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The bones of the body vary in size and shape, but are fundamentally all composed of the same cell types: osteoblasts, osteoclasts, osteocytes, vascular cells, and sometimes marrow cells. Long bones are formed when mesenchymal stem cells (MSCs) give rise to chondrocytes i.e. cartilage cells, and osteoblasts i.e. bone cells. These develop to form layers of bone encasing a cartilagenous core which eventually becomes the marrow cavity. A recent study showed that deleting the Wilms’ tumour gene, Wt1, in adult mice causes a dramatic loss of bone and fat tissue, fat being another derivative of MSCs. This finding led me to ask whether Wt1 expression is involved in bone biology and whether it plays a functional role in the stem or progenitor populations. Wt1 is a transcription factor that acts as a mesodermal / mesenchymal regulator. It acts as a tumour suppressor gene with mutations leading to the eponymous paediatric kidney tumour. However, in adult cancers it has oncogene characteristics, being highly expressed in the tumours of tissues in which it is not normally present. It also plays a pivotal role in the epithelial to mesenchymal transition (EMT) and vice versa in developing heart and kidney, respectively. There is, however, no evidence of its involvement with EMT / MET in adults. Wt1 is expressed in various developing tissues and is particularly vital for kidney development. Due to its involvement as a regulator of EMT / MET during development and the phenotype observed following its deletion in vivo, we hypothesised that Wt1 is expressed in, and required for the function of mesenchymal stem or progenitor cells populations within the bone marrow. A Wt1-GFP knock in mouse was used to show that Wt1 expressing cells are found in the bone marrow, and also for the first time in the bone. The GFP population overlaps with a non-haematopoietic MSC population defined by 3 cell surface markers in the bone and marrow, as well as an osteoblast (OB) progenitor population. Using a tamoxifen inducible CreERT2 showed that Wt1 loss alters the proportion of GFP cells in the bone and marrow cells that overlap with these MSC and OB progenitor markers, but microarrays were needed to assess the functional effects of Wt1 deletion. Microarrays highlighted various pathways that were altered following the in vitro deletion of Wt1 in total bone and marrow culture, as well as the non-haematopoeitic GFP+ and GFP- populations. In bone cells, deleting Wt1 negatively affects various pathways related to MSCs and their derivatives, including collagen biosynthesis, cartilage development and muscle tissue development. Also negatively affected were Wnt signalling regulation and EMT regulation; this is the first time Wt1 has been shown to be involved in EMT in adult cells. These findings were validated using qRT-PCR to show the down regulation of various genes involved in each pathway, showing that as well as being expressed in these populations it is also playing a functional role. Ossification pathways were negatively altered in the cells not expressing Wt1 following the deletion of the gene suggesting that Wt1 may also be acting in a paracrine manner to play its role in bone homeostasis. As well as in adult tissues, Wt1 was found to be expressed during development in the limb tissue of e11.5 to e16.5 mice. Preliminary results show that Wt1 may also have a functional role during bone development, as loss of expression causes a reduction in the percentage of non-haematopoetic MSC cells in the e18.5 hindlimb. As well as this, preliminary lineage tracing experiments suggest that cells found at the bone surface are of Wt1+ origin. This thesis has also highlighted the importance of experimental conditions and controls, particularly for CFU-F assays. CreERT2, loxP sites, tamoxifen, oxygen tension levels, and gender all exert specific effects on colony formation, independent of Wt1 expression. In conclusion, these data identify Wt1 as a key player in bone development and homeostasis. The microarray results led to the conclusion that Wt1 has a functional role in several mesenchymal pathways and highlights various genes that are potential Wt1 targets and should be further investigated using ChIP-Seq methods.

The purpose of this study was to develop a fibrin gel system capable of serving as a three dimensional scaffold for the chondrogenesis of rabbit bone marrow mesenchymal stem cells (BM-MSCs) and to examine the effect of two fibrinolytic inhibitors, aprotinin and aminohexanoic acid, on this system. Rabbit BM-MSCs were obtained from the tibias and femurs of New Zealand white rabbits. After chondrogenic potential of BM-MSCs was verified by pellet culture, 2 x 106 cells were pelleted and suspended in fibrinogen (80mg/ml) and then mixed with equal parts of thrombin (5 IU/ml). The specimen were then divided into four groups: aprotinin control (with aprotinin); aprotinin + transforming growth factor (TGF-beta) (with aprotinin and TGF-beta 1); amino control (with aminohexanoic acid); and amino+TGF-beta (with aminohexanoic acid and TGF- beta1). Each of these groups was further divided into three groups depending on the concentration of the inhibitor. Both of the aprotinin groups received 0.0875, 0.175, or 0.35 TIU/ml of aprotinin and both of the aminohexanoic acid groups were supplemented with 2, 4, or 8 mg/ml of aminohexanoic acid. The gels were harvested and analyzed at 7, 14, and 21 days. All of the aprotinin+TGF-beta groups exhibited a significantly higher aggrecan gene expression than control groups whereas only the amino+TGF-â group treated with 8mg/ml was significantly higher than those of the control groups. In addition, the 0.0875 and 0.175 TIU/ml aprotinin+TGF-beta groups exhibited significantly higher levels of expression than the 2 and 4 mg/ml amino+TGF-beta groups. There were no significant differences among the different concentrations of aprotinin or aminohexanoic acid with or without the treatment of TGF-beta. Similar trends were also seen when the glycosaminoglycan (GAG) content was measured and analyzed. These findings suggest that fibrin gels are a suitable environment for the chondrogenesis of BM-MSCs and that aprotinin in combination with TGF-beta1 is the optimal condition for stimulating BM-MSCs to differentiate into chondrocytes.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains ix, 173 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 163-169).

In this study 12 acute myeloid leukaemia (AML) patients (3 in 1<SUP>st</SUP> complete remission (CR), 3 in 2<SUP>nd</SUP> CR, 3 in early relapse or partial remission and 3 in frank relapse) were treated with continuous infusion of recombinant Interleukin-2 (rIL-2). Adverse reactions among these patients were common. In all patients, there were evidence of lymphocyte activation with subsequent upregulation of the cellular cytotoxic functions following the infusion of rIL-2. Despite this, clinical response among patients treated with active disease remains disappointing, with only 1 patient achieving a 3<SUP>rd</SUP> complete remission after being treated in early 2<SUP>nd</SUP> relapse (marrow blast counts of 10%). The other patients had brief periods of stable disease but died eventually of disease progression. No conclusion however can be drawn from patients treated in complete remission due to the small number of patients entered into the study. In vitro studies were performed in a different cohort of AML patients, at presentation and during complete remission. In all the patients, both the Natural Killer (NK) and Lectin-Dependent Cellular Cytotoxicity (LDCC) activities were significantly reduced when compared to normal healthy controls. Patients in complete remission however had higher values than those studied during active diseases. These findings would suggest a strong rationale for the use of immunotherapy capable of upregulating the NK and LDCC activities, e.g. rIL-2. Further rationale for the use of immunotherapy has been drawn from the findings that leukaemia blast cells of AML are immunogenic, as evidenced by data of T cell activation in these patients and the presence of complement-fixing antibodies for autologous myeloblasts. More importantly no stimulation of the myeloblast proliferation by IL-2 was observed in any of the myeloblasts studied. All these findings would point to a good and safe rationale for the use of rIL-2 in AML patients.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Thesis (M.S.)--Central Connecticut State University, 1999. / Thesis advisor: Kathy Martin-Troy. " ... in partial fulfillment of the requirements for the degree of Master of Science in Biology." Includes bibliographical references (leaves 56-62).

Inaug.-Diss.--Würzburg. / At head of title: Aus der Chirurgischen universitätsklinik zu Würzburg ... Lebenslauf. "Schrifttum": p. 58-65.