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ANDiThrough 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.

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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.

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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.

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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.

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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.

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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.

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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.

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Last Modified: 28-Dec-2012
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