Stem cells
Helena Fulkova
Institute of Animal Science
fulkova.helena@vuzv.cz
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 ? (extraembryonicendoderm), 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
ICM
TE
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 threegerm 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 cellsinto blastocysts
•Teratoma formation – injection of ESCsinto immunodeficient mice (SCID)
Advantages
•In vitro manipulation, large quantities(tissue engineering, genetic manipulations,germ line transmission …)
•Excellent model for random Xchromosome inactivation, generaldifferentiation 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 possiblebiotechnologies 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 transferof embryos to recipient females
•Knock-out – ESCs/pronucleus injection(Zn finger nucleases)
Zinc finger nucleases
•Possible use in KO experiments withoutESCs
•Zinc finger DNA-binding domains + DNA-cleavage 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 zinc-finger 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 leastin vitro … see NIH page for hESCsregistry and rules for submitting a newline)
Thank you for your attention!