Download 26. Stem cells

BIOL 311 Human Genetics
Fall 2006
Lecture: Stem Cells
References: (1) Text pp. 611-613
(2) Vats, A. et al. (2005) Stem cells. Lancet 366, 592-602.
(3) Hook, L., O’Brien, C. and Allsopp, T. (2005) ES cell technology: An introduction to
genetic manipulation, differentiation and therapeutic cloning. Adv. Drug Deliv. Rev. 57,
1904-1917.
(4) Ben-Nun, I. F. and Benvenisty, N. (2006) Human embryonic stem cells as a cellular
model for human disorders. Mol. Cell Endocrin. 252, 154-159.
Lecture outline:
1. Stem cells
a. definitions
b. early embryonic development
c. sources
d. ethical concerns
2. Possible disease applications
a. disease models (human genetic diseases)
b. cellular or tissue therapies (diabetes, CV disease, neurodegenerative disorders)
3. Manipulations of ES cells
a. genetic (recombination, lineage tracing, gene trapping)
b. nuclear transfer (cloning)
c. therapeutic vs. reproductive cloning
4. Issues involving ES cells
Lecture:
1. a. Stem cells: cells which can act as precursors to differentiated cells, but which
retain the capacity for self-renewal.
Totipotent stem cells: all types of body cells can be derived. Capacity of zygote, 2, 4
and 8 cell stage embryos.
Pluripotent stem cells: many cell types can be derived, but not all types. Capacity of
the blastocyst cells.
Multipotent stem cells: only cell types along a particular lineage can be derived. For
example, hematopoietic stem cells can form RBCs, WBCs and lymphocytes.
Early development
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blastocyst  fetus  infant  adult
c. sources
early embryos: human IVF “pre-implantation” embryos; disposition regulated
fetuses: miscarriages, abortions; legal, moral, ethical issues
umbilical cords: permission of family; cord blood banks for fee
adult stem cells: most accessible to physicians, researchers; lack potential of ES or fetal
cells; cells from same individual histocompatible
Possible disease applications
a. disease models
Rats and mice do not get all the same diseases humans do or don’t necessarily have the
same symptoms.
Lesch-Nyhan syndrome
X-linked recessive disorder due to mutation in HPRT1 gene (enzyme in the purine
nucleotide salvage pathway), which leads to overproduction of uric acid. Uric acid
accumulation leads to gout, urinary stones, and neurological symptoms such as self-
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mutilation. Mouse has an enzyme urate oxidase that converts uric acid into allantoin.
Therefore, mice with mutations in their hprt genes do not symptoms associated with the
accumulation of uric acid. The best mouse model system is based on a genetically altered
mouse ES line used to produce a transgenic mouse model. However, a human ES model
might be better for studying the disease and possible therapies.
It may be possible to derive cell lines from non-implanted embryos or adult cells from
individuals with the disease as disease models. Learn to treat the disease in cells, then try
on humans.
b. cellular or tissue therapies
diabetes: lack of regulation of glucose levels in the body by the pancreas
hypothetical therapy: use stem cells that give rise to pancreatic islet cells to
repopulate the pancreas or reprogram them to divide or function properly.
cardiovascular disease: actually a spectrum of diseases that affect the heart and
circulatory system, including high blood pressure, blocked coronary arteries, etc. that can
lead to heart attack and stroke, where the heart and/or blood vessels become damaged.
hypothetical therapy: use stem cells to replace damaged muscle cells of heart,
regrow new arteries and veins where others were damaged.
neurodegenerative diseases:
Alzheimer’s: adult senile dementia
Parkinson’s: loss of motor control;Advocates for stem cell use: Michael J. Fox,
Mohammed Ali, both affected with Parkinson’s.
Spinal cord damage: Loss of neural connections
Christopher Reeve severed his spinal cord in a horseback riding advocate and was an
advocate for stem cell research.
Hypothetical therapy: Use neural stem cell precursors to regrow neural
connections. Stem cells with such potential have been identified, even in adults.
3. Manipulations of ES cells
a. genetic: can insert genes by transfection, electroporation or microinjection
i) recombination
Currently, “knock-out” mice use a technically difficult approach of gene targeting,
homologous recombination of mouse ES cells to create disruptions in the genes of
interest.
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Features: dominant selectable markers, conditional site specific recombination event
(cre/loxP recombination system), can regulate recombination so that it only occurs in
certain cell types.
The same technology could be used to genetically alter human ES cells.
ii) lineage tracking
Can introduce marker genes into ES cells (green fluorescent protein or betagalactosidase) that can be traced when cells are transplanted into tissue to track their fate
or mark their descendents.
iii) gene trapping (also called exon trapping)
Use specific cloning vectors (“gene trap” vectors) to recover genes uniquely expressed in
ES cells.
b. Nuclear transfer (cloning) see Fig. 21.2 of text
The nucleus from a donor somatic cell is injected into an oocyte  the oocyte is allowed
to develop to the blastocyst stage.
When a blastocyst is implanted into the uterus, the goal is a reproductive clone (this is
reproductive cloning).
This technology has been successful for sheep (Dolly), mice, cat, etc.
Not legal or ethically condoned in U.S. for humans.
Therapeutic cloning would use the blastocyst to provide ES cells to create differentiated
cells of a particular type for transplantation.
4. Issues involving ES cells
Include ethical, regulatory, technical (contamination), safety issues.
Pre-implantation embryos or aborted or miscarried fetuses are the potential sources for
ES cells and fetal stem cells, respectively. There is a moratorium in the U.S. on federally
funded research with new embryos or fetuses. Research only unrestricted on the limited
number of human ES cell lines already created.
Problems: Many of these human ES lines don’t grow well for experiments. Also the
normal way to propagate human ES cell lines uses mouse cell “feeder” cultures; if
transplanted into humans, the contaminating mouse materials could initiate an immune
response.
Reaction to this moratorium has led to state initiatives. Stem cell research is allowed in
California and was a significant issue in Missouri in the last election, where voters had to
decide on an amendment supporting therapeutic uses of stem cells.
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Safety is a huge concern in the development of stem cell therapies. These therapies are
mostly designed to be transplantations. Host rejection is a major concern with any kind
of transplantation therapy. Technologies to better match the stem cell antigens with the
patient, suppress the immune response in the patient, or to derive stem cell therapies from
the individual’s own cells need to be worked on.
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