Tendon function is essential for quality of life, yet the pathogenesis and healing of tendinopathy remains poorly understood compared to other musculoskeletal disorders. The aim of regenerative medicine is to replace traditional tissue and organ transplantation by harnessing the developmental potential of stem cells to restore structure and function to damaged tissues. The recently discovered interdependency of cell phenotype and biophysical environment has created a paradigm shift in cell biology. This dissertation introduces a dynamic in vitro model for tendon function, dysfunction and development, engineered to characterize the mechanobiological relationships dictating stem cell fate decisions so that they may be therapeutically exploited for tendon healing. Cells respond to mechanical deformation via a complex set of behaviors involving force-sensitive membrane receptor activity, changes in cytoskeletal contractility and transcriptional regulation. Effective ex vivo model systems are needed to emulate the native environment of a tissue and to translate cell-matrix forces with high fidelity. A naturally-derived decellularized tendon scaffold (DTS) was invented to serve as a biomimetic tissue culture platform, preserving the structure and function of native extracellular matrix. DTS in concert with a newly designed dynamic mechanical strain system comprises a tendon bioreactor that is able to emulate the three-dimensional topography, extracellular matrix proteins, and mechanical strain that cells would experience in vivo. Mesenchymal stem cells seeded on decellularized tendon scaffolds subject to cyclic mechanical deformation developed strain-dependent alterations in phenotype and measurably improved tissue mechanical properties. The relative tenogenic efficacies of adult stem cells derived from bone marrow, adipose and tendon were then compared in this system, revealing characteristics suggesting tendon-derived mesenchymal stem cells are predisposed to differentiate toward tendon better than other cell sources in this model. The results of the described experiments have demonstrated that adult mesenchymal stem cells are responsive to mechanical stimulation and, while exhibiting heterogeneity based on donor tissue, are broadly capable of tenocytic differentiation and tissue neogenesis in response to specific ultrastructural and biomechanical cues. This knowledge of cellular mechanotransduction has direct clinical implications for how we treat, rehabilitate and engineer tendon after injury. / Ph. D.

Osteoarthritis (OA) is a degenerative disease of diarthrodial joints causing pain and loss of joint function. Etiology is heterogeneous, but commonly involves inflammation arising from impairment of normal tissue homeostasis and/or function. A cycle of low-grade inflammation and global tissue degradation causes alteration of tissue morphology and function via primary mechanisms or inability to withstand physiological forces. Current therapies variably ameliorate symptoms but do not modify progression. Mesenchymal stem cells (MSCs) have multi-modal properties but are ineffective in ameliorating equine OA. However, anti-inflammatory activities of bone marrow derived MSCs (BMSCs) are enhanced by three-dimensional spheroid culture so equine BMSC (eBMSC) spheroids could inhibit intra-articular inflammation. The overarching hypothesis is that eBMSCs can be enhanced to produce an allogeneic eBMSC therapy that inhibits intra-articular inflammation. In vitro experiments compared differences in anti-inflammatory phenotype between spheroid and traditionally cultured monolayer eBMSCs, the viability and health of eBMSC spheroids administered through needles, and the effects of allogeneic donor on the anti-inflammatory potential of eBMSC spheroids. A model of equine LPS induced synovitis was used to investigate anti-inflammatory efficacy of spheroid eBMSCs compared to placebo or monolayer eBMSCs in vivo. eBMSCs aggregate into spheroids that have stable stem cell marker expression with increased secretion and gene expression of IL-6 and PGE2, and gene expression of SDF-1 and TSG-6. IFN𝛾 and TNFα were not produced by eBMSC spheroids and IL-10 production varied between individuals. Spheroids maintain higher viability and lower senescence than monolayer eBMSCs after injection through a needle and form in high-throughput culture without detrimental effects on expression of TSG-6, IL-6 and PGE synthases that denote an anti-inflammatory phenotype. Additionally, there is significant variation in this phenotype depending on the eBMSC donor. eBMSC spheroids reduced total nucleated cell counts and objective lameness measurements at peak levels of intra-articular inflammation compared to monolayer cultured eBMSCs in vivo. In summary, spheroids increase anti-inflammatory potential of eBMSCs and are practical for clinical use. Increased anti-inflammatory efficacy was demonstrated in a model of in vivo inflammation. This dissertation provides an understanding of the anti-inflammatory activities of eBMSC spheroids that can be used to develop an OA therapy. / Ph. D.

Vat photopolymerization (VP) is an additive manufacturing (AM) technology that permits the fabrication of parts with complex geometries and feature sizes as small as a few microns. These attributes make VP an attractive option for the fabrication of scaffolds for tissue engineering. However, there are few printable materials with low cytotoxicity that encourage cellular adhesion. In addition, these resins are not readily available and must be synthesized. A novel resin based on 2-acrylamido-2-methyl-1-propanesulfonic acid (NaAMPS) and poly(ethylene glycol) diacrylate (PEGDA) was formulated and printed using VP. The mechanical properties, water content, and high fidelity of the scaffold indicated promise for use in tissue engineering applications. Murine fibroblasts were observed to successfully adhere and proliferate on the scaffolds. The growth, migration, and differentiation of a cell is known to dependent heavily on its microenvironment. In engineered constructs, much of this microenvironment is provided by the tissue scaffold. The physical environment results from the scaffold's geometrical features, including pore shape and size, porosity, and overall dimensions. Each of these parameters are known to affect cell viability and proliferation, but due to the difficulty of isolating each parameter when using scaffold fabrication techniques such as porogen leaching and gas foaming, conflicting results have been reported. Scaffolds with pore sizes ranging from 200 to 600 μm were fabricated and seeded with murine fibroblasts. Other geometric parameters (e.g., pore shape) remained consistent between scaffold designs. Inhomogeneous cell distributions and fewer total cells were observed in scaffolds with smaller pore sizes (200-400 μm). Scaffolds with larger pores had higher cell densities that were homogeneously distributed. These data suggest that tissue scaffolds intended to promote fibroblast proliferation should be designed to have pore at least 500 μm in diameter. Techniques developed for selective placement of dissimilar materials within a single VP scaffold enabled spatial control over cellular adhesion and proliferation. The multi-material scaffolds were fabricated using an unmodified and commercially available VP system. The material preferences of murine fibroblasts which resulted in their inhomogeneous distribution within multi-material scaffolds were confirmed with multiple resins and geometries. These results suggest that multi-material tissue scaffolds fabricated with VP could enable multiscale organization of cells and material into engineered constructs that would mimic the function of native tissue. / Doctor of Philosophy / Vat photopolymerization (VP) is a 3D printing (or additive manufacturing) technology that is capable of fabricating parts with complex geometries with very high resolution. These features make VP an attractive option for the fabrication of scaffolds that have applications in tissue engineering. However, there are few printable materials that are biocompatible and allow cells attachment. In addition, those that have been reported cannot be obtained commercially and their synthesis requires substantial resources and expertise. A novel resin composition formulated from commercially available components was developed, characterized, and printed. Scaffolds were printed with high fidelity. The scaffolds had mechanical properties and water contents that suggested they might be suitable for use in tissue engineering. Fibroblast cells were seeded on the scaffolds and successfully adhered and proliferated on the scaffolds. The growth, migration, and differentiation of cells is influenced by the environmental stimuli they experience. In engineered constructs, the scaffold provides many of stimuli. The geometrical features of scaffolds, including how porous they are, the size and shape of their pores, and their overall size are known to affect cell growth. However, scaffolds that have a variety of pore sizes but identical pore shapes, porosities, and other geometric parameters cannot be fabricated with techniques such as porogen leaching and gas foaming. This has resulted in conflicting reports of optimal pore sizes. In this work, several scaffolds with identical pore shapes and porosities but pore sizes ranging from 200 μm to 600 μm were designed and printed using VP. After seeding with cells, scaffolds with large pores (500-600 μm) had a large number of evenly distributed cells while smaller pores resulted in fewer cells that were unevenly distributed. These results suggest that larger pore sizes are most beneficial for culturing fibroblasts. Multi-material tissue scaffolds were fabricated with VP by selectively photocuring two materials into a single part. The scaffolds, which were printed on an unmodified and commercially available VP system, were seeded with cells. The cells were observed to have attached and grown in much larger numbers in certain regions of the scaffolds which corresponded to regions built from a particular resin. By selectively patterning more than one material in the scaffold, cells could be directed towards certain regions and away from others. The ability to control the location of cells suggests that these printing techniques could be used to organize cells and materials in complex ways reminiscent of native tissue. The organization of these cells might then allow the engineered construct to mimic the function of a native tissue.

Stem cells have the ability to self-renew and to differentiate into a variety of cell types. Stem cells can be found naturally in the body, can be derived from the inner cell mass of blastocysts, or can be made by dedifferentiation of adult cells. Regenerative medicine aims to utilize the potential of stem cells to treat disease and injury. The ability to create stem cell lines from a patient's own tissues allows for transplantation without immunosuppressive therapy as well as patient-specific disease modeling and drug testing. The objective of this study was to use cellular dedifferentiation to create in vitro cell lines with which to study regenerative medicine. First, we used siRNA targeted against myogenin to induce the dedifferentiation of murine C2C12 myotubes into myoblasts. Timelapse photography, immunofluorescence, and western blot analysis support successful dedifferentiation into myoblasts. However, the inability to separate the myotubes and myoblasts prior to siRNA treatment confounded the results. This system has the potential to be used to study mechanisms behind muscle cell regeneration and wound healing, but a better method for separating out the myoblasts needs to be developed before this will be achievable. Second, we used a doxycycline-inducible lentiviral vector encoding the transcription factors Oct4, Sox2, cMyc, and Klf4 to create a line of naive-like porcine induced pluripotent stem cells (iPSCs). This reprogramming vector was verified first in murine cells, the system in which it was developed. Successful production of both murine and porcine iPSC lines was achieved. Both showed alkaline phosphatase activity, immunofluorescence for pluripotency marker (Oct4, Sox2, and Nanog) expression, PCR for upregulation of endogenous pluripotency factors (Oct4, Sox2, cMyc, Klf4, and Nanog), and the ability to form embryoid bodies that expressed markers of all three germ layers. Additionally, we were able to create secondary porcine iPSC lines by exposing cellular outgrowths from embryoid bodies to doxycycline to initiate more efficient production of porcine iPSCs. The secondary porcine iPSCs were similar to the primary porcine iPSCs in their morphology, behavior, alkaline phosphatase expression, and Nanog expression with immunofluorescence. The porcine iPSCs were dependent on doxycycline to maintain pluripotency, indicating that they are not fully reprogrammed. Despite this dependence on doxycyline, this system can be used in the future to study the process of reprogramming, to develop directed differentiation protocols, and to model diseases. / Master of Science

我們假設，將scleraxis (Scx)基因轉導入肌腱來源的幹細胞（TDSC），製成TDSC-Scx細胞系。TDSC-Scx會促進肌腱修復。本研究的目的在於探索Scx促進TDSC成肌腱分化的作用，以及TDSC-Scx對肌腱修復的促進作用。 / 使用慢病毒載體將Scx轉導入TDSCs，不含Scx的空載作為對照也轉導入TDSCs，用載體上帶有的抗性基因，殺稻瘟菌素對細胞進行篩選。分別建成TDSC-Scx和TDSC-Mock細胞系。Scx 的表達分別用定量PCR以及免疫螢光在mRNA和蛋白水準進行鑒定。TDSC-Scx成肌腱，成軟骨和成骨方向的分化能力用定量PCR檢驗。用大鼠臏腱視窗損傷模型進行了細胞移植試驗，測試TDSC-Scx對肌腱損傷的修復作用。實驗分為三組：（1）支架組，（2）空載體組，（3）Scx組。在細胞移植後的第二，四，八周，收集正在修復中的肌腱樣品，進行植入細胞的存留狀態，鈣化，組織學和生物力學測驗。 / TDSC-Scx比TDSC-Mock有更強的成肌腱分化能力。但是，在成軟骨-成骨分化方面，沒有結論。在動物試驗，植入的細胞在第二周仍然可見，但自第四周起，就不見了。在第八周，各組均有個別樣品輕微異位鈣化，但各組別間並無顯著差異。在早期，TDSC-Scx組比空載體組和支架組合得來更好的修復肌腱的能力。 / TDSC-Scx可能促進肌腱損傷的早期修復。 / We hypothesized that transduction of tendon-derived stem cell (TDSC) with scleraxis (Scx) might promote its tenogenic differentiation and promote better tendon repair compared to TDSC without Scx transduction. This study thus aimed to investigate the effect of Scx transduction on the tenogenic differentiation of TDSC and the effect of the resulting cell line in the promotion of tendon repair. / TDSCs were transduced with lentivirus-mediated Scx or empty vector and selected by blasticidin. The mRNA and protein expression of Scx were checked by qRT-PCR and Immuno fluorescence, respectively. The expression of different lineage markers were examined by qRT-PCR. A rat patellar tendon window injury model was used. The operated rats were divided into 3 groups: (1) scaffold-only group, (2) TDSC-Mock group and (3) TDSC-Scx group. At week 2, 4 and 8 post-transplantation, the repaired patellar tendon was harvested for ex vivo fluorescent imaging, vivaCT imaging, histology, or biomechanical test. / TDSC-Scx consistently showed higher expression of tendon-related markers compared to TDSC-Mock. However, the effect of Scx transduction on the expression of chondro-osteogenic markers was less conclusive. The transplanted TDSCs could be detected in the window wound at week 2 but not at week 4. Ectopic ossification was detected in some samples at week 8 but there was no difference among different groups. The TDSC-Scx group promoted early tendon repair histologically and biomechanically compared to the scaffold-only group and the TDSC-Mock group. / TDSC-Scx might be used for the promotion of early tendon repair. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tan, Chunlai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 66-70). / Abstracts also in Chinese. / Chapter Thesis/Assessment Committee --- p.i / Acknowledgment --- p.ii / Publication --- p.vi / Abstract --- p.vii / 摘要 --- p.viii / Chapter Chapter 1 --- Tendon injury and tendon tissue engineering --- p.1 / Chapter 1.1 --- Anatomy of tendon --- p.1 / Chapter 1.2 --- Epidemiology of tendon injury --- p.3 / Chapter 1.3 --- Process and problems of tendon healing --- p.4 / Chapter 1.4 --- Current treatment and cell-based therapy for tendon repair --- p.5 / Chapter 1.5 --- Transcriptional factor Scleraxis and tendon --- p.8 / Chapter 1.5.1 --- Helix-loop-helix (HLH) and bHLH proteins --- p.8 / Chapter 1.5.2 --- Scleraxis --- p.9 / Chapter 1.6 --- Research focus and implications --- p.11 / Chapter 1.7 --- Hypotheses and objectives of this study --- p.12 / Chapter 1.8 --- Clinical significance --- p.13 / Chapter Chapter 2 --- Materials and Methods --- p.14 / Chapter 2.1 --- Study Design --- p.14 / Chapter 2.2 --- Establishment of TDSC-Scx cell line --- p.15 / Chapter 2.2.1 --- Isolation of TDSC and cell culture --- p.15 / Chapter 2.2.2 --- Establishment of TDSC-Scx cell line --- p.17 / Chapter 2.2.2.1 --- Construction of plasmid --- p.17 / Chapter 2.2.2.2 --- Transfection --- p.23 / Chapter 2.2.2.3 --- Infection --- p.24 / Chapter 2.2.2.4 --- Selection --- p.24 / Chapter 2.2.2.5 --- Characterization of TDSC-Scx and TDSC-Mock --- p.25 / Chapter 2.2.2.5.1 --- qRT-PCR --- p.25 / Chapter 2.2.2.5.2 --- Immune-fluorescent (IF) --- p.26 / Chapter 2.2.2.6 --- Lineage marker expression --- p.26 / Chapter 2.2.3 --- Data analysis --- p.29 / Chapter 2.3 --- The effect of TDSC-Scx on healing in a patellar tendon window injury model --- p.30 / Chapter 2.3.1 --- Animal surgery --- p.30 / Chapter 2.3.2 --- Ex Vivo Fluorescence Imaging --- p.32 / Chapter 2.3.3 --- vivaCT --- p.33 / Chapter 2.3.4 --- Histology --- p.33 / Chapter 2.3.5 --- Biomechanical test --- p.35 / Chapter 2.3.6 --- Data analysis --- p.37 / Chapter Chapter 3 --- Results --- p.38 / Chapter 3.1 --- Generation of TDSC-Scx and TDSC-Mock cell lines --- p.38 / Chapter 3.1.1 --- Plasmid --- p.38 / Chapter 3.1.2 --- Cell morphology --- p.38 / Chapter 3.1.3 --- Expression of Scx --- p.38 / Chapter 3.1.4 --- Expression of chondro-/osteo-/tenogenic markers --- p.39 / Chapter 3.2 --- The healing effect of TDSC-Scx on a patella tendon window injury model --- p.40 / Chapter 3.2.1 --- The fate of transplanted cells --- p.40 / Chapter 3.2.2 --- Ossification --- p.40 / Chapter 3.2.3 --- Histology --- p.40 / Chapter 3.2.3.1 --- Fiber arrangement --- p.41 / Chapter 3.2.3.2 --- Cellularity --- p.41 / Chapter 3.2.3.3 --- Cell alignment --- p.42 / Chapter 3.2.3.4 --- Cell rounding --- p.42 / Chapter 3.2.3.5 --- Vascularity --- p.43 / Chapter 3.2.3.6 --- Fiber structure --- p.43 / Chapter 3.2.3.7 --- Hyaline degeneration --- p.43 / Chapter 3.2.3.8 --- Inflammation --- p.44 / Chapter 3.2.3.9 --- Ossification --- p.44 / Chapter 3.2.4 --- Biomechanical properties --- p.44 / Chapter Chapter 4 --- Discussion --- p.55 / Chapter 4.1 --- In vitro --- p.55 / Chapter 4.1.1 --- Scx transduction did not lead to morphological change in TDSCs --- p.55 / Chapter 4.1.2 --- Scx transduction led to higher expression of Scx mRNA --- p.55 / Chapter 4.1.3 --- TDSC-Scx expressed higher levels of tenogenic markers --- p.56 / Chapter 4.2 --- In vivo --- p.58 / Chapter 4.2.1 --- The fate of transplanted cells --- p.58 / Chapter 4.2.2 --- Ossification was vague --- p.58 / Chapter 4.2.3 --- TDSC-Scx promoted tendon healing --- p.58 / Chapter 4.2.4 --- Biomechanical properties --- p.59 / Chapter 4.2.5 --- Clinical consideration --- p.60 / Chapter 4.3 --- Similar studies --- p.61 / Chapter Chapter 5 --- Limitations --- p.63 / Chapter 5.1 --- Direct Scx protein expression and function information unavailable --- p.63 / Chapter 5.2 --- Differentiation assay --- p.64 / Chapter Chapter 6 --- Conclusion --- p.65 / Reference --- p.66 / Appendix --- p.71

Despite a comprehensive mapping of the Parkinson's disease (PD)-related mRNA and protein leucine-rich repeat kinase 2 (LRRK2) in the mammalian brain, its physiological function in healthy individuals remains enigmatic. Based on its structural features and kinase properties, LRRK2 may interact with other proteins involved in signalling pathways. Here, we show a widespread LRRK2 mRNA and/or protein expression in expanded or differentiated human mesencephalic neural progenitor cells (hmNPCs) and in post-mortem substantia nigra PD patients. Using small interfering RNA duplexes targeting LRRK2 in hmNPCs following their differentiation into glia and neurons, we observed a reduced number of dopaminergic neurons due to apoptosis in LRRK2 knockdown samples. LRRK2-deficient hmNPCs exhibited elevated cell cycle- and cell death-related markers. In conclusion, a reduction of LRRK2 expression in hmNPCs severely impaired dopaminergic differentiation and/or survival of dopaminergic neurons most likely via preserving or reactivating the cell cycle.

Cardiovascular disease (CVD) affects more than 70 million Americans and is the number one cause of mortality in the United States. Because the regenerative capacity of adult tissues such as the heart is limited, human embryonic stem cells (hESC) have emerged as a source for potential cardiac therapies. However, despite the use of a variety of biochemical differentiation protocols, current yields of hESC-derived cardiomyocytes (CM) have been low. In the case of hESC-CM, which are inherently electromechanically active, additional forms of inducing a mature cardiac fate have not been fully explored. In order to non-invasively visualize and quantify biochemical, electrical, and mechanical stimulation on hESC-CM differentiation in future studies, a bioreactor imaging system has been developed and is described in this report. / text

The prevalence of neurodegenerative disease in India is rising. Regenerative medicine (RM) is being developed to treat these conditions. However, despite advances in RM application for neurological disorders (NeuroRM), there is a lack of research on clinical translation of NeuroRM technologies in developing countries. Given that India is one of the first nations to translate in this field, much can be learned on challenges and solutions arising during translation. This study identifies stakeholders involved in such translation and outlines roles of each; it describes India’s regulatory environment concerning NeuroRM translation; and discusses the impact of collaboration in clinical translation. Twenty-three face-to-face interviews with clinicians, researchers and policy-makers within India were undertaken and transcripts subjected to thematic analysis. The study demonstrates that clinical translation of NeuroRM within India is taking place robustly, it identifies barriers and good practices being adopted, and provides recommendations based on participants’ experiences.

Cell-based therapies involving adipose-derived stem cells (ASCs) have shown promise in stimulating cardiovascular regeneration, including in the treatment of myocardial infarction (MI) and ischemic heart disease. However, previous studies involving the delivery of ASCs following MI have indicated that therapeutic efficacy has been limited by low survival and/or poor retention of the transplanted cells at the site of injury. To address these limitations, the goal of this thesis was to develop a more effective delivery strategy incorporating an injectable biomaterial combined with chemotactic growth factor delivery to enhance ASC retention within the gel. Working towards future in vivo analysis in a rat model, multilineage characterization studies confirmed that ASCs isolated from the epididymal fat pad of male Wistar rats could differentiate in vitro along the adipogenic, osteogenic, and chondrogenic lineages. Subsequently, the chemotactic response of the rat ASCs (rASCs) to varying concentrations of stromal derived factor-1 α (SDF-1α) and hepatocyte growth factor (HGF) was analyzed using a modified Boyden chamber assay. The results demonstrated that SDF-1α and HGF, at 20, 50, and 100 ng/mL elicited significant migratory responses under normoxic (21%) and hypoxic (5%) culture conditions. RT-PCR analysis was conducted to assess the expression of the two chemotactic growth factors and their associated receptors in the rASCs, and secreted SDF-1α protein expression was quantified by ELISA. Moving towards the development of the biomaterials-based delivery approach, the viability of rASCs encapsulated by photopolymerization in methacrylated glycol chitosan (MGC) hydrogels modified with various degrees of arginine-glycine-aspartic acid (RGD)-peptide modification was examined. More specifically, rASCs were encapsulated in MGC hydrogels with 0%, 4%, and 7% RGD modification and cultured for up to 14 days. Viability staining results indicated that rASC viability was enhanced in the 4% and 7% RGD-modified MGC hydrogels in comparison to the MGC hydrogels with no peptide modification. Pre-loading the gels with 50 ng/mL of SDF-1α had no significant effects on cell viability over 14 days. Overall, the results demonstrate that peptide modification to promote cell adhesion within the MGC hydrogels is key to improving cell viability and thereby improving the therapeutic potential of ASCs. / Thesis (Master, Chemical Engineering) -- Queen's University, 2012-10-24 23:54:37.126

The primary aim of the thesis is to contribute to demonstrating how established and emerging science in the regenerative medicine (RM) domain can be translated into profitable commercial practice, and generate clinically- and cost-effective therapies. It achieves this by exploring and assessing underlying economics, including investment readiness and economic assessment, exploring regulatory and reimbursement frameworks, developing stem cell culture systems and assessing fit with clinical practice. The thesis is the first public domain wide-ranging analysis of business trends in the production, manufacturing and supply segments of the RM industry. It analyses the clinical potential of the domain as well as the translational and commercial challenges facing the industry. The industry is at a turning point as big pharmaceutical companies engage with RM in order to explore technologies as potential therapeutics and discovery tools. This unlocks the industry by confirming an exit path for RM based small- and medium-sized enterprises. Translation has come to be recognised as a core issue in the overall space and translation of regenerative therapies into the clinic is presently challenging, high-risk and expensive. This research addresses the question what are the mechanisms required to enable translation of emerging scientific knowledge into commercially viable clinical RM products? These mechanisms are particularly important as their creation involves and requires major investment decisions, which can determine the success or failure of RM developments and indeed of the companies concerned. The lack of well-established business models and the complexity of the domain suggested a conceptual approach drawing upon relevant literature from product and process development, applied business and revenue models, technological evolution and capital market ingenuity. The research was carried out in two phases. The first phase was concerned with identification of key challenges and mapping the overall industry emergence including emergence of related regulations to provide a context and framework for understanding the domain. Based on the emergence mapping a timeline of key parallel factors was identified, and their inherent connections explored to identify transforming events affecting and influencing multiple factors on the journey to clinical success within a business environment. This creates the reference model. The second phase was concerned with manufacturing a stem cell based therapeutic and applying health economic principles to determine available headroom for investment, cost of goods and return on investment, taking hearing disorders as a case exemplar, and exploring the behaviour of the net present value curve to identify key parameters affecting the economic positioning of this novel regime. A key output of the research is the investment readiness reference model. It integrates key RM business issues against reducing uncertainty and increasing value. The model argues that the complex nature of RM products means that the issues affecting industry emergence and development go well beyond the primarily scientific and technological concerns on which much current research focuses. The performance of RM firms ultimately hinges upon the successful clinical application of their developed products, the key step for creating and realising value, and their ability to deal with the fundamental business issues specific to the area. The framework deals with these business issues, which are investment & technology readiness, business models, organisational challenges, public policy and industry emergence. This thesis explores ideas that may bridge the chasm between the promise and reality of RM i.e. mechanisms to enable late stage translation of RM products. It links technological capability and business models for firms in the domain. Furthermore, it offers a unique perspective on the nature and characteristics of investment readiness and financial assessment, specifically identifying key parameters affecting economic positioning. The key contributions are therefore: New insights into the key challenges involved in realising the commercial potential of cell based therapeutics. Technology road mapping to link fundamental enabling technological capability for developing RM products with robust business plans integrating strategy, technology development and the regulatory and reimbursement framework. A generic investment readiness reference model generated from the enabling technology, value and supply chain structures to identify key indicators and characteristics of industry readiness. A novel experimental programme demonstrating expansion, maintenance and differentiation of human embryonic stem cells by manual and automated methods. New insights into economic positioning by mapping net present value, and economic analysis by estimating available headroom, cost of goods and return on investment for a putative hearing therapeutic.