Support from the P30 application will allow us both to expand our resources and to provide services to the Hematopoiesis research community at reduced cost. Xenotransplantafion studies have been the gold standard for studies of human hematopoietic cells and hematopoietic stem cells for the last twenty years. It is widely acknowledged that many features of blood cells including terminal differentiation and stem cell self renewal can not be fully studies or understood with in vitro methodologies alone Furthermore, studies of normal mouse hematopoiesis do not fully reflect human biology. Thus there is a need for continued and expanding work in xenotransplantation models. Certainly that is one reason why this entire P30 is based entirely on human hematopoietic studies and not focused on the analysis of other species. Studies of diseases of human hematopoiesis are even less well reflected in murine models, and there is a strong demand to expand the use of xenotransplantafion models to study diseases of human hematopoiesis. For such studies, this P30 will weigh heavily on the services of Core D to carry out in vivo studies to understand better the molecular basis of the bone marrow failure (BMF) and myelodysplastic syndrome (MDS) disorders, Cornelia deLange Syndrome (associated with platelet dysfunction), Pearson Disease (associated with erythroid defects) and others. Treatment strategies to improve these outcomes need also be tested in vivo to allow for pharmocokinetic variables and these xenotransplantation services would meet this criteria as well. Finally, the development of cellular therapeutics from hematopoietic stem cells will also need to be tested in vivo. For example. Dr. Poncz will develop techniques for making platelets from in vitro generated megakaryocytes and our services would allow testing of the ability to make platelets from such cells and to offer models to test the biology of the resulting platelets. Drs. Poncz, French and Weiss are also studying the generation of platelets ectopically expressing proteins of value in the care of bleeding, thrombotic and angiogenic disorders and we will provide services to generate models for proof-of-concept using human hematopoietic cells as the beginning material. Xenotransplantation models have become increasingly sophisticated and technically challenging over the last 20 years. Initial work by John Dick's laboratory showed that human hematopoietic stem and progenitor cells would engraft in irradiated severe combined immunodeficiency (SCID) mice but engraftment was inefficient(2,3). Over the ensuing period of time, successive models have been developed with progressively more severe immunodeficiency. Recently, NOD-SCID mice have been crossed to either mice with a mice deficient in RAG recombinase or in IL-2 receptor gamma common chain (IL-2RYC), which is required for the signal transduction of receptors for IL2, IL7, IL15 and other cytokines. The latter mice developed by L. Shultz at the Jackson Laboratory and designated as N0D/SC1D/IL2RYC null (NSG), are the model that we have utilized in our Core facility and forms the basis for most of the proposed work(4). NSG mice have severe deficiencies in T, B and natural killer (NK) cell activity. They engraft human hematopoietic tissue with high efficiency. Unlike earlier strains of mice, they do not develop malignancies that shorten life span, and we have kept animals in our facility engrafted with human hematopoietic tissue for up to 18 months. The downside of this strain is that, because of the immunodeficiency, the animals have low reproductive numbers and require highly sterile facilities for their use. This technical challenge has been met at UPENN through formation of this Xenotransplantation Core facility. As described in more detail below, the animals are kept in HEPA filtered cages within a sterile barrier facility. An irradiator and whole animal imaging machine are housed within the barrier facility. Access to the facility is limited to one lab member per research group as we have shown that increased numbers of researchers or entries into and out of the animal rooms decreases animal health, breeding and engraftment. With these facilities, we have now established the largest breeding colony of NSG mice in the country outside of Jackson Laboratories. We produce 50-60 mice per week and have capacity to at least triple this number if needed by researchers within the P30 or other UPENN investigators. These animals are used for diverse xenotransplantation studies including normal human blood and hematopoietic stem cells as well as human skin, tumor tissues (leukemia, lung, ovarian), and islet cell transplants. To our knowledge, this facility is unique in the country in the size and diversity of xenotransplantation studies, which we manage. These several reasons provide a technical justification for this Core resource. Although the xenotransplantation model is widely accepted as a critical element for studies of normal human hematopoietic cells, many laboratories do not utilize the model. Because of the need for a highly sterile facility, experienced animal care technicians, reduced breeding efficacy and other challenges, maintenance of a high quality xenotransplantation facility is costly and time consuming. For these reasons, it is not cost effective for occasional laboratory users to develop the model. Rather, it is ideally provided by a dedicated Core facility such as we have. By maintaining this model as a Core facility, we are able to provide a resource to investigators on the P30, which would otherwise not be utilized in their studies. The need to develop specialized housing, protocols for establishing engraftment, monitoring engraftment and developing specialized skills for particular uses will be centralized, and the experience gathered will be useful to all of our users. Additionally, there is currently a 3-month delay in receiving new orders of NSG mice from Jackson Laboratories. We can usually provide mice within one to two weeks for small experiments and within one month for larger experiments. Thus, we provide a service, which is available but has limited access commercially. Additionally, we charge $75 per mouse whereas the commercial rate is $110 per mouse. Thus, we provide service at decreased cost compared to commercial sources. In summary, we have an established and experienced Core in xenotransplantation that will allow for efficiency of use, cost savings and sharing of experiential information for establishing and maximizing the expression of multiple lineages that will encourage utilization of all the Investigators on this P30 who have indicated a desire or need to use this Core D as indicated in Table 2 in the APPENDIX. We believe that our Core will enhance synergistic interactions across the UPENN/CHOP campus and will solidify the growth of benign hematopoiesis on campus.
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