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Cloning in Nature. Parthenogenesis. Monozygotic twins (embryo splitting). (incomplete embryo splitting). Egg Cell and Sperm Cells. Pg. 661. Ca 2+ Wave. Metaphase arrest. Ca 2+ and Egg Activation. SEP. “Calcium wave”. Cleavage Stage Embryos. Pre-compaction. Compaction. - PowerPoint PPT Presentation
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Cloning in Nature
ParthenogenesisMonozygotic twins(embryo splitting) (incomplete embryo
splitting)
Egg Cell and Sperm Cells
Pg. 661
Ca 2+ Wave
Metaphase arrest
Ca2+ and Egg Activation
“Calcium wave”
SEP
Cleavage Stage Embryos
Pre-compaction Compaction
Cadherin (Homophilic Cell Adhesion Molecule)
Blastocycst
Uterus
BlastocystInner Cell Mass (ICM)
50 µmTrophoblast
Blastocoel
Pre-implantation Development (~ 7 days in humans)
Mammals Cloned by Embryo Splitting
SheepCattleMousePigHorseRhesus monkey
Blastomere Isolation
“Tetra”
Mammals Cloned by SCNT
SheepMouseCattlePigMule
“Dolly”
“Copy Cat”
HorseGoatRabbitCatDogGaur
Removal of genetic material from oocyte
Transfer of diploid nucleusfrom adult somatic cell or Embryonic cell into“enucleated” oocyte
Cleavage divisions of“reconstructed” embryo
Oocyte Activation
Injected somatic nucleus
CondensedChromosomes Pseudo-pronuclei
SCNT in Mice
Ca 2+ injectioncytochalasin
Removal of genetic material from oocyte
Transfer of diploid nucleusfrom adult somatic cell or Embryonic cell into“enucleated” oocyte
Cleavage divisions of“reconstructed” embryo
Oocyte Activation
Copy Cat Somatic cell donor“mom”
Copy Cat Somatic cell donor“mom”
Inactive X Chromosome (“Barr Body”)
X Chromosome Inactivation
ICM cells ofBlastocyst
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
PluripotentMultipotentUnipotent
Regenerative Medicine
Stem Cells
Transplant into patients
Pluripotent Stem Cells
Mouse Chimeras with Embryonic Stem Cells
Science 282: 1145-1147November 6, 1998
Human Embryonic Stem Cell Colony
Stem Cells
Mouse“feeder”cells
Normal Karyotype in Cultured Human Embryonic Stem Cells
Telomerase Activity
Cultured Cells Remain Undifferentiated
Undifferentiation “Markers”
Oct4 SSAE4
Teratoma
Ectoderm Mesoderm Endoderm(gut epithelium)(bone and cartilage)(squamous epithelium)
Embryonic Stem Cell Signaling Pathways
These Pathways are necessary for maintaining the undifferentiated State
OCT4
Nature 456: 344-349November 20, 2008
Science 318: 1917-1920December 21, 2007
Science 321: 1218-1221August 29, 2008
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
iPS Cell Colony from Patient with ALS
Reprogramming Genes: OCT4, SOX2, KLF4, c-MYC
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Neurons from iPS Cells Derived from ALS patient
Tissues with Adult Stem Cells
Bone marrow/peripheral blood/umbilical cordBlood vesselsBrain/spinal cordSkeletal muscleColonLiverPancreasRetina/corneaSkinDental pulp
Adult Stem Cell
Multipotent
Life-long
Plasticity?
Adult Stem Cells
Multipotent
Progenitor Cells
Stem Cell Niche
Stem Cell
(Common Lymphoid progenitor)
(Common Myeloid progenitor)
Signaling Pathways for Self Renewalin Adult Stem Cells
Signals produced with the HSC niche
Cytkine Signaling via Jak/STAT Pathway
Cytokines in Hematopoiesis
Bone Marrow
(Common Lymphoid progenitor)
(Common Myeloid progenitor)
Myeloid Leukemia
Tumor Growth
(~ 100 days/pop doubling)
(~ 7.5 yrs)
(~ 8 yrs)
(~ 10 yrs)
Myeloid Leukemia
CD34+
Myeloid Leukemia
CD34+
CD34+
Self-renewalProliferation
No Wnt Molecules
(No cell division)
Pathway OFF
INACTIVE
Self-renewing Proliferation
Wnt Target Genes
Cyclin DNanogSOX2c-mycLIN28BMP4FGFMMP
(family of ~ 20 proteins)
Pathway ON
ACTIVE
CONSTANT Proliferation
Wnt Target Genes
Cyclin DNanogSOX2c-mycLIN28BMP4FGFMMP
(family of ~ 20 proteins)
Pathway AlwaysON inCancer
Activating Mutations
WntFRATcatenin
Inactivating Mutations
AxinAPC
Tumor Formation
All Cell Typesin the TumorDifferent Proliferative Potential
Phenotypically Different Cells
Varying Degrees of Differentiation
Different Lifespans
Implications for Cancer Chemotherapy
December 4, 2008
Tumorigenic Potential
~ 0.0001 - 0.1% of Cancer cells are Tumorigenic/Leukemogenic
Assay
Inject tumor cells into SCID miceExamine mice after 8 weeks
Prolonged Observation periodSeverely immunocompromised micePreincubation of injected tumor cells in ECM proteins
Factors Affecting Outcome of Experiment
