Background. Recently rodent and porcine bone marrow stromal cells (MSCs) have been reported to be uniquely immune tolerant. In order to confirm these findings in human cells, we tested the hypothesis that human MSCs are also immune tolerant, such that they can be useful as "universal donor cells" for myocardial regenerative therapy. / Methods. Immunocompetent female rats underwent left coronary ligations (n=90). They were randomized into 3 groups. In Group I, lac-Z labeled male human MSCs were implanted into the peri-infarcted area. In Group II and III isogenic rat MSCs or culture medium were injected respectively. Echocardiography was carried out to assess cardiac function, and the specimens were examined serially for up to 8 weeks with immunohistochemistry, FISH and PCR to examine MSCs survival and differentiation. / Results. Human MSCs were found to survive within the rat myocardium without immunosuppression. This was confirmed by PCR and FISH test. No cellular infiltration characteristic of immune rejection was noted. Some of these cells appeared to express cardiomyocyte-specific markers such as troponin-Ic and connexin-43. Furthermore, the implanted MSCs significantly contributed to the improvement in ventricular function and attenuated LV remodeling. / Conclusions. Human MSC survived within this xenogeneic environment, and contributed to the improvement in cardiac function. Our findings support the feasibility of using these cells as "universal donor cells" for xeno- or allo-geneic cell therapy, as they can be tested, prepared and stored well in advance for urgent use. Allogeneic MSCs from healthy donors may be particularly useful for severely ill or elderly patients whose own MSCs could be dysfunctional. / Plusieurs études ont récemment démontré la tolérance immunologiquedes cellules souches stromales (CSS) issues de rongeurs et de porcinés. Pour confirmer cesrésultats chez les cellules humaines, l'étude actuelle évalue l'effet des CSS humaines sur larégénération du myocarde chez des rats immunocompétents et étudie la possibilité d'utiliserces CSS comme « donatrices universelles» à la suite d'un infarctus.

CBL proteins are E3 ubiquitin ligases and adaptor proteins. The mammalian homologs – CBL, CBL-B and CBL-3 show broad tissue expression; accordingly, the CBL proteins play roles in multiple cell types. We have investigated the function of the CBL-B protein in hematopoietic cells and fibroblasts. The causative agent of chronic myeloid leukemia (CML) is BCR-ABL. This oncogenic fusion down-modulates CBL-B protein levels, suggesting that CBL-B regulates either the development or progression of CML. To assess the involvement of CBL-B in CML, bone marrow transduction and transplantation (BMT) studies were performed. Recipients of BCR-ABL-infected CBL-B(-/-) cells succumbed to a CML-like myeloproliferative disease with a longer latency than the wild-type recipients. Peripheral blood white blood cell numbers were reduced, as were splenic weights. Yet despite the reduced leukemic burden, granulocyte numbers were amplified throughout the animals. As well, CBLB(-/-) bone marrow (BM) cells possessed defective BM homing capabilities. From these results we concluded that CBL-B negatively regulates granulopoiesis and that prolonged latency in our CBL-B(-/-) BMT animals was a function of perturbed homing.To develop an in vitro model to study CBL-B function we established mouse embryonic fibroblasts (MEFs) deficient in CBL-B expression. Transduction of the wild-type and CBL-B-deficient MEFs with BCR-ABL did not confer transformation; nevertheless, the role of CBL-B in fibroblasts was evaluated. The CBL-B(-/-) MEFs showed enhanced chemotactic migration toward serum in both Transwell migration and time-lapse video microscopy studies. The biochemical response to serum was extensively evaluated leading to the development of a model. We predict that CBL-B deficiency either: (a) augments GRB2-associated binding protein 2 (GAB2) phosphorylation leading to enhanced extracellular signal-regulated kinase (ERK) and protein kinase B (PKB / Akt) signaling, or (b) alleviates negative control of Vav3 resulting in stimulation of Rho effectors. In either case, our results reveal a negative regulatory role for CBL-B in fibroblast migration. The two studies detailed herein expand our knowledge of CBL-B function. They strongly suggest that CBL-B can modulate granulocyte proliferation and point toward a role for CBL-B in the motility of numerous cell types.

Objective: Injuries to the marrow cavity result in rapid bone formation followed by regeneration of the marrow. It is not known whether this process is affected by age, although the quality of marrow is markedly different in young and old animals. To test if marrow restoration differs with age, we used the rat tibial bone marrow ablation model, which has been used to examine calcification, osteointegration of metal implants, and remodeling of bone graft substitutes. Methods: Marrow was ablated in the left tibia of seven rats (rNu/rNu) per time point. At 0,7,14,21,28,35 and 42 days post-surgery, treated tibias and contralateral tibias were harvested and fixed in buffered formalin. Both tibias were scanned using microCT and trabecular and cortical BVF/TV calculated. Mid-sagittal sections of decalcified bones were stained with H&E and BVF/TV calculated. Results: MicroCT analysis of 1-month animals showed increased bone formation on day-7 and on day-21 the marrow was restored. Increased bone was seen in 3-month animals on day-7 and day-14, but it was significantly less than in 1-month rats. By day-21, trabecular bone was reduced by 50%. 10-month animals had less trabecular bone at day-7 and 14, but bone remained in the medullary canal through day-1. Histomorphometry indicated that bone formation peaked at day-7 in 1-month rats with remodeling underway by day-14. Bone formation in 3-month rats also peaked at day-7, but restoration occurred by day-21. However, in 10-month rats, peak bone occurred on day-14, with remodeling on day-28. Conclusions: Aged animals produced less primary bone than younger animals and remodeling was initiated later. Differences in micro-CT and histomorphometric analyses may reflect a reduction in calcification of the osteoid in the 10-month old animals. (Supported by Boston Scientific, Inc.)

Thesis (M.A.)--University of Hawaii at Manoa, 2004. / Includes bibliographical references (leaves 97-107). / ix, 107 leaves, bound ill. 29 cm

Bone marrow-derived mesenchymal stem cells (MSC) have been shown to provide neuroprotection after transplantation into the injured nervous system. The present thesis investigates whether adult human and rat MSC differentiated along a Schwann cell lineage could increase their expression of neurotrophic factors and promote regeneration after transplantation into the injured peripheral nerve and spinal cord. Human and rat mesenchymal stem cells (hMSC and rMSC) expressed characteristic stem cell surface markers, mRNA transcripts for different neurotrophic factors and demonstrated multi-lineage differentiation potential. Following treatment with a cocktail of growth factors, the hMSC and rMSC expressed typical Schwann cells markers at both the transcriptional and translational level and significantly increased production of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). Age and time in culture are of relevance for clinical settings and growth-promoting effects of hMSC from young donors (16-18 years) and old donors (67-75 years) were compared. Undifferentiated hMSC from both young and old donors increased total neurite length of cultured dorsal root ganglion (DRG) neurons. Differentiation of hMSC from the young donors, but not the eldery donors, further enhanced the neurite outgrowth. Undifferentiated hMSC were cultured for eleven weeks in order to examine the effect of in vitro expansion time on neurite outgrowth. hMSC from the young donors maintained their proliferation rate and their ability to enhance neurite outgrowth from DRG neurons. Using a sciatic nerve injury model, a 10mm gap was bridged with either an empty tubular fibrin glue conduit, or conduits containing hMSC, with and without cyclosporine treatment. Cells were labeled with PKH26 prior to transplantation. At 3 weeks after injury the conduits with cells and immunosuppression increased regeneration compared with an empty conduit. PKH26 labeled human cells survived in the rat model and the inflammatory reaction could be suppressed by cyclosporine. After cervical C4 hemisection, BrdU/GFP-labeled rMSC were injected into the lateral funiculus rostral and caudal to the spinal cord lesion site. Spinal cords were analyzed 2-8 weeks after transplantation. Transplanted MSC remained at the injection sites and in the trauma zone for several weeks and were often associated with numerous neurofilament-positive axons. Transplanted rMSC induced up-regulation of vascular endothelial growth factor in spinal cord tissue rostral to the injury site, but did not affect expression of brain-derived neurotrophic factor. Although rMSC provided neuroprotection for rubrospinal neurons and significantly attenuated astroglial and microglial reaction, cell transplantation caused aberrant sprouting of calcitonin gene-related peptide immunostained sensory axons in the dorsal horn. In summary these results demonstrate that both rat and human MSC can be differentiated towards the glial cell lineage, and show functional characteristics similar to Schwann cells. hMSC from the young donors represent a more favorable source for neurotransplantation since they maintain proliferation rate and preserve their growth-promoting effects in long-term cultures. The data also suggest that differentiated MSC increase expression of neurotrophic factors and support regeneration after peripheral nerve and spinal cord injury.

It is a well established phenomenon that the epithelial lining of the urinary bladder (urothelium) when implanted into skeletal muscle induces ectopic ossification. However, despite numerous observations, this reaction is poorly understood. This research further studied this reaction by - (a) demonstrating the reaction in a suitable small animal model; (b) attempting to induce the reaction by implanting urothelial cells purified by cell culture techniques; and (c) comparing the bone forming reaction induced by implanted urothelium to the reaction induced by implanting Bone Marrow Stem Cells (BMSC's) and Osteophyte Stem Cells (OSC's). By demonstrating newly formed bone after the implantation of guinea pig urothelium into the skeletal muscle of a Severe Combined Immuno-Deficient Mouse (SCID-Mouse) this research demonstrated that a suitable small animal model had been established. This is despite inherent difficulties (particularly bacterial contamination) associated with establishing a primary cell culture of guinea pig urothelial cells. Additionally, the intramuscular ectopic osteoinductive potential of human BMSC's (hBMSC's) in the SCID-mouse has also been demonstrated. Confirming that the injection of cultured cells in suspension is an adequate intramuscular delivery technique, this research demonstrates that hBMSC's induce ectopic ossification by non-immunological means. This research has demonstrated a number of differences between urothelium induced ectopic ossification and ectopic ossification induced by BMSC's, suggesting they are two separate processes. This is important because the chemotaxis and subsequent osteogenic differentiation of BMSC's has previously been one of the more popular postulated mechanisms of urothelium induced ectopic ossification. Finally, this research has demonstrated the ectopic osteoinductive potential of stem cells isolated from the marrow of human osteophytes (human Osteophyte Stem Cells, hOSC's). This observation has not been previously reported, and will hopefully provide a valuable contribution to a body of knowledge that has important ramifications in both the treatment of osteoarthritis, and the use of BMSC's in tissue engineering.

Low back pain is a common cause of disability and work inability, often associated with intervertebral disc degeneration. The current understanding of disc degeneration is limited and none of the available treatments is entirely effective. The work described herein investigates potential strategies for the biological herapeutic restoration of disc degeneration. Firstly, an in vitro study to investigate the effects of BMP-7 on human discal cellular viability was performed. Cultured cells were treated with TNF-a or H202 to induce apoptosis, resulting in the down regulation of extracellular matrix proteins, decreased cell viability, morphological changes and activation of caspase-3; however, addition of BMP-7 alone prevented the observed effects, demonstrating the ability of BMP-7 to prevent apoptosis of human disc cells in vitro. Secondly, the differentiation potential of stem cells towards disc-like cells was studied. Rodent mesenchymal stem cells (rMSCs) were cultured alone or co-cultured with rat disc tissue. Differentiation potential of rMSCs was evaluated by mRNA and protein expression, cellular function and morphological studies. The co-culture conditions led to the expression of chondrocytic markers in rMSCs, whereas rMSCs cultured alone did not express the chondrocytic markers. Cellular contact between the co-cultured rMSCs and the discal tissue were observed. This study demonstrated that rMSCs can differentiate into functional disc-like cells in a tissue influenced co-culture environment. Finally, the survival and differentiation of CD34+ or CD34?? bone marrow (hBM) cells, in an intra-discal xenogeneic transplantation rat model was investigated. Human CD34+ or CD34?? cells were isolated, fluorescent-labelled and injected into rat coccygeal discs. The survival of transplanted cells was confirmed by fluorescent positive cells as well as a human nuclear specific marker. Interestingly, CD34?? cells survived until day 42 in the injected discs, and differentiated into cells ex:pressing a chondrocytic phenotype. In contrast, CD34+ cells could not be detected by day 21. This data suggests that transplanted hBM CD34?? cells, in contrast to CD34+ cells, were able to survive and differentiate within the intervertebral disc. Together, the results of these studies can both encourage and contribute to the basis of potential biological therapies in the restoration of intervertebral disc degeneration.

Bibliography: leaves 271-325. / xxxiii, 325, [249] leaves, [23] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The date presented in this thesis is directed toward the molecular characterisation of cell surface molecules (CSMs) that mediate interactions between human haemopoietic progenitor cells (HPC) and cells of the bone marrow (BM) stroma. The research focuses on the role of selectins in the regulation of haemopoiesis, the identification and molecular characterisation of novel structures expressed at the surface of primitive human HPC and cultured BM stromal cells, the molecular characterisation of the antigen identified by the mAb HCC-1 which delineates a subset of the CD34+ cell population, and the molecular cloning of a novel mucin-like transmembrane glycoprotein termed MGC -24v. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1997?

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 132-142.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.