Stem Cell Biology I

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Stem Cell Biology I

• Defining and isolating stem cells

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What is a stem cell?

• A cell from which other types of cells can
  develop.
• An undifferentiated cell that divides to give
  rise to specialized cells.
• An undifferentiated active somatic cell that
  undergoes division and gives rise to other stem
  cells or to cells that differentiate to form
  specialized cells.
• A cell that undergoes asymmetric cell division,
  giving rise to one stem cell and one partially
  differentiated cell

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Types of stem cells

• A totipotent cell can give rise to an entire
  organism
• A pluripotent cell can give rise to any of an
  organisms cells, but cannot form the trophoblast
  (e.g., placenta) necessary for the organism to
  grow in utero
• A multipotent cell can give rise to more than one
  differentiated cell type, but not all of them

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Types of stem cells

• Embryonic Stem cells (ES cells)?
• Pluripotent
• Embryonic Germ cells (EG cells)?
• Pluripotent
• Adult Stem cells (AS cells)?
• Multipotent
• NO STEM CELLS ARE TOTIPOTENT NO CLONING
  HAPPENS HERE THE ONLY TOTIPOTENT CELLS ARE A
  FERTILIZED EGG AND IMMEDIATE DAUGHTER CELLS

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Stages of early development Where ES cells come
from
2 Cell
4 Cell
Blastocyst
16 Cell
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How embryonic stem cells are isolated

• http//www.sumanasinc.com/scienceinfocus/sif_stemc
  ells.html
• For more information of human embryonic stem
  cells and research, visit the FAQ at NIH
  http//stemcells.nih.gov/info/faqs.asp

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Somatic Cell Nuclear Transfer (SCNT)?

• http//learn.genetics.utah.edu/units/cloning/whati
  scloning/
• aka Therapeutic Cloning because implanted
  embryo can develop into new organism (e.g., Dolly
  the sheep)?
• Transfer of G0 nucleus, females only for
  autologous transfer (no immune reaction)?

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Different types of stem cells

• http//gslc.genetics.utah.edu/units/stemcells/scty
  pes/

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Examples of Adult Stem Cells
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In case you missed it
Fertilized egg up to 8-cell stage
Embryonic stem cells
(MULTIPOTENT)?
Adult stem cells
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Fluorescence Activated Cell Sorter (FACS)?

• http//www.unsolvedmysteries.oregonstate.edu/flow_
  cytometry_06.shtml

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Reverse-Transcriptase Polymerase Chain Reaction
(RT-PCR)?

• http//www.bio.davidson.edu/courses/Immunology/Fla
  sh/RT_PCR.html

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DNA Microarray

• http//www.bio.davidson.edu/courses/genomics/chip/
  chip.html

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Story Time

• Recently, four transcription factors (Oct4,
  Sox2, c-myc, and Klf4) were shown to be
  sufficient to reprogram mouse fibroblasts to
• undifferentiated, pluripotent stem cells termed
  induced pluripotent stem (iPS) cells
• but the observation that the expression of c-Myc
  causes death and differentiation of human ES
  cells suggests that combinations of factors
  lacking this gene are required to reprogram human
  cells

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• To identify candidate reprogramming factors, we
  compiled a list of genes with enriched expression
  in human ES cells relative to that of myeloid
  precursors and prioritized the list based on
  known involvement in the establishment or
  maintenance of pluripotency Taken from DNA
  microarray experiment comparing hES vs. myeloid
  (adult) stem cells
• We then cloned these genes into a lentiviral
  vector to screen for combinations of genes that
  could reprogram the differentiated derivatives of
  an OCT4 knock-in human ES cell line generated
  through homologous recombination
• By testing subsets of the 14 initial genes, we
  identified a core set of 4 genes, OCT4, SOX2,
  NANOG, and LIN28, that were capable of
  reprogramming human ES cellderived somatic cells
  with a mesenchymal phenotype

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We next tested whether OCT4, SOX2, NANOG, and
LIN28 are sufficient to reprogram primary,
genetically unmodified, diploid human fibroblasts.
Cell surface markers (proteins) expressed by
hES cells
Infected fibroblasts
Control fibroblasts
Infected fibroblasts
Control fibroblasts
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RT-PCR confirmation that exogenous genes are
expressed exogenous-specific primers vs.
endogenous primers
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Fig. 4. Pluripotency of iPS(IMR90) cells injected
into the hind-limb muscle of two mice. (A) Neural
tissue (ectoderm) (B) cartilage (mesoderm) (C)
gut epithelium (endoderm). Scale bars, 0.1 mm.
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Successful use of stem cells regrow damaged
heart muscle and blood vessels
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