published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

Hematopoietic stem cells (HSCs) transplantations are widely used for the treatment of hematological and non-hematological disorders in clinic. Successful transplantation requires sufficient number and efficient homing of HSCs. Many studies have focused on developing an effective strategy to expand functional HSC population. Some regulatory molecules have been recently shown great promise for controlling the amplification of HSCs. In these dissertation studies, I first aim to identify gene(s) and their allelic variants contributing to strain-specific difference in HSC numbers between C57BL/6 (B6, low) and DBA/2 (D2, high) mice by using a classic forward genetic approach. Firstly, 3 quantitative trait loci (QTL) on chromosome (Chr) 3,5 and 18 were mapped by linkage analyses and confirmed in congenic mice. Secondly, Chr.3 QTL affected several HSC number-related biological processes. The D2 allele increased cycling and self-renewal whereas it decreased apoptotic rates of HSCs. Both actions conspired to increase HSC population size. Lastly, a small number of differentially-expressed genes was identified in Chr.3 congenic HSCs by a microarray-based candidate gene method, and the differential expression of one candidate, latexin, was found to relate to HSC number variations. Our studies report the strong evidence for the potential functions of latexin in HSC number regulation, and they are important for understanding molecular mechanisms of stem cell regulation and developing effective stem cell expansion strategies for clinical applications. In the second part of my studies, I studied homing and engraftment capabilities of HSCs. By using functional assays for progenitor and stem cells, I first reported the absolute homing efficiencies of murine young or old donor cells into young or old recipient mice. The results indicated that homing of primitive hematopoietic cells was not efficient and significantly decreased by aging of donors and recipients. The proliferation and differentiation states of HSCs were also impaired by homing itself, as well as by donors' and recipients age. Moreover, the hematopoietic reconstitution dynamics following transplantation were also affected by aging. Together, these findings will provide useful information for clinical applications especially when older individuals increasing serve as stem cell donors for elderly patients.

It is well documented that both quantitative and qualitative changes in the murine hematopoietic stem cell (HSC) population occur with age. In mice, the effect of aging on stem cells is highly strain-specific, thus suggesting genetic regulation plays a role in HSC aging. In C57BL/6 (B6) mice, the HSC population steadily increases with age, whereas in DBA/2 (D2) mice, this population declines. Our lab has previously mapped a quantitative trait locus (QTL) to murine chromosome 2 that is associated with the variation in frequency of HSCs between aged B6 and D2 mice. In these dissertation studies, I first aim to characterize the congenic mouse model which was generated by introgressing D2 alleles in the QTL onto a B6 background. Using a surrogate assay to mimic aging, I analyzed the cell cycle, apoptotic and self-renewal capabilities of congenic and B6 HSCs and show that D2 alleles in the QTL affect the apoptotic and selfrenewal capabilities of HSCs. In the second aim of these studies, I used oligonucleotide arrays to compare the differential expression of B6 and congenic cells using a population enriched for primitive stem and progenitor cells. Extensive analysis of the expression arrays pointed to two strong candidates, the genes encoding Retinoblastoma like protein 1 (p107) and Sorting nexin 5 (Snx5). B6 alleles were associated with increased p107 and Snx5 expression in old HSCs therefore both genes were hypothesized to be positive regulators of stem cell number in aged mice. Finally, in the third aim of these studies, I show that the individual overexpression of p107 and Snx5 in congeic HSCs increases day35 cobblestone area forming cell (CAFC) numbers, therefore confirming their roles as positive regulators of HSC number in vitro. These studies uncover novel roles for p107 and Snx5 in the regulation of HSC numbers and provide additional clues in the complex regulation of HSC aging.

The toll skeletal muscle injury, resulting in significant muscle mass loss, has on the patient reaches far more than physical and emotional, as the tolls are financial as well. Approximately more than 3 billion dollars is spent on the initial medical costs and on subsequent disability benefits, following a volumetric muscle loss. Skeletal muscle has a robust capacity for self-repair; this propensity for repair is hindered when skeletal muscle loss is larger than 20% of the total mass of the muscle. Previous work in our lab, has shown functional and morphological improvements following the cellular therapy, with mesenchymal stem cells (MSC), as well as with nerve relocation to the extracellular matrix (ECM). To further observe the regenerative properties of the above treatments, a defect weighing approximately 307 ± 3.7 mg wet weight and measuring approximately 1x 1cm² was removed from the lateral gastrocnemius (LGAS) of male Sprague Dawley rats. Additionally, the medial branch of the plantar nerve was then relocated and implanted to the middle of the ECM. Seven days post injury bone-marrow derived mesenchymal stem cells were injected directly into the implant using a PEGylated Fibrin hydrogel (PEG). Following 56 days of recovery, partial functional restoration was observed in the LGAS ECM seeded with MSC and implanted with the plantar nerve. The LGAS produced 86.3 ± 5.8% of the contralateral LGAS, a value that was significantly higher than ECM implantation alone (p <.05). The implanted ECM seeded with MSC and implanted with the plantar nerve showed significant increases in blood vessel density and myofiber content (p <.05). The data suggest that a volumetric injury can be repaired by neurotization of an implanted muscle-derived ECM seeded with MSCs. / text