STEM CELL PRODUCTION CORE

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Mesenchymal stem cells or marrow stromal cells (MSCs) are a subset of adult stem cells from bone marrow. These cells are of medical and therapeutic interest because they have been shown to differentiate into osteoblasts, adipocytes, chondrocytes, myocytes, astrocytes, oligodendrocytes and neurons. Due to their inherent plasticity, these cells have the potential to be useful for the treatment of a large number of genetic diseases. Dr. Bunnell has successfully defined the requirements for the expansion and characterization of rhesus macaque MSCs. The Stem Cell Production Core Facility (SCPC) focuses on generation, maintenance and distribution of nonhuman primate MSCs. We routinely prepare MSCs from rhesus macaque bone marrow and adipose tissue samples. We presently have a bank pf MSC cell lines generated from over 100 rhesus macaques of varying age prepared and ready for distribution. The SCPC presently has a large impact not only on the Gene Therapy Program, but on other divisions such as Comparative Pathology within the TNPRC, but also Departments and Centers within the Tulane Health Sciences Center and the Pennington Biomedical Research Center and other research labs nationally. Produce rhesus macaque stem cell preparations for collaborative research projects. For this we plan to use the techniques developed over the last funding period to isolate stem cells (MSCs, NSPCs and ESCs) to expand the bank of various stem cell types isolated from animals of various ages (fetal, newborn and adults) and sexes of animals. The cells will be used to generate large cell banks for use internally or externally by collaborating scientists. Additionally, we will work with scientists interested in performing autologous transplantation studies by generating MSC banks from specific animals for their research programs. Continue the characterization of nonhuman primate stem cells. Our goal in these studies will be to delineate the cellular and molecular events that take place during maturation and/or differentiation of the various stem cell populations. Scientists in the division will continue to compare the biologic properties of the various stem cells in vitro. Assays mediating in vitro differentiation specific to rhesus stem cells that will maximize the differentiation and proliferation of MSCs and their progeny will be generated. One focus of the in vitro characterization will be the differentiation along neural lineages for all of the stem cells described. In order to accomplish the treatment of Krabbe's disease, stem cells will need to undergo differentiation into oligodendrocytes. A significant effort will be made towards the development and application of differentiation protocols to accomplish this goal. Fully develop assays for the detection of the engraftment of stem cells. Most importantly, the division will play a significant role in the generation of assays for the assessment of engraftment of nonhuman primate stem cells in vivo in both murine and nonhuman primate models. Over the last funding period, the division established technologies for the routine detection of NHP cells in murine models using antibodies to human nuclear antigen, which readily permits the detection of NHP cells. The division has in place staining for BrdU and direct immunofluorescence assessment of EGFP. Over the next funding period, the division will need to focus on the detection of NHP cells in NHP tissues, using technologies such as in situ hybridization for the Y chromosome or detection of cells using single nucleotide polymorphisms (SNPs). This will provide us an opportunity to collaborate with the Genetics and Genome Banking core in the Division of Veterinary Medicine and the DNA Microarray and Expression Core in Bacteriology and Parasitology on the application of SNPs for stem cells detection, as these cores will be developing SNP technologies for the testing of parental lineages. Combining stem cell and gene transfer technologies for use in the rhesus Krabbe's disease model. Our goal is to characterize the requirements for effective gene delivery and expression in stem cells and their differentiated progeny both in vitro and in vivo. The gene transfer efficiency of our various vector systems for nonhuman primate stem cells will be characterized. This will permit us the opportunity to generate banks of stem cells labeled with reporter gene elements, EGFP or beta-galactosidase. More importantly, we will assess the requirements that provide for high levels and protracted duration of gene expression in rhesus MSCs both in vitro and in vivo following transplantation.