| |
 Through
improved understanding of normal developmental processes,
it is possible to gain insights into the mechanisms of developmental
disorders. PDC investigators are actively studying stem
cell potentials, preimplantation embryo development, germline
development, fetal and postnatal mammalian development,
and mechanisms of aging. Current investigations are aimed
at establishing cell-based therapies to treat developmental
disorders, including neurodegenerative diseases, diabetes,
ADHD, autism, cerebral palsy, and pediatric disorders.
Transgenesis is a powerful tool for evaluating gene function
and modeling human diseases. Pioneering research by PDC
investigators resulted in the world’s first transgenic
non-human primate (ANDi). To meet the PDC’s mission
of using translational animals models to bring biomedical
research from the lab bench to the bedside, a variety of
methods are currently used or under development for producing
transgenic mice, rats and nonhuman primates.
Fetal and postnatal development
Dr. Ozolek is a Fellow in Pediatric Pathology who previously
practiced neonatology both in the United States Air Force
and in civilian private practice. His clinical research
within Neonatal-Perinatal Medicine focused on describing
the effects of in utero prenatal hypoxia-ischemia on the
hematologic and central nervous systems. In the realm of
Pediatric Pathology, his attention has turned to the possibility
of cellular therapy for fetal hypoxic-ischemic central nervous
system injury, in particular, prenatal white matter injury
or periventricular leukomalacia (PVL), a condition that
can result in cerebral palsy.
Dr. Ozolek has characterized the proliferation, survival,
and differentiation along neuronal and glial lineages of
human embryonic stem cells (hESCs) grown under two different
feeder conditions at three time points. Additionally, he
has described the preferential movement and differentiation
of hESCs towards human glioblastoma tumor cells both in
vitro using a newly developed mobility assay and in vivo
after injection of glioblastoma cells and hESCs into different
locations within the mouse brain. A side project has included
the characterization of cellular elements within the developing
primate brain using a variety of neuronal and glial lineage
expression markers. In the near future, the injection of
stem cells alone and in combination with scaffolding matrices
into a rodent model of PVL to repair injured CNS tissue
will be possible.
[top]
Germline development
The germline exhibits the complete cycle of developmental
potency. Eggs and sperm can be considered totipotent because
they contribute the female and male components of the zygote,
the premier and totipotent cell in mammalian development.
Primordial germ cells obtained from mid-gestation fetuses
give rise to pluripotent embryonic germ (EG) cells in vitro;
and unipotent spermatogonial stem cells produce and maintain
spermatogenesis throughout postnatal life. Therefore, studying
germline development can reveal the mechanisms of developmental
potency through biological, genetic, and proteomic analyses.
Current studies are focused on deriving and characterizing
new EG cell lines, deriving gametes from ES or EG cells
in vitro, and studying the biology of adult type spermatogonial
stem cells in the postnatal testis. Molecular mechanisms
of developmental potency will be revealed through our studies
of germ cells at different stages of development.
[top]
Aging
Hutchinson-Gilford Progeria Syndrome (HGPS, commonly called
“Progeria”) is a devastating disease that causes
young children to undergo accelerated aging. HGPS recapitulates
most of the pathologies of normal aging, causing affected
children as young as five years to develop widespread atherosclerosis
that includes the coronary arteries and aorta, resulting
in death by heart attack or stroke in their early teens.
Recently, the genetic basis for HGPS was shown to be caused
by a mutation in a gene called Lamin A (LMNA). Although
the genetic basis of HGPS has now been found, the biological
pathways that lead from the mutation to the broad manifestations
of aging are unknown. To address this problem, PDC is developing a mouse model of HGPS to study
the pathological changes that occur over time to cells,
tissues, and organs in this disease.
[top]
Therapeutic Cloning for Neurodegenerative Diseases
Our unique expertise in somatic cell nuclear transfer in
primates and its application to murine models of PD and
human and primate ES cell differentiation, has led us to
evaluate whether dopaminergic (DA) neurons generated after
somatic cell nuclear transfer from primates will improve
the survival and function of DA neuron grafts towards a
first proof-of-principle application of therapeutic cloning
in primates. Parkinson’s disease (PD) is considered
a prime application for cell therapeutic approaches in the
CNS due to the defined and extensive loss of midbrain  DA
neurons in PD patients at time of diagnosis. Recent studies
from our group have demonstrated that hES cells can be differentiated
into cells with midbrain DA neuron characteristics. However,
there are concerns that the successful treatment of (PD)
with hES derived DA neuron transplants could be hampered
by poor cell survival, loss of DA phenotype, deregulated
growth of transplanted cells, and/or immunological incompatibilities
between the transplanted cells and the host environment.
Most studies using hES derived DA neurons involved xenografting
human cells in rodent hosts, animals of highly divergent
anatomical scale and developmental pace. Transplantation
into primate hosts ameliorates this issue and more directly
assesses critical parameters for DA neurons survival and
function. Short term analysis of cell survival and fate,
typically required for such studies, cannot be easily carried
out in primates (due to obvious cost and primate welfare
concerns). However, we have developed imaging strategies
in primates based on MRI and microPET technology that can
be used to monitor the grafted cells non-invasively and
repeatedly in individual monkeys in vivo.
Twinned embryos have been split with genetically identical
ES cell lines established and marked with transgenic reporters
so that eventually they can be traced within their infant
and adult twin sibling. The availability of genetically
identical primates will help investigations on the environmental
contribution to various genetic diseases, innovative transplantation
strategies, and vaccine research. While nuclear transfer
techniques in primates continue to prove challenging, the
splitting of embryos is gaining acceptance within several
regional primate research centers, as a reliable means to
make identical twins, triplets and even quadruplets. The
first report of this approach in nonhuman primates was published
by Chan et al., 2000. This technique is being used for chimera
construction to investigate both primate ES cell potentials
and also autoimmunity during diabetes.
[top]
Stem Cell Therapy for Diabetes Type I
The major goal of the Pediatric Research Section of the
University of Pittsburgh Diabetes Institute is to find tolerogenic
strategies that will allow transplant of allogeneic pancreatic
islets into diabetic children without the need for an immunosuppressive
regimen to protect the graft. Different promising approaches
seem to be working well in the mouse model of the disease.
However, the transfer of protocols from rodents to humans
is not a trivial task, and all of the parameters must be
finely adapted and pre-tested. We are testing our conviction
that an intermediary step into nonhuman primates will facilitate
this transition. Allogenic transplants can be immediately
performed, so to study the effects of our tolerization protocols
against immune rejection. The technology of cloning by embryo
splitting, chimeric embryo construction and possibly transgenesis
in primates is allowing us to generate animals carrying
human histocompatibility antigens that predispose to type
1 diabetes. These animals may develop the disease and allow
us to also study the autoimmune component of type 1 diabetes
active also against the transplanted allogeneic beta cells.
This research is partially funded by an F32 training grant
from NIDDK (Driving b-cell differentiation from ESCs) awarded
to Dr. A. Ben-Yehudah.
[top]
Macaque Models for ADHD, Autism, Cerebral Palsy and Pediatric
Disorders
The PDC is also contributing towards primate models for
attention deficit hyperactivity disorder (ADHD)and especially
autism. Dr. Hewitson has recently garnered funding to coordinate
an international investigation into the genetic and environmental
causes of autism – the still mysterious developmental
disorder that may well be reaching epidemic proportions.
Studies include MRI and PET imaging coupled with cognitive
testing of infants to look at potential brain anomalies,
as well as measurements of immune function. For this research,
Dr. Hewitson relies heavily on the Primate NICU and Infant
Primate Laboratory (IPL), housed in the PDC vivarium. The
IPL determines if pre- or post-natal manipulations produce
abnormalities in health, growth, behavior, perceptual-motor
abilities, social-emotional behavior, and/or cognitive-learning
skills in the resultant offspring from birth through the
second postnatal year. This research is supported by a grant
from Medical Interventions for Autism awarded to Dr. L.
Hewitson.
The effects of pre-natal methyl mercury exposure on lymphocyte
function in infants is also under investigation. Methyl
mercury, a known neurotoxin, has been shown to cause a variety
of developmental effects. Exposure during pregnancy simultaneously
exposes the developing fetus and can result in neurological
problems. A recent report by the EPA suggested that more
than one child in six born in the United States could be
at risk for developmental disorders because of in utero
mercury exposure. Mercury can also act as an immunosuppressant
resulting in increased susceptibility to infections. The
mechanism by which mercury acts as an immunotoxin is not
well understood. These studies will therefore determine
whether ‘environmental’ exposure to methyl mercury
during pregnancy, alters lymphocyte phenotypes (assessed
in cord blood samples at birth) predisposing the infants
to be more susceptible to infection. This work is supported
by a pilot grant from the University of Pittsburgh Center
for the Environmental Basis of Human Disease awarded to
Dr. L. Hewitson.
Rhesus monkeys born after a variety of assisted reproductive
techniques (ARTs) are also being monitored for development
based on recent concerns over the health of ART offspring
(Bowen et al., 1998; Hardy et al., 2002). The Infant Primate
Research Laboratory (IPRL) at the PDC studies the postnatal
development of normal and medically at-risk macaque monkeys.
The staff assesses parameters of health, growth, and behavioral
development from birth through 24 months. These measures
have identified deficiencies and retarded development produced
by both naturally occurring conditions and experimental
treatments. Infants born after a variety of assisted reproductive
techniques (ARTs) such as intracytoplasmic sperm injection
and embryo splitting have been carefully monitored. In the
first comparisons of 13 ART (IVF, ICSI and embryo split)
offspring and 15 control offspring delivered at roughly
the same gestational age (158 days) several differences
were noted. All ART offspring displayed less nonsocial positive
behavior (exploration and play) than the controls. The ICSI
offspring showed the most social negative behavior. No differences
in frequencies of nonsocial positive behaviors were noted
although all ART groups showed higher frequency of social
positive behavior than controls. These results, while preliminary
because of the small number of offspring but statistically
significant, also showed that the offspring produced by
embryo splitting exhibit behaviors consistent with attention
deficit, hyperactivity, disorder (ADHD). ADHD is a disorder
that falls within the autism spectrum, which is a significant
neurological and psychiatric concern. The incidence of autism
and related disorders is reaching epidemic proportions and
nonhuman primates may be invaluable for addressing the biomedical
basis of this complex disorder.
[top]
Transgenesis
The most direct way to study genes and their functions
in mammalian development is through evaluation of transgenic
or knockout models. In addition, transgenic animal models
of human diseases can accelerate the discovery of therapeutic
interventions.
Nonhuman primate transgenesis is perhaps a quarter century
behind mouse transgenics, and primates pale as a research
resource compared to mice. However, primate models are compelling
for the rapid acceleration of discoveries on the molecular
basis of disease in humans and also because the human genome
is identifying primate specific biological processes. Consequently,
nonhuman primates provide the only close relative on which
humane and responsible experiments can be performed. The
PDC has achieved expression of randomly inserted transgenes
in nonhuman primates (Chan, A.W.S., et al., 2001) and, while
this is a far cry from the multitude of strategies perfected
in mice, it is a technique that is already important for
some translational studies. For example, in collaborations
with Dr. Steve DeKosky, Dr. Navara is producing a transgenic
nonhuman primate Alzheimer’s model with over-expression
of double Swedish mutations of APP, as has been performed
by studies in mice (Games, D., et al., 1995).
Rodent models provide invaluable tools for studying mammalian
development and human disease, and have the advantage that
they are experimentally tractable and have relatively low
cost. While techniques for producing transgenic and knockout
mice are well established, transgenesis in rats is inefficient
and knockout technologies are not available due to the lack
embryonic stem cells. PDC investigators maintain several
lines of transgenic mice and rats and employ a variety of
established and developing techniques for modifying the
germlines of rodents. These techniques include pronuclear
injection, ES cell chimeras, retrovirus-mediated gene transfer
into unfertilized eggs followed by ICSI, TransgenICSI (plasmid
attached to sperm that is later injected into oocytes) (Chan,
A.W.S., et al., 2000), male germline stem cell-mediated
transgenesis (Nagano et al., 2001; Orwig et al., 2002),
and somatic cell mediated transgenesis by SCNT into oocytes.
Our progress in rodents will accelerate the discovery and
development of transgenic and knockout methods in nonhuman
primates. Each species presents unique challenges and opportunities
for biomedical discovery and the development of mouse, rat,
and nonhuman primate models will facilitate the translation
of basic biological findings from the lab bench to the bedside.
[top]
|
