Transcript Embryonic Stem Cells

Blastocyst
The stage at which embryonic and extra-embryonic tissues are
segregated (about 5 days post fertilization).
Trophoblast
ICM
Consists of:
1. The Inner Cell Mass (ICM) which will give rise to the embryo
2. The trophoblast which will give rise to the placenta
Culturing ES cells
Human ES cell grown on a monolayer
of mouse embryonic fibroblast cells
known as “feeder cells”.
Feeder Cells are slowly being replaced by culture media
with specific factors required for maintaining ES cell fate:
Leukemic Inhibitory Factor (LIF)
Bone Morphogenetic Protein (BMP)
Both are secreted by feeder cells and/or are present in
serum used in media.
Lodish et al., Molecular Cell Biology
How can you be sure they are really ES cells on the plate?
1) Allow the cells to differentiate spontaneously on a Petri plate. They will do
this spontaneously when they get crowded.
2) In liquid suspension, allow the cells to spontaneously form embryoid bodies
with three germ layers.
3) Inject the cells into a mouse (or other mammal) with a suppressed immune
system and observe the formation of teratomas.
ES Cell Differentiation
(neurons)
Embryoid Bodies
Teratoma
Growth factors induce stem cell differentiation
Research into…
factors that
promote/repress
differentiation
…greatly increases the
potential for successful stem
cell therapy.
Gilbert, Tyler, Zackin: Bioethics (2005)
Embryonic Stem Cells (ES cells)
ICM
Embryonic Stem Cells (ES cells)
The Knockout Mouse
Transgenic Technology using ES Cells
TLR5 knockout
prone to
obesity
WT
Gene Modification in
Mice
-orKnockout Mouse
Technology
First transgenic mouse: 1977
First Knockout mouse: 1989
The Knockout Mouse – targeted gene inactivation
Tsai et al., 2007
Targeted Gene Inactivation (Knockout)
Homologous Recombination with Positive/Negative Selection
neor confers resistance to an antibiotic
(Geneticin, aka G418).
Tsai et al., 2007
tk (Herpes Simplex Virus thymidine
kinase) phosphorylates ganciclovir
and blocks DNA synthesis.
G418
Griffiths et al., An Introduction to Genetic Analysis
Introduction of Transgene into ES cells in Culture
Transduction: use of a viral vector to
insert DNA (complications from virus)
Transfection: use calcium phosphate
precipitate of DNA and add to cells in a
monolayer, some take up DNA
Lipofection: use liposomes as a vector to
introduce DNA, fusion at cell membrane
Electroporation: use electric current to
transiently create small pores in cell
membrane that DNA can pass through
Cloning Gun for electroporation
Tritech Research
Chimeric Mice
Archeology Museum Florence
Chimera vs. Mosaic
The offspring will be…
Strain: 129 (Brown, A/A)
Mate with Males
(Black a/a)
Strain: C57B10/6J (Black, a/a)
Genotype?
Chimera
Lodish et al., Molecular Cell Biology
Chimeric mice are mated to
Black mice.
ES cell derived progeny are
Brown (Brown is dominant
to Black).
Not all Brown progeny carry
the KO allele.
Screen with…?
How long to get a
homozygote?
Lodish et al., Molecular Cell Biology
Knockin
Mouse
Tis21::GFP Knockin
Southern Blot
GFP expression in
Central Nervous
System
Haubensak et al., 2003, PNAS
Somatic Cell Nuclear Transfer (SCNT)
Early experiments in the 1960’s
showed some success with cell
reprogramming with amphibians.
Wilmut et al., 1997
First successful SCNT involving
adult mammalian differentiated
donor cells  “Dolly”
“Dolly”
SCNT – somatic cell was fused with an enucleated oocyte.
Egg donor and nucleus donor were from different types of
sheep (Blackface and Dorset).
Gilbert, Tyler, Zackin: Bioethics (2005)
Cloning
Anne McLaren, 2001 (UK):
One week after the news that a sheep had been cloned, a poll was conducted .
90% of people thought that human cloning should be banned
67% of people thought that cloning animals was unacceptable
56% of people said they would not eat the meat of a cloned animal
Reasons for Cloning
1. Reproductive cloning: creating a genetically identical individual
2. Therapeutic cloning: creating genetically identical ES cells for medical
purposes
Cloning Humans
Hwang Woo-suk and Moon Shin-yong of Seoul National University have
developed a line of human Embryonic Stem Cells (ES cells) by cloning
adult human somatic cells (Hwang et al., 2004).
RETRACTED – he fabricated data
Many other labs have reported success with SCNT for therapeutic
purposes only.
Gilbert, Tyler, Zackin: Bioethics (2005)
Stem cell therapy
Lewis, Human Genetics
March 9, 2009 - Obama repeals policy preventing federal tax money
from funding embryonic stem cell research.
"In recent years when it comes to stem cell research, rather than furthering discovery, our government has
forced what I believe is a false choice between sound science and moral values...
In this case, I believe the two are not inconsistent. As a person of faith, I believe we are called to care for
each other and work to ease human suffering. I believe we have been given the capacity and will to pursue
this research — and the humanity and conscience to do so responsibly.”
- Obama, 3.9.09
At present, research on human SCNT may not
be conducted with federal funds but there is
no federal ban on privately funded research in
this direction.
Some states have issued a ban on human SCNT
for reproduction and others have issued the
ban for reproduction or therapy.
Stem Cells for Treating Disease
Drugs to treat
symptoms
Existing cell
lines, aborted
embryos, IVF
“leftovers”
ES cells differentiate
into functional
neurons
Parkinson’s Disease Patient
SCNT to create ES
cells to differentiate
into functional
neurons
Gene therapy:
correct genetic
defect with various
strategies
iPS cells
differentiate into
functional neurons
FDA Approved ES Cell Therapy Clinical Trials
As of Spring 2012…..
2010
Starsgardt and Age-Related Macular Degeneration (cause blindness)
Advanced Cell Technology
12 patients
2009
Spinal Cord Injuries
Geron Corporation (since has dropped stem cell division of company)
10 patients