Embryonic Stem Cells

Question 1

Thomson et al, 1998

Answer 1

First humans ESC line protocol published:

1. Embryos donated w/informed consent from IVF.
2. Immunosurgery to remove trophectoderm
3. Plate on feeders
4. After 9-15 days, disperse and replate
5. Self renewal is fibroblast feeder dependent, but LIF independent (different to mouse, they do grow in LIF).

Question 2

Evans and Kaufman, 1981

Answer 2

First mouse ESC line protocol published:

1. Mouse blastocysts induced to enter diapause
2. Flush delayed blastocysts and culture in groups of 6
3. Trophoblast grows out, ICM picked
4. ICM disaggregated and re-plated
5. Cells propagate on fibroblast feeders

Question 3

Gold Standard for Pluripotency

Answer 3

Can functionally replace all cell types in the developing organism, ie, can form the 3 germ layers

Question 4

NIH Criteria for Pluripotency

Answer 4

1. Teratoma formation
2. Capable of unlimited self renewal
3. Expression of TFs and cell surface markers

Question 5

Brivanlou et al, 2003

Answer 5

Published standards for hESCs potency assay:
- transplantation of cells into nonhuman tissue and assess ability to incorporate into that tissue.

• transplant into nonhuman blastocyst (essentially chimera formation). Has to be transient, remove embryos during gestation as not ethical to produce human chimeras

Question 6

EC vs ES

Answer 6

1. ES have more powerful colonising ability
2. Greater differentiation potential
3. Retain stable karyotype for longer
4. greater chimera efficiency (>50%)
5. Greater contribution to the germ line

Question 7

Markers and TFs of Murine ESCs

Answer 7

SSEA1, CD9, Oct-4, Sox-2, Nanog, Telomerase, Alkaline phosphatase

Question 8

Other Embryonic Stem Cells

Answer 8

Epistem
Trophoblast
Extra embryonic Endoderm
FGF/Activin/Bio

Question 9

Evans and Kauffman state successful mouse embryonic culture depends on:

Answer 9

1. Exact stage at which the pluripotent cells exist in the embryo
2. Explantation of a sufficient number of these cells
3. Tissue culture in conditions suited to multiplication, not differentiation

Question 10

Essential Characteristics of hESCs

Answer 10

1. Derived from the pre or peri implantation embryo
2. Prolonged undifferentiated proliferation
3. Stable developmental potential to form derivatives of the 3 germ layers even after prolonged culture

Question 11

Number of hESC lines produced by original Thomson et al, 1998 protocol?

Answer 11

5 - H1, H7, H9, H13 and H14.

20% of embryos will produce stem cell lines

Now there are over 1000 cell lines, 16 derived at UoM.

Question 12

Morphology

Answer 12

1. Small and rounded
2. Prominent nucleolus
3. Defined colonies with high nucleus to cytoplasm ratio
4. Colonies have smooth boundaries - if they are ragged, they are starting to differentiate.

Question 13

Markers and TFs of Human ESCs

Answer 13

SSEA3 (SSEA1 is a differentiation marker in human), Tra 1-60, Tra 1-81, Alkaline phosphatase, Oct-4, Nanog, Sox-2

Question 14

Characterisation of Human ESCs

Answer 14

1. Normal karyotype (although do tend to get duplications in chromosomes 12 and 17)
2. High telomerase
3. Cell surface markers
4. Differentiate if removed from feeders (ie, ins serum containing media)
5. Produce teratomas in SCID mice

Question 15

Variations in Human ESCs

Answer 15

1. Derived from diverse genetic backgrounds over every continent (whereas mouse 129 strain is homogenous).
2. Various culture protocols used to develop blastocysts
3. Manner of derivation differs
4. Culture/passage protocols vary

Question 16

Hobner et al, 2004

Answer 16

Showed that mouse ES cells could form oocytes

Question 17

Hayashi et al, 2012

Answer 17

Showed that the oocytes produced from mouse ES cells could go through the whole reproductive cycle