Transcript Stem Cells in Research Promises and Pitfalls Denise Inman, PhD University of Washington Department of Neurosurgery.

Stem Cells in Research
Promises and Pitfalls
Denise Inman, PhD
University of Washington
Department of Neurosurgery
Overview
• What are stem cells?
– How do embryonic and ‘adult’ stem cells differ?
– How are different types of stem cell lines created?
• Stem cells in research and medicine
• Alternatives to the embryo
Early Development
Fertilized egg
Totipotent: Can become any cell in body
or placenta
Pluripotent: Can become any cell in body
Totipotent
stem cells
Fate Decision
Pluripotent
stem cells
Multipotent: Can become any cell within
a specific germ layer or cell lineage
Blastocyst
Embryonic stem cells come from inner cell mass of blastocyst.
Fate Decision
Implantation
Gastrulation
Multipotent
stem cells
Primary Germ Cells
Endoderm (inner)
Mesoderm (middle)
Ectoderm (outer)
Embryonic Stem Cell Characteristics
•Not committed to a specific fate
•Pluripotent—can differentiate into
specialized cell types
•Self-renewing
Courtesy of James Thomson,
U. Wisconsin-Madison
endoderm mesoderm
ectoderm
Is that an Embryonic Stem Cell?
The true potential of
stem cells can only be
assessed retrospectively
SCID Mouse:
Severe Combined
ImmunoDeficiency
Embryonic stem cells injected into a SCID mouse will
grow into teratomas, tumors of the germ cell layers.
Individual ESCs under the correct conditions will
make many different cell types.
Stem Cells: From Embryonic to Adult
Embryonic stem cells are those
removed from the blastocyst before
the fate decision from
pluripotentiality to multipotentiality.
Adult stem cells are those
multipotential cells that persist in
fully developed tissues. These cells
never differentiated into the mature
cell types of the tissues in which
they reside.
http://www.brown.edu/Courses/BI0032
Adult Stem Cells
• Multipotential
– Make cells within a specific lineage
• Not differentiated
• Rare
• Self-replicating
Neural stem cells in culture. One cell is extending a process.
Adult Stem Cells – Bone Marrow
Major repository of
adult stem cells
-Hematopoeitic
-Mesenchymal
Give rise to immune
system cells
Constant turnover
NIH: stemcells.nih.gov/ info/basics/basics4.asp
Stem Cell Phenotype
Shihabuddin, et al., J. Neuroscience 20(23) 8727-8735, 2000
Fate dictated by environment…
Neural stem cells
Neurons
Astrocytes
Oligodendrocytes
Oligodendrocyte Progenitor Cells
Stem cells placed in spinal
cord become glial cells…
Stem cells placed in brain become neurons…
Re-cap: What are stem cells?
• Embryonic and adult stem cells
– Obtained at different developmental stages
– Different potential
• Pluripotent versus Multipotent
– Sensitive to environment
Overview
• What are stem cells?
– How do embryonic and ‘adult’ stem cells differ?
– How are different types of stem cell lines created?
• Stem cells in research and medicine
– How do scientists work with stem cells?
• In situ labeling
• Primary culture
• Cell lines
– Promises and perils of stem cells
• Alternatives to the embryo
Cell Lines
• Cells under propagation
• All cells are genetically identical
• Can be frozen and stored
Plate
Exponential Growth
Remove from plate
Replate at lower density
Culturing Embryonic Stem Cells
Obtain stem cells from
In Vitro Fertilization
Sperm
+
Somatic Cell Nuclear Transfer
Oocyte without nucleus
Oocyte
Inject nucleus from
adult somatic cell
Fertilized egg
Blastocyst
Blastocyst
1. Remove inner cell mass
2. Put cells in dish with feeder layer
3. Cells divide
Origins of ES Cell
Lines
• Excess IVF embryos
New England Journal of Medicine,
Wellcome Trust
• Therapeutic Cloning (somatic cell nuclear
transfer)
– Donor oocyte+somatic cell nucleus
– Cells have characteristics of nuclear donor
– Lines representing different diseases
– Individualized lines: non-immunogenic to donor
Roslin Institute http://www.roslin.ac.uk/library/
Somatic Cell Nuclear
Transfer
Challenging: In cloned cell lines, about 4% of
genes function abnormally, owing to departures
from normal activation or expression of certain
genes
-Imprinting, methylation state
Removing the egg nucleus
before transferring a somatic
cell nucleus
Limited success: ~25 percent of
nuclear transfers led to a
blastocyst; 35 percent of
blastocysts led to establishment
of cell lines
Patient-specific embryonic stem cells derived
from human SCNT blastocysts.
Science 308(5729):1777-1783, 2005.
hES Cell Lines in the US
• Most, if not all, of the stem cell lines are
contaminated with mouse feeder layer proteins.
• These cells will never be used in clinical
application.
• Considerable biological variability across cell
lines.
• Increased culturing can cause ES cells to
accumulate epigenetic and genetic changes,
altering their ability to form different types of
cells.
Promises and Perils of Stem Cells
What’s at stake?
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Embryonic stem cells in therapy
Cloning
Adult stem cells in therapy
Beyond cell replacement
Beyond the embryo
What can ESCs do for you?
• Theoretically
– Replace damaged, diseased cells
– Gene therapy
• Genetically manipulated hES cells might serve as
vectors to carry and express genes in target organs
following transplantation in the course of gene
therapy
Why Clone?
Therapeutic and Reproductive Cloning
• Human protein production
– Produce human protein-based medicine in
milk from transgenic cows
• α-1-antitrypsin for cystic fibrosis
• Transplants without immune response
– Organ rejection or graft-vs-host disease
Therapeutic Cloning
How Promising are Adult Stem Cells?
• Bone marrow transplants
– Hematopoeitic stem cell transfer
• Difficulty maintaining control once in vivo
– Niche dictates phenotype
– Plasticity
Adult Stem Cell Clinical Trials
• Bone marrow stem cells from self or
allogeneic (sibling) transplant
– after chemotherapy for myeloma, glioma,
leukemia, lymphoma, neuroblastoma, lung
cancer
– sickle cell anemia, liver disease, autoimmune
disorders, vascular disease
• Mesenchymal stem cells for myocardial
infarction
Potential Beyond Cell Replacement
• Exploring disease mechanisms
– study how basic cellular mechanisms are disrupted or
changed by disease proteins
• Drug discovery
– High-throughput assays will identify targets. For
example, using mouse ES cell-derived neural cells for
an assay to screen Alzheimer's disease
• Genetic screening
• Toxicology testing
Overview
• What are stem cells?
– How do embryonic and ‘adult’ stem cells differ?
– How are different types of stem cell lines created?
• Stem cells in research and medicine
• Alternatives to the embryo
Beyond the Embryo
• The President’s Council for Bioethics
– White Paper published May 2005
– http://bioethics.gov/reports/white_paper/text.html
ESCs without the E
• De-differentiation
– Requires aid of special cytoplasmic factors
obtained from oocytes (or from pluripotent
embryonic stem cells)
•Obtainable from any adult
•Immunocompatible
•Some success with muscle, liver, blood
Issues:
How far back can dedifferentiation go?
Muscle cells
Multipotent
progenitors
ESCs without the E
• Remove single cell from 6-8 cell embryo
– Spin-off of preimplantation diagnosis
Remove cell
Establish cell line
Issues:
Is there harm in removing a cell?
Could a cell line be established with one cell?
Is cell at this stage totipotent?
Embryo
ESCs without the E
• Removal from dead embryo
– Early IVF embryos (roughly 4-8 cells) that
have spontaneously died. Normal-appearing
blastomeres in cleavage-arrested, mosaic
embryos.
Issues:
Can markers of organismic death be found?
Can pluripotent stem cells be derived from
dead embryos?
If so, will they be chromosomally (and
otherwise) normal?
Parthenogenesis
Establish cell line
Oocyte
Blastocyst
• Biochemically trick a human oocyte into thinking it has been
fertilized.
• Treated eggs divide to the blastocyst stage (50-100 cells), at which
point stem cells can presumably be derived.
• The “parthenogenetic” (that is, unfertilized but still developing)
blastocyst-like entity is assumed by most to lack the potential for
development as a human being.
ESCs without the E
• Bio-engineered embryo-like artifacts
– Embryos engineered to lack the essential
elements of embryogenesis but still capable of
some cell division and growth
Altered Nuclear Transfer
Remove altered nucleus to oocyte
Somatic cell
Oocyte
Cell Division
Blastocyst
Embryo
ESCs without the E
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De-differentiation
Single cell removal from embryo
Removal from dead embryo
Parthenogenesis
Bio-engineered embryo-like artifact
Creative thinking, possible solutions to an ethical dilemma.
Research has yet to determine if one or more of these proposals are possible.
Recent Research
• RNAi was used to change expression of a
gene in a hESC line.
– Stem Cells 23(3):299–305, 2005
• hESCs driven to develop into motor
neurons.
– Nature Biotechnology 23:215-221, 2005.
Recent Research
• Mesenchymal stem cells injected into rat heart
increased pumping capacity and vessel growth
after heart attack.
– Journal of Clinical Investigation 115:326–338, 2005.
• “Stembrids” were made —one ESC was
enucleated and then given the nucleus from an
adult somatic cell.
– Not shown that the resulting “stembrid” would be
immunologically acceptable to the adult somatic cell
donor.
Summary
• Stem cells
• Embryonic vs. Adult
• IVF, SCNT
• Therapeutic cloning and immune matching
• Much scientific progress, but therapies are
not yet directly translated from research
• Greatest potential contribution from mechanistic
studies in ESCs
• Embryonic alternatives need more development
Conclusion
• Stem cells are complicated: scientifically,
ethically, legally. The best way to approach
them is with education.
• Working with stem cells is one of the most
important opportunities of our time.