Download Stem cells

Stem cells
Helena Fulkova
Institute of Animal Science
[email protected]
Why stem cells?
• Genetic manipulation: Transgenics
(knock-in/knock-out)
• Tissue therapy
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
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
– loss of sex chromosomes
- trisomy of chromosome 8
… a BIG problem for possible
biotechnologies and tissue therapy
FISH – chrom X, chrom 8
Normal
Abnormal
Induced Pluripotent Stem cells –
iPS cells
• Possible application – cell therapy
• Induction of ES-like cells from cell cultures
• Viral transduction or transfection
Problems
• Highly inefficient
• Manipulation of oncogenes (cancer-like cells –
c-myc/klf4/p53)
• No ESCs conditions – no iPS cell culture
…impractical for tissue engeneering
• Worse differentiation
Transgenics
• Knock-in – ESCs/pronucleus injection
(random integration, no of copies?)
→ chimera production/breeding or transfer
of embryos to recipient females
• Knock-out – ESCs/pronucleus injection
(Zn finger nucleases)
Zinc finger nucleases
• Possible use in KO experiments without
ESCs
• Zinc finger DNA-binding domains + DNAcleavage domains (Fok I)
• Possible to use without ESCs step
Geurts AM, Cost GJ, Freyvert Y, et al. (July 2009). "Knockout rats via embryo microinjection of zincfinger nucleases". Science 325 (5939): 433.
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!