Download Stem cells

Stem cells
Helena Fulkova
Institute of Animal Science
[email protected]
Stem cells
• „Totipotent“ – zygote (2-cell stage embryo)
• „Pluripotent“ – embryonic stem cells
• „Multipotent“ (Unipotent) – adult stem cells
Stem cells II
• Division
- Asymmetric
(1 stem cell + 1 differentiated cell)
– Symmetric (2 stem cells)
Stem cells III
• From embryos – ESC (embryonic), TSC
(trophoblast), XEN cells ? (extraembryonic
endoderm), Epi SC (epiblast - postimplantation)
• Adult – testicular, ovarial ???, tissue specific
(skin, liver…), mesenchymal (bone marrow,
adipose tissue, peripheral blood …)
• iPS cells – induced pluripotent stem cells
Embryonic stem cells
• First differentiation – blastocyst (ICM vs. TE)
– Dependent upon Oct4 vs. Cdx2 expression
TE
ICM
Oct4
Cdx2
DAPI
merge
DAPI
Nanog
ESCs – embryonic stem cells
• Human, mouse, Rhesus monkey (rabbit,
rat?)
• From ICM cells
• Expression:
– intacellular (Oct3/4 (Pou5f1), Nanog,
Sox2 …)
- cell surface (SSEA1 – mo, SSEA4 – hu,
TRA-1-60 and TRA-1-81 – hu)
Derivation and culture
• Feeders vs. Feeder- free system
(MEFs, STOs, SNLs vs. Gelatin, Matrigel,
3T3 cell matrix …)
DAPI
SSEA1
Derivation and culture II
• LIF (Leukemia inhibitory factor) – Mo
• BMP – Mo
• FGF – Hu (LIF independent)
• Activin (inhibin A) /Nodal - Hu
• FCS (ES tested) or KOSR
Differentiation - pluripotency
• The ability to differentiate into all three
germ layers – ectoderm, mesoderm,
endoderm (in vitro and in vivo)
• Lineage specific markers:
– Meso (muscles – skeletal, cardiac, blood …)
– Ecto (skin, neuronal cells - CNS …)
– Endo (digestive tube + derivatives)
In vitro differentiation
• Mostly through EBs formation
βIII tubulin
TROMA 1
DAPI
MF20
In vivo – not applicable to human!
• Chimera production – injection of ES cells
into blastocysts
• Teratoma formation – injection of ESCs
into immunodeficient mice (SCID)
Advantages
• In vitro manipulation, large quantities
(tissue engineering, genetic manipulations,
germ line transmission …)
• Excellent model for random X
chromosome inactivation, general
differentiation mechanism
• Hope for cell (tissue) based therapy - Hu
Problems
• Very sensitive cells – fast differentiation
• Unstable karyotype
– XX lines (loss of one X chromosome)
- trisomy of chromosome 8
… a BIG problem for possible therapy
FISH – chrom X, chrom 8
Normal
Abnormal
Epigenetic properties of ESCs
• Bivalent domains (H3K4, H3K27
methylation) – promoters of tissue specific
genes
• Global hypomethylation
• Chromatin loosely organised =
„hyperdynamic (hyperplastic) chromatin“
(good donors for SCNT…)
• Both X chromosomes active
Epigenetic mechanism of
differentiation
• Stabilization of histone modifications in
promoter regions
• Methylation of promoters of pluripotencyassociated genes
• Changes in genome organisation, random
X chromosome inactivation
…. Implication for SCNT and iPS
technology
Meshorer et al., 2006, Dev Cell
Li et al., 2007, Mol Cell Biol
Possible application:
Therapeutic cloning – cell therapy
• Somatic Cell Nuclear Transfer
– Donor (any somatic cell from a patient)
– Recipient (oocyte – Hu or other specie =
ISNT)
No problem with tissue rejection!
Disadvantages
• Lack of human oocytes
• SCNT does not work in humans (?)
• ISNT – poor compatibility of cytoplasm
and nucleus (poor reprogramming,
mtDNA, embryonic genome activation …)
ISNT
ISNT II
B X B 96h
P x B 96h
Trophoblast stem cells - TSCs
• Derived from TE (Cdx2, Eomes)
• FGF4 + heparin (MEF conditioned
medium)
• Chimeras – only trophoblast lineage
• Imprinted X-inactivation
• Differentiation into giant cells
XEN cells – Extra embryonic
endoderm
• True stem cells?
• From ICM, on MEFs in TE culture media
(RPMI + FGF4, Heparin)
• Chimeras – only extra-embryonic
endoderm lineages
• Imprinted X-inactivation
Epiblast stem cells - EpiSCs
• Mouse, Rat – Activin/Nodal signalling
(Human ES-like)
• Postimplantation embryo – epiblast
• Oct4/Nanog/Sox2 expression …
• Monolayer morphology
• Chimera – no contribution
• Teratomas - Yes
Induced Pluripotent Stem cells –
iPS cells
• Possible application – cell therapy
• Induction of ES-like cells from cell cultures
• Viral transduction or transfection
Good laboratory practice
• Cell culture
• ESC characterization
Cell culture
• Dedicated area – restricted access
• Keep a good record of lines (lines,
clones…)
• Use cell culture tested reagents
(ESC tested)
• Mycoplasma testing
ESCs characterization
• Karyotype (every 5th passage)
• Markers of pluripotency (IF, RT PCR)
• Differentiation (all 3 germ layers – at least
in vitro … see NIH page for hESCs
registry and rules for submitting a new
line)
Thank you for your attention!