Embryonic Stem Cells Flashcards
Thomson et al, 1998
First humans ESC line protocol published:
- Embryos donated w/informed consent from IVF.
- Immunosurgery to remove trophectoderm
- Plate on feeders
- After 9-15 days, disperse and replate
- Self renewal is fibroblast feeder dependent, but LIF independent (different to mouse, they do grow in LIF).
Evans and Kaufman, 1981
First mouse ESC line protocol published:
- Mouse blastocysts induced to enter diapause
- Flush delayed blastocysts and culture in groups of 6
- Trophoblast grows out, ICM picked
- ICM disaggregated and re-plated
- Cells propagate on fibroblast feeders
Gold Standard for Pluripotency
Can functionally replace all cell types in the developing organism, ie, can form the 3 germ layers
NIH Criteria for Pluripotency
- Teratoma formation
- Capable of unlimited self renewal
- Expression of TFs and cell surface markers
Brivanlou et al, 2003
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
EC vs ES
- ES have more powerful colonising ability
- Greater differentiation potential
- Retain stable karyotype for longer
- greater chimera efficiency (>50%)
- Greater contribution to the germ line
Markers and TFs of Murine ESCs
SSEA1, CD9, Oct-4, Sox-2, Nanog, Telomerase, Alkaline phosphatase
Other Embryonic Stem Cells
Epistem
Trophoblast
Extra embryonic Endoderm
FGF/Activin/Bio
Evans and Kauffman state successful mouse embryonic culture depends on:
- Exact stage at which the pluripotent cells exist in the embryo
- Explantation of a sufficient number of these cells
- Tissue culture in conditions suited to multiplication, not differentiation
Essential Characteristics of hESCs
- Derived from the pre or peri implantation embryo
- Prolonged undifferentiated proliferation
- Stable developmental potential to form derivatives of the 3 germ layers even after prolonged culture
Number of hESC lines produced by original Thomson et al, 1998 protocol?
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.
Morphology
- Small and rounded
- Prominent nucleolus
- Defined colonies with high nucleus to cytoplasm ratio
- Colonies have smooth boundaries - if they are ragged, they are starting to differentiate.
Markers and TFs of Human ESCs
SSEA3 (SSEA1 is a differentiation marker in human), Tra 1-60, Tra 1-81, Alkaline phosphatase, Oct-4, Nanog, Sox-2
Characterisation of Human ESCs
- Normal karyotype (although do tend to get duplications in chromosomes 12 and 17)
- High telomerase
- Cell surface markers
- Differentiate if removed from feeders (ie, ins serum containing media)
- Produce teratomas in SCID mice
Variations in Human ESCs
- Derived from diverse genetic backgrounds over every continent (whereas mouse 129 strain is homogenous).
- Various culture protocols used to develop blastocysts
- Manner of derivation differs
- Culture/passage protocols vary
Hobner et al, 2004
Showed that mouse ES cells could form oocytes
Hayashi et al, 2012
Showed that the oocytes produced from mouse ES cells could go through the whole reproductive cycle