Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 199-227).

A new paradigm has evolved in which multiple regulatory interactions between the skeletal and hematopoietic systems have been identified. Previous studies have demonstrated that megakaryocytes (MK) play a dual role in skeletal homeostasis by stimulating osteoblast proliferation and simultaneously inhibiting osteoclast (OC) development. Here we identify a novel regulatory pathway in which the main MK growth factor, thrombopoietin (TPO), directly regulates osteoclastogenesis. To study the role of TPO in OC development, spleen or bone marrow (BM) cells (2x10[exponent]6 cells/ml) or BM macrophages (BMM, 1x10[exponent]5 cells/ml) from C57BL/6 mice , as a source of OC precursors, were cultured with M-CSF (30 ng/ml) and RANKL (50 ng/ml) to induce OC formation. TPO (0.1-1000 ng/ml) and/or primary MK (0-0.5%), derived from C57BL/6 fetal livers, were titrated into these cultures and OC were identified as tartrate resistant acid phosphatase positive (TRAP+) giant cells with >3 nuclei. There was a significant, up to 15-fold reduction in OC formed when MK were added to all OC generating cultures, p < 0.001. Moreover, if OC generating cultures did not contain MK or MK progenitors, TPO treatment significantly enhanced OC formation up to six-fold, p < 0.01. This data demonstrates that MK are responsible for the inhibition of OC formation and that in cultures containing MK or MK progenitors such as BM or spleen cells, that TPO acts indirectly to inhibit OC formation by stimulating megakaryopoiesis, whereas in the absence of MK or MK progenitors TPO directly enhances OC formation. This conclusion is further supported by Real-Time PCR data which demonstrates that OC progenitors express c-mpl, the TPO receptor, albeit at low levels when compared to expression of c-mpl on MK. Finally, we have begun to dissect the c-mpl signaling pathway in OC progenitors. We have found that TPO induces tyrosine phosphorylation of several specific cellular proteins in the JAK/STAT pathway. Thus, TPO acts in a somewhat paradoxical manner by inhibiting OC formation through the stimulation of MK, while simultaneously playing a direct role in enhancing osteoclastogenesis.

published_or_final_version / Pathology / Master / Master of Philosophy

The alteration of insulin-like growth factor I (IGF-I) and its binding proteins (IGFBP) and their effects on proliferation and differentiation of murine bone marrow-derived macrophage (BMM) precursors were investigated. Bone marrow cells exposed to 20% L929-fibroblast conditioned medium (LCM) were cultured in serum-free medium for 24 h. Western ligand blotting (WLB) analysis detected four bands in all samples. 41-kDa and 30-kDa bands were detected after 12 h and remained constant during BMM differentiation. The 28-kDa and 25-kDa proteins were almost undetectable until day 2, but accumulated significantly from day 3 to day 7. Immunoblotting analysis verified these two bands as IGFBP-4. Northern blotting analyses detected both IGFBP-4 and IGFBP-3 mRNA in the cells. The 41-kDa protein was postulated to be IGFBP-3 in a glycosylated form. The identity of the 30-kDa band is not known. Northern blotting analysis showed that IGF-I mRNA level increased in a time-dependent manner until day 3, and decreased thereafter during BMM differentiation. The effect of IGF-I and its analogs on cell proliferation was studied by ($ sp3$H) thymidine incorporation. IGF-I and its analogs enhanced cell proliferation of freshly isolated bone marrow cells. Both IGF-I and long R$ sp3$ IGF-I, but not des(1-3)IGF-I, continued to exert a stimulating effect on day 1, although to a lesser extent. The effect of IGF-I and its analogs on BMM differentiation was studied by checking morphology, non-specific esterase-1 (NSE-1) activity, and mannose receptor expression. No significant differences in morphology and NSE-1 activity were observed among the treatment groups. There was no difference of mannose receptor expression on day 4 between the IGF-I group and the control cells, whereas long R${ sp3}$ and des(1-3)IGF-I increased the receptor number by 260% and 228% respectively, with less increased K$ rm sb{d}$ values. (Abstract shortened by UMI.)

Acceleration of the regeneration process is necessary for severe full-thickness skin injuries, such as burns, trauma or chronic ulcers. The aim of this dissertation was to investigate the influence of bone marrow-derived stem cells on skin regeneration using full-thickness wound model in BALB/c mouse. Lin¯ cells were isolated and identified. Regenerative properties of purified cells were evaluated in vitro. Wound healing after Lin¯ cells transplantation was examined histologically and cytokine gene expression was determined by quantitative RT-PCR. The results revealed that Lin¯ population is heterogeneous and contains distinct undifferentiated cell types. In vitro experiments showed that skin tissue extracellular matrix components enhance proliferation and migration of Lin¯ cells. Histologic analysis indicated that inhibition of inflammation, re-epithelization, formation of skin appendages and extracellular collagen, were most effective after cell transplantation with type I collagen. Quantitative analysis revealed that transplanted Lin¯ cells decrease expression of proinflammatory cytokine TNF-α and increase anti-inflammatory IL-10 expression. Also, gene expression of growth factors TGF-β and VEGF, which enhance regeneration, was improved in different healing phases. The results obtained provide additional knowledge for possible therapeutic application of stem/progenitor cells in difficult-to-heal wounds. / Odos žaizdų regeneracijos skatinimas yra aktualus pažeidus didelį paviršiaus plotą bei giliuosius audinio sluoksnius. Šio darbo tikslas buvo ištirti kaulų čiulpų Lin¯ ląstelių įtaką odos regeneracijai in vivo, taikant BALB/c linijos pelių visų odos sluoksnių pažeidimo modelį. Darbo metu buvo išskirtos bei identifikuotos Lin¯ ląstelės. Jų regeneracinės savybės buvo įvertintos in vitro. Buvo atlikta histologinė gyjančio audinio analizė bei nustatyti citokinų genų raiškos pokyčiai. Rezultatai parodė, kad Lin¯ ląstelių populiacija yra heterogeniška, sudaryta iš nediferencijuotų ląstelių. Nustatyta, kad odos tarpląstelinės medžiagos komponentai skatina Lin¯ ląstelių proliferaciją ir migraciją in vitro. Histologinė analizė parodė, kad po ląstelių transplantacijos I tipo kolageno gelyje anksčiausiai prasideda uždegimo slopinimas, greičiausiai baigiamas reepitelizacijos procesas, atsinaujinusioje dermoje susiformuoja tvarkingai išsidėsčiusios pagalbinės odos struktūros bei natyviam audiniui būdingas tarpląstelinės medžiagos kolagenas. Atlikus kiekybinę analizę nustatyta, kad po Lin¯ ląstelių transplantacijos sumažėja uždegiminio citokino TNF-α raiška bei padidėja priešuždegiminio IL-10 raiška. Skirtingose gijimo stadijose padidėja augimo veiksnių, skatinančių audinio regeneraciją, TGF-β ir VEGF genų raiška. Gauti rezultatai papildo žinias, reikalingas vystyti metodus, skirtus sutrikusio gijimo žaizdų regeneracijos skatinimui.

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.

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?

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

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2007. / Title from first page of PDF file (viewed Feb. 18, 2009). Includes bibliographical references.