Stem cells

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Stem cells

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

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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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Nanog
DAPI
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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
• XX lines (loss of one X chromosome)
• - trisomy of chromosome 8
• a BIG problem for possible therapy

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Normal
Abnormal
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Epigenetic properties of ESCs

• Bivalent domains (H3K4, H3K27 methylation)
  promoters of tissue specific genes
• Global hypomethylation
• Chromatin loosely organised hyperdynamic
  (hyperplastic) chromatin (good donors for SCNT)
• Both X chromosomes active

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Epigenetic mechanism of differentiation

• Stabilization of histone modifications in
  promoter regions
• Methylation of promoters of pluripotency-associate
  d genes
• Changes in genome organisation, random X
  chromosome inactivation
• . Implication for SCNT and iPS technology

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Li et al., 2007, Mol Cell Biol
Meshorer et al., 2006, Dev Cell
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Possible application Therapeutic cloning cell
therapy

• Somatic Cell Nuclear Transfer
• Donor (any somatic cell from a patient)
• Recipient (oocyte Hu or other specie ISNT)
• No problem with tissue rejection!

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Disadvantages

• Lack of human oocytes
• SCNT does not work in humans (?)
• ISNT poor compatibility of cytoplasm and
  nucleus (poor reprogramming, mtDNA, embryonic
  genome activation )

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ISNT
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ISNT II
B X B 96h
P x B 96h
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Trophoblast stem cells - TSCs

• Derived from TE (Cdx2, Eomes)
• FGF4 heparin (MEF conditioned medium)
• Chimeras only trophoblast lineage
• Imprinted X-inactivation
• Differentiation into giant cells

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XEN cells Extra embryonic endoderm

• True stem cells?
• From ICM, on MEFs in TE culture media (RPMI
  FGF4, Heparin)
• Chimeras only extra-embryonic endoderm lineages
• Imprinted X-inactivation

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Epiblast stem cells - EpiSCs

• Mouse, Rat Activin/Nodal signalling (Human
  ES-like)
• Postimplantation embryo epiblast
• Oct4/Nanog/Sox2 expression
• Monolayer morphology
• Chimera no contribution
• Teratomas - Yes

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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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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!