver 
pancreas
27
Q

what diseased fibroblasts have induced pluripotent cells been derived from?

A

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
28
Q

induction of pluripotency - what can we use this for?

A

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

29
Q

several diseases have now been modelled and tested with drugs.

what diseases and what are the genes?

A 
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
30
Q

how can embryonic stem cells be used to treat disease?

A

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

MSc - embryo exam (13 decks)

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