This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.The goal of this project is to investigate whether stem cells from bone marrow can regenerate liver tissue. We have made good progress against our year 3 5 benchmarks in 2005 as described below. Benchmark 1. Stem Cell Isolation and T-cell Purging Our stem cell transplantation studies are at very high risk for graft versus host disease, GVHD, and graft rejection. To prevent GVHD and/or rejection of the stem cell graft by the female recipients, we have now further modified the engineering of the stem cell grafts by an additional purging step in order to eliminate the contaminating immuno-competent T cells and other antigen presenting cells to engineer a stem cell graft consisting of the most primitive or morphologically indistinguishable stem cell populations or 'blast' cells as this cell population is believed to represent the most rare/pluripotent stem cells capable of multiorgan engraftment.Benchmark 2: Real Time Quantitative PCR The 272 base pair portion of the macaca mulatta sex determing region Y(SRY) gene was cloned into a p CR II-TOPO plasmid. The plasmid insert was confirmed by sequencing and PCR was performed using the primer set that will be used for the RT-PCR. This plasmid will be used to create the RT-PCR standard curve, allowing the determination of the absolute SRY positive cells number (male cells) in a given cell sample(male and female cells mixture). We are going to label the primer and probe with FAM/TAMRA dye and run the samples on Applied Biosystems 7700 System Detector. The data generated from this study has been demonstrated to effectively detect Y chromosome containing cells and has been published.Benchmark 3: Combined FISH for the 'Y' chromosome SRY probe with CD34 and other tissue markers for liver, lung and kidney Our goal is to establish a stem cell transplantation model for liver regeneration and multi-organ engraftment. We have used the 'Y' chromosome as a marker for 'stem cell tracking' and determining the engraftment of 'Y+' stem cells in various tissues and organs of female recipients. We are using combined fluorescence immuno-hybridization, FISH. Last year we were only successful in detecting the stem cell factor receptor, CD117(ckit), in liver only. Over the past year, we have confirmed and expanded on last years studies in liver sections by optimized the reaction conditions and further testing different clones of CD34 antibodies in combination with, CD117 and CK8/18 on other rhesus monkey paraffin embedded tissues including lungs, kidney, and gut. Benchmark 4: Pilot Study: large scale GCSF and SCF primed bone marrow harvests, PKH-fluorescence labeling, and cryopreservation of PKH labeled mononuclear cells for autologous transplantation. Due to the high risk of mortality and morbidity associated with unrelated mismatched allogeneic stem cell transplantation originally proposed, the Institutional Animal Care Users Committee (IACUC) at the TNPRC, has recommended and approved a 'Pilot' Study utilizing autologous stem cells in combination with all of the entire transplant protocols including radiation, immunosuppressive regimens, etc and partial hepatectomy proposed in the original main study utilizing unrelated allogeneic stem cells. The results from the Pilot study will allow us to safely manage immunocompromised monkeys that will be transplanted with mismatched allogeneic stem cells. To this end, this past year, we have performed individual autologous stem cell harvests primed with GCSF and SCF. The stem cells have been labeled and cryopreserved and ready for transplant.
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