published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

惡性腫瘤嚴重威脅著人類的身體健康，其治療也成為人類關注的焦點。傳統的化學療法和放射療法由於缺乏特異性，取得療效的同時往往也帶來較大的毒副作用。隨著對腫瘤發生發展分子機制認識的不斷深入，腫瘤的基因治療已成為攻克和治愈腫瘤最具希望和挑戰的研究領域。近年來研究發現骨髓間充斥幹細胞(MSCs)可被募集至腫瘤或損傷部位并參與腫瘤生長或組織修復，研究證明間充斥幹細胞通過靜脈注入帶瘤鼠（比如乳腺癌、膠質瘤、結腸癌及黑色素瘤）體內后，特異性的分佈于生長中的腫瘤中。這種特異性向腫瘤組織趨化轉移的特性使得骨髓間充斥幹細胞成為腫瘤基因靶向治療的載體的理想細胞。酶蛋白基因如單純皰疹病毒胸苷激酶(HSV-TK)可以使一些無毒或低毒的前藥轉化為強細胞毒性物質，殺死腫瘤細胞。我們前期實驗結果表明，通過遺傳改造后的表達TK基因的MSCs在GCV的存在下，具有殺傷腫瘤細胞抑制腫瘤生長的能力。但沒有改造的MSCs遷移至腫瘤之後可能會分化成成纖維細胞或者腫瘤基質細胞等支持腫瘤生長，但其命運和影響到底如何，我們怎麼樣進一步促進其向腫瘤的遷移以提高殺傷腫瘤的效率是本研究需要解決的問題。 / 本研究擬採用免疫螢光組織化學技術和分子生物學等技術研究和觀察MSCs對腫瘤（以乳腺癌，前列腺癌為例）的趨化過程及其在腫瘤生長中的作用，在在此基礎上研究促進攜帶HSV-TK自殺基因的MSCs的腫瘤靶向性細胞治療策略，採用分子和細胞生物學等方法評估其對荷瘤鼠體內腫瘤殺傷的原理，為利用TK-MSCs腫瘤的靶向治療奠定基礎。 / 研究結果顯示體外共培養的條件下，小鼠骨髓間充斥幹細胞可促進小鼠乳腺癌細胞增長，且增長速度同培養體系中間充斥幹細胞數目呈正相關。將兩種細胞混合注射于裸鼠體內，相比共注射小鼠皮膚成纖維細胞，間充斥幹細胞可促進體內腫瘤生長。使用人胚胎骨髓間充斥幹細胞和前列腺癌細胞可得出類似的效果。將腫瘤組織切片分析發現間充斥幹細胞促進體內腫瘤細胞增殖的同時，提高了腫瘤組織內血管密度。體外實驗發現共培養前列腺癌細胞和間充斥幹細胞可促進血管生成且在間充斥細胞內同血管增生相關的蛋白表達量都有相應提高，進一步證實間充斥幹細胞可能通過促進血管增生從而促進腫瘤生長。另外，我們利用人胚胎來源的骨髓間充斥幹細胞建立了穩定表達TK自殺基因的細胞系，且在GCV的存在下具有抑制腫瘤生長的能力。為了促進它們向腫瘤遷移的能力，我們用多柔比星預處理腫瘤細胞，和沒處理過的對照組相比，能增強對表達TK的間充斥幹細胞的招募能力。且在聯合利用多柔比星和TK的條件下，腫瘤生長能得到較大程度的抑制，這種抑制作用强於單獨使用多柔比星和表達自殺基因的間充斥幹細胞系統。初步認為是多柔比星的處理能增強腫瘤組織內炎性介質的分泌從而增強間充斥幹細胞的遷移達到增強自殺基因系統殺死腫瘤細胞的目的。 / 總的來說，雖然間充質幹細胞對腫瘤的生長存在一定的促進作用，但我們仍能對其進行遺傳改造，且在其它抗腫瘤藥的配合下達到最大的抗腫瘤效果。 / Eradication of cancer, especially when it has metastasized is extremely difficult and conventional cancer therapies are simply unable to specifically target tumors/cancers, thus causing unwanted side effects and complications. Recently, it has been shown that bone marrow mesenchymal stem cells (MSCs) are able to migrate specifically to tumors and contribute to the formation of tumor-associated stroma. These properties make MSCs good candidates as anti-tumor agent delivery vehicles and lead to a great deal of interest in the possibility of genetically modifying MSCs to express anticancer molecules and using them as specific targeted anticancer agents. We and others have showed that MSCs have the ability to migrate towards various cancer cells including breast, colon, fibrosarcoma and prostate cancer cells. Suicide gene therapy is widely used in cancer gene therapy. When stably infected with herpes simplex virus thymidine kinase gene by lentivirus, TK-MSCs maintained their MSCs characters and tumor tropism potential and significantly inhibited tumor growth, in the presence of the pro-drug ganciclovir (GCV). Improve MSCs homing to tumor tissue as anti-tumor gene therapy vehicles and maximizing their tumor killing effects is highly warranted. Furthermore, MSCs interact with tumor cells in numerous ways, which have the potential to support or suppress tumor growth. Therefore the fate and role of MSCs engrafted in tumor sites need to be clarified in order to making better use of these cells as anti-cancer agent delivery vehicles. / The aims of the current study are: (1) to study the role and fate of MSCs homed into the tumors; (2) to establish human bone marrow MSCs that stably express the TK genes; (3) to investigate the methods that enhance the anti-tumor efficiency of TK-MSCs. / In this study, bone marrow-derived mesenchymal stem cells from mice or human fetus were isolated and characterized. Effects of BM-MSCs on tumor cell proliferation in vitro were analyzed in a co-culture system with mouse breast cancer cell 4T1 cells. Both co-culture with BM-MSCs and treatment with MSC-conditioned medium led to enhanced growth of 4T1 cells. Co-injection of 4T1 cells and MSCs into nude mice led to increased tumor size compared with injection of 4T1 cells alone. Identical experiments using human prostate cancer cell DU145 cells and hBM-MSCs instead of 4T1 cells and mBM-MSCs yielded similar results. Compared with tumors induced by injection of cancer cells alone, tumor vessel area was greater in tumors from co-injection of 4T1 or DU145 with BM-MSCs, which correlated with decreased central tumor necrosis and increased tumor cell proliferation. Furthermore, both conditioned medium from co-cultures of hBM-MSCs and DU145 cells or hBM-MSCs alone was able to induce angiogenesis in human umbilical vein endothelial cells (HUVEC). When hBM-MSCs exposed to DU145 cells environment, the expression of markers associated with neovascularization (α-SMA, VEGF, TGF-β and IL6) were increased. Together, these results indicate that MSCs promote tumor growth both in vitro and in vivo and suggest that tumor promotion in vivo may be attributable in part to enhanced angiogenesis. / Immortalized human fetal bone marrow-derived MSCs (hfBMSCs) expressing herpes simplex virus thymidie kinase was established by conventional lentiviral transduction method. Functional expression of TK was evaluated by cytotoxicity in the presence of its prodrug GCV. SV40-TK-hfBMSCs exhibited comparable proliferation, surface phenotype expression, multi-differentiation potential and tumor-tropic migration ability as hfBMSCs. By measurement of tumor volume, repeated injection of the SV40-TK-hfBMSCs and subsequent consecutive GCV administration could suppress tumor growth in DU145 or PC3 human prostate tumor xenograft nude mice model without causing weight loss. However, its clinical applications are still limited. Alternative strategies have been pursued in this study by the use of combination therapy with cytotoxic chemotherapy to improve the overall efficacy of the TK-hfBMSCs/GCV system. / TK-hfBMSCs/GCV was evaluated alone or combined with low-dose doxorubicin in human prostate carcinoma DU145 xenografts in nude mice, testing for effects on local growth and overall survival. Tissues were evaluated through immunofluorescence and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining (TUNEL) for treatment effects on tumor cell proliferation and apoptosis. Transwell migration assay was used to access the migration ability of TK-hfBMSCs to tumor cells upon doxorubicin treatment and caspase-3 activity was conducted for test the tumor cells apoptosis under TK-hfBMSCs/GCV, doxorubicine, or combination of the two compound treatments respectively. Only minimal growth inhibition was observed in DU145 after treatment with TK-hfBMSCs/GCV or doxorubicin alone at doses and time points as indicated. In contrast, the combination of both agents resulted in a significant growth inhibition. Caspase-3, plays a central role in the execution-phase of cell apoptosis, was increased by TK-hfBMSCs/GCV or doxorubicine and also to a much greater extent by the combination treatment. Treatment by TK-hfBMSCs/GCV resulted in only a slight decrease in tumor growth compared with controls. Treatment with low-dose doxorubicin alone resulted in a small, nonstatistically significant decrease in tumor growth; In contrast, combined low-dose doxorubicin and TK-hfBMSCs/GCV was markedly inhibitory compared with control, doxorubicin alone, or TK-hfBMSCs/GCV alone. During the whole treatment process, no significant weight loss was observed. Furthermore, combined therapy induced increased area of necrosis, significant apoptosis and decreased tumor cell proliferation in treated tumors. Taken together, low dosage of doxorubicin could be used in combination with TK-hfBMSCs based suicide gene therapy. / In conclusion, we have demonstrated that BM-MSCs could increase the growth of human prostate cancer and mouse breast cancer. The promotion effect may partly attribute to the increased expression of pro-angiogenic factors in BM-MSCs in tumor microenvironment and subsequent enhancement in angiogenesis and tumor growth. The current study also suggests combination of TK-hfBMSCs/GCV and doxorubicin was more effective in inhibiting prostate cancer cells growth than TK-hfBMSCs/GCV or doxorubicin alone. Although many problems need to be resolved for further application, our study provided the possibility of a new strategy of suicide gene-based therapy accompanied by low dosage of chemotherapy in treating prostate cancer. Therefore MSCs were described as a “double-edged sword in their interaction with tumors. However, if MSCs are suitably engineered with anticancer genes they could be employed as a valuable “single-edged sword“ against cancers. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zhang, Ting. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 120-158). / Abstracts also in Chinese. / ACKNOWLEDGEMENT --- p.ii / PUBLICATIONS --- p.vii / ABSTRACT --- p.xiii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Mesenchymal stem cells (MSCs) --- p.2 / Chapter 1.2 --- Tumor microenvironment and involvement of MSCs in tumor establishment --- p.5 / Chapter 1.3 --- Tumors-tropic characteristics of MSCs --- p.15 / Chapter 1.4 --- Impact of MSCs on in vivo tumors --- p.21 / Chapter 1.5 --- In vivo imaging demonstrating MSCs tumor-homing potentials --- p.25 / Chapter 1.6 --- Evidence for use of MSCs as anti-tumor agents delivery vehicles --- p.26 / Chapter 1.7 --- Homing strategies to enhance efficacy and safety of MSCs therapy --- p.32 / Chapter 1.8 --- Summary --- p.35 / Chapter CHAPTER 2 --- Hypotheses, Objectives and Study Design --- p.35 / Chapter 2.1 --- Hypothesis --- p.35 / Chapter 2.2 --- Objective --- p.36 / Chapter 2.3 --- Study design --- p.37 / Chapter CHAPTER 3 --- Bone Marrow-derived Mesenchymal Stem Cells Promote Growth and Angiogenesis of Breast and Prostate Tumors (Study I) --- p.40 / Chapter 3.1 --- Materials and Methods --- p.40 / Chapter 3.2 --- Results --- p.49 / Chapter 3.3 --- Discussion --- p.64 / Chapter 3.4 --- Conclusions --- p.67 / Chapter CHAPTER 4 --- Immortalized human fetal bone marrow-derived mesenchymal stem cell expressing anti-tumor suicide gene for anti-tumor therapy in vitro and in vivo (Study II) --- p.68 / Chapter 4.1 --- Materials and Methods --- p.68 / Chapter 4.2 --- Results --- p.73 / Chapter 4.3 --- Discussion --- p.85 / Chapter CHAPTER 5 --- Enhanced antitumor effects by combination therapy using mesenchymal stem cell expressing anti-tumor suicide gene and Doxorubicin in a xenograft mouse model (Study III) --- p.89 / Chapter 5.1 --- Materials and Methods --- p.89 / Chapter 5.2 --- Results --- p.97 / Chapter 5.3 --- Discussion --- p.111 / Chapter CHAPTER 6 --- General discussion and conclusions --- p.116 / Chapter 6.1 --- General discussion --- p.116 / Chapter 6.2 --- General conclusions --- p.119 / FUNDING --- p.120 / REFERENCE --- p.120

Lin, Ka Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-122). / Abstracts in English and Chinese. / Abstract (In English) --- p.ii / Abstract (In Chinese) --- p.iii / Acknowledgment --- p.V / Abstracts of Publications --- p.vi / Abbreviations --- p.viii / List of Figures --- p.ix / List of Table --- p.X / Contents --- p.xi / Chapter Chapter I --- Introduction --- p.Page / Chapter 1.1 --- Pluripotent Stem Cell --- p.1 / Chapter 1.1.1 --- Characteristic of pluripotent stem cells --- p.1 / Chapter 1.1.2 --- Origin of pluripotent stem cells --- p.1 / Chapter 1.1.2.1 --- Embryonic carcinoma cells --- p.2 / Chapter 1.1.2.2 --- Embryonic stem cells --- p.2 / Chapter 1.1.2.3 --- Epiblast stem cells --- p.2 / Chapter 1.1.2.4 --- Embryonic germ cells and adult germline stem cells --- p.3 / Chapter 1.1.2.5 --- Induced pluripotent stem cells --- p.3 / Chapter 1.1.3 --- Pluripotency in Embryonic Stem Cells --- p.4 / Chapter 1.1.3.1 --- Extrinsic signal governing pluripotency --- p.5 / Chapter 1.1.3.1.1 --- LIF signaling --- p.5 / Chapter 1.1.3.1.2 --- BMP signaling --- p.6 / Chapter 1.1.3.1.3 --- Other signaling pathways --- p.6 / Chapter 1.1.3.2 --- Intrinsic sternness factors --- p.7 / Chapter 1.1.3.2.1 --- Oct4 Expression in Embryonic Stem cells --- p.7 / Chapter 1.1.3.2.2 --- Sox-2 Expression in Embryonic Stem Cells --- p.8 / Chapter 1.1.3.2.3 --- Nanog Expression in Embryonic Stem Cells --- p.9 / Chapter 1.1.3.2.4 --- "Transcriptional Regulation of Oct-4, Nanog and Sox-2 in Embryonic Stem Cells" --- p.10 / Chapter 1.1.3.2.5 --- Others pluripotent genes --- p.11 / Chapter 1.1.3.2.5.1 --- Utfl --- p.11 / Chapter 1.1.3.2.5.2 --- Rexl --- p.11 / Chapter 1.1.3.2.5.3 --- Esrrb --- p.12 / Chapter 1.1.3.2.5.4 --- Eras --- p.12 / Chapter 1.1.3.2.5.5 --- Tell --- p.12 / Chapter 1.1.3.2.5.6 --- Dnm3tl --- p.13 / Chapter 1.1.3.2.5.7 --- Dppa3 --- p.13 / Chapter 1.1.3.2.5.8 --- Dppa4 --- p.14 / Chapter 1.1.3.2.5.9 --- Dppa5 --- p.14 / Chapter 1.1.3.2.5.10 --- Klf2 --- p.15 / Chapter 1.2 --- Somatic cell reprogramming --- p.16 / Chapter 1.2.1 --- Definition of reprogramming --- p.16 / Chapter 1.2.2 --- The history of reprogramming --- p.16 / Chapter 1.2.2.1 --- Reprogramming by nuclear transfer --- p.17 / Chapter 1.2.2.2 --- Reprogramming by fusion with ES or EC cells --- p.18 / Chapter 1.2.2.3 --- Reprogramming with defined factor --- p.19 / Chapter 1.3 --- Induced pluripotent stem cells --- p.20 / Chapter 1.3.1 --- Transcription factor used for reprogramming to iPS cells --- p.20 / Chapter 1.3.1.1 --- Klf4 --- p.20 / Chapter 1.3.1.2 --- c-Myc --- p.21 / Chapter 1.3.2 --- Cornerstone of iPSC generation --- p.22 / Chapter 1.3.3 --- Major events in the reprogramming process --- p.23 / Chapter 1.3.4 --- Gene delivery systems for ips cell generation --- p.26 / Chapter 1.3.5 --- Culture system for embryonic stem cells and iPSC --- p.28 / Chapter 1.3.4.1 --- Feeder and serum used cell culture system --- p.28 / Chapter 1.3.4.2 --- Serum-free culture condition --- p.29 / Chapter 1.3.5 --- Differentiation potential of iPSC --- p.30 / Chapter 1.3.5.1 --- In vitro stringency tests --- p.30 / Chapter 1.3.5.2 --- In vivo stringency test --- p.30 / Chapter 1.3.5.3 --- In utero stringency test --- p.31 / Chapter 1.4 --- Mouse Lewis lung carcinoma-D 122 --- p.32 / Chapter 1.5 --- Dendritic cell vaccine in cancer immunotherapy --- p.33 / Chapter 1.5 --- Green Fluorescence protein Reporters --- p.35 / Chapter 1.5.1 --- GFP reporters in embryos and stem cell --- p.35 / Chapter 1.5.2 --- copGFP --- p.35 / Chapter 1.6 --- Aim of study --- p.36 / Chapter Chapter II --- Methods and Materials / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Synthetic oligos used in polymerase chain reaction (PCR) --- p.37 / Chapter 2.1.2 --- DNA clones used in the study --- p.39 / Chapter 2.1.3 --- Materials for DNA manipulation --- p.39 / Chapter 2.1.4 --- Materials for RNA manipulation --- p.39 / Chapter 2.1.5 --- Antibodies --- p.40 / Chapter 2.1.6 --- Kits --- p.41 / Chapter 2.1.7 --- Bacteria strain and culture reagents 41 / Chapter 2.1.8 --- Culture media and reagents --- p.42 / Chapter 2.1.8.1 --- General culture media and reagents --- p.42 / Chapter 2.1.8.2 --- Traditional ES medium --- p.42 / Chapter 2.1.8.3 --- Feeder-free Serum-free ESGRO medium --- p.42 / Chapter 2.1.9 --- Cell lines used --- p.43 / Chapter 2.1.10 --- Instrumentation --- p.43 / Chapter 2.2 --- Methods --- p.44 / Chapter 2.2.1 --- Cell culture --- p.44 / Chapter 2.2.1.1 --- Routine cell culture --- p.44 / Chapter 2.2.1.2 --- Resuscitation and culture from frozen stock --- p.44 / Chapter 2.2.1.3 --- Passage of cells --- p.44 / Chapter 2.2.1.4 --- Cryopreservation of cells --- p.45 / Chapter 2.2.1.5 --- Mouse ES cells culture --- p.45 / Chapter 2.2.1.5.1 --- Passage and maintenance of SNL --- p.45 / Chapter 2.2.1.5.2 --- Inactivation and plating of SNLs (Feeder preparation) --- p.45 / Chapter 2.2.1.5.3 --- Cryopreservation (freezing) of SNLs --- p.46 / Chapter 2.2.1.6 --- Mouse ES cells culture in feeder-free culture conditions --- p.46 / Chapter 2.2.1.6.1 --- Preparation of gelatin coated plates --- p.46 / Chapter 2.2.1.6.2 --- Thawing mouse ES cells --- p.46 / Chapter 2.2.1.6.3 --- Passage of mouse ES cells --- p.47 / Chapter 2.2.1.6.4 --- Freezing mouse ES cells --- p.47 / Chapter 2.2.1.7 --- ES cells differentiation-Formation of embryoid bodies (EBs) --- p.47 / Chapter 2.2.1.8 --- Direct differentiation by retinoic acid --- p.48 / Chapter 2.2.1.9 --- Generation of iPS --- p.48 / Chapter 2.2.2 --- Cell transfections --- p.48 / Chapter 2.2.2.1 --- Lipofectamine 2000 transfection --- p.48 / Chapter 2.2.2.2 --- Nucleofection --- p.49 / Chapter 2.2.2.2.1 --- Optimization of nucleofection --- p.49 / Chapter 2.2.2.2.2 --- Nucleofection condition --- p.49 / Chapter 2.2.3 --- Nucleic acid --- p.49 / Chapter 2.2.3.1 --- Genomic DNA isolation --- p.49 / Chapter 2.2.3.2 --- Restriction Enzyme Digestion --- p.50 / Chapter 2.2.3.3 --- RNA and genomic DNA quantification --- p.50 / Chapter 2.2.3.4 --- Reversed transcription polymerase chain reaction (RT-PCR) --- p.50 / Chapter 2.2.3.4.1 --- RNA isolation and Reverse transcription (RT) --- p.50 / Chapter 2.2.3.4.2 --- Polymerase chain reaction (PCR) --- p.51 / Chapter 2.2.3.4.3 --- Real-time polymerase chain reaction (qRT- PCR) --- p.52 / Chapter 2.2.3.5 --- Agarose gel electrophoresis --- p.53 / Chapter 2.2.3.6 --- Genomic PCR for bisulfite sequencing --- p.53 / Chapter 2.2.4 --- Bacteria and Plasmid preparation --- p.54 / Chapter 2.2.4.1 --- Preparation of competent cells --- p.54 / Chapter 2.2.4.2 --- Heat-shock transformation --- p.54 / Chapter 2.2.4.3 --- Midi prep of plasmid --- p.54 / Chapter 2.2.5 --- Cell Staining --- p.55 / Chapter 2.2.5.1 --- Alkaline phosphatase staining --- p.55 / Chapter 2.2.5.2 --- Immunofluorescence --- p.55 / Chapter 2.2.6 --- Flow cytometry --- p.56 / Chapter 2.2.7 --- Animal Handling --- p.56 / Chapter Chapter III --- Results / Chapter 3.1 --- Generation of Nanog-reporter-GFP-D 122 --- p.57 / Chapter 3.2 --- Nucleofection optimization for D122 --- p.60 / Chapter 3.3 --- Generation ofD122-iPS --- p.65 / Chapter 3.3.1 --- Plasmid construct used in the study --- p.65 / Chapter 3.3.2 --- Protocol of D122-iPS generation --- p.67 / Chapter 3.3.3 --- Reprogramming Efficiency of D12´2ؤreNanog cells --- p.69 / Chapter 3.4 --- Expression of pluripotency markers upon reprogramming --- p.70 / Chapter 3.4.1 --- Alkaline Phosphatase staining --- p.70 / Chapter 3.4.2 --- Nanog-GFP expression --- p.72 / Chapter 3.4.3 --- Pluripotency gene expression upon reprogramming --- p.74 / Chapter 3.4.4 --- GFP positive D122 reNanog Colonies --- p.79 / Chapter 3.5 --- Characterization of the D122-iPS-lC --- p.80 / Chapter 3.5.1 --- Morphology of D122-iPS-lC --- p.80 / Chapter 3.5.2 --- Pluripotency gene expression --- p.82 / Chapter 3.5.3 --- Pluripotency markers SSEA-1 and Oct4 --- p.85 / Chapter 3.5.4 --- Bisulfite genomic sequencing --- p.88 / Chapter 3.5.5 --- Differentiation of the D122-iPS-lC --- p.90 / Chapter 3.5.5.1 --- Embryoid body formation by hanging drop --- p.90 / Chapter 3.5.5.2 --- Retinoic acid induced differentiation --- p.92 / Chapter Chapter IV --- Discussion / Chapter 4.1 --- General Discussion --- p.96 / Chapter 4.1.1 --- Cancer immunotherapy and dendritic cells --- p.96 / Chapter 4.1.2 --- Dendritic vaccine and tumor antigen --- p.97 / Chapter 4.1.3 --- Induced pluripotent stem cell technology and dendritic cells --- p.98 / Chapter 4.1.4 --- Tumor antigen presentation and dendritic cells --- p.98 / Chapter 4.1.5 --- D122 and cancer immunotherapy --- p.99 / Chapter 4.1.6 --- Method to introduce transcription factors for reprogramming --- p.100 / Chapter 4.1.7 --- Kinetics of reprogramming --- p.101 / Chapter 4.1.8 --- Culture condition for reprogramming D122_reNanog --- p.102 / Chapter 4.1.9 --- Reprogramming efficiency --- p.103 / Chapter 4.1.10 --- Establishment of D122-iPS-lC --- p.103 / Chapter 4.1.11 --- Differentiation of D122-iPS-1C --- p.104 / Chapter 4.2 --- Future Work --- p.106 / Chapter 4.3 --- Conclusion --- p.107 / Chapter Chapter V --- Bibliography --- p.108 / Appendix --- p.124

Bone loss from the cranio-maxillofacial region is a major clinical problem affecting patients worldwide. Conventional treatment strategy includes the use of autogenous or allogeneic bone, biomaterials, and osteogenic growth factors. However, there has been no effective therapy for most cases so far. Stem cell-based gene therapy is the latest research method with possible applications in humans. The present study aims to (1) characterize rabbit mesenchymal stem cells (MSCs) relating to growth pattern, surface antigens, and the potential for multi-differentiation; (2) determine the transduction efficiency and duration of recombinant adeno-associated virus2 carrying enhanced green fluorescent protein (rAAV2EGFP) reporter gene in rabbit MSCs and study the effects of rAAV2EGFP transduction on stem cells’ phenotype and capacity of multi-differentiation; (3) evaluate the differentiation characteristics of rabbit MSCs following recombinant adeno-associated virus 2 carrying bone morphogenetic protein 2 gene (rAAV2BMP2) transduction; (4) investigate whether MSCs transduced by rAAV2BMP2 could successfully induce bone regeneration in rabbit critical-size cranial defects. MSCs were isolated from bone marrows of rabbit tibias and cultured. Cell counting and colony-forming assays demonstrated that growth rates of MSCs dropped substantially with increasing passages. Flow cytometry on MSCs at passage 1 showed that cells expressed high level of CD49a and low level of CD44 as well as stage-specific embryonic antigen 4 (SSEA4). Multi-differentiation and reverse transcriptase-polymerase chain reaction (RTPCR) tests demonstrated that rabbit MSCs were capable to differentiate into osteocytes, chondrocytes and adipocytes. Immunofluorescence microscopy showed that rabbit MSCs produced a series of hematopoietic growth factors, including stem cell factor (SCF), vascular endothelial growth factor-A (VEGFA) and granulocyte macrophage colony-stimulating factor (GMCSF). Subsequently, rabbit MSCs were transduced with rAAV2EGFP in vitro. By comparing the transduction efficiency with different doses of rAAV2EGFP particles, multiplicity of infection (MOI) of 1 x 10 4 was identified as an optimal parameter for the transduction of rAAV2 in rabbit MSCs. Fluorescent microscopy demonstrated long-term expression of EGFP in rabbit MSCs after transduction both in vitro and in vivo. In addition, cell proliferation assay, adipogenic induction test and flow cytometry showed that rAAV2EGFP transduced MSCs exhibited a similar pattern with non-transduced cells on the cell growth, capacity of adipogenic differentiation and expression of surface antigens, indicating that rabbit MSCs maintain their stem cell properties after rAAV2EGFP transduction. / published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Aging of the hematopoietic system promotes various immune and systemic disorders and is driven in-part by dysfunction of life-long self-renewing hematopoietic stem cells (HSC). Autophagy is required for the benefit associated with activation of conserved longevity signaling programs and is essential for HSC function in response to various stressors. With age, some HSCs basally increase autophagy flux and maintain inert metabolic activity. This autophagy-activated subset is responsible for the residual regenerative capacity of old stem cells, but the mechanisms promoting autophagy activation in HSC aging remain unknown. Here, we demonstrate that autophagy is a response to chronic inflammation in the aging HSC niche. Chronic inflammation impairs glucose metabolism in young and old HSCs (oHSC) by impeding AKT-FOXO intracellular signaling networks. We find that autophagy enables metabolic adaptation of oHSCs to non-glucose energy substrates for functional maintenance. Notably, water-only fasting transiently further activates autophagy in oHSCs, and upon refeeding normalizes glucose uptake and glycolytic flux as well as regenerative output. Our results demonstrate that inflammation-driven glucose hypometabolism impairs oHSC regenerative capacity, that autophagy activation metabolically adapts oHSCs to an inflamed niche, and that autophagy is a modulable node to restore glycolytic and regenerative capacity during stem cell aging.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2007. / SUPERFICIAL digital flexor tendon injury has a serious negative impact on the competitive horse industry. Injured horses require up to a year of rest for recovery and likelihood of re-injury upon return to normal activity is as high as 80 %. Tendon healing requires (a) production of collagen by fibroblasts, to provide tensile strength and elasticity to the tendon, (b) minimisation of restrictive fibrosis, which compromises tendon gliding function and (c) minimisation of peritendinous adhesions. We review conventional treatments for tendon healing before exploring stem cell application as a therapeutic alternative. We promote the use of hematopoietic and mesenchymal stem cells derived from adult peripheral blood - as opposed to bone marrow-derived stem cells or embryonic stem cell sources - and review published research output in this regard. In conclusion, we outline our research objectives and present and discuss our results in the chapters that follow. Mononuclear cells - consisting of hematopoietic stem cells, mesenchymal stem cells and leucocytes – were isolated from the peripheral blood of sheep and horses through red blood cell lysis and blood plasma extraction. Cell counts and propidium iodide dye exclusion viability tests were conducted on the cell pellets. Sheep sub samples were tested for CD45 expression and horse sub samples for CD4 and CD11a/18 cell surface markers by flow cytometry for characterisation purposes. In both cases, separate sub samples were incubated with matched immunoglobulin (IgG) isotypes, conjugated to fluorescein isothiocyanate (FITC), to serve as controls. For the culture of mononuclear cells, 4.5 x 106 cells were selected for autologous sheep injections, 3 x 106 CD45- cells for allogeneic sheep injections (the latter excluding leucocytes that may induce an immune response) and 72 x 106 cells for horse injections. These cells were incubated with bromo-deoxyuridine (BrdU), cultured and subsets were extracted for a second round of cell counts and viability tests before being resuspended in blood plasma. For the horse samples an additional 1 x 106 mononuclear cells were incubated until reaching 60 % confluence and tested for myogenic differentiation. Low cell mortality and lack of fluorescence from IgG-FITC controls reflected effective protocols and a lack of false positive results. The fact that the equine cell population differentiated into myotubes verified the presence of mesenchymal stem cells in injections. We tested whether surgical incisions or collagenase injections best mimicked naturally occurring tendon injuries and compiled macroscopic and microscopic descriptions of tendon injury sites at seven weeks post-injury. The superficial digital flexor tendons of 27 sheep received an incision, a collagenase injection or a saline control injection. After one week a number of sheep were sacrificed while the remainder received further saline treatment and were sacrificed after another seven weeks. Tendons were examined through clinical observations, image analysis of maximum tendon diameter, mechanical testing and histological sectioning of affected tissues. Collagenase-induced injury resembled tendonitis more closely than surgically-induced injury. Collagenase-injured tendons (a) induced lengthier lameness in affected limbs, (b) were more swollen and difficult to palpate, (c) assumed the bow appearance characteristic of natural injury, (d) experienced extensive haemorrhage due to collagen lysis, (e) had decreased elasticity and capacity to carry loads and stress, (f) displayed decreased stiffness due to collagen fibre disruption and (g) developed severe inflammation. After seven weeks injured tendons displayed increased vascularisation in the areas of haemorrhage and in the adjacent collagen matrix. High inflammation rates and low collagen levels however still persisted. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of 27 sheep. After one week these tendons received treatment with a control saline solution, autologous peripheral blood mononuclear cells (MNCs) or allogeneic peripheral blood CD45- MNCs. Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analyses of maximum tendon diameter, mechanical tests and histological investigations. Tendons treated with MNCs displayed an improvement in echogenicity and fibre linearity, higher and more organised collagen levels, stronger mechanical properties and less swelling. Although these improvements were not always significant, they provided strong evidence to suggest marked healing benefits over a longer time period. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of four horses. After one week these tendons received treatment with either a control saline solution or autologous peripheral blood mononuclear cells (MNCs). Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analysis of maximum tendon diameter and histological investigations. Tendons treated with MNCs displayed significant improvements in fibre linearity in the direct vicinity of the lesion, as well as recovery rate thereof, and experienced less swelling when compared with their untreated counterparts. Healing trends suggested that, given a longer period of observation post-injury, more significant improvements may become apparent. Human adipose tissue is known be an easily accessible and high yielding source of multipotent mesenchymal stem cells. These stem cells could potentially be used for therapeutic advancement of tendon regeneration. Our first goal was to examine the in vitro myogenic differentiation potential of adipose-derived, adherent mononuclear cells (MNCs) from six adult sheep. The second goal was to characterise the population of cells isolated through various available ovine specific, non-mesenchymal stem cell surface markers, namely, CD1, CD31, CD34 and CD45. After incubation, only four of the six MNC cultures started to proliferate. These four cultures all exhibited high myogenic differentiation ability. The isolated cell populations did not express any of the non-mesenchymal stem cell specific cell surface markers. In conclusion, our data suggests that peripheral blood stem cells and adipose-derived stem cells are important candidate cell types for therapeutic application to improve tendon repair in horses and sheep. Sufficient time must be allowed following injury and prior to stem cell treatment (at least one month) and a controlled exercise program should be followed posttreatment. A larger sample size is required and at least six months of recovery before macroscopic and histological repair can be analysed more accurately and conclusively. Ultrasonography should be carried out on a continuous basis, as it is a non-invasive method of monitoring change over time.

Indiana University-Purdue University Indianapolis (IUPUI) / Human induced pluripotent stem (iPS) cells have the unique ability to differentiate into 200 or so somatic cell types that make up the adult human being. The use of human iPS cells to study development and disease is a highly exciting and interdependent field that holds great promise in understanding and elucidating mechanisms behind cellular differentiation with future applications in drug screening and cell replacement studies for complex and currently incurable cellular degenerative disorders. The recent advent of iPS cell technology allows for the generation of patient-specific cell lines that enable us to model the progression of a disease phenotype in a human in vitro model. Differentiation of iPS cells toward the affected cell type provides an unlimited source of diseased cells for examination, and to further study the developmental progression of the disease in vitro, also called the “disease-in-a-dish” model. In this study, efforts were undertaken to recapitulate the differentiation of distinct retinal cell affected in two highly prevalent retinal diseases, Usher syndrome and glaucoma. Using a line of Type III Usher Syndrome patient derived iPS cells efforts were undertaken to develop such an approach as an effective in vitro model for studies of Usher Syndrome, the most commonly inherited disorder affecting both vision and hearing. Using existing lines of iPS cells, studies were also aimed at differentiation and characterization of the more complex retinal cell types, retinal ganglion cells (RGCs) and astrocytes, the cell types affected in glaucoma, a severe neurodegenerative disease of the retina leading to eventual irreversible blindness. Using a previously described protocol, the iPS cells were directed to differentiate toward a retinal fate through a step-wise process that proceeds through all of the major stages of neuroretinal development. The differentiation process was monitored for a period of 70 days for the differentiation of retinal cell types and 150 days for astrocyte development. The different stages of differentiation and the individually derived somatic cell types were characterized by the expression of developmentally associated transcription factors specific to each cell type. Further approaches were undertaken to characterize the morphological differences between RGCs and other neuroretinal cell types derived in the process. The results of this study successfully demonstrated that Usher syndrome patient derived iPS cells differentiated to the affected photoreceptors of Usher syndrome along with other mature retinal cell types, chronologically analogous to the development of the cell types in a mature human retina. This study also established a robust method for the in vitro derivation of RGCs and astrocytes from human iPS cells and provided novel methodologies and evidence to characterize these individual somatic cell types. Overall, this study provides a unique insight into the application of human pluripotent stem cell biology by establishing a novel platform for future studies of in vitro disease modeling of the retinal degenerative diseases: Usher syndrome and glaucoma. In downstream applications of this study, the disease relevant cell types derived from human iPS cells can be used as tools to further study disease progression, drug screening and cell replacement strategies.