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

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines promising advances in cell-based modelling and therapies

3D human liver tissue from pluripotent stem cells displays stable phenotype in vitro and supports compromised liver function in vivo.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to the shortage of donor organs. Developing renewable sources of human liver tissue is therefore attractive. Pluripotent stem cell-derived liver tissue represents a potential alternative to cadaver derived hepatocytes and whole organ transplant. At present, two-dimensional differentiation procedures deliver tissue lacking certain functions and long-term stability. Efforts to overcome these limiting factors have led to the building of three-dimensional (3D) cellular aggregates. Although enabling for the field, their widespread application is limited due to their reliance on variable biological components. Our studies focused on the development of 3D liver tissue under defined conditions. In vitro generated 3D tissues exhibited stable phenotype for over 1 year in culture, providing an attractive resource for long-term in vitro studies. Moreover, 3D derived tissue provided critical liver support in two animal models, including immunocompetent recipients. Therefore, we believe that our study provides stable human tissue to better model liver biology 'in the dish', and in the future may permit the support of compromised liver function in humans

Fluid shear stress modulation of hepatocyte like cell function

Freshly isolated human adult hepatocytes are considered to be the gold standard tool for in vitro studies. However, primary hepatocyte scarcity, cell cycle arrest and the rapid loss of cell phenotype limit their widespread deployment. Human embryonic stem cells and induced pluripotent stem cells provide renewable sources of hepatocyte-like cells (HLCs). Despite the use of various differentiation methodologies, HLCs like primary human hepatocytes exhibit unstable phenotype in culture. It has been shown that the functional capacity can be improved by adding back elements of human physiology, such as cell co-culture or through the use of natural and/or synthetic surfaces. In this study, the effect of fluid shear stress on HLC performance was investigated. We studied two important liver functions, cytochrome P450 drug metabolism and serum protein secretion, in static cultures and those exposed to fluid shear stress. Our study demonstrates that fluid shear stress improved Cyp1A2 activity by approximately fivefold. This was paralleled by an approximate ninefold increase in sensitivity to a drug, primarily metabolised by Cyp2D6. In addition to metabolic capacity, fluid shear stress also improved hepatocyte phenotype with an approximate fourfold reduction in the secretion of a foetal marker, alpha-fetoprotein. We believe these studies highlight the importance of introducing physiologic cues in cell-based models to improve somatic cell phenotype

Serum Free Production of Three-Dimensional Human Hepatospheres from Pluripotent Stem Cells

The video component (running time: 06:57) of this article can be found at https://www.jove.com/video/59965/Copyright © 2019 The Author(s) Creative Commons Attribution 3.0 License. The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores. Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.UK Regenerative Medicine Platform (MRC MR/L022974/1); Chief Scientist’s Office (TCS/16/37)

Three-dimensional cell culture: From evolution to revolution

Recent advances in the isolation of tissue-resident adult stem cells and the identification of inductive factors that efficiently direct differentiation of human pluripotent stem cells (hPSCs) along specific lineages have facilitated the development of high-fidelity modelling of several tissues in vitro. Many of the novel approaches used have employed self-organising three-dimensional (3D) culturing of organoids, which offer several advantages over conventional two-dimentional platforms. Organoid technologies hold great promises for modelling diseases and predicting the outcome of drug responses in vitro. Here, we outline the historical background and some of the recent advances in the field of 3D organoids. We also highlight some of the current limitations of these systems and discuss potential avenues to further benefit biological research using 3D modelling technologies.GSK and Novartis and a Brunel University London Scholarship awar