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 Blastocyst Inner Cell Mass (ICM) 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 Horse Mouse Goat Cattle Rabbit Pig Cat Mule Horse Goat Rabbit Cat Dog Gaur “Dolly” “Copy Cat”

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 Mesoderm Endoderm (squamous epithelium) (gut epithelium) (bone and cartilage)

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 Reprogramming Genes: OCT4, SOX2, KLF4, c-MYC

Neurons from iPS Cells Derived from ALS patient

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+

Pathway OFF No Wnt Molecules INACTIVE (No cell division) Self-renewal Proliferation (No cell division)

Pathway ON Cyclin D Nanog SOX2 c-myc LIN28 BMP4 FGF MMP (family of ~ 20 proteins) Pathway ON ACTIVE Wnt Target Genes Cyclin D Nanog SOX2 c-myc LIN28 BMP4 FGF MMP Self-renewing Proliferation

Pathway Always ON in Cancer Cyclin D Nanog SOX2 c-myc LIN28 BMP4 FGF (family of ~ 20 proteins) Pathway Always ON in Cancer Activating Mutations Wnt FRAT catenin Wnt Target Genes Cyclin D Nanog SOX2 c-myc LIN28 BMP4 FGF MMP Inactivating Mutations Axin APC CONSTANT Proliferation

Tumor Formation All Cell Types in the Tumor Phenotypically Different Cells Varying Degrees of Differentiation All Cell Types in the Tumor Different Proliferative Potential Different Lifespans

Implications for Cancer Chemotherapy

December 4, 2008

are Tumorigenic/Leukemogenic 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