published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Meng, Fanbiao. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 114-138). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

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

Indiana University-Purdue University Indianapolis (IUPUI) / The Cre/loxP system is a tool for targeted recombination of DNA. For applying Cre recombinase-mediated genome modifications, there is a requirement for reliable, high-fidelity, and specific transgenic expression of the Cre recombinase. This study focuses on the reliability of different bone cell specific Cre models in the Cre/loxP system. In this study, DMP1-Cre transgenic mouse which has a transgene driven by DMP1 promotor that allows Cre-expression only in late stage osteoblasts and osteocytes was used. Ctsk-Cre mouse with a driven by Ctsk promoter was used so that only osteoclasts would undergo Cre-mediated recombination. E2A-Cre mouse where the Cre recombinase is driven by a global promoter E2A was also included in this study as a control line to test the Cre reporter line Ai9. Dmp1-Cre, Ctsk-Cre and E2A-Cre mice were crossed to the fluorescent Cre-reporter line—Ai9, which harbors a floxed stop codon, followed by the fluorophoremTomato, inserted into the Rosa26 locus. This construct is expected to give red fluorescence when it recombines with Cre-expressing mouse cells and no fluorescence in non-recombinant mouse cells. Double positive (Ai9+/Cre+) offspring selected by PCR were perfused, and 5mu-m thick section of bone and soft tissues were examined for red fluorescent expression. Cre positive cells were quantitated using ‘ImageJ’ software program. The DMP1-vi Cre mouse results showed significant expression in the targeted osteocytes and osteoblasts. In addition, skeletal muscle tissue also showed significant Cre- expression. Ctsk-Cre mice showed significant expression in targeted osteoclasts. But brain tissue was positive in Cre-expression. Bone-Cre mouse models are expected to express Cre recombinase only in their respective bone cells and they have been used for gene deletion studies in bone cells. However, this study has revealed that the bone cell specific Cre mouse models DMP1-Cre and Ctsk-Cre have unexpected expression in muscle and brain respectively. In order to use these models for targeted gene deletion in bone cells, further testing and studies have to be conducted.

A population of stromal cells that retains osteogenic capacity in adult bone (adult bone stromal cells or aBSCs) exists and is under intense investigation in relation to osteogenesis and relevant pathology. aBSCs may be different from their embryonic or neonatal counterparts, and are influenced by species-/age-specific and other factors. Mice heterozygous for a null allele of prkar1a (Prkar1a+/-, a gene encoding for cyclic adenosine mono-phosphate (cAMP)-dependent regulatory subunit of protein kinase A (PKA), developed bone lesions that resembled fibrous dysplasia (FD) originated from cAMP-responsive osteogenic cells. Prkar1a +/- mice were crossed with mice heterozygous for catalytic subunit Calpha (Prkaca+/-), the main PKA activity-mediating molecule and generated mouse model with double heterozygosity for prkar1a and prkaca (Prkar1a +/-Prkaca+/-). Unexpectedly, Prkar1a+/-Prkaca+/- mice developed a large number of osseous lesions starting at 2--3 months of age that varied from the rare chondromas in the long bones and the ubiquitous osteochondrodysplasia of tail vertebral bodies to the occasional sarcoma in older animals. Cells from these lesions were fibroblast- and FD-like, and almost always originated from an area proximal to the growth plate and adjacent to endosteal surface of the periosteum; they expanded gradually in the bone marrow space. These cells expressed osteogenic cell markers, showed higher PKA activity that was mostly type II (PKA-II) and display an alternate pattern of catalytic subunit expression, and surprisingly possessed higher cAMP levels. In addition, markers of bone synthesis and lysis were increased. Gene expression profiling not only confirmed an early (progenitor) osteoblastic nature for these cells but also showed a signature that was indicative of mesenchymal-to-epithelial (MET) transition and increased Wnt signaling, particularly the brachyury expression. These studies show that a specific subpopulation of aBSCs can be stimulated in adult bone by PKA-II and altered Calpha activity, generating the only available germline mutant mouse model of a disorder that has similarities to human FD. Along with previous data, these studies also suggest that the effects of cAMP signaling on osteogenesis and stromal cell maintenance and proliferation in mice are age-, bone-, site- but also PKA-type and catalytic subunit-specific. / Parts of the work have been published in Proceedings of the National Academy of Sciences of the United States of America 2010; 107(19):8683--8. / Tsang, Kit Man. / Advisers: Constantine A. Stratakas; Kwak-Pui Fung. / Source: Dissertation Abstracts International, Volume: 72-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 144-183). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Indiana University-Purdue University Indianapolis (IUPUI) / Signal Transducer and Activator of Transcription 3 (Stat3) is a transcription factor expressed in bone and joint cells that include osteoblasts, osteocytes, osteoclasts, and chondrocytes. Stat3 is activated by a variety of cytokines and growth factors, including IL-6/gp130 family cytokines. These cytokines not only regulate the differentiation of osteoblasts and osteoclasts, but also regulate proliferation of chondrocytes through Stat3 activation. In 2007, mutations of Stat3 have been confirmed to cause a rare human immunodeficiency disease – Job syndrome which presents skeletal abnormalities like: reduced bone density (osteopenia), scoliosis, hyperextensibility of joints, and recurrent pathological bone fractures. Changes in the Stat3 gene alter the structure and function of the Stat3 proteins, impairing its ability to control the activity of other genes. However, little is known about the effects of Stat3 mutations on bone cells and tissues. To investigate the in vivo physiological role of Stat3 in bone homeostasis, osteoblast/osteocyte-specific Stat3 knockout (KO) mice were generated via the Cre-LoxP recombination system. The osteoblast/osteocyte-specific Stat3 KO mice showed bone abnormalities and an osteoporotic phenotype because of a reduced bone formation rate. Furthermore, inactivation of Stat3 decreased load-driven bone formation, and the disruption of Stat3 in osteoblasts suppressed load-driven mitochondrial activity, which led to an elevated level of reactive oxygen species (ROS) in cultured primary osteoblasts. Stat3 has been found to be responsive to mechanical stimulation, and might play an important role in mechanical signal transduction in osteocytes. To investigate the role Stat3 plays in mechanical signaling transduction, osteocyte-specific Stat3 knockout (KO) mice were created. Inactivation of Stat3 in osteocytes presented a significantly reduced load-driven bone formation. Decreased osteoblast activity indicated by reduced osteoid surface was also found in osteocyte-specific Stat3 KO mice. Moreover, sclerostin (SOST) protein which is a critical osteocyte-specific inhibitor of bone formation, its encoded gene SOST expression has been found to be enhanced in osteocyte-specific Stat3 KO mice. Thus, these results clearly demonstrated that Stat3 plays an important role in bone homeostasis and mechanotransduction, and Stat3 is not only involved in bone-formation-important genes regulation in the nucleus but also in mediation of ROS and oxidative stress in mitochondria.