The biliary tree is a series of ductular tissues responsible for the drainage of bile produced by the liver and pancreatic secretions from the pancreas. The biliary tree is affected by a diversity of life- threatening diseases collectively called cholangiopathies. Cholangiopathies show regionalization, with some diseases such as biliary atresia predominantly targeting extrahepatic bile ducts (EHBDs) outside of the liver. Despite this, little is known on whether anatomical location within the biliary tree contributes to differences in functionality of biliary epithelium, especially in the EHBD compartment. Additionally, reports have demonstrated the possibility for in vitro culture of bile duct stem/progenitor cell organoids from both intrahepatic (IHBD) and EHBD sources. The relation of these organoid systems to each other, and to their tissue of origin, is largely unknown. In this dissertation, I address these major questions by combining transcriptional analyses and in vitro culture of human bile duct organoids derived from primary IHBD and EHBD epithelium. First, I show that in vitro organoids can be derived from four regions of the human biliary tree: gallbladder, common bile duct, pancreatic duct, and intrahepatic bile ducts. Characterization of these organoids demonstrated expression of adult stem cell (LGR5/PROM1) and ductal (KRT19/KRT7) markers suggesting these cultures contained cells with a biliary stem/progenitor phenotype. Further, I show that IHBD organoids are distinct from EHBD organoids requiring different conditions for sustained growth. Using RNA-Sequencing, I demonstrate that primary tissues from different regions of the extrahepatic biliary tree display unique expression profiles and identify novel tissue-specific markers. I also show that only a limited number of these tissue specific differences are maintained in the in vitro organoids and that the organoids are very different from their tissue of origin. Finally, I demonstrate that IHBD, but not EHBD organoids, express a low-level of hepatocyte-specific markers under differentiation conditions. Taken together, the work in this dissertation has uncovered regional specific markers for different anatomical regions of the human biliary tree. Further, I demonstrate that major differences exist between IHBD organoids and EHBD organoids in vitro and discover that only IHBD organoids have the capacity to express hepatocyte markers under differentiation conditions. Ultimately, these results may help to identify new targets for therapeutic development for cholangiopathies and regenerative medicine. They have also provided important insight to the understanding of both basic biliary physiology and also the field of biliary stem/progenitor cell organoids.

Epigenetic mechanisms are of fundamental importance for resolving and maintaining cellular identity. The mechanisms regulating muscle stem and progenitor cell identity have ramifications for understanding all aspects of myogenesis. The epigenetic mechanisms regulating muscle stem cells are therefore important aspects for understanding the regulation of muscle regeneration and maintenance. Important roles for the trithorax H3K4 histone methyltransferase (HMT) MLL1 have been established for early embryogenesis, and for hematopoietic and neural identity. Here, using a conditional Mll1 knockout (KO), we find that in vivo, MLL1 is necessary for efficient muscle regeneration, and for maintenance and proliferation of muscle stem and progenitor cells. Loss of Mll1 in cultured myoblasts reveals an essential role for expression of the myogenic specification gene Pax7. Mll1 KO results in a minor decrease in Pax7 mRNA and a strong decrease of Pax7 protein. While MLL1 was found to bind the Pax7 promoter, Mll1 KO results in a minor decrease of H3K4me3 at Pax7, supporting a recognized non-HMT role for Mll1 at Pax7. Microarray analysis of mRNA expression in Mll1 KO myoblasts finds that Myf5 is the most strongly downregulated of all genes, unexpectedly, mRNA expression of previously identified MLL1 targets are unaffected by loss of MLL1 in myoblasts. Pax7 activates Myf5 expression through recruitment of a H3K4 HMT, and in Mll1 KO myoblasts expression of, and H3K4me3 at Myf5 is lost. Exogenous Pax7 rescues Myf5 expression and H3K4me3 at Myf5 in the absence of MLL1, indicating that Myf5 expression is conditional on Pax7, but not MLL1. We also show that Myf5 DNA is methylated in non-myogenic cells, and in satellite stem cells that have never expressed Myf5, but is not methylated in satellite cells that are committed to the myogenic lineage, indicating that demethylation of Myf5 may be a fundamental step in myogenic commitment. Intriguingly, Myf5 promoter DNA becomes remethylated in Mll1 KO myoblasts. This work finds that Pax7 expression and myogenic identity is partly dependent on MLL1 expression. Further, evidence is uncovered that myogenic commitment is initiated by demethylation of Myf5. These findings add to the understanding of the epigenetic mechanisms that regulate and define muscle stem cells.

Introduction. Safe closure of gastric luminal defects remains a big challenge for development of gastric endoscopic surgery. The aims of this thesis are to assess the effect and efficiency of Eagle Claw VIII (endoscopic suturing device)and adipose tissue-derived mesenchymal stem cells (ADMSCs) for closure and enhancing healing of gastric luminal defects. / Methods and Results. 1. Endoscopic suturing is superior to endoclips for closure of gastrotomy after NOTES. A 2cm linear incision on the body of porcine stomach was closed by hand suturing, Eagle Claw VIII or endoclips, respectively (n=17 for each group). The results indicated that all gastrotomies were successfully closed. Closure time was significantly longer in Eagle Claw VIII group. Bursting pressure of gastrotomies for Eagle Claw VIII was significantly higher than endoclips, but lower than hand suturing. Besides, both Eagle Claw VIII and endoclip closure encountered significantly technical challenges. This study suggested that Eagle Claw VIII had potential for endoscopic closure of gastrotomies, but need further refinement. / 2. ADMSCs for Acceleration of Healing of Sutured Gastric Perforation(SGP). ADMSCs were isolated and expanded in vitro, and characterized by stromal differentiations and cell surface markers. A 2cm SGP was produced on gastric body of rats. 5×10⁶ ADMSCs were transplanted into SGP by local injection (LI-ADMSCs) or topical spraying (TS-ADMSCs). Healing of SGP was assessed. LI-ADMCs significantly decreased peritoneal adhesion and wound dehiscence, and increased bursting pressure of SGP, when comparing to other experimental groups. Histologic analysis indicated that SGPs in LI-ADMSCs group had more re-epithelialization and collagen regeneration, and less inflammation. Expression of TGF-β1 was up-regulated, while IL-6 was down-regulated in LI-ADMSCs group, when comparing to fibrin and control groups. This study suggested that local injection of ADMSCs is an effective approach for accelerating the healing of SGP. / 3. Promoting Effect of ADMSCs on Healing of Gastric Ulcer is abrogated by NSAIDs. Gastric ulcer model in rats was successfully produced by using 70% acetic acid. A total of 1×10⁷ ADMSCs was locally injected into ulcer lesion. Ulcer area was measured at different time points. Therapeutic potentialof ADMSCs was assessed when NSAIDs was simultaneously administrated. The results demonstrated that ADMSCs significantly decreased ulcer area. Histologic assessment indicated that ADMSCs increased re-epithelialization, angiogenesis and collagen deposition, and suppressed inflammation. Transplanted ADMSCs homed into gastric ulcer lesion and differentiated into endothelial and smooth muscle cells. In addition, ADMSCs treatment increased the gene expressions for wound healing, and activated COX-2-PGE₂ and Erk1/2-MAPK signaling pathways. Repeated administration of Indomethacin reduced cell proliferation and angiogenesis, and eliminated ADMSCs-induced ulcer healing on day 10. The results suggested that ADMSCs promoted the healing of peptic ulcer, which is eliminated by NSAIDs. / Conclusions. Endoscopic suturing by Eagle Claw VIII is feasible for closure of gastrotomy, when comparing to endoclips. ADMSC promotes the healing of gastric luminal defects including SGP and ulcer. The promoting effect of ADMSC is PGE₂-dependent, and attenuated by NSAIDs. These evidences implied that combined use of endoscopic suturing and ADMSCs is a helpful approach for safe closure of gastrotomy and gastric perforation. / 引言：胃傷口癒合是胃消化內鏡手術發展的障礙之壹。本課題之目的是評價和探索Eagle Claw VIII和脂肪幹細胞(ADMSCs)縫合和促進胃內傷口癒合的效果和作用。 / 方法和結果：1. 內鏡縫合器Eagle Claw VIII閉合經胃自然腔道手術後傷口的效果評價體外豬胃體上造2cm的胃傷口模型，使用手工縫合、內鏡下Eagle Claw VIII縫合或內鏡夾閉合胃傷口；每組17個樣本。本研究提示所有胃傷口均成功閉合。Eagle Claw VIII縫合胃傷口時間顯著長於其他兩組的閉合時間。Eagle Claw VIII縫合的胃傷口破裂壓顯著高於內鏡夾閉組，但是明顯低於手工縫合組。此外，內鏡縫合和夾閉都面臨較大的技術難度。本研究提示Eagle Claw VIII有臨床運用的潛在價值，但需要進壹步改進。 / 2. 局部移植脂肪幹細胞促進胃穿孔癒合的實驗性研究：建立大鼠2cm胃體穿孔模型，局部註射或傷口表面塗抹法移植ADMSCs，觀察胃傷口癒合情況。局部註射移植ADMSCs顯著減輕胃傷口粘連和裂開發生率，增加胃傷口破裂壓。組織學分析提示ADMSCs治療促進傷口上皮和肉芽組織再生，抑制炎癥反應。此外，局部註射ADMSCs增加TGF-β1抑制IL-6表達。本研究提示局部註射移植ADMSCs是促進胃穿孔傷口癒合的有效方法。 / 3. 局部移植脂肪幹細胞促進胃饋瘍癒合的實驗性研究：使用70%醋酸建立大鼠胃體饋瘍模型；饋瘍病竈內局部註射移植1×107 ADMSCs。研究提示第10和15天ADMSCs顯著減小饋瘍面積。組織學研究提示ADMSCs增加饋瘍傷口上皮和血管再生，促進膠原蛋白分泌和抑制炎癥反應。移植的ADMSCs能夠在饋瘍病竈內成活，並分化成血管內皮細胞和平滑肌細胞。ADMSCs顯著提高促傷口癒合相關基因表達水準。此外，ADMSCs啟動COX-2-PGE2和Erk1/2-MAPK信號通路。第10天，和對照組相比，引哚美辛/ADMSCs組潰瘍病竈內細胞增殖和血管再生顯著降低、饋瘍癒合延遲。本研究提示脂ADMSCs促進胃饋瘍癒合；非甾體抗炎藥顯著減弱ADMSCs的促胃饋瘍癒合作用。 / 結論：與內鏡夾閉相比，Eagle Claw VIII內鏡縫合胃創口有可行性。ADMSCs促進胃穿孔和饋瘍癒合，且依賴於前列腺素E2；引哚美辛抑制前列腺素E2合成從而抑制ADMSCs促胃組織癒合之效能。本研究提示聯合使用內鏡縫合器和ADMSCs是促進胃傷口癒合的潛在有效方法。 / Thesis Ph.D. Chinese University of Hong Kong 2014. / Includes bibliographical references (leaves 152-162). / Abstracts also in Chinese. / Title from PDF title page (viewed on 11, October, 2016). / Liu, Liu. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Though it is well known that tobacco related products can cause prenatal maldevelopment, very little is known on how tobacco products affect bone tissue as it develops in the embryo. Identifying which chemicals can induce the greatest harm to the prenatal skeletal system is an improbable task as there are over 7,000 chemicals in tobacco smoke alone. We hypothesized that the Toxicological Priority Index (ToxPI) program can be used to rank osteogenic cytotoxicity potential to aid in the assessment of what chemicals out of the thousands can cause osteogenic differentiation inhibition. ToxPI aggregates information from various assays and incorporates them into visual “pie charts” which allow chemicals to be ranked against each other by given parameters. The larger the pie chart the greater likelihood of potential effects and vice versa. Seventeen tobacco chemical constituents were ranked using ToxPI and those chemicals with pie charts (0 To assess the ability of ToxPI to correctly predict maldevelopment in silico eight compounds were then tested in vitro: four of them being ToxPi positive and the other four having null predicted effects. To verify the predictions, human embryonic stem cells were differentiated into osteoblasts and exposed to various concentrations of each compound. Cell viability was measured via MTT assay in conjunction with a calcium assay to measure osteogenic differentiation. In addition, adult human feeder fibroblasts cell viability in response to exposure was measured. ToxPI positive predictions (xin vitro, caused differentiation inhibition. Together our data suggests that ToxPi might be useful to identify strongly inhibitory chemicals based on their cytotoxicity but might also give false negative results for chemicals that cause differentiation inhibition at sub-toxic levels.

Prostate cancer is the most common cancer occurring in the men in USA and Europe. According to CDC, incidence of Prostate cancer in African American men in the year 2008 was 234.6 cases per 100,000 compared to 150 cases per 100,000 in Caucasian men, reasons for this disparity remain unclear. Castration resistant prostate cancer is an advanced form of prostate cancer with poor survival rates. 10-20% of prostate cancer patients develop metastatic castration resistant prostate cancer (CRPC) within approximately 5 years of follow-up. Androgen deprivation therapy which is at the center of metastatic prostate cancer is often impeded by development of CRPC. Previous studies have demonstrated that prostatic androgen concentration ranging between 10-25 percent in the treated patients versus the untreated could still continue AR signaling. Previous in vitro studies have demonstrated higher tumor homing potential in normal adipose derived mesenchymal stem cells (ADMSC) from African American patient compared to ADMSC derived from a Caucasian patient when grown in prostate cancer cell condition media. This study attempts to exploit this tumortropicity of ADMSC for selective delivery of alpha keto reductases in the metastasized prostate cancer cells to hydrolyse DHT and other androgens into weaker androgens. Enriched ADMSC were plated in a 6 well plate and were co-transfected with transfected with AKRC14 and GFP. Gene expression was confirmed by PCR and WB. ADMSCs are capable of expressing AKR1C14 on transfection with plasmid. Stem cells expressing AKR1C4 open the avenues for furthering therapeutic strategies in metastatic CRPC by hydrolyzing the androgens. / 1 / Manish Ranjan

Pas de résumé disponible / Use of stem cells in regenerative medicine has attracted great interest in the past decade. Muscle stem cells such as satellite cells were shown to regenerate skeletal muscle tissue after injury and to contribute to muscle growth. These properties have raised an enormous interest in using satellite cells for the therapy of skeletal muscle wasting disorders where the intrinsic stem cell population is unable to repair muscle tissue. However, better understanding of the mechanisms controlling satellite cell lineage progression and self-renewal is crucial to exploit the power of these cells in combating myopathic conditions. In the studies described here, the mechanisms regulating the in vivo behavior and maintenance of quiescence of Drosophila Adult Muscle Precursors (AMPs) that share several properties with the vertebrate satellite cells are analyzed. We show that undifferentiated embryonic AMPs display homing behavior and that their survival depends on the somatic muscles. We observe that AMPs establish direct contact with muscle fibers by sending thin filopodia and that this AMP-muscle interaction is crucial for AMPs spatial positioning. Larval muscles also play an important role in promoting the AMP cell proliferation. They achieve this by secreting Drosophila Insulin like peptide 6 (dIlp6) that activate the AMPs from their quiescent state and induce proliferation during the end of the second larval instar. We also demonstrate that Notch acts downstream of Insulin pathway and positively regulates proliferation of AMPs via dMyc. In the second part of the thesis manuscript we report that the affected formation ofadult muscles impacts on persisting abdominal larval templates. In this section role of the Notch signaling pathway in specification of the Adult founder cells is also demonstrated. Finally, we report generation of new tools for the cell type specific genome wide approaches that can be applied to identify global gene expression profiles in quiescent versus activated AMPs. Together these studies identified several new features of AMPs and enhance our understanding on the processes regulating stem cells homing, quiescence and reactivation.

Human umbilical cord blood (HUCB) cells consist of a heterogeneous population of cells, rich in hematopoietic stem and progenitor cells. These cells have been used in the treatment of various nonmalignant and malignant hematopoietic diseases. With in the last few years HUCB cells have been used in pre-clinical animal models of brain and spinal cord injuries, in which functional recovery has been shown. The properties of cord blood cells that could be important in cell transplantation (repair or replacement) of CNS injury or disease are currently being evaluated. The major focus of this study was to determine whether HUCB cells would migrate to ischemic tissue extracts. In addition, factors that may be inducing the cells to migrate were examined by identifying the cytokines or chemokines present in the ischemic tissue extracts. The secondary focus was to establish whether cultured HCUB cells are releasing cytokines and chemokines (in vitro) in response to their environment. The results of these studies showed that HUCB cells migrate to ischemic tissue in a time dependent manner. In which there is a 48 to 72 hour window of opportunity for the delivery of HUCB cells to the ischemic brain. In addition, the cord blood cells were shown to release cytokines that may be aiding in the behavioral recovery seen in the transplantation studies. The results from this study are promising in that the current 3-hour therapeutic window for the treatment of stroke victims, using approved anticoagulant treatment, may be extended with the use of cord blood cell therapy with the peak at 48 hours.

Infusion of the "mononuclear fraction" of human cord blood cells (HUCBC), which is composed of immature blood cells and hematopoietic progenitors, is known to reduce neurobehavioral deficits in rats subject to middle cerebral artery occlusion (MCAO). When MCAO rats are infused with 106 cells 24 hours after the induction of the stroke, their motor function improves. To extend these findings, we first examined the behavioral recovery of MCAO rats in the presence of increasing doses of HUCBC. The recovery in behavioral performance seen with measurements of spontaneous activity and motor deficits, depended on the amount of cells delivered, with 106 HUCBC being the threshold for significant behavioral recovery. Measurements of the ischemic volume revealed an inverse relationship between HUCBC dose and damage volume, which reached significance at the higher HUCBC doses (107 and 3-5x107 cells). Moreover, investigation of the distribution of the intravenously injected cells showed that HUCBC were localized to the injured brain hemisphere and the spleen. Given these findings, we hypothesized that there may be a role of HUCBC in the modulation of the peripheral or brain-localized immune response that is normally evoked after stroke. Results on the effect of HUCBC infusion on splenocytes indicated that HUCBC treatment prevented the alterations in splenocyte type (CD8+ depletion) and function (T-cell suppression) induced by stroke. In particular, examination of cytokine production from splenocyte cultures of HUCBC-treated MCAO rats revealed increased production of IL-10 and decreased production of IFNgamma relative to MCAO rats. Microglia (immunostained with a CD11b antibody) and B cells (identified with the B220 cell marker) that were increased after MCAO were dramatically decreased after HUCBC treatment. Proinflammatory cytokines such as TNF-alpha, IL-1beta and IL-2 were upregulated after MCAO surgery and their expression was abrogated after HUCBC infusion. All these findings indicate that the action of HUCBC may be specifically related to the modulation of the stroke-induced inflammatory response, providing a possible mechanism by which cord blood cells have been repeatedly reported to promote functional recovery from ischemic injury.

Abstract not available

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.