1. Stem cells Helena Fulkova Institute of Animal Science fulkova.helena@vuzv.cz

2. Why stem cells? Genetic manipulation: Transgenics (knock-in/knock-out) Tissue therapy

3. Stem cells „Totipotent“ – zygote (2-cell stage embryo) „Pluripotent“ – embryonic stem cells „Multipotent“ (Unipotent) – adult stem cells

4. Stem cells II Division - Asymmetric (1 stem cell + 1 differentiated cell) –Symmetric (2 stem cells)

5. 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

6. Embryonic stem cells First differentiation – blastocyst (ICM vs. TE) –Dependent upon Oct4 vs. Cdx2 expression ICM TE

7. Oct4Cdx2 DAPI merge

8. 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)

9. Derivation and culture Feeders vs. Feeder- free system (MEFs, STOs, SNLs vs. Gelatin, Matrigel, 3T3 cell matrix …) DAPISSEA1

10. 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

11. 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)

12. In vitro differentiation Mostly through EBs formation βIII tubulin TROMA 1 DAPI MF20

13. In vivo – not applicable to human! Chimera production – injection of ES cells into blastocysts Teratoma formation – injection of ESCs into immunodeficient mice (SCID)

15. 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

16. 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

17. FISH – chrom X, chrom 8 Normal Abnormal

18. Induced Pluripotent Stem cells – iPS cells Possible application – cell therapy Induction of ES-like cells from cell cultures Viral transduction or transfection

21. 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

22. 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)

23. Zinc finger nucleases Possible use in KO experiments without ESCs 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."Knockout rats via embryo microinjection of zinc- finger nucleases"

24. Good laboratory practice Cell culture ESC characterization

25. Cell culture Dedicated area – restricted access Keep a good record of lines (lines, clones…) Use cell culture tested reagents (ESC tested) Mycoplasma testing

26. 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)

27. Thank you for your attention!