Transcript The end of the beginning for pluripotent stem cells Peter

The end of the beginning
for pluripotent stem cells
Peter J. Donovan* & John Gearhart†
*Kimmel Cancer Center, Thomas Jefferson University, Philadelphia,
Pennsylvania
†The Institute of Cell Engineering, Johns Hopkins University School
of Medicine, Baltimore, Maryland
Jason Ip
Graduate Student
“If we do the work that we can do
in this country, the work that we
will do when John Kerry is
president, people like Christopher
Reeve will get up out of that
wheelchair and walk again”
-Senator John Edwards
October 12, 2004
Background
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Pluripotent stem cells
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def. cells in a stem cell line
capable of differentiating
into several different final
differentiated types
First recognized in
teratocarinomas
Primordial germ layers


The ectoderm, endoderm,
and mesoderm, are the
three major cell lineages
Formed during gastrulation
(cell migration resulting in
cleavage)
Background
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The three types of embryonic
tissue

Embryonic stem (ES)
 Embryonic germ (EG)
 Embryonal carcinoma (EC)

Attributes of EC, ES, and EG
cells

Transcription factor Oct4
 Alkaline Phosphatase
 Telomerase
…Upregulation sustains
pluripotency
Agenda

The Science of Pluripotency
 Developmental
potential
 The basic biology of human development
 Embryonic stem cells vs. adult stem cells

Bringing stem cells to the clinic
 Expansion
and differentiation
 Safety considerations in cell-based therapies
 The future of stem cells
The Science of Pluripotency
Developmental potential
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Assessment in three
independent assays
1.
2.
3.
In vitro differentiation
in a Petri dish
Differentiation into
teratomas or
teratocarcinomas
within
histocompatible mice
In vivo differentiation
within blastocoel
cavity of a preimplantation embryo
Developmental potential

Directing differentiation
 Manipulation
of cellular environment
Growth of cells at high density
 Growth of cells on different types of feeder cells
 Addition of growth factors
 Growth on crude or defined ECM substrates

…differentiation varies and lacks robustness
 Suspended

three dimensional aggregation
Development of embryoid bodies
Developmental potential

Embryoid bodies
 Capable
of
differentiating into any
of the three primordial
germ layers
 Germ layer cells are
multipotent, as
opposed to pluripotent
The basic biology of human
development

Pluripotent stem cells can…
 Aid
in deciphering developmental geneexpression
Survival
 Proliferation
 Differentiation
 Migration

 Lend
insight to tumorigenesis and genetic
diseases
Embryonic stem cells vs. adult
stem cells

Ethical consideration
 What

marks the beginning of life?
Main differences
 The
number of potential derivatives;
embryonic > adult

Feasibility
 Lack
of publication on adult stem cell
research (add more?)
Bringing stem cells to the clinic
Successful transplantations of
mouse ES cells
1.
2.
3.
4.
Cardiomyoctes form stable and functional
grafts
Glial precursors interact with host neurons to
replenish lost myelin in the brain and spinal
cord
Embryoid bodies differentiate into neurons in
the spinal cord, promoting motor recovery
Insulin-producing cell line implanted into mice
resulted in normalized glycemia
Successful transplantations of
mouse ES cells

Rats with motor injury and stroke treated with
neuronal cells derived from human EC cells
resulted in partial recovery of motor function
?
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Successful transplantations of
mouse ES cells
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Transplanted cells are
replacing lost cells such as
neurons or glia
Transplanted cells providing
factors facilitating the
regeneration of host cells
Cell-based therapies may be useful in
abating the effects of injury and disease
Stem cell expansion and
differentiation

Requirements leading to clinical therapy
Growth in large quantities
2. Controlled homogeneous differentiation
3. Histocompatibility
1.

Limitations
Stem cell survival in long-term culture
2. Stem cell genetic mutations
1.
Stem cell expansion and
differentiation

Possible solutions
 Progenitor cells
 Advantages

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

Disadvantages

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
Derived from embryo bodies
Easier to grow and expand
Possess normal karyotype
Limited self-renewal capability
Can be unipotent or
multipotent
Example:

Neural progenitors can be
formed from human ES cells
in high density culture,
become neurons
Stem cell expansion and
differentiation
 Genomics


Microarray technology can
reveal expression of growth
factors, growth-factor
receptors, and cell-adhesion
molecules
Expression profiles allow for
optimal conditioning of stem
cell growth environment
Safety considerations in cell-based
therapies

Three key safety issues:
Histocompatibility
2. Tumorigenesis
3. Infection from serum-containing culture
1.
Safety considerations in cell-based
therapies

Histocompatibility
 Immune suppression
 Slows immune response
 Nonspecific
 Tolerance induction
 Antigen-induced
 Specific
 Embryo-derived compatible cells
 Therapeutic Cloning (somatic cell nuclear transfer)
 Genetic recombination of existing stem cell lines to match
patient
Safety considerations in cell-based
therapies
Therapeutic cloning
Genetic Recombination
Safety considerations in cell-based
therapies

Tumorigenesis
 Conflicting
arguments
Imprinted genetic loci are erased in EG cell lines
 EG cell lines behave normally in chimeras

 Key
questions
How can cells be ensured to migrate to designated
sites?
 At what stage of differentiation should
transplantation occur? (hmmm?)

Safety considerations in cell-based
therapies

Infection from serumcontaining culture
 Infection
caused by blood-borne
bacteria
 Serum contains necessary
growth factors
 Human ES cells require fetal
calf serum or conditioned
medium via mouse feeder cells
for growth
The future of stem cells
Mouse ES cells have contributed much to
our understanding of embryogenesis
 Prospects

 Stem
cell therapeutics
 Deeper understanding of human growth and
development

Treatment on non-human primates likely
to be a next step before use in the clinic
References
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http://www.biology-online.org/dictionary.asp
http://www.csa.com/discoveryguides/stemcell/images/pluri.jpg
http://en.wikipedia.org/wiki/Image:Cloning_diagram_english.svg
http://alignmap.com/wp-content/Graphics/JohnEdwards(098).jpg
http://upload.wikimedia.org/wikipedia/commons/d/d4/Cell_differentiation.gif
http://www.bio.miami.edu/dana/pix/gastrulation.jpg
http://abcnews.go.com/Health/wireStory?id=4313450
http://www.brown.edu/Courses/BI0032/adltstem/stem-cell.gif
http://www.wormbook.org/chapters/www_germlinegenomics/germlinegenom
icsfig1.jpg
http://static.howstuffworks.com/gif/stem-cell-therapeutic.gif
http://regentsprep.org/Regents/biology/units/reproduction/crossingover.gif
http://omegascientific.com/catalog/images/fetal-bovine-fam.jpg