In this revised competing application, the Principal Investigator seeks to define the relationship of cell cycle status in hematopoietic stem cells and their ability to engraft during transplantation.
Four Specific Aims are enumerated. In the first specific aim, the Principal Investigator plans to identify the cytokines which induce an engraftment defect. During the previous tenure of this grant, Dr. Quesenberry demonstrated that hematopoietic stem cells cultured in the presence of a cocktail of cytokines including IL-1, IL-3, IL6 and stem cell factor display a substantially reduced capacity to engraft in non-marrow ablated recipients. Having developed a non- myelosuppressive model of marrow transplantation in which large numbers of male donor cells are infused for several days into female recipients, the Applicant was successful in demonstrating a very high engraftment efficiency in such animals. However, when marrow cells were first cultured in the presence of a number of cytokines which act to expand putative stem and progenitor cell numbers, the ability of these cells to contribute to long-term hematopoiesis was essentially eliminated. As this non-conditioning model of stem cell transplantation would find great use in a setting of gene therapy, the Applicant proposes to determine the cytokine or cytokines responsible for the induced effect. Initial studies will be directed towards defining the amount of time cells need to be exposed to these proliferative cytokines in order to induce the engraftment defect, and refinement in the methodology will also be addressed by studying two additional systems which potentially will eliminate any barrier to transplantation (male into female might engender an immunologic intolerance). The Investigators will study Ly 5.1 into Ly 5.2 congenic transplants, and a ROSA 26 transplant model in which beta galactosidase can be tracked. These refinements should optimize the model for the subsequent specific aims, which attempt to elucidate the relationship of cell cycle status to engraftment and any experimental manipulation which might impact favorably upon this defect. In the second Specific Aim, the Investigator plans to generate both whole marrow cells and rhodaminelo/Hoechstlo fractions and using either growth factor stimulation or isoleucine deprivation attempt to develop a synchronized population of cells to test whether the transplantation defect is restricted to a specific point in the cell cycle. The Investigator will monitor cell cycle using FACS analysis and will then perform transplantations with cells throughout the cell cycle. It is hoped but not fully expected that cells that transit an entire cell cycle might provide the most useful information. In the third Specific Aim, the Investigator plans to identify a cocktail of cytokines which are capable of maintaining primitive stem cell engraftment potential. If the Investigator's hypothesis is correct, these should be cytokines which maintain viability but fail to induce cell cycle progression. To the basic cytokine cocktail of IL-11, IL-3, IL-6 and """"""""steel"""""""" factor, modulation using different concentrations of cytokines (based on the finding that low levels of cytokines can support survival, whereas high levels support proliferation), additional cytokines which appear to act in relatively primitive cells including LIF, TGF beta, IL-1 alpha, and MIP-1 alpha, and different adherent stromal layers including Dexter cultures and the cell line TCI will also be tested for effects on cell maintenance or expansion without an associated engraftment defect. Finally, in the fourth Specific Aim, the goal will be to develop in vitro techniques to reverse cytokine-induced engraftment defects. If successful, the Investigator hopes that such manipulation will allow marrow cells to be transiently stimulated to allow retroviral integration, followed by their manipulated return to quiescence, thereby repairing the engraftment defect and thus allowing for engraftment of the cells in which retroviral gene therapy had occurred. Once again, combinations of cytokines will be assessed by following their effects on short-term clonogenic cells populations (HPP- CFC and LPP-CFC) and most importantly, on the ability to engraft in the long-term setting.
| Colvin, Gerald A; Berz, David; Liu, Liansheng et al. (2010) Heterogeneity of non-cycling and cycling synchronized murine hematopoietic stem/progenitor cells. J Cell Physiol 222:57-65 |
| Aliotta, Jason M; Keaney, Patrick; Passero, Michael et al. (2006) Bone marrow production of lung cells: the impact of G-CSF, cardiotoxin, graded doses of irradiation, and subpopulation phenotype. Exp Hematol 34:230-41 |
| Quesenberry, Peter J; Colvin, Gerald; Abedi, Mehrdad (2005) Perspective: fundamental and clinical concepts on stem cell homing and engraftment: a journey to niches and beyond. Exp Hematol 33:9-19 |
| Quesenberry, Peter J; Colvin, Gerald A; Abedi, Mehrdad et al. (2005) The stem cell continuum. Ann N Y Acad Sci 1044:228-35 |
| Nowakowski, Grzegorz S; Dooner, Mark S; Valinski, Helen M et al. (2004) A specific heptapeptide from a phage display peptide library homes to bone marrow and binds to primitive hematopoietic stem cells. Stem Cells 22:1030-8 |
| Dooner, Mark; Cerny, Jan; Colvin, Gerald et al. (2004) Homing and conversion of murine hematopoietic stem cells to lung. Blood Cells Mol Dis 32:47-51 |
| Quesenberry, Peter J; Abedi, Mehrdad; Aliotta, Jason et al. (2004) Stem cell plasticity: an overview. Blood Cells Mol Dis 32:1-4 |
| Cerny, Jan; Quesenberry, Peter J (2004) Chromatin remodeling and stem cell theory of relativity. J Cell Physiol 201:1-16 |
| Lum, Lawrence G; Fok, Hubert; Sievers, Richard et al. (2004) Targeting of Lin-Sca+ hematopoietic stem cells with bispecific antibodies to injured myocardium. Blood Cells Mol Dis 32:82-7 |
| D'Hondt, Lionel; McAuliffe, Christina; Damon, Jeffrey et al. (2004) Circadian variations of bone marrow engraftability. J Cell Physiol 200:63-70 |
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