有證據顯示基質細胞在腫瘤的發生發展中可以發揮重要的作用，基質細胞可以提供適宜腫瘤細胞增殖的腫瘤微環境。腫瘤相關成纖維細胞是一種特殊的與腫瘤生成高度相關的基質細胞。而通过我们的论证，小鼠胚胎成纖維細胞可以作為一種腫瘤相關成纖維細胞的細胞模型。 / 腫瘤誘導因子（TIF）是本實驗室在成瘤實驗中發現的一種新的倉鼠CXC 趨化因子。基于蛋白質序列的分析，TIF 属于Gro CXC 趨化因子家族。這個家族主要通過激活其受體CXCR2 來發揮作用。為了研究TIF 在腫瘤發生中的作用，我們在CHO-K1 細胞中建立了過表達TIF 的穩定細胞株。 / 我們發現共同注射的永生化MEF 與過表達TIF 的D12 細胞導致了腫瘤生長的抑制。為了研究這種現象，重組TIF 蛋白在大腸桿菌中表達，并且用鎳柱進行了提純。純化的蛋白被用于處理CHO-K1 細胞與永生化MEF。我們發現高水平的TIF 可以導致CXCR2 下游的Erk 磷酸化水平下降。其可能的機制為CXCR2 在高水平的TIF 作用下的脫敏作用。同時高水平TIF 可以導致永生化MEF 中CD133 水平的下降。因此，CXCR2 脫敏為TIF 導致腫瘤抑制的可能機制。 / Lines of evidence indicate that stromal cell is one of the determinants in tumor formation by providing a favorable microenvironment for the growth of cancer cells. Cancer associated fibroblast (CAF) is a special form of stromal cells which are shown to be derived from bone marrow. Upon reaching the tumor, the bone marrow-derived mesenchymal stem cells differentiate into CAF, which secrets various growth factors and cytokines to promote cancer growth. Furthermore, genetic study shows that CAF displays p53 mutations and other genetic changes. / Tumor induced factor (TIF) is a CXC chemokine that is originally identified from a xenograft tumor. Sequence analysis suggests TIF is a family member of the Gro CXC chemokines, and exerts its cellular function via activating CXCR2 receptors. In order to investigate the functional roles of TIF, a stable cell line over-expressing TIF in hamster CHO-K1 was established. / To explore the cancer-stromal interactions in xenograft, mouse embryonic fibroblast (MEF) were used as a study model for CAF. MEF was sub-cultured by a conventional protocol that was used for developing the NIH3T3 cells. Based on the growth patterns and expressions of cell markers, growth of MEF can be divided into three stages: the early stage, the senescent stage and the immortalized stage. Our results suggested that MEF might mirror the various developmental stages of CAF. / To examine the contributions of MEF in tumorigenesis, CHO-K1 cells and MEF were co-injected into nude mice. Intriguingly, MEF that in senescent and immortalized stages, rather than in early stage, promoted tumor formation. A possibility arose that the contribution of senescent and immortalized MEF in promoted tumorigenesis may due to CD133 and CXCL1, as the expression of CD133 and CXCL1 in senescent and immortalized MEF were higher than that of MEF in early stage. Moreover, as MEF could gradually develop into a fibroblast promoted tumor formation, MEF could be used as a crucial model to illustrate the origination and development of CAF. / Surprisingly, in nude mice co-injected with immortalized MEF with TIF-overexpressing D12 cells, suppression instead of promotion of tumor growth was found. In order to explore the underlined mechanism of tumor suppression, recombinant TIF protein was purified based on a bacterial expression system. Using purified TIF protein to treat CHO-K1 cells and MEF, it was found that low concentration of TIF promoted Erk phosphorylation but high concentration of TIF suppressed it, which might resulted from desensitization of CXCR2 receptors. Reduction of Erk phosphorylation resulted in decreased proliferation in CHO-K1 cells and alleviated expression of CD133 in MEF, which could be the mechanisms for TIF-induced tumor suppression in nude mice. / Taken together, a CAF model was established to examine the function of TIF in tumor-fibroblast interactions. Mechanistic studies indicated that TIF-induced tumor suppression in nude mice was mediated via desensitization of CXCR2 receptors by high concentration of TIF in the tumor microenvironment. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qi, Wei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 189-206). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Chapter Chapter 1 --- General Introduction / Chapter 1.1 --- Tumorigenesis --- p.4 / Chapter 1.1.1 --- Virus transformation --- p.4 / Chapter 1.1.2 --- Proto-oncogene and oncogene --- p.5 / Chapter 1.1.3 --- Tumor suppressor gene --- p.7 / Chapter 1.1.4 --- Epigenetic alteration --- p.9 / Chapter 1.1.5 --- Cancer stem cell --- p.11 / Chapter 1.1.6 --- Tumor microenvironment --- p.14 / Chapter 1.2 --- Cancer associated fibroblast (CAF) --- p.17 / Chapter 1.2.1 --- Markers for CAF --- p.17 / Chapter 1.2.2 --- CAF and normal fibroblast --- p.20 / Chapter 1.2.3 --- CAF, a important player in tumor growth --- p.22 / Chapter 1.2.4 --- CAF and angiogenesis --- p.23 / Chapter 1.2.5 --- CAF and tumor invasion --- p.25 / Chapter 1.3 --- Chemokine --- p.27 / Chapter 1.3.1 --- Structure of chemokine --- p.27 / Chapter 1.3.2 --- Chemokine and cell Recruitment --- p.30 / Chapter 1.3.3 --- Chemokine and tumor microenvironment --- p.30 / Chapter 1.4 --- Tumor Induced Factor and its induced tumor suppression --- p.38 / Chapter 1.5 --- The aims of the project --- p.47 / Chapter Chapter Two --- Purification of Tumor Induced Factor / Chapter 2.1 --- Introduction --- p.49 / Chapter 2.2 --- Materials --- p.52 / Chapter 2.2.1 --- Chemical --- p.52 / Chapter 2.2.2 --- Enzyme --- p.52 / Chapter 2.2.3 --- Antibody --- p.52 / Chapter 2.3 --- Method --- p.53 / Chapter 2.3.1 --- Overview of protein expression system --- p.53 / Chapter 2.3.2 --- Purification of Trx-His₆-S-TIF protein --- p.54 / Chapter 2.3.3 --- BCA assay --- p.60 / Chapter 2.3.4 --- SDS-PAGE --- p.60 / Chapter 2.3.5 --- Western blotting --- p.61 / Chapter 2.3.6 --- Preparation of pET28/His₆-Sumo-TIF bacterial expression vector --- p.62 / Chapter 2.3.7 --- Optimization of culture condition for BL21 expressed His₆-Sumo-TIF protein --- p.67 / Chapter 2.3.8 --- Purification of His₆-Sumo-TIF protein --- p.68 / Chapter 2.3.9 --- Homology model of TIF --- p.68 / Chapter 2.4 --- Results --- p.69 / Chapter 2.4.1 --- Purification of Trx-His₆-S-TIF --- p.70 / Chapter 2.4.2 --- Optimization of purification protocol of His₆-Sumo-TIF --- p.71 / Chapter 2.4.3 --- Large scale purification of mature TIF --- p.75 / Chapter 2.4.4 --- Homology modeling of TIF --- p.80 / Chapter 2.5 --- Discussion --- p.83 / Chapter Chapter 3 --- Three Stages Hypothesis / Chapter 3.1 --- Introduction --- p.86 / Chapter 3.2 --- Material --- p.93 / Chapter 3.2.1 --- Chemical --- p.93 / Chapter 3.2.2 --- Enzyme --- p.93 / Chapter 3.2.3 --- Animal --- p.93 / Chapter 3.2.4 --- Antibody --- p.94 / Chapter 3.3 --- Methods --- p.95 / Chapter 3.3.1 --- Isolate MEF from 13.5 days mouse embryo --- p.95 / Chapter 3.3.2 --- Culture of MEF following 3T3 protocol --- p.96 / Chapter 3.3.3 --- X gal staining --- p.96 / Chapter 3.3.4 --- Analysis of MEF cell size and complexity by flow cytometry --- p.98 / Chapter 3.3.5 --- MTT assay --- p.98 / Chapter 3.3.6 --- Analysis of CD133 by flow cytometry --- p.99 / Chapter 3.3.7 --- ROS detected by DCFH-DA fluorescent probe --- p.99 / Chapter 3.3.8 --- Double staining of cancer stem cell marker and ROS fluorescent probe --- p.100 / Chapter 3.3.9 --- Reverse transcription --- p.101 / Chapter 3.3.10 --- Analysis CXCL1 mRNA expression level by PCR --- p.102 / Chapter 3.3.11 --- Gelatin zymography --- p.103 / Chapter 3.3.12 --- In-vivo tumorigenicity assay --- p.104 / Chapter 3.4 --- Results --- p.106 / Chapter 3.4.1 --- Three Stages of MEF --- p.106 / Chapter 3.4.2 --- X gal staining --- p.106 / Chapter 3.4.3 --- Flow cytometric analysis of cell diameter and cellular complexity of MEF --- p.109 / Chapter 3.4.4 --- MTT assay --- p.109 / Chapter 3.4.5 --- CD 133 expression of MEF detected by flow cytometry --- p.110 / Chapter 3.4.6 --- Reactive oxygen species of MEF detected by flow cytometry --- p.118 / Chapter 3.4.7 --- The level of ROS and CD133 of MEF detected by flow cytometry stimultaneously --- p.121 / Chapter 3.4.8 --- TIF treatment reduces the small CSC subpopulation in senescent stage MEF --- p.124 / Chapter 3.4.9 --- Increased CXCL1 expression in senescent stage and immortalized stage MEF --- p.125 / Chapter 3.4.10 --- Matrix metalloproteinase 2 activities in different stages of MEF . --- p.129 / Chapter 3.4.11 --- In vivo tumorigenicity assay --- p.130 / Chapter 3.5 --- Discussion --- p.133 / Chapter Chapter Four --- Biphasic Effect of TIF in Cancer-Fibroblasts Interaction / Chapter 4.1 --- Introduction --- p.140 / Chapter 4.2 --- Material --- p.143 / Chapter 4.2.1 --- Chemical --- p.144 / Chapter 4.2.2 --- Kit and Instrument --- p.144 / Chapter 4.2.3 --- Antibody --- p.144 / Chapter 4.3 --- Method --- p.145 / Chapter 4.3.1 --- Purification of TIF-His₆-Flag --- p.145 / Chapter 4.3.2 --- Western blotting to detect purified TIF-His₆-Flag --- p.145 / Chapter 4.3.3. --- Measurement of cell proliferation by cell counting --- p.145 / Chapter 4.3.4 --- MTT assay --- p.146 / Chapter 4.3.5 --- Western blotting to detect pErk and total Erk --- p.146 / Chapter 4.3.6 --- Soft agar assay --- p.148 / Chapter 4.3.7 --- Gelatinase detection --- p.148 / Chapter 4.3.8 --- Wound healing assay --- p.149 / Chapter 4.3.9 --- Colony formation assay --- p.149 / Chapter 4.3.10 --- Detection of CD133 by flow cytometry --- p.150 / Chapter 4.4 --- Results --- p.151 / Chapter 4.4.1 --- Purification of TIF-His₆-Flag --- p.151 / Chapter 4.4.2 --- Reduced cell proliferation of D12 in long time culture --- p.153 / Chapter 4.4.3 --- Reduced metabolic activities of D12 cells in time culture --- p.155 / Chapter 4.4.4. --- TIF-CXCR2-pErk signal axis in CHO cells --- p.155 / Chapter 4.4.5 --- Bigger colonies formed by D12 cells in soft agar assay --- p.161 / Chapter 4.4.6 --- TIF-CXCR2-pErk-MMP9 signal pathway in D12 cells --- p.162 / Chapter 4.4.7 --- Reduced migration of D12 cells --- p.164 / Chapter 4.4.8 --- Reduced cell invasion of D12 cells --- p.165 / Chapter 4.4.9 --- Reduced colony number of D12 cells in colony formation assay --- p.168 / Chapter 4.4.10 --- Bi-phasic “bell shape“ bi-phasic response on Erk activation of TIF in CHO-K1 cells --- p.169 / Chapter 4.4.11 --- Bi-phasic “bell shape“ effect of TIF to pErk in immortalized MEFs --- p.172 / Chapter 4.4.12 --- Reduced CD133 in immortalized MEF by high concentration of TIF --- p.173 / Chapter 4.5 --- Discussion --- p.177 / Chapter Chapter Five --- General Discussion / Chapter 5.1 --- Project Summary --- p.183 / Chapter 5.2 --- Significances of the project --- p.185 / Chapter 5.3 --- Future work --- p.188

前言：間充質幹細胞容易擴增並且能分化為成骨細胞、軟骨細胞和脂肪細胞，並且能對炎症、感染和損傷做出反應，並且遷移到相應的組織部位。這些特性使間充質幹細胞成為骨骼組織工程學中非常重要的細胞來源。外周血間充質幹細胞是一種存在於血液中的間充質幹細胞，而主要的間充質幹細胞存在與骨髓中，被稱之為骨髓間充質幹細胞。在我們實驗室之前的研究中通過DNA微陣列發現外周血間充質幹細胞中很多基因的表達與骨髓間充質幹細胞有很大區別。這其中的一些基因可能參與調控間充質幹細胞的分化和歸巢，我們從中挑選了三個變化比較明顯的基因--CRBP1, N-cadherin和 SOX11做進一步研究。本研究的目的在於研究CRBP1, N-cadherin和 SOX11在骨髓間充質幹細胞分化和遷移中的作用及相關機理。 / 方法：培養的骨髓間充質幹細胞來源於6-8周大小的SD大鼠。細胞的表型經過多分化潛能測試（成骨分化，成脂分化和成軟骨分化）和流式細胞儀檢驗。克隆大鼠的CRBP1, N-cadherin和SOX11基因到慢病毒載體。而且還設計了針對CRBP1和 N-cadherin的shRNA及非特異性對照shRNA。慢病毒由暫態轉染293FT細胞產生。細胞遷移實驗採用了BD Falcon的細胞遷移系統（cell culture insert）。實驗採用了定量PCR、免疫共沉澱、western雜交和雙螢光報告檢驗。對於體內實驗，細胞經感染帶有不同基因的病毒後，種植到Si-TCP材料並移植到裸鼠皮下。8周後，收集樣品進行組織學和免疫組織學分析。最後，我們建立了大鼠的股骨開放式骨折模型，並在4天后將SOX11基因修飾的間充質幹細胞通過心臟注射打到大鼠體內。4周後，收集股骨骨折樣品並進行microCT、力學測試和組織學分析。 / 結果：CRBP1過表達能夠促進骨髓間充質幹細胞的成骨分化潛能，並能抑制其成脂分化。進一步的機理研究表明CRBP1可以通過與RXRα的蛋白相互作用抑制RXRα誘導的β-catenin降解，從而維持β-catenin和磷酸化-ERK1/2在較高的水準，導致間充質幹細胞成骨能力增強；N-cadherin過表達可以促進間充質幹細胞的遷移，但是卻通過下調β-catenin和磷酸化ERK1/2抑制其成骨分化。過表達SOX11可以通過增強BMP信號通路促進三系分化。SOX11還可以通過啟動CXCR4的表達來促進細胞遷移。最後，在大鼠的股骨開放骨折模型上通過系統注射，我們證明穩定過表達SOX11的間充質幹細胞遷移到骨折部位的數量明顯增加。這些細胞到達骨折部位以後可以起始骨痂的鈣化，促進骨折的修復。 / 結論：本研究證明CRBP1, N-cadherin 和SOX11具有調節骨髓間充質幹細胞遷移和/或分化的功能。這些基因也許會成為幹細胞治療的新靶點。系統注射SOX11基因修飾的骨髓間充質幹細胞對於骨折修復可能具有較好的療效。本研究初步研究了CRBP1, N-cadherin 和SOX11在間充質幹細胞中的作用，為探討以間充質幹細胞為基礎的組織工程的某些新臨床應用提供了一些線索。 / Introduction: Mesenchymal stem cells (MSCs) can be easily harvested, expanded, and have the capability of differentiating into osteoblasts, chondrocytes and adipocytes, and they can home to various tissues in response to stimuli such as inflammation, infection and injuries. MSCs are therefore valuable cell source for musculoskeletal tissue engineering. Peripheral blood-derived MSCs (PB-MSCs) are one kind of MSCs that reside in peripheral blood, whereas the main source of MSCs is bone marrow-derived MSCs (BM-MSCs). In our previous study, we found many genes were differentially expressed in the PB-MSCs compared to their counterpart BM-MSCs demonstrated by microarray analysis, among which the effects of CRBP1, SOX11 and N-cadherin on MSCs in terms of migration and differentiation are studied. / Methods: BM-MSCs and PB-MSCs were cultured from 6-8 weeks SD rats. The phenotypes of MSCs were characterized by tri-lineage (adipo-, osteo- and chondrogenic) differentiation and flow cytometry analysis. The genes encoding rat CRBP1, SOX11 and N-cadherin were cloned into lentiviral vectors respectively. shRNAs targeting CRBP1, N-cadherin, and one nonspecific shRNA were designed. Pseudo-lentivirus was produced by transient transfection of 293FT cells. Cell migration was examined using transwell insert culture system. Quantitative RT-PCR, CO-IP, western blot and dual-luciferase assay were employed in the studies. For in vivo study, MSCs transduced with different genes were seeded on Si-TCP scaffolds and implanted subcutaneously in nude mice. 8 weeks later, the samples were collected for histological and immunohistological analysis. Finally, an open femoral fracture model was established in 8-week old SD rats, SOX11-modified MSCs were injected at four days after fracture. At 4-week after MSCs injection, the femurs were collected for microCT, mechanical test and histological analysis. / Results: For CRBP1gene, our results showed that CRBP1 overexpression promoted osteogenic differentiation of BM-MSCs, while inhibited their adipogenic differentiation. We demonstrated that CRBP1 promoted osteogenic differentiation by inhibiting RXRα-induced β-catenin degradation through physical interactions, and maintaining β-catenin and pERK1/2 at higher levels. For N-cadherin gene, we found that N-cadherin overexpression promoted MSCs migration, and suppressed osteogenic potential of MSCs through inhibiting ERK and β-catenin signaling pathways. For SOX11 gene, we demonstrated that SOX11 overexpression enhanced the adipo-, osteo- and chondrogenic differentiation of BM-MSCs, through enhancing BMP signaling pathways. The migration capacity of BM-MSCs was also enhanced when Sox-11 was overexpressed, through activating CXCR4 expression. Finally, in the open femur fracture model we demonstrated that a larger number of SOX11-overexpressing BM-MSCs migrated to the fracture site, initiated earlier callus ossification and improved bone fracture healing quality. / Conclusions: This study demonstrated that CRBP1, N-cadherin and SOX11 gene can regulate the migration and/or differentiation potentials of BM-MSCs. These genes may become new therapeutic targets in stem cell therapy applications. Systemic administration of genetically modified SOX11-overexpressing BM-MSCs may be useful in promoting fracture healing. Overall, this study defined some unknown functions of CRBP1, N-cadherin and SOX11 in MSCs and shed the lights on some novel therapeutic implications for MSCs-based tissue engineering. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Xu, Liangliang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 128-144). / Abstract also in Chinese. / Declaration --- p.i / Abstract --- p.ii / 摘要 --- p.v / Acknowledgements --- p.vii / Chapter 1 --- p.1 / Introduction --- p.1 / Chapter 1.1 --- Mesenchymal stem cells --- p.2 / Chapter 1.1.1 --- Characteristics of mesenchymal stem cells --- p.2 / Chapter 1.1.2 --- Bone marrow- and peripheral blood-derived MSCs --- p.4 / Chapter 1.1.3 --- Other tissue-derived MSCs --- p.5 / Chapter 1.2 --- Adipogenesis of MSCs --- p.6 / Chapter 1.3 --- Chondrogenesis of MSCs --- p.7 / Chapter 1.4 --- Osteogenesis of MSCs --- p.8 / Chapter 1.4.1 --- Regulators of osteogenesis --- p.9 / Chapter 1.4.2 --- Stratergies for improving bone tissue engineering --- p.11 / Chapter 1.5 --- Signaling pathways involved in osteogenesis --- p.13 / Chapter 1.5.1 --- ERK signaling pathway --- p.14 / Chapter 1.5.2 --- Wnt signaling pathway --- p.15 / Chapter 1.5.3 --- BMP signaling pathway --- p.17 / Chapter 1.6 --- Migration of MSCs --- p.20 / Chapter 1.7 --- Fracture healing --- p.22 / Chapter 1.8 --- Clinical application of MSCs --- p.23 / Chapter 1.8.1 --- BM-MSCs vs. PB-MSCs --- p.24 / Chapter 1.8.2 --- Autologous vs. Allogeneic MSCs transplantation --- p.25 / Chapter 1.9 --- Scope of the present study --- p.26 / Chapter 1.9.1 --- CRBP1 --- p.26 / Chapter 1.9.2 --- N-cadherin --- p.27 / Chapter 1.9.3 --- SOX11 --- p.27 / Chapter 1.10 --- Experimental scheme --- p.29 / Chapter 2 --- p.31 / Comparison between PB-MSCs and BM-MSCs --- p.31 / Chapter 2.1 --- Chapter introduction --- p.32 / Chapter 2.2 --- Materials and methods --- p.33 / Chapter 2.2.1 --- Cell culture --- p.33 / Chapter 2.2.2 --- Flow cytometry --- p.33 / Chapter 2.2.3 --- Adipogenic differentiation --- p.34 / Chapter 2.2.4 --- Osteogenic differentiation --- p.34 / Chapter 2.2.5 --- RNA Extraction and Real-time PCR --- p.34 / Chapter 2.3 --- Results --- p.35 / Chapter 2.3.1 --- Morphology of PB-MSCs --- p.35 / Chapter 2.3.2 --- Cellular surface markers of BM-MSCs and PB-MSCs --- p.36 / Chapter 2.3.3 --- Multi-differentiation potential of BM-MSCs and PB-MSCs --- p.38 / Chapter 2.3.4 --- Target genes expression in BM-MSCs and PB-MSCs --- p.39 / Chapter 2.4 --- Discussion and future work --- p.40 / Chapter 3 --- p.41 / Role of CRBP1 in Differentiation and Migration of MSCs --- p.41 / Chapter 3.1 --- Chapter introduction --- p.42 / Chapter 3.2 --- Materials and methods --- p.46 / Chapter 3.2.1 --- Chemicals --- p.46 / Chapter 3.2.2 --- Isolation and culture of BM-MSCs --- p.46 / Chapter 3.2.3 --- RNA Extraction and Real-time PCR --- p.47 / Chapter 3.2.4 --- Plasmid construction, transfection, production of lentivirus and infection --- p.48 / Chapter 3.2.5 --- Osteogenic differentiation --- p.50 / Chapter 3.2.6 --- Adipogenic differentiation --- p.50 / Chapter 3.2.7 --- Western blot --- p.51 / Chapter 3.2.8 --- Immunofluorescence labeling and fluorescence microscopy --- p.52 / Chapter 3.2.9 --- Cell migration assay --- p.52 / Chapter 3.2.10 --- Ectopic bone formation assay --- p.52 / Chapter 3.2.11 --- Statistical analysis --- p.53 / Chapter 3.3 --- Results --- p.53 / Chapter 3.3.1 --- Transducing BM-MSCs with lentivirus carrying CRBP1 or shRNAs --- p.53 / Chapter 3.3.2 --- CRBP1 accelerates osteogenesis of BM-MSCs via enhancing ERK1/2 and β-catenin pathways --- p.56 / Chapter 3.3.3 --- CRBP1 stabilizes β-catenin by inhibiting RXRα-induced degradation --- p.58 / Chapter 3.3.4 --- CRBP1 inhibits adipogenesis of BM-MSCs --- p.61 / Chapter 3.3.5 --- CRBP1 overexpression has no effect on MSCs migration potential --- p.63 / Chapter 3.3.6 --- CRBP1 promotes ectopic bone formation in vivo --- p.64 / Chapter 3.4 --- Discussion --- p.66 / Chapter 3.5 --- Future work --- p.73 / Chapter 4 --- p.74 / Role of N-cadherin in Differentiation and Migration of MSCs --- p.74 / Chapter 4.1 --- Chapter introduction --- p.75 / Chapter 4.2 --- Materials and methods --- p.78 / Chapter 4.2.1 --- Chemicals --- p.78 / Chapter 4.2.2 --- Isolation and culture of BM-MSCs --- p.78 / Chapter 4.2.3 --- Plasmid construction, transfection, production of lentivirus and infection --- p.79 / Chapter 4.2.4 --- Osteogenic differentiation and ALP activity assay --- p.81 / Chapter 4.2.5 --- Western blot --- p.81 / Chapter 4.2.6 --- Ectopic bone formation assay --- p.82 / Chapter 4.2.7 --- Statistical analysis --- p.82 / Chapter 4.3 --- Results --- p.83 / Chapter 4.3.1 --- Expression of N-cadherin during osteogenesis in MSCs --- p.83 / Chapter 4.3.2 --- N-cadherin overexpression inhibits osteogenesis through suppressing β-catein and ERK1/2 signaling pathways --- p.84 / Chapter 4.3.3 --- N-cadherin silencing increases osteogenesis through enhancing β-catenin and ERK1/2 signaling pathways --- p.86 / Chapter 4.3.4 --- N-cadherin promotes migration of MSCs --- p.87 / Chapter 4.3.5 --- Cellular surface markers of SV40-immortalized MSCs --- p.89 / Chapter 4.3.6 --- N-cadherin inhibits ectopic bone formation in vivo --- p.89 / Chapter 4.4 --- Discussion --- p.91 / Chapter 4.5 --- Future work --- p.94 / Chapter 5 --- p.96 / Role of SOX11 in Differentiation and Migration of MSCs --- p.96 / Chapter 5.1 --- Chapter introduction --- p.97 / Chapter 5.2 --- Materials and methods --- p.105 / Chapter 5.2.1 --- Plasmid construction, transfection, production of lentivirus and infection --- p.105 / Chapter 5.2.2 --- Cell culture --- p.106 / Chapter 5.2.3 --- Luciferase reporter gene assay --- p.106 / Chapter 5.2.4 --- Osteogenic differentiation and ALP activity assay --- p.106 / Chapter 5.2.5 --- Adipogenic differentiation --- p.107 / Chapter 5.2.5 --- Chondrogenic diffferentiation --- p.107 / Chapter 5.2.6 --- Western blot --- p.108 / Chapter 5.2.7 --- RNA Extraction and Real-time PCR --- p.108 / Chapter 5.2.8 --- Cell migration --- p.110 / Chapter 5.2.9 --- Ectopic bone formation --- p.110 / Chapter 5.2.10 --- Fracture healing model and analysis --- p.111 / Chapter 5.2.11 --- Statistical Analysis --- p.112 / Chapter 5.3 --- Results --- p.112 / Chapter 5.3.1 --- SOX11 is upregulated during osteogenesis of BM-MSCs --- p.112 / Chapter 5.3.2 --- SOX11 promotes adipogenesis in BM-MSCs --- p.113 / Chapter 5.3.3 --- SOX11 promotes migration of BM-MSCs --- p.114 / Chapter 5.3.4 --- SOX11 promotes osteogenesis in BM-MSCs --- p.115 / Chapter 5.3.5 --- SOX11 promotes chondrogenesis of MSCs --- p.117 / Chapter 5.3.6 --- Mechanisms of how SOX11 regulates differentiation and migration of MSCs --- p.118 / Chapter 5.3.7 --- SOX11-modified MSCs promote bone fracture healing in an open femur fracture rat model --- p.122 / Chapter 5.4 --- Discussion --- p.126 / Chapter 5.5 --- Future work --- p.131 / Appendix --- p.153

As células mesenquimais estromais multipotentes (CMM) são na atualidade uma fonte atrativa para aplicações na engenharia de tecidos e na terapia celular. Devido à baixa disponibilidade nos tecidos (0,01%-0,0005%) e às elevadas doses necessárias para uma infusão (aproximadamente 106 células/Kg paciente) tornou-se necessário o desenvolvimento de tecnologias de expansão in vitro, eficientes e de custo reduzido, que permitam a obtenção de CMM com manutenção das características funcionais (diferenciação e inibição da proliferação de linfócitos), imunofenotípicas e citogenéticas. As CMM são células aderentes, ou seja, necessitam de um substrato sólido para se aderir e proliferar. O procedimento convencional de expansão em garrafas estáticas, geralmente envolve um processo laborioso em que não há correto controle e monitoramento dos parâmetros de cultivo e possui uma maior susceptibilidade à contaminação devido à excessiva manipulação para atingir o número ideal de células. Além disso, este tipo de cultivo não permite uma produção em larga escala. Em função disso, o presente trabalho foi proposto com o objetivo de desenvolver um bioprocesso escalonável, economicamente viável e eficiente para expansão de CMM derivadas da medula óssea em microcarregadores. Para isso, as células foram cultivadas em microcarregador Cyotdex 3, em frasco spinner com o meio -MEM suplementado com 15% de SFB. Foram avaliadas neste trabalho, a adesão celular aos microcarregadores, crescimento, metabolismo, recuperação celular final e avaliação das propriedades funcionais e imunofenotípicas pré e pós cultivo, comparando ao cultivo já estabelecido em garrafas estáticas. De maneira geral, os resultados obtidos mostraram que foi possível expandir CMM utilizando a tecnologia de microcarregadores. A análise do metabolismo celular mostrou que não houve exaustão de nutrientes importantes como glicose e glutamina durante o cultivo, tampouco formação dos subprodutos lactato e amônia em concentrações inibitórias. As células recuperadas após a expansão mantiveram as características imunofenotípicas e funcionais. A produção média (n=10) foi de aproximadamente 4,9x105 cel/mL. Como o sistema utilizado permite o escalonamento, se utilizássemos um biorreator de 1L, seria possível a produção de aproximadamente 5x108 células que seriam suficientes para tratar mais de 3 pacientes de até 70Kg na dose de 2x106 células/Kg. Para expansão da mesma quantidade de células na forma tradicional seriam necessárias 135 garrafas de 175 cm2 com um custo total de expansão duas vezes superior à estimativa do custo de expansão utilizando microcarregadores. / Multipotent mesenchymal stromal cells are currently an attractive source for applications in tissue engineering and cell therapy. Due to the low availability in tissues (0,01%-0,0005%) and the high doses necessary for an infusion (about 106 cells/Kg patient), it has become necessary the development of effective and low cost technologies for in vitro expansion that enable to obtain MSC with maintenance of functional (differentiation and inhibition of lymphocytes proliferation), immunophenotypic and cytogenetics characteristics. MSC are adherent cells, i.e., they need a solid substrate to adhere and proliferate. The conventional procedure for expansion in static flasks normally involves a laborious process in which there is no suitable control and monitoring of the cultivation parameters besides presenting a higher susceptibility to contamination due to excessive manipulation to reach the ideal amount of cells. Moreover, this kind of cultivation does not allow a large scale production. For this reason, this work was proposed with the objective to develop a low cost, effective and scalable bioprocess for expansion of bone marrow-derived MSC in microcarriers. Cells grew on microcarriers Cyotdex 3, in spinner flasks with the -MEM medium supplemented with 15% FBS. We evaluated the cell adhesion to microcarriers, growth, metabolism, final cell recovery, and the functional and immunophenotypic properties before and after cultivation, comparing them with the cultivation already established in static flasks. In general, the results obtained showed that it was possible to expand MSC using microcarriers technology. The analysis of the cell metabolism showed that there was no depletion of important nutrients such as glucose and glutamine during cultivation, neither formation of lactate and ammonia subproducts in inhibitory concentrations. The cells recovered after the expansion kept the immunophenotypic and functional characteristics. The mean production (n=10) was about 4,9x105 cel/mL. As the system used allows the scale-up, if we had used a bioreactor of 1L it would had been possible to produce approximately 5x108 cells that would be enough to treat more than three patients of up to 70kg with a dose of 2x106 cells/kg. For the expansion of the same amount of cells in the traditional way, it would be necessary 135 T-flasks of 175 cm2 with total cost twice higher than the estimate cost of expansion using microcarriers.

A esclerose múltipla é uma doença inflamatória crônica desencadeada por células T autorreativas contra antígenos proteicos da mielina. A encefalomielite experimental autoimune é o modelo murino mais utilizado para o estudo da EM. As tubas uterinas e o útero de camundongos fêmeas são órgãos ricos em células mesenquimais que são pouco utilizadas em estudos. Dessa forma, no presente projeto, caracterizamos a obtenção dessa população e avaliamos sua capacidade imunossupressora utilizando o modelo de EAE. Observamos que o tratamento é capaz de modular o perfil de linfócitos T CD4+ durante sua ativação nos linfonodos, induzindo o direcionamento para a subpopulação Tr1 e atenuando as Th1 e Th17. Assim, houve uma diminuição do número de células infiltrantes no SNC associado a uma menor ativação de células da microglia. Em conjunto, demostraramos que as meMSC utilizadas como tratamento são capazes de atrasar o desenvolvimento da EAE e, portanto, evidenciando o caráter imunomodulador das MSCs derivadas do endométrio, chamando a atenção para seu potencial terapêutico. / Multiple sclerosis is a chronic inflammatory disease triggered by autoreactive T cells against myelin protein. Experimental Autoimmune Encephalomyelitis is the most commonly used murine model for the study of MS. The uterine tubes and uterus of female mice are organs rich in mesenchymal cells which are rarely used. Thus, in the present work, we characterized the extraction of this population and evaluated its immunosuppressive capacity using the EAE model. We observed that the meMSC treatment is capable of modulating the CD4 T lymphocyte profile during its activation in the lymph nodes, inducing the expansion of the Tr1 subpopulation and attenuating Th1 and Th17. Consequently, there was a decrease in the number of infiltrating cells in the CNS associated with a reduction of microglial activation. Taken together, our results demonstrated that the meMSCs used as treatment are capable of delaying the development of EAE, therefore, evidencing its immunomodulatory features drawing attention to its therapeutic potential.

As distrofias musculares progressivas (DMP) são um grupo de doenças genéticas hereditárias caracterizadas pela degeneração progressiva e irreversível da musculatura esquelética. A distrofia muscular de Duchenne (DMD) é a forma mais comum e mais grave de DMP, com prevalência de 1 a cada 3500 a 5000 meninos. Em geral, a perda da ambulação ocorre entre 9 a 12 anos e complicações respiratórias e cardíacas podem levar ao óbito a partir da segunda década. A pesquisa em terapia celular iniciou-se com o objetivo de reverter ou diminuir a progressão do processo distrófico através do repovoamento do músculo com células normais. Atualmente, acredita-se em um benefício terapêutico com base nas propriedades anti-inflamatórias, anti-fibróticas e imunomodulatórias das células tronco adultas (CTA). As CTAs mesenquimais são bastante heterogêneas quanto à sua composição celular o que ocasiona inconsistência de resultados. Por isso, a caracterização e separação de sub-populações através de marcadores específicos e o enriquecimento de culturas de CTA com um subtipo celular de interesse pode aumentar a robustez e o efeito das terapias. Uma dessas subpopulações é o pericito que, ao contrário das CTAs mesenquimais, foi bem descrito quanto à sua localização e função in vivo. Além disso, pericitos derivados de tecido adiposo humano aumentaram a sobrevida de camundongos duplo mutantes para distrofina e utrofina (dko). Dessa forma, este trabalho pretendeu comparar o potencial terapêutico de CTAs mesenquimais e pericitos de um mesmo tecido adiposo em camundongos dko. Conseguimos confirmar o resultado anterior, mostrando que os pericitos tendem a melhorar a sobrevida de animais tratados, sendo ainda melhores do que células mesenquimais, mas a melhora perdura somente durante o tratamento. A sobrevida é maior no começo do tratamento, sugerindo que o quanto antes o tratamento for iniciado, com animais mais jovens e sintomas mais leves, melhor poderá ser o resultado. Outras perguntas a serem pesquisadas na tentativa de melhorar o efeito terapêutico da terapia celular com pericitos são: número de injeções, quantidade de células a serem injetadas, tempo de tratamento e idade das células \"doadoras\" / Progressive muscular dystrophies (PMD) are inherited genetic diseases characterized by progressive muscle loss and weakness. Duchenne muscular dystrophy (DMD) is the most common and aggressive form of PMD, with incidence of 1 in every 3500-5000 boys. In general, patients with DMD are confined to wheelchairs around 9-12 years of age and death occurs due to respiratory and heart dysfunction after the second decade. Cell therapy research at first aimed to recover or slow down the dystrophic process by repopulating the patient\'s muscle with normal cells. However, nowadays it is believed also that therapeutic benefits occur by the anti-inflammatory, anti-fibrotic and immunomodulation properties of mesenchymal stem cells (MSC). MSC are constituted by an heterogeneous cell population and therefore, cell sorting of the subpopulation cell of interest is being done routinely. By doing this enrichment, the effect can be more robust and powerful. One of the cell populations of interest for research is pericyte, which are cells well defined regarding their in vivo function and location, as opposed to MSC. Besides that, pericytes derived from adipose tissue were successful in increasing survival of double knockout mice for dystrophin and utrophin (dko). The present work aimed to compare the therapeutic potential of MSC and pericytes derived from the same adipose tissue sample in the dko mouse model. We confirmed our previous results, showing that pericytes tend to improve the survival of treated mice, and are even better than MSC from the same source but the trend was statistically significant only during the treatment period. Additionally, we also observed that the survival was better in the beginning of treatment, suggesting that earlier treatment may lead to a better therapeutic effect. In an attempt to increase the therapeutic effect of these procedure other questions to be asked are: the number of injections and number of cells per injection, the duration of the treatment and the \"age\" of the donor cells

One of the leading causes of death and hospital stays in the United States, myocardial infarction (MI) occurs when coronary blockages lead to downstream ischemia in the myocardium. Following the MI, the heart activates a number of pathways to repair or remodel the infarcted zone. Endothelial cells respond to ischemia by de-differentiating to form neovasculature and myofibroblasts. The resident cardiac differentiable stem cells (CDCs) are recruited via local cytokines and chemokines to the infarct zone where they too differentiate into myofibroblasts. Mesenchymal stem cells (MSCs) of the bone marrow respond to circulating factors by immobilizing to the heart and differentiating down cardiac lineages. In regenerative medicine approaches, these processes are exploited to augment the resident supply of reparative cells. Clinical trials to transplant cardiac stem cells into MI zones have been met with mixed results. When CDCs are harvested from autologous or type-matched donors, the cells are prepared with a minimum of manipulations, but the yield is quite small. Conversely, MSCs from bone marrow are highly proliferative, but the manipulations in culture required to trigger cardiac differentiation have been found to transform the cell into a more immunogenic phenotype. In addition, there is a dearth of in vivo evidence for the fate of transplanted cells. Currently, intracardiac echocardiographs are used to assess the infarcted area and to guide delivery of stem cell transplants. However, this modality is invasive, short-term, and does not image the transplanted cells directly. In this project, I addressed these shortcomings with a regenerative medicine and bioimaging approach. Our lab has developed multimodal nanoparticles based on a core of mesoporous silica, functionalized with fluorescein or tetramethylrhodamine isothiocyanate for visibility in fluorescent microscopy, Gd2O3 for magnetic resonance imaging (MRI), and trifluoropropyl moieties for ultrasound applications. After establishing in vitro models of cardiac stem cells using CDCs and MSCs, the particles were implemented and characterized in vitro. At a concentration of 125 μg/mL in culture, the particles are highly biocompatible, and labeled cells were found to be fluorescent, echogenic, and detectable with MRI in prepared agar phantoms. Ex vivo mouse hearts, first mounted in agar phantoms, then left in situ, were implemented as a model for guided delivery using ultrasound and follow-up cell tracking with MRI. These results in this project demonstrate the feasibility of this highly novel and practical approach. Additional studies will be carried out to evaluate the biocompatibility and retention versus clearance in live animal models, prior to the carrying out of true pre-clinical models for myocardial infarction.

Background: Directing autogenous Mesenchymal Stem Cell (MSC) to defect sites has a great promise in bone regeneration. We designed a MSC specific, bone affinity peptide (E7HA7) by conjugating E7 with a polyglutamate hydroxyapatite (HA) binding motif. We sought to characterize the in-vivo releasing pattern and bioactivity of E7HA7. Methods: HA discs were coated with fluorescent labeled peptides E7HA7, E7HA2 or E7 were subcutaneously implanted in Sprague Dawley rats. In an ectopic bone formation model was used to test the in-vivo bioactivity of E7HA7 conjugated to DBM. Results: E7HA7 showed slower peptide release from scaffolds in comparison to other groups, being statistically significant at week 2 compared to E7, and to E7HA2 at week 4 and 8. In ectopic model, the medians for new bone formation in each group were: iDBM=0.041mm3, iDBM-E7=0.071mm3, aDBM=0.138mm3, and aDBM-E7=0.192mm3. Conclusions: Conjugation of E7 to polyglutamate bone binding domain showed slow releasing kinetics and osteoinductive potential.

In cases of compromised bone remodeling like osteoporosis, insufficient osseointegration occurs and results in implant failure. Implant retention relies on proper secondary fixation, which is developed during bone remodeling. This process is disrupted in metastatic bone diseases like osteoporosis. Osteoporosis is characterized low bone mass and bone strength resulting from either accelerated osteoclast-mediated bone resorption or impaired osteoblast-mediated bone formation. These two processes are not independent phenomena. In fact, osteoporosis can be viewed as a breakdown of the cellular communication connecting bone resorption to bone formation. Because bone remodeling occurs at temporally generated specific anatomical sites and at different times, local regulators that control cross-talk among the cells of the BRU are important. Previous studies show Ti implant surface characteristics like roughness, hydrophilicity, and chemistry influence the osteoblastic differentiation of human MSCs and maturation of OBs. Furthermore, microstructured Ti surfaces modulate the production of factors shown to be important in the reciprocal communication necessary for the maintenance of healthy bone remodeling. Semaphorin signaling proteins are known to couple the communication of osteoblasts to osteoclasts and are capable of stimulating bone formation or bone resorption depending on certain cues. Implant surface properties can be optimized to exploit these effects to favor rapid osseointegration in patients with osteoporosis.

As a novel therapy for neurodegenerative diseases, transplantation of multipotent mesenchymal stromal cells (MSCs) requires extensive optimization in animal models before being implemented in clinical trials. It is a goal of our laboratory to understand the mechanism of action of these cells and to improve their therapeutic efficacy. To address these goals, this study aims to optimize the cell dosage, cell type, administration route and timing, and/or donor age for stem cell therapy in two mouse models of demyelinating diseases: globoid cell leukodystrophy (GLD; Krabbe’s disease) and experimental autoimmune encephalomyelitis (EAE). GLD is a neurodegenerative lysosomal storage disease caused by the deficiency of galactocerebrosidase (GALC). Accumulation of toxic byproducts in myelin producing oligodendrocytes leads to the demyelination of neurons and increase in brain inflammation. The twitcher mouse model of GLD was used to test the therapeutic effects of MSCs after injection through intracerebroventricular (ICV) or intraperitoneal (IP) routes. Weekly MSC IP injections and single IP GALC-transduced MSC injections were performed. Other twitcher mouse cohorts received temporal vein (TV) or intracerebral (IC) injections of GALC-containing adeno-associated virus serotype 9 (AAV9-GALC) with or without IP MSC injections. All GLD affected mice treated with peripheral MSC and/or vector therapy had extended lifespans with improved motor function. The ameliorating effects of MSCs were related to their potent anti-apoptotic and anti-inflammatory effects on the peripheral and central nervous systems. These results indicate a promising future for peripheral administration of MSCs and vectors as non-invasive, adjunct therapies for patients affected with GLD. A similar study was performed using the EAE mouse model of multiple sclerosis (MS), which is a demyelinating disease due to an autoimmune reaction to myelin. The results demonstrated that biological age of the donor reduces the ability of MSCs to alleviate symptoms and improve pathology in the EAE mouse model. Upon transplantation, the young, but not old, MSCs provided neuroprotective effects through immunomodulation and remyelination in the central nervous system (CNS). The age-related therapeutic differences corroborate recent findings that biologic aging occurs in stem cells and highlight the potential need for allogeneic transplantation of MSCs in older MS patients. / acase@tulane.edu

Human Immunodeficiency Virus -1 infects CD4+ cells, and the subsequent loss of these cells cause Acquired Immune Deficiency Syndrome. Highly active antiretroviral therapy (HAART) is crucial to control viremia in the clinical management of AIDS/HIV infection; however, drug regimens are complex, expensive, and require life-long intervention with potential side effects. Current conventional anti-HIV drugs target different phases of the HIV life cycle and can be categorized as nucleoside or nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, entry inhibitors (co-receptor antagonists and fusion inhibitors), and integrase inhibitors(II). Enfuvirtide (Fuzeon, or T-20) is the first fusion inhibitor approved by the FDA and has substantial side effects and drug delivery issues with most patients developing some local injection site reaction. The subcutaneous application of enfuvirtide and its short half-life, which requires twice daily administration, has disadvantages in patients who are already burdened by complex oral therapy. To overcome these drug issues, we propose an alternative method to administer the HIV-1 peptide fusion inhibitor C46. Stem cells can be a vehicle for delivering genes to specific tissues in the body and their therapeutic delivery systems are extensively used in cancer research. For many years, restoration of blood and immune system function has been used as a component in the care of cancer patients who have been treated with chemotherapeutic agents. Mesenchymal Stem Cells (MSCs) have been demonstrated as a delivery vehicle for gene therapy applications based on their ability to engraft and home to inflamed tissues. MSCs are multi-potent and have immunological function in several human diseases. To investigate MSCs immune suppressive ability in HIV infection system, we will evaluate the crosstalk between MSCs and HIV infection immune-modulatory network. / acase@tulane.edu