Stem cells

Helena FulkovaInstitute of Animal Sciencefulkova.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 ? (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

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 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 + 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 least in vitro … see NIH page for hESCs registry and rules for submitting a new line)

Thank you for your attention!