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Transcript 
Cloning in Nature
Parthenogenesis
Monozygotic twins
(embryo splitting)
(incomplete embryo
splitting)
Egg Cell and Sperm Cells
Pg. 661
Ca 2+ Wave
Metaphase arrest
Ca2+ and Egg Activation
SEP
“Calcium wave”
Cleavage Stage Embryos
Pre-compaction
Compaction
Cadherin (Homophilic Cell Adhesion Molecule)
Blastocycst
Uterus
Inner Cell Mass
(ICM)
Blastocyst
Blastocoel
Trophoblast
50 µm
Pre-implantation Development
(~ 7 days in humans)
Mammals Cloned by Embryo Splitting
Sheep
Cattle
Mouse
Pig
Horse
Rhesus monkey
Blastomere Isolation
“Tetra”
Mammals Cloned by SCNT
Sheep
Mouse
Cattle
Pig
Mule
“Dolly”
“Copy Cat”
Horse
Goat
Rabbit
Cat
Dog
Gaur
Removal of genetic material
from oocyte
Transfer of diploid nucleus
from adult somatic cell or
Embryonic cell into
“enucleated” oocyte
Oocyte Activation
Cleavage divisions of
“reconstructed” embryo
SCNT in Mice
Injected
somatic nucleus
Condensed
Chromosomes
Pseudo-pronuclei
Ca 2+ injection
cytochalasin
Removal of genetic material
from oocyte
Transfer of diploid nucleus
from adult somatic cell or
Embryonic cell into
“enucleated” oocyte
Oocyte Activation
Cleavage divisions of
“reconstructed” embryo
Copy Cat
Somatic cell donor
“mom”
Copy Cat
Somatic cell donor
“mom”
Inactive X Chromosome
(“Barr Body”)
X Chromosome Inactivation
ICM cells of
Blastocyst
Copy Cat
Somatic cell donor
“mom”
Neural Plasticity
Environment has a significant role
in establishing and maintaining
neural connections in the brain
Stem Cell Concept
Pluripotent
Multipotent
Unipotent
Regenerative Medicine
Stem Cells
Transplant into patients
Pluripotent Stem Cells
Mouse Chimeras with Embryonic Stem Cells
Science 282: 1145-1147
November 6, 1998
Human Embryonic Stem Cell Colony
Stem Cells
Mouse
“feeder”
cells
Normal Karyotype in Cultured
Human Embryonic Stem Cells
Cultured Cells Remain Undifferentiated
Telomerase Activity
Undifferentiation “Markers”
Oct4
SSAE4
Teratoma
Ectoderm
(squamous epithelium)
Mesoderm
(bone and cartilage)
Endoderm
(gut epithelium)
Embryonic Stem Cell Signaling Pathways
OCT4
These Pathways are necessary for maintaining the undifferentiated State
Nature 456: 344-349
November 20, 2008
Science 318: 1917-1920
December 21, 2007
Science 321: 1218-1221
August 29, 2008
iPS Cell Colony from Patient with ALS
QuickTime™ and a
TIFF (L ZW) d eco mpres sor
are nee ded to s ee this picture.
Reprogramming Genes: OCT4, SOX2, KLF4, c-MYC
Neurons from iPS Cells Derived from ALS patient
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Tissues with Adult Stem Cells
Bone marrow/peripheral blood/umbilical cord
Blood vessels
Brain/spinal cord
Skeletal muscle
Colon
Liver
Pancreas
Retina/cornea
Skin
Dental pulp
Adult Stem Cell
Life-long
Multipotent
Plasticity?
Adult Stem Cells
Multipotent
Progenitor Cells
Stem Cell Niche
Stem Cell
(Common Myeloid progenitor)
(Common Lymphoid progenitor)
Signaling Pathways for Self Renewal
in Adult Stem Cells
Signals produced with the HSC niche
Cytkine Signaling via Jak/STAT Pathway
Cytokines in Hematopoiesis
Bone Marrow
(Common Myeloid progenitor)
(Common Lymphoid progenitor)
Myeloid Leukemia
Tumor Growth
(~ 10 yrs)
(~ 8 yrs)
(~ 7.5 yrs)
(~ 100 days/pop doubling)
Myeloid Leukemia
CD34+
Myeloid Leukemia
CD34+
CD34+
No Wnt Molecules
Pathway
OFF
INACTIVE
(No cell division)
Self-renewal
Proliferation
Pathway
ON
(family of ~ 20 proteins)
ACTIVE
Wnt Target Genes
Self-renewing
Proliferation
Cyclin D
Nanog
SOX2
c-myc
LIN28
BMP4
FGF
MMP
Pathway
Always
ON in
Cancer
(family of ~ 20 proteins)
Activating Mutations
Wnt
FRAT
catenin
Wnt Target Genes
Inactivating Mutations
Axin
APC
CONSTANT
Proliferation
Cyclin D
Nanog
SOX2
c-myc
LIN28
BMP4
FGF
MMP
Tumor Formation
Phenotypically Different Cells
Varying Degrees of Differentiation
Different Proliferative Potential
Different Lifespans
All Cell Types
in the Tumor
Implications for Cancer Chemotherapy
December 4, 2008
Tumorigenic Potential
Assay
Inject tumor cells into SCID mice
Examine mice after 8 weeks
~ 0.0001 - 0.1% of Cancer cells
are Tumorigenic/Leukemogenic
Factors Affecting Outcome of Experiment
Prolonged Observation period
Severely immunocompromised mice
Preincubation of injected tumor cells in ECM proteins
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