Stem cell technologies as a branch of regenerative medicine are becoming increasingly popular as the science behind it evolves. Therefore, it is important that the regulatory framework pertaining to stem cell technologies be well defined and appropriate to prevent unethical and unscrupulous behaviour on the part of medical practitioners, which gives rise to stem cell tourism. South African legislation pertaining to stem cell technology is regarded as inadequate and dissonant with the Constitution, exacerbating the problem of stem cell tourism and denying patients access to certain stem cell therapies, which ultimately can be viewed as an infringement of their constitutional rights. The United Kingdom (UK) provides a clear-cut regulatory framework, which is not only centred around consent and patient safety but is also conducive to production of stem cell therapies. For such reasons, this dissertation finds the UK framework to be an appropriate benchmark against which the South African regulatory framework can be evaluated. By means of comparison and elaborating on the biology of stem cells in addition to pertinent ethical principles, legislation and human rights of both South Africa and the UK, an argument will be made out that South African legislation pertaining to stem cell therapy and related matters is wanting. Furthermore, analysis will be made of the definition of biological medicine as put forward by the Medicines and Related Substances Control Act 101 of 1965 to conclude that certain stem cell therapies are best excluded from such a definition as such stringent requirements and protocols encumbers access to stem cell therapies and inflates costs. Lastly, remedial measures are proposed to remedy these injustices by proposing for the institution of a specialist adivisary committee to oversee stem cell and related activities. Key Words: Regenerative Medicine; Stem Cells; Stem Cell Regulation; National Health Act; Medicines and Related Substances Control Act; Advanced Therapy Medicinal Product; Human Tissue Authority; Human Fertilisation and Embryology Authority; HTA; HFEA; Medicine and Healthcare Products Regulatory Agency; MHRA; European Medicines Agency; Tissue-engineered Products; Doctor-Patient Relationship; Medical Innovation Bill 2014; Experimental Treatments; Innovative Therapy; Hospital Exemption; Informed Consent; Special Exemption; Autologous Stem Cell Therapy; Stem Cell Transplants; Gene Therapy Advisory Committee. / Dissertation (LLM)--University of Pretoria, 2017. / Public Law / LLM / Unrestricted

<p>The pancreas is a vital organ that secretes key metabolic hormones and digestive enzymes. In pancreatic ductal adenocarcinoma (PDAC), one of the leading causes of cancer-related death in the world, limited advances in diagnosis or therapies have been made over decades. Key features of PDAC progression include an elevated matrix stiffness and an increased deposition of extracellular matrices (ECM), such as hyaluronic acid (HA). Understanding how cells interact with components in the tumor microenvironment (TME) as PDAC progresses can assist in developing diagnostic tools and therapeutic treatment options. In recent years, hydrogels have proven to be an excellent platform for studying cell-cell and cell-matrix interactions. Utilizing chemically modified and naturally derived materials, hydrogel networks can be formed to encompass not only the components, but also the physicochemical properties of the dynamic TME. In this work, a dynamic hydrogel system that integrates multiple click chemistries was developed for tuning matrix physicochemical properties in a manner similar to the temporally increased matrix stiffness and depositions of HA. Subsequently, these dynamic hydrogels were used to investigate how matrix stiffening and increased HA presentation might affect survival of PDAC cells and their response to chemotherapeutics. </p>

Understanding the regenerative capacity and the role of human AT2s in the distal lung is imperative for defining alveolar response to injury and disease. Additionally, due to human AT2 expression of COPD genome wide association study (GWAS) genes, they are an especially relevant cell type to study the disease. Here we apply CRISPR-interference (CRISPRi) to reduce the expression of COPD GWAS gene, ADGRG6, to interrogate its function in induced pluripotent stem cell-derived type 2 alveolar epithelial cells (iAT2s). We find that decreased expression of ADGRG6 in iAT2s caused disruption to iAT2 cell polarity, organization of the actin cytoskeleton, and establishment of tight junctions. In addition, ADGRG6 knockdown (kd) causes a hyperproliferative phenotype. Finally, we find that ADGRG6-kd may contribute to dysregulation of tight junction formation in the presence of cigarette smoke.

Laryngeal cancer affects tens of thousands worldwide every year. The standard of care of surgical resection, chemotherapy, and/or radiation therapy results in significant quality of life deficits including reliance on tracheostomy tubes, loss of voice, and inability to swallow. There are no therapeutic options that restore a functional larynx so that patients can live a more normal life. Laryngeal reconstruction using tissue engineering strategies offers the potential to solve this problem. Laryngeal anatomy is complex with multiple tissue types and therefore engineering approaches require consideration of a multi-layer, interfacial tissue design. Our strategy to overcome these challenges involves the use of advanced bio fabrication techniques where type I oligomeric collagen alone or in the presence of autologous stem cells is used to custom-make the cartilage, skeletal muscle, and mucosal layer of the larynx. This doctoral research project begins by describing the development of the tissue engineered skeletal muscle with aligned collagen matrix and autologous muscle progenitor cells induced to express motor endplates. Next, using this engineered muscle plus the cartilage layer developed by a colleague; we implanted the myochondral engineered construct in a rat hemilaryngectomy model. In this study we saw host-implant integration with no inflammatory foreign body response, neo cartilage and muscle formation, and some return of laryngeal function on the reconstructed side. Next, we worked to scale-up these technologies for use in a porcine model. The pig larynx is more similar in size and function to the human larynx and allows for a full thickness defect to be created. Using confined compression, we created 4-mm thick acellular and cellular cartilage constructs, as well as a 0.5-mm thick acellular mucosal layer. A 1-cm diameter muscle layer containing autologous muscle progenitor cells was created using flow alignment and cultured to induce expression of motor endplates before implantation. Tissue constructs were subjected to mechanical property analyses as well as PCR analysis to describe the differential gene expression by component cells within muscle and cartilage constructs. Each layer was individually sutured into a pig hemilaryngectomy model. The pigs recovered well from the surgery, were eating, had no difficulty breathing, and no aspiration events. At 2 months, respiratory epithelium had completely healed over the implant and was vascularized and had areas of submucosal gland growth. The motor endplate expressing muscle implants showed new, organized muscle ingrowth while the acellular implants showed a relative paucity of new, disorganized muscle. This work represents a significant advancement in the field of laryngeal reconstruction and is a first of its kind to use scalable tissue engineering technologies designed to specifically meet each layer’s functional criteria.

Background: Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. Methods: In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficial digital flexor tendons. Additional tendon specimens underwent manually performed freeze-thaw cycles based on an established procedure. Tendon decellularization was completed by using non-ionic detergent treatment (Triton X-100). Effectiveness of decellularization was assessed by residual nuclei count and calculation of DNA content. Cytocompatibility was evaluated by culturing allogeneic adipose tissue-derived mesenchymal stromal cells on the tendon scaffolds. Results: There were no significant differences in decellularization effectiveness between samples decellularized by the automated freeze-thaw procedure and samples that underwent manual freeze-thaw cycles. Further, we inferred no significant differences in the effectiveness of decellularization between two different cooling and heating rates applied in the automated freeze-thaw process. Both the automated protocols and the manually performed protocol resulted in roughly 2% residual nuclei and 13% residual DNA content. Successful cell culture was achieved with samples decellularized by automated freeze-thawing as well as with tendon samples decellularized by manually performed freeze-thaw cycles. Conclusions: Automated freeze-thaw cycles performed by using a liquid nitrogen-based controlled rate freezer were as effective as previously described manual freeze-thaw procedures for decellularization of equine superficial digital flexor tendons. The automation of this key procedure in decellularization of large tendon samples is an important step towards the processing of large sample quantities under standardized conditions. Furthermore, with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine, the automation of key procedural steps is highly required to develop manufacturing processes under GMP conditions.

Indiana University-Purdue University Indianapolis (IUPUI) / Dry age-related macular degeneration (AMD) is a progressive neurodegenerative disorder characterized by geographical atrophy of the retinal pigment epithelium (RPE), causing irreversible central vision loss. Systemically-delivered bone marrow-derived cells (BMDCs), programmed to RPE-like cells via expression of human RPE65, regenerate damaged RPE and preserve vision in murine models of retinal degeneration. RPE65 rapidly activates adenylate cyclase (AC), which then activates endogenous Rpe65 and RPE-associated marker Cralbp. Previous studies expressed RPE65 from an integrating lentiviral vector (ILV), which is an unnecessary safety risk due to the potential for insertional mutagenesis, as long- term expression of RPE65 is not required for BMDC programming. Here, we developed a 3rd generation integrase-defective lentiviral vector (IDLV) for programming both murine and human BMDCs to RPE-like cells, reducing insertional mutagenesis risk and expanding the protocol to include human cells. We enhanced IDLV3-RPE65 infection of murine and human BMDCs by preloading concentrated vector on RetroNectin at MOI 50, and infecting with low-speed centrifugation, increasing RPE65 mRNA levels from ~12-fold to ~25-fold (p<0.05). IDLV3-RPE65 infection initiates expression of endogenous Rpe65 mRNA expression in murine BMDC and Cralbp/CRALBP mRNA in both murine and human BMDCs, indicating programming to RPE-like cells. Inhibiting AC in RPE65infected BMDCs abrogated expression of the endogenous genes, confirming the role of AC activation in programming. Critically, IDLV3-RPE65-infected murine BMDCs are recruited to and incorporate into to the RPE layer, and preserve vision in murine models of retinal degeneration. We conclude that BMDCs programmed with IDLV3-RPE65 successfully prevent retinal degeneration progression and are appropriate for testing in human cells, with a view to move into human clinical trial for the treatment of dry AMD. This approach significantly increases the safety of the therapy and is, to the best of our knowledge, the first application of a single IDLV in the generation of therapeutic cells from adult stem cells.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12971号 / 論医科博第2号 / 新制||医科||5(附属図書館) / 32409 / 新制||医科||5 / 横浜市立大学大学院国際総合科学研究科バイオ科学専攻 / (主査)教授 長船 健二, 教授 中畑 龍俊, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20972号 / 医博第4318号 / 新制||医||1026(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 渡邊 直樹, 教授 江藤 浩之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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For years, neonates have been "therapeutic orphans" and denied the benefits of clinical research because most therapeutic options are usually tested in the adult population. Most treatments and interventions have not been explored, and there is room potential rigorous, evidence-based clinical trials towards diseases specific to this population. Regenerative medicine holds great promise by potentially offering new ways of treating incurable diseases. However, bench to bedside translations often fail due to low recruitment rate. Thus, there is a need for effective interventions to increase trial participation and execution to help accelerate neonatal research. Behaviour theories could help to better understand trial participation, screening and recruiting behaviours, inform fit-for-purpose interventions, and assist in building cumulative evidence. There is a lack of clarity on the barriers and enablers to clinical trial participation from important stakeholder groups; NICU parents and research staff members. Study 1 reports findings on identified barriers and enablers that might affect parents' decision-making to participate in an early phase mesenchymal stromal cell (MSC) therapy trial including concerns with safety, efficacy and expected outcomes, but were willing to consider consenting to the trial after watching the animated video and having altruistic consideration. Study 2 reports findings on identified barriers and enablers that might affect research personnel recruitment to a multi-centre MSC trial which include: having cautious hope about the trial, importance of coordination with the clinical staff and study team and optimizing the study flow. Due to the challenging context of the study, the participants prefer to have clinicians involved with the recruitment. Lasty, Study 3 reports findings on identified barriers and enablers to screening potential patients for an adeno- associated viral vector gene therapy trial. Physicians were optimistic about the treatment but were concerned about the safety, feasibility, the expected outcomes of the treatment, and available resources (personnel, equipment, funding). Many expressed the need for support from clinical professionals prior to approaching parents and highlighted variability in screening roles. The resulting comprehensive set of factors helps to identify priorities for future research and provide insights towards developing novel interventions for neonatal research.