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Stem cells

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Stem cells Helena Fulkova Institute of Animal Science fulkova.helena_at_vuzv.cz – PowerPoint PPT presentation

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Title: Stem cells


1
Stem cells
  • Helena Fulkova
  • Institute of Animal Science
  • fulkova.helena_at_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

TE
ICM
7
Oct4
Cdx2
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 )

DAPI
SSEA1
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

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

14
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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
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

19
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20
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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.

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!
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