Lecture 8 - Pluripotent Stem Cells

Question 1

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

Answer 1

• Self-renewal - making a copy of oneself.
• Make a range of cell types (Potency).
• Convert to a different cell types (Differentiation)

Question 2

The features of a stem cell allows them to :

Answer 2

• Build Embryos, and Tissues (Development).

* Repair Tissues (Regeneration)

Question 3

how many stem cells can be isolated from early mammalian embryos?

Answer 3

several different

–> They have different properties – even though some are derived from the same tissues

Question 4

Different types of stem cells:

Answer 4

• Extra embryonic stem cells (XEN)
• Trophoblast stem cells (TS)
• Mouse embryonic stem cells (mES)
• Human embryonic stem cells (hESC)
• Epiblast stem cells (EpiSC)

Question 5

Pluripotent stem history:

Answer 5

• Stevens 1954: Teratomas in 129 mice
• PIerce, 1964: EC cell are stem cells
• inject single cell
• Ephrussi 1968, Cells lines from EC cells
• Brinster 1974 Papaioannou et al 1975 Mintz and Illmensee 1975: EC cells resemble PSCs
• Martin Evans - 1981 Gail Martin - 1981: Mouse ES cells
• Jamie Thomson 1998, Human ES cells

Question 6

what are the properties of ES Cells

Answer 6

• Derived from inner cell mass (ICM) of blastocysts.
• Non transformed. – but high telomerase activity.
• Indefinite proliferative potential – therefore high amplification potential.
• Stable diploid karyotype (46,XX or 46, XY for human)
• Clonogenic – ie the whole population can re-derived from a single cell. (BUT hES cells have low clonogenic capacity, what does this mean?)
• Pluripotent – make all 3 germ layers.
• Carry particular markers (surface markers, genes)
• Incorporation into chimeras. (not practical for human ES cells into mouse embryos OR into human embryos !)

Question 7

are mouse ES cells primed or naive?

Answer 7

NAIVE (dont express lineage markers)

Question 8

Are mouse EpiSC primed or naive

Answer 8

primes - express lineage markers

Question 9

are human PSCs primed/naive

Answer 9

primed (express lineage markers)

Question 10

human ES cell derivation:

Answer 10

-Growth in 'IVF' medium 
=Hatched blastocyst 
-immunosurgery 
=Anti-'trophectoderm' 
\+ compliment = ES cell

Question 11

how to characterise pluripotent stem cells

Answer 11

• surface markers
• genetic
• epigenetic
• gene expression
• Functional either leading to clonogenic assays OR differentiation

Question 12

Hows to characterise pluripotent stem cells: SURFACE MARKERS

Answer 12

Antibodies can be raised against surface markers. These antibodies can be used to characterise cells.
Why?
•Sensitivity and selectivity – single cell detection
•Analysis of mixtures of cells
•Manipulation of cell mixtures – cell isolation or elimination •Function
Analysis
•Immunofluorescence – in situ; flow cytometry •Immunohistochemistry

Question 13

Hows to characterise pluripotent stem cells: Imprinting

Answer 13

imprinting: dosage control of certain genes
Genes are either
(i) Biallelically expressed
OR
(ii) Maternally or
(iii) Paternally imprinted
This is to control the dose of expression.
IF imprinting is lost you can get growth defects

Question 14

How to characterise pluripotent stem cells. ES cell should make

Answer 14

all the cell of the body

Question 15

test for pluritpotency:

Answer 15

Teratoma formation

–> If cells are pluripotent, cells from ALL 3 germ layers should be present

Question 16

Human ES cells have strong/poor single-cell re-plating efficiency

Answer 16

POOR

Question 17

pluripotent stem cells from mouse and humans though derived from similar stages of development can have…

Answer 17

different characteristics 
-such as:
-molecular (gene expression)
-surface markers 
-epogenetic 
& functional (includes differentiation potential and single-cell re-plating ability)