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