Transcript 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!