Stem cells

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Stem cells

• Helena Fulkova
• Institute of Animal Science
• fulkova.helena_at_vuzv.cz

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Why stem cells?

• Genetic manipulation Transgenics
  (knock-in/knock-out)
• Tissue therapy

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Stem cells

• Totipotent zygote (2-cell stage embryo)
• Pluripotent embryonic stem cells
• Multipotent (Unipotent) adult stem cells

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Stem cells II

• Division
• - Asymmetric (1 stem cell 1 differentiated
  cell)
• Symmetric (2 stem cells)

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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

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Embryonic stem cells

• First differentiation blastocyst (ICM vs. TE)
• Dependent upon Oct4 vs. Cdx2 expression

TE
ICM
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Oct4
Cdx2
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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)

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Derivation and culture

• Feeders vs. Feeder- free system
• (MEFs, STOs, SNLs vs. Gelatin, Matrigel, 3T3
  cell matrix )

DAPI
SSEA1
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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

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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)

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In vitro differentiation

• Mostly through EBs formation

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In vivo not applicable to human!

• Chimera production injection of ES cells into
  blastocysts
• Teratoma formation injection of ESCs into
  immunodeficient mice (SCID)

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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

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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

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Normal
Abnormal
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Induced Pluripotent Stem cells iPS cells

• Possible application cell therapy
• Induction of ES-like cells from cell cultures
• Viral transduction or transfection

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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

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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)

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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.

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Good laboratory practice

• Cell culture
• ESC characterization

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Cell culture

• Dedicated area restricted access
• Keep a good record of lines (lines, clones)
• Use cell culture tested reagents (ESC tested)
• Mycoplasma testing

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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)

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Thank you for your attention!