Clinical Recovery from CNS Damage

After decades of focusing on how to alleviate and prevent recurrence of acute CNS injuries, the emphasis has finally shifted towards repairing such devastating events and rehabilitation. This development has been made possible by substantial progress in understanding the scientific underpinnings of recovery as well as by novel diagnostic tools, and most importantly, by emerging therapies awaiting clinical trials. In this publication, several international experts introduce novel areas of neurological reorganization and repair following CNS damage. Principles and methods to monitor and augment neuroplasticity are explored in depth and supplemented by a critical appraisal of neurological repair mechanisms and possibilities to curtail disability using computer or robotic interfaces. Rather than providing a textbook approach of CNS restoration, the editors selected topics where progress is most imminent in this labyrinthine domain of medicine

Crop Production

Humans are depended on crops for food, fibber and more recently for fuel. Demands for crop production in rising because of increasing population, change in food habits and biofuel consumption. The book focus on challenges, progress and prospects of crop production. It comprises of vast array of topics including latest agronomics practices for different crops to enhance productivity, mitigate the challenges imposed by climate change, improve water use efficiency, factors controlling dormancy, optimum use of fertilizers etc. This volume will serve as an excellent resource for students and researchers interested and working in the area of sustainable crop production

Characterisation of STRO-1 expression on human mesenchymal stem cells and identification of putative cancer stem cells in osteosarcoma : prevention by micronutrients

It is becoming increasingly more common to use culture expanded human mesenchymal stem cells (hMSCs) in regenerative medicine due to their low incidence in vivo. However, their successful application is hampered by a lack of selective markers to positively identify the expanded multipotent cells. This study aimed to characterise STRO-1 antigen as a potential biomarker of multipotency on cultured bone marrow derived hMSCs. In an attempt to identify the nature of this antigen, two techniques were implemented: peptide phage display technology and a microarray based approach. Changes in the expression of STRO-1 were investigated during culture expansion of hMSCs. STRO-1 expression positively correlated with cellular morphology and multilineage potential, whereby senescent cells down-regulated STRO-1 antigen and exhibited decreased adipogenic and osteogenic potential. Furthermore, STRO-1 was found to be heterogeneously expressed on hMSC populations and enrichment followed by lineage specific induction of the STRO-1BRIGHT fraction resulted in enhanced adipogenic and osteogenic differentiation potential. The expression of STRO-1 antigen was further characterised as a marker of differentiation, whereby differentiating cells were found to down-regulate STRO-1. A cellular hierarchy in hMSC population was therefore proposed based on STRO-1 status, with the highest STRO-1 expressive cells representing the multipotent subset. In an attempt to identify the epitope that STRO-1 IgM antibody recognised, peptide phage display technology was used as solid and liquid phase panning systems but the approach yielded no promising peptide candidate. Subsequently, comparative gene expression microarray analysis of osteosarcoma cell lines (143B, CAL72, G-292, HOS, MG-63, Saos-2 and U-2-OS) was implemented and a list of eight potential candidate genes encoding STRO-1 antigen was selected. This work ultimately led to the identification of putative cancer stem cells (CSCs) in seven osteosarcoma cell lines initially based on STRO-1 expression. With MG-63 strongly expressing STRO-1, the maintenance of MSC-like properties by STRO-1 expressing cell lines was investigated. A heterogeneous pattern of osteogenic differentiation was observed between and within the cell lines. Closer inspection revealed a cellular hierarchy comprising of holoclones and paraclones, with the holoclones representing the putative CSCs in osteosarcoma. Overall, this thesis addressed the fields of regenerative medicine as well as oncology by proposing STRO-1 antigen as a marker of multipotency on hMSCs and osteosarcoma holoclonal cells as the putative cancer stem cell targets for anti-cancer drug development.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Characterisation of STRO-1 expression on human mesenchymal stem cells and identification of putative cancer stem cells in osteosarcoma: prevention by micronutrients

It is becoming increasingly more common to use culture expanded human mesenchymal stem cells (hMSCs) in regenerative medicine due to their low incidence in vivo. However, their successful application is hampered by a lack of selective markers to positively identify the expanded multipotent cells. This study aimed to characterise STRO-1 antigen as a potential biomarker of multipotency on cultured bone marrow derived hMSCs. In an attempt to identify the nature of this antigen, two techniques were implemented: peptide phage display technology and a microarray based approach. Changes in the expression of STRO-1 were investigated during culture expansion of hMSCs. STRO-1 expression positively correlated with cellular morphology and multilineage potential, whereby senescent cells down-regulated STRO-1 antigen and exhibited decreased adipogenic and osteogenic potential. Furthermore, STRO-1 was found to be heterogeneously expressed on hMSC populations and enrichment followed by lineage specific induction of the STRO-1BRIGHT fraction resulted in enhanced adipogenic and osteogenic differentiation potential. The expression of STRO-1 antigen was further characterised as a marker of differentiation, whereby differentiating cells were found to down-regulate STRO-1. A cellular hierarchy in hMSC population was therefore proposed based on STRO-1 status, with the highest STRO-1 expressive cells representing the multipotent subset. In an attempt to identify the epitope that STRO-1 IgM antibody recognised, peptide phage display technology was used as solid and liquid phase panning systems but the approach yielded no promising peptide candidate. Subsequently, comparative gene expression microarray analysis of osteosarcoma cell lines (143B, CAL72, G-292, HOS, MG-63, Saos-2 and U-2-OS) was implemented and a list of eight potential candidate genes encoding STRO-1 antigen was selected. This work ultimately led to the identification of putative cancer stem cells (CSCs) in seven osteosarcoma cell lines initially based on STRO-1 expression. With MG-63 strongly expressing STRO-1, the maintenance of MSC-like properties by STRO-1 expressing cell lines was investigated. A heterogeneous pattern of osteogenic differentiation was observed between and within the cell lines. Closer inspection revealed a cellular hierarchy comprising of holoclones and paraclones, with the holoclones representing the putative CSCs in osteosarcoma. Overall, this thesis addressed the fields of regenerative medicine as well as oncology by proposing STRO-1 antigen as a marker of multipotency on hMSCs and osteosarcoma holoclonal cells as the putative cancer stem cell targets for anti-cancer drug development

Diagnosis and Treatment of Small Bowel Disorders

Over the last few decades, remarkable progress has been made in understanding the aetiology and pathophysiology of diseases and many new theories emphasize the importance of the small bowel ‘ecosystem’ in the pathogenesis of acute and chronic illness. Emerging factors such as microbiome, stem cells, innate intestinal immunity and the enteric nervous system along with mucosal and endothelial barriers have key role in the development of gastrointestinal and extra-intestinal diseases. Therefore, the small intestine is considered key player in metabolic disease development, including diabetes mellitus, and other diet-related disorders such as celiac and non-celiac enteropathies. Another major field is drug metabolism and its interaction with microbiota. Moreover, the emergence of gut-brain, gut-liver and gut-blood barriers points toward the important role of small intestine in the pathogenesis of common disorders, such as liver disease, hypertension and neurodegenerative disease. However, the small bowel remains an organ that is difficult to fully access and assess and accurate diagnosis often poses a clinical challenge. Eventually, the therapeutic potential remains untapped. Therefore, it is due time to direct our interest towards the small intestine and unravel the interplay between small-bowel and other gastrointestinal (GI) and non-GI related maladies

Development of a novel in vitro 3D model to identify cancer genes via insertional mutagenesis

This thesis was submitted for the degree of Doctorate of Philosophy and awarded by Brunel University LondonThe aim of the presented research project was to develop a personalised in vitro based human model to test the genotoxicity of gene therapy viral vectors. For this purpose, human induced pluripotent stem cells (hiPSCs) and human induced pluripotent stem cell-derived 3D heps were used in combination with a number of lentiviral and adeno-associated viral vectors and molecular analysis was performed to detect insertion sites (IS) to assess the predictive power of 3D heps model to predict insertional mutagenesis. In this study, two previously designed plasmid were used, one of which contained the U3 region of the LTR from the wild type (pHV) and considered as “Unsafe” vector, and the other was a self-inactivating (SIN) lentiviral vector with no U3 region of the LTR (pHR) and used as a “Safe” vector. Initially, high-titre production of both lentiviral vectors was achieved in the HEK293 cell line. The titre was calculated using flow cytometry analysis. The recombinant AAV serotype-2 vectors were kindly provided by our collaborators in Australia. These vectors constructed with “Clean ITR” with strong and weak promoters, driving the green fluorescent protein (GFP) expression. In parallel, P106i, as an integration-free hiPSCs, was expanded and fully characterised, using flow cytometry, immunostaining and qPCR analysis. Expression of pluripotency cell surface markers such as SSEA4, TRA-1-81 and TRA-1-60 was detected in more than 90% of the cells using flow cytometry. The expression of POU domain class 5 transcription factor 1 (POU5F1 or OCT4) and SRY-Box Transcription Factor 2 (SOX2) as two major transcription factors regulating pluripotency were also confirmed at gene and protein levels by quantitative polymerase chain reaction (qPCR) and immunostaining, respectively. The high level of pluripotency markers confirmed the pluripotent state and the identity of P106i, which was used in this study. Then, a recently published protocol to generate phenotypically stable 3D heps from human embryonic stem cells (hESCs) were amended and optimised to generate 3D heps from hiPSC efficiently. This protocol addresses issues surrounding previous 3D protocols, such as scalability and long-term in vitro phenotypic stability. Notably, hiPSC-derived 3D heps displayed liver functions for an extended period. Standard characterisation tests were performed on day 20 of differentiation using a range of molecular and cell biology techniques, including immunofluorescence analysis of liver-specific markers such as HNF4-alpha and secretion of serum albumin (ALB) and alpha-fetoprotein (AFP) using ELISA assay. The result revealed a high level of ALB and a low level of AFP in hiPSC-derived 3D heps compared to conventional 2D hepatocyte-like cells. Following characterisation, hiPSCs and 3D heps were transduced with recombinant AAV serotype-2 vectors (at MOI 1E5) and lentiviral vectors (at MOI 20). Modifications were made to enhance transduction efficiency in 3D heps. The outer layer of the 3D heps exhibited the highest level of GFP expression, which transduced with rAVV serotype-2 vectors. In 3D heps transduced with pHR and pHV lentiviral vectors, the same pattern was observed; however, a higher level of GFP expression was observed in cells transduced with the pHR lentiviral vector. The result of successful transduction was confirmed by PCR analysis. hiPSCs were also transduced with pHR and pHV lentiviral vectors at two different time points, and the result was confirmed via PCR analysis. In order to see the effect of gene expression in the proximity of viral insertion at the individual cell level, single-cell cloning (SCC) was performed on hiPSCs which were transduced with pHR and pHV lentiviral vectors. The results showed positive GFP expression in hiPSCs, confirming transduction. The DNA and RNA of the single-cell clones, hiPSCs and 3D heps were collected and sent for downstream analysis of the insertion site (IS). Upon viral integration, it is crucial to detect IS in the setting of clonal dominance. For bioinformatic analysis, lentiviral IS was retrieved by EPTS/LM-PCR and CIS were detected in P106i and 3D heps samples. This result revealed an overall decrease in IS overtime in transduced cells for both lentiviral vectors by comparison of the days 3 and 30 time points. The number of identified IS in 3D heps transduced with pHR and pHV lentiviral was low compared to P106i cells. There was also an apparent reduction in IS in P106i cells transduced with pHR and pHV lentiviral vectors over time, suggesting possible cell death during propagation and culture expansion. Following EPTS/LM-PCR, identification of cancer-related genes IS, and CIS were performed in P106i cells and 3D heps. The result indicates that in P106i, the number of proto-oncogenes was higher in samples transduced with pHV lentiviral vectors, suggesting the possible effect of the full LTR vector on the number of cancer-related genes. In addition, polyclonality of pHV and pHR was observed in analysed single-cell clones of P106i. However, in clone G transduced with pHV lentiviral vector, the gene LINC01249 exhibited 93.4% of the viral sequence count. In addition, despite the polyclonality of the samples, the qPCR result revealed that the genes near the IS have increased in level of relative gene expression. In conclusion, the 3D heps provide a valuable in vitro tool to assess genotoxicity associated with viral vectors