Dissertations / Theses on the topic 'Regenerative medicine (incl. stem cells)'
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Prasongchean, W. "Somatic stem cells : properties and potential for regenerative medicine." Thesis, University College London (University of London), 2011. http://discovery.ucl.ac.uk/1336076/.
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Stem cells play fundamental roles in embryonic development, tissue homeostasis and have great potential in regenerative medicine. The main aims of this study were: i) to elucidate the properties and neural differentiation potential of somatic stem cells from different sources focusing on the analysis of stem cells with low immunogenicity and/or suitable for autologous cell therapy, amniotic fluid (AFSC) and adipose tissue-derived stem cells (ADSC), respectively; ii) to investigate a putative neural stem/progenitor cell niche in the choroid plexus (CP), organ that plays crucial roles in cerebrospinal fluid secretion and brain homeostasis. Unlike previously suggested, I found that AFSCs do not harbour significant neurogenic potential, as assessed by treatment with neurogenic small molecules, transplantation onto hippocampal organotypic cultures and within the chick nervous system. However, in a severe embryonic injury model grafted AFSCs reduced haemorrhage and significantly increased embryo survival via paracrine mechanisms. I then established and characterized ADSCs cultures derived from the fat of paediatric patients. They expressed markers of embryonic stem cells, mesenchymal and neural tissues, and displayed significant plasticity, as indicated by their ability to differentiate both into bone and cartilage upon appropriate stimulation, to home into the chick nervous system, and to be relatively rapidly reprogrammed to “induced pluripotent stem cells”. Altogether, though ADSCs seem more plastic than AFSCs, both provide valuable tools for developing novel therapeutic approaches and analyzing cell phenotype modulation. Finally, I showed the presence of neural precursors, neuroblasts and neuron-like cells within the CP in different species by analysis of neural markers and BrdU incorporation in vivo and in organotypic cultures, and demonstrated innervation of the CP at early developmental stages. Altogether these findings suggest the existence of a neural regulatory network within the CP that may play a crucial role in modulating its function in the developing and post-natal brain.
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Wong, J. W. "Centrifugal recovery of embryonic stem cells for regenerative medicine bioprocessing." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/16358/.
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In order to realise the potential of embryonic stem (ES) cells as a regenerative medicine, it is crucial that economical, robust and scalable bioprocesses be established. Because bioprocesses irrevocably define the safety and efficacy of any biologically derived product, an understanding of the the impact of the engineering environment on ES cells is sought. This thesis uses murine ES cells as a mimic for ES cell types that will be used in cell based regenerative medicine applications to examine the bioprocessing impact of centrifugal recovery cells. A micro scale-down approach was used to examine the effects of centrifugal force, centrifugation time and process temperature on both the yield and biological characteristics of cells subjected to batch centrifugation. When subjected to centrifugation, mES cell loss and cell damage does not appear to occur during the settling or cell pelleting. In general, 5-25% of cells are lost during pellet resuspension to recover the centrifuge cells. The level of cell loss is determined by a combination of centrifugal force, centrifugation time and process temperature. The extent of damage of the remaining cells (i.e. cells not lost during resuspension) is minimised at lower processing temperatures. It is hypothesised that at low processing temperatures, cell loss is minimised due to weak cell-to-cell contact and are thus less susceptible to damage caused by the shear environment generated to disperse the collected cell pellet. The concept of Windows of operations was also applied to evaluate an optimal set of centrifuge operating conditions that results in minimal cell loss and cell damage. The process visualisation tool indicates that operating the centrifuge at 5-9 mins x 300-500 g will result in maximum cell recovery at 4, 21 and 37oC process temperatures. The influence of centrifugation on the biological characteristics of mES cells revealed changes in proliferative capacity, pluripotency and differentiation status when exposed to varying levels of centrifugal force. mES cells exposed to increasing levels of centrifugal force up to 2,000 g progressively lost pluripotency. The pluripotency potential of cells exposed to 3,000 g of centrifugal force was not significantly different from un-centrifuged mES cells. Differentiating mES cells exposed to increasing levels of centrifugal force exhibited increased cell proliferation and a possibility of early induction of endoderm and mesoderm differentiation. Although limited in some areas, the results strongly suggest that restricting exposure to no more than low levels of centrifugal force is necessary to safeguard the stability of the desired mES cell characteristics. Overall, the insight gained from the work accomplished serves to create and establish an awareness of the challenges faced within the arena of whole cell bioprocessing for regenerative medicines.
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Chu, Pui Kei Carol. "The bioprocessing of stem cells for use in regenerative medicine." Thesis, University College London (University of London), 2009. http://discovery.ucl.ac.uk/17422/.
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Stem cells hold great therapeutic potential in regenerative medicine due to their self-renewal capacity and their ability to differentiate into a wide range of cell lineages. This thesis is focused on the characterisation of both a pluripotent and a more specialised multipotent stem cell source. These studies involve the optimisation of essential parameters for cell expansion and differentiation. Initially, human mesenchymal stem cells (hMSCs) were used as a model system for cell expansion studies. This was followed by more advanced studies using mouse embryonic stem cells (mESC). The results obtained in this research revealed that hMSCs inoculated at low densities resulted in larger fold expansion than those inoculated at high densities. However, at low (<100 cells/cm^2) and high (10000 cells/cm^2) inoculation densities, the expression of STRO-1, a surface marker present on hMSC was lost very rapidly. The optimal inoculation density was found to be 5000 cells/cm^2. Prolonged in vitro culture of hMSCs caused a reduction in the cell proliferative potential and STRO-1 antigen expression. Therefore to preserve the ‘stemness' properties of hMSCs, expansion needs to be limited to low passage levels. The optimal fetal bovine serum concentration for hMSCs expansion was found to be at 15%. The differentiation of mESC was studied using the embryoid body (EB) system. The results obtained indicated that the size of the EB and activin A, a ligand that activates the nodal signaling pathway have profound effects on mESC lineage commitment and thus on bioprocessing. Quantitative reverse transcription-polymerase chain reaction analysis revealed that activin A enhanced and accelerated endoderm formation within small EBs, and delayed the loss of pluripotency of undifferentiated cells within large EBs. In addition, activin A has an inhibitory effect on neuroectodermal differentiation. Small EBs treated with activin A displayed a minimal neuroectodermal gene expression whereas large treated EBs displayed a higher level of expression. Finally, various in vitro culture systems for mESC differentiation were examined. These include static, rotary suspension and combined culture systems. This research provides an insight into some of the parameters affecting the growth kinetics and developmental characteristics of hMSCs and mESCs. The findings reported may help facilitate the development of successful stem cell bioprocess design essential for the scaling and manufacture of safe, efficacious and cost effective human therapies.
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Perruisseau-Carrier, Claire. "Neuronal commitment of Umbilical Cord Mesenchymal Stem Cells for brain regenerative medicine." Thesis, Lyon 1, 2013. http://www.theses.fr/2013LYO10192.
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De nos jours, aucune prévention ou aucun remède efficace n'existe pour guérir les maladies du cerveau humain. Les cellules souches représentent un grand espoir pour la réparation et la régénération des tissus neuraux endommagés. L'objectif de cette thèse est d'évaluer la capacité des cellules souches du cordon ombilical humain (hUC MSCs) à se différencier en neurones, pour une thérapie cellulaire appliquée au cerveau. Nous avons isolé, multiplié et caractérisé les hUC MSCs naïves à l'échelle des gènes et des protéines. Ensuite, les e_ets sur l'expansion des hUC MSCs et leur différenciation neuronale de différents paramètres ont été évalués par qPCR et marquages immunologiques principalement: milieux et matrices de culture, oxygénation, culture en 3D, ainsi que divers facteurs et molécules tels que les microARNs. Les résultats montrent que les hUC MSCs prolifèrent mieux sans sérum et en conditions de normoxie du cerveau (1-5 % O2). Les hUC MSCs naïves semblent préparées à devenir des neurones à l'échelle des gènes et des protéines, mais pas suffisamment pour supporter leur complète différenciation. L'introduction de microARNs requiert des améliorations pour réguler efficacement les voies de signalisation des hUC MSCs. Au cours de cette étude, nous avons identifé les paramètres favorisant l'expansion des hUC MSCs dans des conditions compatibles avec la clinique. Cependant, une question reste ouverte: les hUC MSCs sont-elles capables de vraie transdifferentiation en neurones fonctionnels malgré les controverses? Des recherches supplémentaires sont nécessaires, mais cette étude constitue une première étape vers l'utilisation des hUC MSCs en médecine régénératrice du cerveauNowadays, no effective prevention or cure of human brain diseases is available. Stem cells hold great promise for the repair and regeneration of damaged neural tissues. This thesis aims to evaluate the potency of human umbilical cord mesenchymal stem cells (hUC MSCs) to be committed to the neuronal lineage, for brain cell-based therapy. To achieve this goal, naive hUC MSCs were isolated, expanded, and characterized at the gene and protein level, while particularly focusing on the neuronal lineage and clinical-grade culture conditions. Then, several parameters were investigated for hUC MSCs proliferation and neuronal commitment, including media, coatings, 3D culture, hypoxia, chemicals and molecules. Growth curves drawings, qPCRs, and immunostainings were used among other methods for identifying the best conditions for hUC MSCs expansion, differentiation, culture in 3D, and microRNAs delivery. The results indicate that hUC MSCs better proliferate in serum-free media and brain's normoxia condition (1-5 % O2). Naive hUC MSCs appear primed for neuronal fate at gene and protein level, but not su_ciently to support their neuronal di_erentiation. microRNAs delivery requires further improvement to efficiently promote neuronal signaling pathways in hUC MSCs. Along this study we identified the best parameters for hUC MSCs expansion in clinical-grade conditions. However, a question still remains: are hUC MSCs capable of full transdifferentiation towards functional neurons despite all controversies? Additional work is needed, but this study is a first step towards answering this question, bringing more clues to make transplantation of hUC MSCs for brain regenerative medicine closer
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Sutha, Ken. "Osteoinductive material derived from differentiating embryonic stem cells." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/51722.
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The loss of regenerative capacity of bone, from fetal to adult to aged animals, has been attributed not only to a decline in the function of cells involved in bone formation but also to alterations in the bone microenvironment that occur through development and aging, including extracellular matrix (ECM) composition and growth/trophic factor content. In the development of novel treatments for bone repair, one potential therapeutic goal is the restoration of a more regenerative microenvironment, as found during embryonic development. One approach to creating such a microenvironment is through the use of stem cells. In addition to serving as a differentiated cell source, pluripotent stem cells, such as embryonic stem cells (ESCs), may possess the unique potential to modulate tissue environments via local production of ECM and growth factors. ESC-produced factors may be harnessed and delivered to promote functional tissue regeneration. Such an approach to generate a naturally derived, acelluar therapy has been employed successfully to deliver osteoinductive factors found within adult bone, in the form of demineralized bone matrix (DBM), but the development of treatments derived instead from developing, more regenerative tissues or cells remains attractive. Furthermore, the derivation of regenerative materials from an ESC source also presents the added benefit of eliminating donor to donor variability of adult, cadaveric tissue derived materials, such as DBM. Thus, the objective of this project was to examine the osteoinductive potential harbored within the embryonic microenvironment, in vitro and in vivo. The osteogenic differentiation of mouse ESCs as embryoid bodies (EBs) was evaluated in response to phosphate treatment, in vitro, including osteoinductive growth factor production. The osteoinductivity of EB-derived material (EBM) was then compared to that of adult tissue-derived DBM, in vivo. Phosphate treatment enhanced osteogenic differentiation of EBs. EBM derived from phosphate treated EBs retained bioactive, osteoinductive factors and induced new bone formation, demonstrating that the microenvironment within osteogenic EBs can be harnessed in an acellular material to yield in vivo osteoinductivity. This work not only provides new insights into the dynamic microenvironments of differentiating stem cells but also establishes an approach for the development of an ESC-derived, tissue specific therapy.
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Srivastava, Sapna. "The potential of human adipose derived stem cells for myocardial regenerative therapy." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95088.
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Background: Cell therapy using Human Bone Marrow Stem Cells (HBMSCs) has been shown to improve heart function after a myocardial infarction. The harvesting technique involved with bone marrow stem cells is invasive and yields a low cell number. There is now an increasing interest in Human Adipose Derived Stem Cells (HADSCs) as they are abundant and readily accessible from liposuction material. The present study was undertaken to investigate if HADSCs are superior than HBMSCs in myocardial regenerative therapy. Results: Both HADSCs and HBMSCs proliferated in a time dependent manner, however, the proliferative ability of HADSC was greater than HBMSCs. In addition, both cells differentiated to the osteoblast lineage confirming their multipotency when treated with induction medium. Furthermore, treatment of both cells with 5-AC resulted in positive immunostaining of cardiac markers, troponinI and connexin 43, however the expression of these markers was enhanced in HADSCs. This was further confirmed by western blot analysis, however 5-AC treatment did not exhibit cell contraction or multinucleation. In addition, these results were further confirmed by our in vivo study. Both cells were injected in the heart of a rat model of myocardial infarction and was monitored for ejection fraction (EF) and fractional shortening (FS) for 24 hours, 3 weeks and 6 weeks post-surgery. The cardiac function of the rats treated with stem cells was improved as demonstrated by increase in EF and FS, however, a greater improvement was seen with HADSCs compared with HBMSCs. This notion is further substantiated by our studies on left ventricular infarct size measurement, showing that HADSC are more potent in reduction of the infarct compared to HBMSCs. Conclusion: The data suggest that HADSCs may prove to be a more ideal alternative for regenerative therapy in the future.La thérapie cellulaire à l'aide de cellules souches humaines de la moelle osseuses (CSHMOs) a été démontré d'améliorer la fonction cardiaque après un infarctus du myocarde. La technique de récolte des CSHMOs est pourtant invasive et donne un nombre de cellules viables faible. Il y a maintenant un intérêt croissant dans les cellules souches humaines dérivés du tissu adipeux (CSHTAs), car elles sont abondantes et facilement accessibles à partir des amas de graisses provenant des chirurgies de liposuccion. La présente étude a été menée pour vérifier si les CSHTAs sont supérieures aux CSHMOs dans la thérapie régénératrice du myocarde. Résultats: Les CSHTAs ainsi que les CSHMOs ont proliféré dans une manière temps dépendante, cependant, la capacité proliférative des CSHTAs était supérieure à celle des CSHMOs. De plus, les deux types de cellules souches ce sont différenciées en lignée ostéoblastique, affirmant leur capacité multipotent lorsqu'elles sont traitées avec le milieu d'induction. En outre, le traitement des deux types de cellules souches avec le 5-AC a entraîné l'immunomarquage positif de troponin I et de connexine 43, marqueurs cardiaques, cependant l'expression de ces marqueurs était plus robuste dans les CSHTAs. Cela a été confirmé par analyse d'immunobuvardage de type Western, cependant les cellules traité au 5-AC ne présentait pas de contraction des cellules ou le développement de plusieurs noyaux. En plus, ces résultats ont été confirmés par nos études in vivo. Les deux types de cellules ont été injectées dans le cur d'un modèle de rat d'infarctus du myocarde et a été suivie pour la fraction d'éjection (FE) et la fraction de raccourcissement (FR) pour 24 heures, 3 semaines et 6 semaines post-chirurgie. La fonction cardiaque des rats traités avec les cellules souches a été améliorée, fait démontré par l'augmentation de l'FE et le FR, cependant, une plus grande amélioration de ce
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Yasin, Mohammed. "Non-regenerative benefits of adult bone marrow derived stem cells for myocardial protection." Thesis, Queen Mary, University of London, 2013. http://qmro.qmul.ac.uk/xmlui/handle/123456789/8701.
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Ischaemic heart disease is the most common cause of mortality in the western hemisphere and it is rapidly becoming the leading cause of death globally. Moreover, therapeutic interventions by cardiologists and cardiac surgeons frequently subject the heart to acute I/R injury, which in itself can cause mortality. Recent investigations of adult stem cells have primarily focused on their regenerative potential for chronic ischaemic heart disease. In this thesis, I have investigated the hypothesis that adult bone marrow derived stem cells are cardioprotective in acute regional myocardial I/R injury. In a rat model of left anterior descending coronary artery (LAD) reversible occlusion and reperfusion, I demonstrate that an intravenous bolus of adult bone marrow derived (1) bone marrow mononuclear (BMNNC) and (2) mesenchymal stem cells (MSC) upon reperfusion can attenuate infarct size. This effect is comparable to ischaemic preconditioning (IPC), which is the gold standard for cardioprotection. Next, I demonstrated the mechanisms for adult stem cell cardioprotection are principally anti-apoptotic and depend upon stem cell secreted factors to (1) activate phosphatidylinositide 3-kinase (PI3)/Akt cell survival kinase-signaling pathway (2) inhibit glycogen synthase kinase-3β (3) inhibit p38MAPK (4) inhibit nuclear translocation of p65NF-κB. 7 Proteomic analysis of myocardium subjected to I/R and treated with either BMMNC or BMMNC derived supernatant (BMS) upon reperfusion demonstrated higher expression of a whole host of pro-survival proteins. These were notably (1) 14-3-3-ε protein (2) anti-oxidant peroxiredoxin-6 (3) heat shock protein (HSP) αB-crystallin, HSP72, HSP tumour necrosis factor receptor-1 associated protein, and HSP ischaemia responsive protein-94 (4) glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (5) mitochondrial aconitase and mitochondrial voltage-dependent anionselective channel protein-1. Thereafter, I investigated the mobilization of endogenous bone marrow stem cells and trafficking to the ischaemic myocardium by stromal cell derived factor-1 (SDF-1) /chemokine, receptor type 4 (CXCR4) signaling. I demonstrate high up-regulated expression of CXCR4 and CD26 in BMMNC following IPC, which might have a role in IPC-mediated cardioprotection. Finally, and in concordance with this finding I demonstrate that both IPC and an exogenous MSC bolus upon reperfusion can synergize to abolish acute myocardial I/R injury.
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Williams, Kaylyn Renee. "In Vitro Models of Cellular Dedifferentiation for Regenerative Medicine." Thesis, Virginia Tech, 2018. http://hdl.handle.net/10919/83715.
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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
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Mohrman, Ashley E. "Regenerative Medicine Approaches to Spinal Cord Injury." University of Akron / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=akron1491495476427594.
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Singh, Pawanbir. "Enabling late-stage translation of regenerative medicine based products." Thesis, Loughborough University, 2010. https://dspace.lboro.ac.uk/2134/6060.
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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.
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Wang, Yinxiang, and 王胤祥. "Induction of cells with osteo-chondrogenic potential by transcription factor-mediated reprogramming process." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/207993.
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Skeletal system plays a crucial role in our life. Skeletal diseases and disorders unlike cancer, are not fatal, but affect the quality of our life. Cell-based therapeutic strategies to generate targeted desired cell types for repair or replacement of damaged skeletal tissues are ideal regenerative medicines. Because of the heterogeneous cell types generated from embryonic and mesenchymal stem cells, the ability of progenitor population to differentiate into a target cell type appear to be a better alternative for tissue regeneration. Osteo-chondroprogenitors uniquely co-expressing Sox9 and Runx2 with dual differentiation potential to become chondrocytes and osteoblasts is a progenitor cell which is suitable for cell based therapy of bone disease. Therefore, developing effective strategies to generate sufficient quantities of osteo-chondroprogenitors are essential.
Toward this, we took advantage of two lineage conversion approaches. The first strategy was to interrogate the ability of osteoblasts to be reprogrammed into induced pluripotent stem (iPS) cells and another one was to use defined transcription factors to induce chondrocyte lineage from skin fibroblasts. The selection of osteoblasts is based on the fact that it is originally derived from osteo-chondroprogenitor lineage and the stochastic events of iPS induction might revert osteoblasts first to their progenitor state before becoming pluripotent. The second approach is based on a previous report using three transcription factors (Sox9, Klf4 and c-Myc) to reprogramme skin fibroblasts into chondrocyte lineage. Our aim is to examine whether osteo-chondroprogenitors would be formed during the two reprogramming processes using Sox9-EGFP knock-in mice as a reporter.
We reasoned that osteoblasts can be reprogrammed into iPS cells by four Yamanaka’s factors with pluripotency as shown by their ability to form teratomas and contribute to chimeric embryos. However base on the limitation of selector marker of osteo-chondroprogenitor we still cannot capture this progenitor during iPS reprogramming. And because of the pluripotency potential, pluripotent reprogramming approach also brings high risk of teratoma formation. Therefore our second objective was performed to examine whether osteo-chondroprogenitors would be formed during lineage reprogramming. Transient appearance of Sox9-EGFP/Runx2+ve cells was observed in the intermediate stage of over 14 days of chondrocyte lineage induction from skin fibroblasts by Sox9, klf4 and c-Myc. Cells expressing Sox9-EGFP/Runx2+ve showed typical molecular markers of osteo-chondroprogenitors. In vitro and in vivo differentiation assays demonstrated that Sox9-EGFP/Runx2+ve cells can differentiate predominantly into osteoblasts and chondrocytes. Taken together our data indicate that cells with osteo-chondrogenic potential could be generated by defined transcription factors-mediated reprogramming processes.published_or_final_version
Biochemistry
Doctoral
Doctor of Philosophy
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TAKEI, YOSHIFUMI, KENJI KADOMATSU, KAORI YASUDA, and NAOSHI KOIDE. "ESTABLISHMENT AND OPTIMAL CULTURE CONDITIONS OF MICRORNA-INDUCED PLURIPOTENT STEM CELLS GENERATED FROM HEK293 CELLS VIA TRANSFECTION OF MICRORNA-302S EXPRESSION VECTOR." Nagoya University School of Medicine, 2012. http://hdl.handle.net/2237/16033.
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Sunohara, Tadashi. "MicroRNA-based separation of cortico-fugal projection neuron-like cells derived from embryonic stem cells." Kyoto University, 2020. http://hdl.handle.net/2433/253176.
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Youngstrom, Daniel W. "Mesenchymal Stem Cell Mechanobiology and Tendon Regeneration." Diss., Virginia Tech, 2015. http://hdl.handle.net/10919/64422.
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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.
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Hoover, Brett A. "Smart Cellector: A Proposal for the Development and Commercialization of a Cellular Imaging, Analysis and Processing Technology for Application in Regenerative Medicine." Case Western Reserve University School of Graduate Studies / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=case1295655205.
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Hackett, Simon Marc. "Addressing the immunological barriers to regenerative medicine : differentiation and characterisation of dendritic cells derived from induced pluripotent stem cells." Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.644988.
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Induced pluripotent stem cells (iPS cells) have shown great promise in the newly-developing field . of regenerative medicine. Recently, however; there have been conflicting reports regarding their immunogenicity, even when derived in an autologous fashion, due to the ectopic expression of certain developmental antigens. Dendritic cells (DC) are professional antigen presenting cells and therefore serve as potential tools for modulating the immune response, particularly with respect to acceptance of stem cell-derived tissues. We hypothesised that DC differentiated from iPS cells, so-called iPS, may have the capacity to induce tolerance to tissues differentiated from the same parent iPS cell line. As a first step towards this goal, we have derived iPS cells from both embryonic and adult mouse fibroblasts, developed protocols for their differentiation along the DC lineage and characterised the resulting DC both phenotypically and functionally, using bone-malTow derived-DC (bmDC) for comparison. Furthermore, their potential for inducing tolerance as a function of regulatory T cell induction has also been explored. Our data suggest that, although ipDC are able to function in a similar manner to bmDC both in vitro and in vivo, they display important differences with respect to cytokine production and MHC class II expression which may reflect their early embryonic origin and render them pro-tolerogenic. One application for this novel population of DC might be the induction of tolerance to autologous iPS cell-derived tissues. In order to assess the need for tolerance under such circumstances, we have investigated the reported immunological rejection of iPS cells when transplanted syngeneically. Although we were unable to coroborate previous findings showing immunological rejection of iPS cells under such circumstances, we were able to show that the formation of teratomas from iPS cells is a stochastic and very variable process, unlike their generation from ES cells. Importantly, we were able to exclude the involvement of any antigen-specific component to the failure of teratoma survival by inducing blanket tolerance to iPS cell-derived tissues, using a cocktail of non-depleting monoclonal antibodies specific for CD4, CD8 and CD40L. This is the first repOlted study deriving dendritic cells from mouse iPS cells and demonstrating their extensive characterisation. Additionally, the work presented in this thesis adds to the growing body of evidence of the immunological propelties of iPS cells with our work demonstrating that they appear not to attract an adaptive immune response but, however, show significant heterogeneity with respect to their ability to engraft in vivo.
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Miller, Angela. "Peptide based hydrogels in the study of mesenchymal stem cells for the purposes of regenerative medicine." Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6880/.
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Regenerative medicine is a vastly expanding subject area, with a number of different strategies and substrates being studied to ultimately create a model for repairing diseased and injured tissue. Stem cells are a promising cell type in this field as they are known to differentiate into a number of cell types and contribute to normal cell repair. However, despite their potential as a useful cell choice in the field of regenerative medicine, stem cell based therapies have limited potential due to their ability to form tumours when implanted in the human body, problems arising with immunogenicity and the difficulty in obtaining adequate cell numbers for transplantation. To overcome these problems, many research groups are interested in using biomimetic substrates and scaffolds to mimic the architecture, chemical composition and stiffness properties of the in vivo cell niche and various human tissues, but in an in vitro setting. By doing so, the MSC behaviour can be studied and the differential lineages examined allowing the desired substrate to be tuned to obtain the desired cellular outcome. In this work, hydrogels composed of Fmoc diphenylalanine and Fmoc Serine have been characterised and used to study changes in Mesenchymal stem cell (MSC) responses in a two and three dimensional state. The results obtained from this work demonstrate that such hydrogels support MSC growth and produce a mixed phenotype population which differentiates over time. A further hydrogel paired with collagen has shown promise in promoting MSC differentiation down the osteogenic lineage and has potential for the future study in maxillofacial reconstruction models.
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Popielarczyk, Tracee. "Homing and Differentiation of Mesenchymal Stem Cells in 3D In Vitro Models." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/78789.
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Mesenchymal stem cells (MSCs) have great potential to improve clinical outcomes for many inflammatory and degenerative diseases through delivery of exogenous MSCs via injection or cell-laden scaffolds and through mobilization and migration of endogenous MSCs to injury sites. MSC fate and function is determined by microenvironmental cues, specifically dimensionality, topography, and cell-cell interactions. MSC responses of migration and differentiation are the focus of this dissertation. Cell migration occurs in several physiological and pathological processes; migration mode and cell signaling are determined by the environment and type of confinement in three-dimensional (3D) models.
Tendon injury is a common musculoskeletal disorder that occurs through cumulative damage to the extracellular matrix (ECM). Studies combining nanofibrous scaffolds and MSCs to determine an optimal topographical environment have promoted tenogenic differentiation under various conditions. We investigated cellular response of MSCs on specifically designed nanofiber matrices fabricated using a novel spinneret-based tunable engineered parameters production method (STEP). We designed suspended and aligned nanofiber scaffolds to study cellular morphology, tendon marker gene expression, and matrix deposition as determinants for tendon differentiation.
The delivery and maintenance of MSCs at sites of inflammation or injury are major challenges in stem cell therapies. Enhancing stem cell homing could improve their therapeutic effects. Homing is a process that involves cell migration through the vasculature to target organs. This process is defined in leukocyte transendothelial migration (TEM); however, far less is known about MSC homing. We investigated two population subsets of MSCs in a Transwell system mimicking the vasculature; migrated cells that initiated transmigration on the endothelium and nonmigrated cells in the apical chamber that failed to transmigrate. Gene and protein expression changes were observed between these subsets and evidence suggests that multiple signaling pathways regulate TEM.
The results of these experiments have demonstrated that microenvironmental cues are critical to understanding the cellular and molecular mechanisms of MSC response, specifically in homing and differentiation. This knowledge has identified scaffold parameters required to stimulate tenogenesis and signaling pathways controlling MSC homing. These findings will allow us to target key regulatory molecules and cell signaling pathways involved in MSC response towards development of regenerative therapies.Ph. D.
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Jackson, JeShaune D. Jackson. "Bench to Bone: Commercializing a Cellular Therapeutic for Regenerative Medicine." Case Western Reserve University School of Graduate Studies / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1521472107740449.
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Sheard, Jonathan. "Screening and improving the safe provision of mesenchymal stem cells in regenerative medicine : an in vitro study." Thesis, Aston University, 2016. http://publications.aston.ac.uk/30085/.
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A number of studies have suggested that mesenchymal stem cells (MSCs) may undergo genetic alterations and spontaneous malignant transformation to form tumour cells, or at least can become contaminated with other cell types following extended periods in culture. Possible transformation or contamination of MSCs during cell culture expansion prior to their use in transplantation therapies is a risk, which should be taken into account. There is a continued need for the development of improved tools for monitoring safety and release criteria of cells intended for cell-based therapies. In order to help address these risks, this thesis aimed to develop improved safety measures in MSC-based cell therapies. Initially, this was investigated through the use of microscopic imaging and image analysis platforms to screen, characterise and distinguish between cultures of non-transformed MSCs and MSC-derived tumour cells, i.e. the osteosarcoma cell lines, SAOS2 and MG63, as well as cells derived from a chondrosarcoma. High content screening (HCS) and live-cell imaging and analysis platforms were used to enable these experiments. Phenotypic features that distinguished the normal versus malignant cell types were identified, including immunoreactivity for the proliferation-associated Ki67 antigen and pluripotency marker Oct4, as well as significant differences in nuclear morphology. These findings help inform release criteria for therapeutic MSCs. To further potentially improve the safety of MSC-based therapies, research was also performed to address the possibility that tumour cells in MSC cultures might remain undetected, thereby still providing a risk in MSC transplantations. Novel combinatorial regimes of potential anti-tumour drugs, i.e. bezafibrate, medroxyprogesterone, and valproic acid (termed V-BAP) were tested in vitro for their effects on MSCs versus SAOS2 and MG63 cells. At determined concentrations, these drugs were shown to significantly inhibit the growth of the osteosarcoma cells, but had little effect on MSCs. Thus, this thesis has made inroads into improved safety of MSC-based therapies by (i) demonstrating the application of imaging-based cell screening platforms to help characterise MSC cultures intended for cell transplantation, and (ii) identifying a novel drug regime that selectively targets osteosarcoma cells whilst having little effect on MSCs. The findings on V-BAP also have application in anti-tumour treatments for osteosarcoma.
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Lazin, Jamie Jonas. "The effect of age and sex on the number and osteogenic differentiation potential of adipose-derived mesenchymal stem cells." Thesis, Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/34696.
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It has been shown that stem cells exist within adult adipose tissue. These stem cells are named adipose-derived mesenchymal stem cells (ASCs), are derived from the mesoderm, and can differentiate into a number of cells including osteoblasts, chondrocytes, and adipocytes. However, before these cells can be used clinically it is important that we understand how factors like age, sex, and ethnicity affect ASC number and potential. Additionally, since men and women vary in their distribution of adipose tissue, it will be important to see if the ideal source of ASCs is different for each sex. The goal of this study was to assess how age and sex affects ASCs. We used flow cytometry to investigate how age and sex affected the number of ASCs in adipose tissue. Additionally, we plated these cells in culture and treated them with an osteogenic media (OM) with the intention of pushing them towards osteoblast differentiation. The purpose of this was to see if age or sex affected the potential of the ASCs to undergo osteogenesis in culture. For this study we used real-time PCR and biochemical assays to look at markers of early and late osteogenic differentiation. Finally, we used immunohistochemistry to demonstrate where in adipose tissue the CD73 and CD271 positive cell population exists. It is our hope that this work will shed light on how age and sex affect ASCs so that clinicians can optimize their ASC harvest depending on the patient's physiology.
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Nair, Rekha. "Acellular matrices derived from differentiating embryonic stem cells." Diss., Georgia Institute of Technology, 2009. http://hdl.handle.net/1853/37170.
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Embryonic stem cells (ESCs) can differentiate into all somatic cells, and as such, are a promising cell source for therapeutic applications. In vitro, ESCs spontaneously differentiate via the aggregation of cells into embryoid bodies (EBs), which recapitulate aspects of early embryogenesis and harbor a unique reservoir of cues critical for tissue formation and morphogenesis. Embryonic healing responses employ similar intrinsic machinery used for tissue development, and these morphogenic cues may be captured within the EB microenvironment. Recent studies have shown that when injected into injury or defect models in vivo, ESCs synthesize and secrete extracellular factors that ultimately contribute to repair, suggesting that these molecules may be as important for regenerative therapies as functional differentiation of the cells. The overall objective of this project was to develop novel acellular matrices derived from differentiating ESCs undergoing morphogenesis. The central hypothesis was that embryonic matrices contain complex mixtures of extracellular factors that, when isolated, retain bioactivity and enhance wound healing in an adult environment.
The overall objective was accomplished by: (1) investigating the production of extracellular matrix (ECM) by differentiating ESCs as a function of differentiation time; (2) assessing the ability of solvents to efficiently decellularize EBs; and (3) evaluating the healing response elicited by acellular matrices derived from EBs in a murine dermal wound healing model. Endogenous ECM synthesis by EBs varied with time and was associated with specific differentiation events. Novel techniques were developed to effectively remove cell components from EBs in order to extract complex, bioactive acellular matrices. EB-derived acellular matrices significantly enhanced the healing of excisional dermal wounds in mice, indicating the potency of extracellular factors synthesized by ESCs. All together, these studies demonstrate that acellular matrices derived from ESCs retain morphogenic factors capable of influencing tissue repair. In addition, this work lays the foundation for future studies to further examine the functional role of endogenous matrix molecules on ESC differentiation and to evaluate the utility of a stem cell-derived matrix for a variety of regenerative medicine applications.
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Coffin, Spencer. "Extending the Window of Use for Human Mesenchymal Stem Cell Seeded Biological Sutures." Digital WPI, 2015. https://digitalcommons.wpi.edu/etd-theses/510.
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Cell therapy, including human mesenchymal stem cell (hMSC) therapy, has the potential to treat different pathologies, including myocardial infarctions (heart attacks). Biological sutures composed of fibrin have been shown to effectively deliver hMSCs to infarcted hearts. However, hMSCs rapidly degrade fibrin making cell seeding and delivery time sensitive. To delay the degradation process, we propose using aprotinin, a proteolytic enzyme inhibitor that has been shown to slow fibrinolysis. This project investigated the effects of aprotinin on hMSCs and suture integrity. Viability of hMSCs incubated with aprotinin, examined using a LIVE/DEAD stain, was similar to controls. No differences in proliferation, as determined by Ki-67 presence, and were observed. hMSCs incubated in aprotinin differentiated into adipocytes, osteocytes, and chondrocytes, confirming multipotency. CyQuant assays were used to determine the number of cells adhered to fibrin sutures. The number of adhered cells was increased through aprotinin supplementation at Days 2, 3, and 5 time points. To examine the effect of aprotinin on suture integrity, sutures were loaded to failure to determine ultimate tensile strength (UTS) and modulus (E). Sutures exposed to aprotinin had higher UTS and E when compared to sutures exposed to standard growth media. Degradation of fibrin was quantified using an ELISA to quantify fibrin degradation products (FDP) and by measuring suture diameter. Fibrin sutures incubated in aprotinin had larger diameters and less FDP compared to the controls, confirming decreased fibrinolysis. These data suggest that aprotinin can reduce degradation of biological sutures, providing a novel method for extending the implantation window and increasing the number of cells delivered for hMSC seeded biological sutures.
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Nsiah, Barbara Akua. "Fluid shear stress modulation of embryonic stem cell differentiation." Diss., Georgia Institute of Technology, 2012. http://hdl.handle.net/1853/47552.
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Vascularization of tissue-engineered substitutes is imperative for successful
implantation into sites of injury. Strategies to promote vascularization within tissue-engineered constructs have focused on incorporating endothelial or endothelial progenitor cells within the construct. However, since endothelial and endothelial progenitor cells are adult cell types and limited in number, acquiring quantities needed for regenerative medicine applications is not feasible. Pluriopotent stem cells have been explored as a cell source for tissue-engineered substitutes because of their inherent ability to differentiate into all somatic cell types, including endothelial cells (ECs). Current EC differentiation strategies require laborious and extensive culture periods, utilize large quantities of expensive growth factors and extracellular matrix, and generally yield heterogenous populations for which only a small percentage of the differentiated cells are ECs. In order to recapitulate in vivo embryonic stem cell (ESC) differentiation, 3D stem cell aggregates or embryoid bodies (EBs) have been employed in vitro. In the developing embryo, fluid shear stress, VEGF, and oxygen are instructive cues for endothelial differentiation and vasculogenesis. Thus, the objective of this work was to study the effects of fluid shear stress pre-conditioning of ESCs on EB endothelial differentiation and vasculogensis. The overall hypothesis is that exposing ESCs to fluid shear stress prior to EB differentiation will promote EB endothelial differentiation and vasculogenesis. Pre-conditioning ESCs with fluid shear stress modulated EB differentiation as well as endothelial cell-like cellular organization and EB morphogenesis. To further promote endothelial differentiation, ESCs pre-conditioned with shear were treated with VEGF. Exposing EBs formed from ESCs pre-conditioned with shear to low oxygen resulted in increased production of VEGF and formation of endothelial networks. The results of this work demonstrate the role that physical forces play in modulating stem cell fate and morphogenesis.
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Dahal, Shataakshi. "Stem Cells Based Elastic Matrix Regeneration for Small Abdominal Aortic Aneurysms (AAAs) Repair." Cleveland State University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=csu1599137475237285.
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Dahal, Shataakshi. "Stem Cells Based Elastic Matrix Regeneration for Small Abdominal Aortic Aneurysms (AAAs) Repair." Cleveland State University / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=csu1599137475237285.
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Bogers, Sophie Helen. "Turning Round: Optimizing the Anti-Inflammatory Properties of Equine Bone Marrow Derived Mesenchymal Stem Cells for Osteoarthritis Through Three-Dimensional Culture." Diss., Virginia Tech, 2017. http://hdl.handle.net/10919/81746.
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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.
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Francis, Michael. "RECAPITULATING OSTEOBLASTOGENESIS WITH ELECTROSPUN FIBRINOGEN NANOFIBERS AND ADIPOSE STEM CELLS AND ELECTROSPINNING ADIPOSE TISSUE-DERIVED BASEMENT MEMBRANE." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/2025.
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To repair, replace, or regenerate damaged or diseased tissue has been a long-standing, albeit elusive, goal in medical research. Here, we characterize patient-derivable mesenchymal stem cell types, termed adipose-derived stem cells (ASCs). These cells, which can be derived from liposuction fat and lipoaspirate saline, are sources for patient-derivable extracellular matrix (ECM), fibrinogen (Fg) and adipose tissue extracellular matrix, and may prove useful for synthesizing new bone tissue analogues in vitro. Traditionally and rapidly isolated ASCs were thoroughly characterized as multipotent, having osteogenic, adipogenic, and chondrogenic differentiation potential, and they exhibited comparable proliferative lifespans. These ASCs also shared an indistinguishable immunophenotype when compared to bone marrow-derived mesenchymal stem cells, suggesting that these cells are an excellent source for bone following tissue engineering experimentation. In order to synthesize bone ex-vivo, electrospun scaffolds of fibrinogen (Fg), polydioxanone (PDO), and Fg:PDO blends were seeded with early passage ASCs, fibroblasts, or osteosarcoma cells and were maintained for 21 days in osteogenic or regular growth media. Constructs were analyzed both histologically and molecularly for evidence of osteoblastogenesis. Using SEM, the appearance of regular, mineralized-appearing structures were found in osteogenic-induced ASC seeded scaffolds beyond 14 days, only in the scaffolds containing Fg. Further, at 21 days of culture, Fg scaffolds with ASCs in osteogenic media became hard and brittle. Robust new collagen synthesis and matrix remodeling were observed on all Fg scaffolds, the levels of which were elevated over time. Pronounced mineralization was found throughout bone-induced ASC scaffolds, while control scaffolds (BJ foreskin fibroblasts) showed no mineral deposition (although they did demonstrate excellent cellularity). Analysis of gene expression (qRT-PCR) indicated that electrospun Fg supported osteoblastogenesis through the upregulation of alkaline phosphatase and osteocalcin gene expression. To confirm our gene expression results, osteogenic-induced ASCs on Fg scaffolds were also shown to secrete osteocalcin in the extracellular matrix, a key marker in osteoblastogenesis. Thus, electrospun Fg is an excellent material for ASC growth, proliferation, and osteogenic differentiation, providing an ideal system for furthering basic bone model-based research and for advancing regenerative medicine. In addition to establishing Fg as a source of scaffolding, we developed and characterized a novel method for isolating and subsequently electrospinning adipose tissue matrix. Because adipose ECM contains many primordial matrix proteins important for embryonic development and regeneration (such as laminin, type IV collagen, and fibronectin), adipose ECM may prove to be an autologous tissue engineering matrix and stem cell culture substrate. We show here that adipose tissue ECM can, in fact, be electrospun into a nanofiberous mesh, histologically shown to contain connective tissue, collagens, elastic fibers/elastin, proteoglycans, and glycoproteins in the newly synthesized matrix. We also show that this novel electrospun adipose tissue scaffold is capable of supporting stem cell growth. Taken together, experiments using ASCs cultured on extracellular matrices of electrospun Fg or adipose ECM present an excellent framework for future advances in regenerative medicine therapeutics and research.
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Chan, Alexander K. C. "Development of characterisation and quality potency assays for human mesenchymal stem cells." Thesis, Loughborough University, 2016. https://dspace.lboro.ac.uk/2134/22977.
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Regenerative medicine and cell therapies hold great potential to treat a variety of medical conditions. Product characterisation of these therapies is particularly difficult as they pose regulatory challenges due to donor heterogeneity and the lack of standardised lot release tests that can reliably predict in vivo function. Human mesenchymal stem cells (hMSCs), also called multipotent stem cells or mesenchymal stromal cells, are a viable option in cell therapies due to their immunosuppressive and pro-angiogenic functions. Currently there are no standardised methods or potency assays to quantify these properties. To address this, five individual hMSCs lines from different donors were created and characterised based upon growth rate, differentiation capability and extracellular surface protein expression. A novel multiparameter flow cytometry method to characterise the cells based upon extracellular surface markers was developed that supports high-throughput and high-content analyses. Three candidate lines were taken forward and assessed in multiple in vitro bioassays that examined the hMSC immunosuppressive response to a defined inflammatory environment, effect on T-cell proliferation, and effect on a mixed lymphocyte population. Next, the angiogenic properties were assessed using human umbilical vein endothelial cells (HUVECs) tube formation as a model for cardiac regeneration. This involved utilising automated time lapse microscopy techniques coupled with image analysis software to quantify endothelial to tube formation. Further analysis of the hMSC secretome revealed differences in the levels of pro-angiogenic cytokines such as vascular endothelial growth factor, hepatocyte growth factor and IL-8. Significant differences in angiogenic potency were found between the hMSC lines. This thesis highlights the need to develop specific assays that reflect the intended clinical action. Taken together, these quantitative approaches provide valuable tools to measure hMSC quality and potency, and supports continued efforts to improve characterisation strategies for cellular therapies.
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Dosier, Christopher R. "Bone tissue engineering utilizing adult stem cells in biologically functionalized hydrogels." Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/47678.
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Repair of large bone defects remains a clinical challenge for orthopedic surgeons. Current treatment strategies such as autograft and allograft are limited by the amount of available tissue in the case of the former, and failure of revascularization effecting engraftment in the case of the latter. Tissue engineering offers an alternative approach to this challenging clinical problem. The general principle of tissue engineering for bone regeneration prescribes delivery of osteoinductive factors to induce an endogenous response within the host to repair a defect that will not normally heal. One such tissue engineering approach is cell based therapy and this is attractive in the cases of patients with a lack of endogenous osteoprogenitors cells due to volumetric loss of tissue/ageing.
Stem cell therapy has emerged as a possible alternative to current treatment modalities, however many challenges to clinical translation remain. Central to these challenges for bone tissue engineering are lingering questions of which cells to use and how to effectively deliver those cells. The goal of this thesis was to elucidate more effective ways to enhance bone repair utilizing adult stem cells. First, we investigated adipose derived stem cells (ADSCs) as a viable cell source for bone tissue engineering. Upon isolation, adipose derived stem cells are a heterogeneous population of multipotent cells predisposed to adipogenic differentiation. We developed an enrichment protocol that demonstrated the osteogenic potential of ADSCs can be enhanced in a dose dependent manner with resveratrol, which had been demonstrated to up-regulate Runx-2 expression. This enrichment strategy produced an effective method to enhance the osteogenic potential of ADSCs while avoiding cell sorting and gene therapy techniques, thus bypassing the use of xenogenic factors to obtain an enriched source of osteoprogenitor cells. This protocol was also used to investigate differences between human and rat ADSCs and demonstrated that rat ADSCs have a higher osteogenic potential than human ADSCs in vitro.
The second major thrust of this thesis was to develop an injectable hydrogel system to facilitate bone formation in vivo. Both a synthetic and a naturally based polymer system was investigated, the results of which demonstrated that the naturally based alginate hydrogel was a more effective vehicle for both cell viability in vitro and bone formation in vivo. Our results also demonstrated that despite the ability to increase the osteogenic potential of ADSCs in vitro with resveratrol treatment, this was insufficient to induce bone formation in vivo. However, the inclusion of bone marrow mesenchymal stem cells (BMMSCs) in BMP-2 functionalized alginate hydrogels resulted in significantly greater mineralization than acellular hydrogels. Finally, the effect of timing of delivery of therapeutics to a non-healing segmental bone defect in the femur was investigated. We hypothesized that delivery of biologics after the initial inflammation response caused by injury to the host tissue would result in greater regeneration of tissue in terms of newly formed bone. Contrary to our initial hypothesis, these experiments demonstrated that delayed implantation of therapeutics has a detrimental effect on the overall healing response. It was, however, demonstrated that the inclusion of BMMSCs results in greater bone volume regenerated in the defect site over acellular hydrogels.
In conclusion, this work has rigorously investigated the use of adipose derived stem cells for bone tissue engineering, and further produced an injectable hydrogel system for stem cell based bone tissue engineering. This work also demonstrated that the inclusion of adult stem cells, specifically BMMSCs, can enhance the regeneration response in a non-healing bone defect model relative to acellular hydrogel.
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André, Emilie. "Combination of nano and microcarriers for stem cell therapy of Huntington's disease : new regenerative medicine strategy." Thesis, Angers, 2015. http://www.theses.fr/2015ANGE0047/document.
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La combinaison de biomatériaux et cellules souches, a pour but de protéger des cellules endommagées et de ralentir la progression des maladies neurodégénératives, comme la maladie de Huntington (MH). Les cellules souches mésenchymateuses et particulièrement une sous-population, les cellules MIAMI, ont déjà démontré leur efficacité dans la maladie de Parkinson. Il est cependant essentiel d’améliorer leur différenciation neuronale, leur survie et évaluer leur sécrétome. L’objectif principal de ce travail fut de proposer une stratégie innovante de médecine régénératrice pour la MH associant cellules souches, nano et micro médecines. Pour l’évaluer, un nouveau modèle animale ex vivo de la MH a été mis en place. Nous avons ensuite développé et optimisé deux nano-vecteurs, des nanocapsules lipidiques et des nanoparticules solides de SPAN, et les avons associés à un inhibiteur de REST qui est un facteur de transcription qui empêche la différenciation neuronale. La transfection de ce siREST a montré une amélioration du phénotype neuronal. Ces cellules ainsi modifiées furent ensuite induites vers un phénotype GABAergic grâce à des facteurs de croissance. Puis elles ont été associées à un support 3D, les microcarriers pharmacologiquement actif (MPA) permettant une meilleure intégration des cellules après greffe. Les MPA sont des microsphères ayant une surface biomimétique de laminine et libérant de façon contrôlée un facteur trophique le « brain derived neurotrophic factor » (inducteur d’un phénotype neuronal et neuro-protecteur). Des résultats prometteurs ont été obtenus, encourageant à continuer l’évaluation de cette stratégie in vivo dans des modèles génétiques de la MHThe combination of biomaterials and stem cells aims to protect damaged cells and slow the progression of neurodegenerative diseases such as Huntington's disease(HD). Mesenchymal stem cells, particularly a subpopulation known as MIAMI cells, have already demonstrated their effectiveness in Parkinson's disease. However, it is essential to improve their neuronal differentiation, survival, and to assess their secretome. The main objective of this work was to propose an innovative regenerative medicine strategy for HD by combining stemcells, micro and nano medicines. To perform this assessment, a new ex vivo animal model of HD has been set up. We then developed and optimized two nanovectors,lipid nanocapsules and solid SPAN nanoparticles,carrying an inhibitor of REST a transcription factor, which prevents neuronal differentiation. The transfection of this siREST showed an improvement in the neuronal phenotype. These modified cells were then induced into a GABAergic phenotype through growth factors. They were then associated with a 3D support, the pharmacologically active microcarriers (PAM) allowing a high rate of engraftment. The PAM are microspheres which have a biomimetic surface of laminin and release a trophic factor BDNF, brain derived neurotrophic factor (inducer of a neural phenotype and neuroprotective) in a controlled manner. Promising results were obtained, further encouraging continuing the evaluation of this strategy in vivo in genetic models of HD
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Webber, Nicholas R. "Technology and Commercial Assessment of a Tissue Regenerating Drug in the Regenerative Medicine Market." Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1402249985.
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Chilton, Jamie Meredith. "Investigation of the limitations of viral gene transfer to murine embryonic stem cells." Diss., Atlanta, Ga. : Georgia Institute of Technology, 2008. http://hdl.handle.net/1853/29745.
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Thesis (Ph.D)--Biomedical Engineering, Georgia Institute of Technology, 2008.Committee Chair: Joseph Le Doux; Committee Member: Anthanassios Sambanis; Committee Member: David Archer; Committee Member: Michelle LaPlaca; Committee Member: Steve Stice; Committee Member: Todd McDevitt. Part of the SMARTech Electronic Thesis and Dissertation Collection.
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Nakamura, Sou. "Expandable Megakaryocyte Cell Lines Enable Clinically Applicable Generation of Platelets from Human Induced Pluripotent Stem Cells." Kyoto University, 2015. http://hdl.handle.net/2433/202779.
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Bargehr, Johannes. "The role of human embryonic stem cell-derived epicardium in myocardial graft development." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/276112.
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Reisbig, Nathalie A. "Synovial Extracellular Matrix and Synovial Mesenchymal Stem Cells are Chondrogenic In Vitro and In Vivo." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543150403002824.
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Drury-Stewart, Danielle Nicole. "Controlling the microenvironment of human embryonic stem cells: maintenance, neuronal differentiation, and function after transplantation." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45967.
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Precise control of stem cell fate is a fundamental issue in the use of human embryonic stem (hES) cells in the context of cell therapy We examined three ways in which the microenvironment can be controlled to alter hES cell behavior, providing insight into the best conditions for maintenance of pluripotency and neural differentiation in developmental and therapeutic studies. We first examined the effects of polydimethylsiloxane (PDMS) growth surfaces on hES cell survival and maintenance of pluripotency. Lightly cured, untreated PDMS was shown to be a poor growth surface for hES cells. Some of the adverse effects caused by PDMS could be mitigated with increased curing or UV treatment of the surface, but neither modification provided a growth surface that supported pluripotent hES cells as well as polystyrene. This work provides a basis for further optimizing PDMS for hES cell culture, moving towards the use of microdevices in establishing precise control over stem cell fate. The second study explored the use of an easily constructed diffusion-based device to grow hES cells in culture on a defined, physiologic oxygen (O₂) gradient. We observed greater hES cell survival and higher levels of pluripotency markers in the lower O₂ regions of the gradient. The greatest benefit was observed at O₂ levels below 5%, narrowing the potential optimal range of O₂ for the maintenance of pluripotent hES cells. Finally, we developed a small molecule-mediated adherent and feeder-free neural differentiation protocol that reduced the cost and time scale for in vitro differentiation of neural precursors and functional neurons from human pluripotent cells. hES cell-derived neural precursors transplanted into a murine model of focal ischemic stroke survived, improved neurogenesis, and differentiated into neurons. Transplant also led to a more consistent and measurable sensory recovery after stroke as compared to untransplanted controls. This protocol represents a potentially translatable method for the generation of CNS progenitors from human pluripotent stem cells.
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Bratt-Leal, Andrés Miguel. "Biomaterial integration within 3D stem cell aggregates for directed differentiation." Diss., Georgia Institute of Technology, 2011. http://hdl.handle.net/1853/45934.
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The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation.
The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs.
Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.
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Rafiq, Qasim Ali. "Developing a standardised manufacturing process for the clinical-scale production of human mesenchymal stem cells." Thesis, Loughborough University, 2013. https://dspace.lboro.ac.uk/2134/12335.
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Human mesenchymal stem cells (hMSCs) are a promising candidate for cell-based therapies given their therapeutic potential and propensity to grow in vitro. However, to generate the cell numbers required for such applications, robust, reproducible and scalable manufacturing methods need to be developed. To address this challenge, the expansion of hMSCs in a microcarrier-based bioreactor system was investigated. Initial studies performed in T-flask monolayer cultures investigated the effect of key bioprocess parameters such as dissolved oxygen concentration (dO2), the level of medium exchange and the use of serum-free media. 20 % dO2 adversely impacted cell proliferation in comparison to 100 % dO2, whilst FBS-supplemented DMEM was found to be the most consistent and cost-effective cell culture medium despite the advances in serum-free cell culture media. Several microcarriers were screened in 100 mL agitated spinner flasks where Plastic P102-L was selected as the optimal microcarrier for hMSC expansion given the high cell yields obtained, its xeno-free composition and effective harvest capacity. The findings from the initial small-scale studies culminated in the successful expansion of hMSCs on Plastic P102-L microcarriers in a fully equipped 5 L stirred-tank bioreactor (2.5 L working volume), the largest reported volume for hMSC microcarrier culture to date. A maximum cell density of 1.68 x 105 cells/mL was obtained after 9 days in culture; further growth was limited by the low glucose concentration and lack of available surface area. A novel, scalable harvesting method was also developed, allowing for the successful recovery of hMSCs. Importantly, harvested hMSCs retained their immunophenotype, multipotency and ability to proliferate on tissue culture plastic.
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Nakane, Takeichiro. "Impact of Cell Composition and Geometry on Human Induced Pluripotent Stem Cells-Derived Engineered Cardiac Tissue." Kyoto University, 2018. http://hdl.handle.net/2433/232090.
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Rönsch, Kathleen. "Patterning of stem cells during limb regeneration in Ambystoma mexicanum." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-232386.
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Axolotl uniquely generates blastema cells as a pool of progenitor/stem cells to restore an entire limb, a particular property that other organisms, such as humans, do not have. What underlies these differences? Is the main difference that cells residing at the amputation plane (in the stump) undergo reprogramming processes to re-enter the embryonic program, which allows developmental patterning to start, or are there fundamental differences? There is also a significant debate about whether regeneration occurs via stem cell differentiation or by dedifferentiation of mature limb tissue. The aim of my thesis was to address following questions: Are the cells in the blastema reprogrammed or differentiated to regenerate? Are the blastema cells genetically reactivated de novo during regeneration? How does the amputated limb exactly know which part of the limb needs to be regenerate?
Using a novel technique of long-term genetic fate mapping, my team demonstrated that dedifferentiation in regenerated axolotl muscle tissue does not occur. Instead, PAX7+ satellite cells indeed play an important role during muscle regeneration in the axolotl limb. Surprisingly, this is in contrast to the newt, which regenerates muscle cells through a dedifferentiation process. Therefore, there is a fundamental difference that underlies the regenerative mechanism ((Sandoval-Guzman et al., 2014) [KR1]). This demonstrates that there is an unexpected diversity and flexibility of cellular mechanims used during limb regeneration, even among two closely related species. Finally, if one salamander species uses a mammalian regenerative strategy (Cornelison and Wold, 1997; Collins et al., 2005) involving stem cells and another uses a dedifferentiative strategy, this raises the question of whether there are other fundamental aspects of regeneration that could also be anomalous. This hypothesis is promising since there could be more than one possible mechanism to induce mammalian regeneration.
The process of limb regeneration in principle seems to be more similar to those of limb development as historically assumed. We showed molecularly that embryonic players are reused during regeneration by reactivating the position- and tissue-specific developmental gene programs by using the newly isolated Twist sequences as early blastema cell markers ((Kragl et al., 2013) [KR2]). To gain insights into the molecular mechanisms of the P/D limb patterning in general, it was crucial to study the early patterning events of the resident progenitor/stem cells by using the specific blastema cell marker HoxA as a positional marker along the proximo-distal axis. Our HOXA protein analysis using high molecular and cellular resolution as well as transplantation assays demonstrated for the first time that axolotl limb blastema cells acquire their positional identity in a proximal to distal sequence. We found a hierarchy of cellular restrictions in positional identities. Amputation at the level of the upper arm showed that the blastema harbors cells, which convert to lower arm and hand. We observed ((Roensch et al., 2013) [KR3]) for the first time that intercalation- the intermediate element (lower arm) arises later from an interaction between the proximal and distal cells identities- does not occur. Intercalation, which has been an accepted model for a long time, is not the patterning mechanism underlying normal (without any manipulation) limb regeneration that is unique to axolotl. We further demonstrated, using the Hox genes as markers that positional identity is cell-type specific since their effects were confirmed to be present in the lateral plate mesoderm- derived cells of the limb.
As our knowledge about limb blastemas expands concerning cell composition and molecular events controlling patterning, the similarity to development is becoming more and more clear. My work has resolved many ambiguities surrounding the molecularly identification of different types of blastema cells and how P/D limb patterning occurs during regeneration in comparison to development. It has highlighted the importance of combining high-resolution methods, such as in situ hybridizations, single-cell PCR (sc-PCR) of individual dissociated blastema cells and genetic labeling methods with grafting experiments to map cell fates in vivo.
In addition to understanding the processes of regeneration, another long-term goal in the regenerative medicine field is to identify key molecules that trigger the regeneration of tissues. Recently, my colleague Takuji Sugiura (Sugiura et al., 2016) observed that an early event of blastema formation is the secretion of molecules like MLP (MARCKS-like protein), which induces wound-associated cell cycle re-entry. Such findings further increase the enthusiasm of biologists to understand the underlying principles of regeneration. By building our knowledge of the molecules and pathways that are involved in tissue regeneration, we increase the possibility of identifying a way to ‘activate’ regenerative processes in humans and thus reach the final goal of regenerative medicine, which is to use the concepts of cellular reprogramming, stem cell biology and tissue engineering to repair complex body structures.
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Carpenedo, Richard L. "Microsphere-mediated control of embryoid body microenvironments." Diss., Georgia Institute of Technology, 2010. http://hdl.handle.net/1853/33948.
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Embryonic stem cells (ESCs) hold great promise for treatment of degenerative disorders such as Parkinson's and Alzheimer's disease, diabetes, and cardiovascular disease. The ability of ESCs to differentiate to all somatic cell types suggests that they may serve as a robust cell source for production of differentiated cells for regenerative medicine and other cell-based therapeutics. In order for ESCs to be used effectively in clinical settings, efficient and reproducible differentiation to targeted cell types must be demonstrated. The overall objective of this project was to engineer microenvironmental control over differentiating ESCs through the formation of embryoid bodies (EBs) uniform in size and shape, and through the incorporation of morphogen-containing polymer microspheres within the interior of EBs. The central hypothesis was that morphogen delivery through incorporated polymer microspheres within a uniform population of EBs will induce controlled and uniform differentiation of ESCs.
Rotary suspension culture was developed in order to efficiently produce uniform EBs in high yield. Compared to static suspension culture, rotary suspension significantly improved the production of differentiating cells and EBs over the course of 7 days, while simultaneously improving the homogeneity of EB size and shape compared to both hanging drop and static EBs. The diffusive transport properties of EBs formed via rotary suspension were investigated using a fluorescent, cell permeable dye to model the movement of small morphogenic molecules within EBs. Confocal microscopy, cryosections and EB dissociation all demonstrated that the dye was not able to fully penetrate EB, and that the larger EBs at later time points (7 days) retarded dye movement to a greater extent than earlier EBs (days 2 and 4). Polymer microspheres capable of encapsulating morphogenic factors were incorporated into EBs in order to overcome the diffusional limitations of traditional soluble delivery. The size of microspheres, microsphere coating, microsphere to cell ratio, and rotary mixing speed were all observed to influence incorporation within EBs. The use of microsphere-mediated delivery within EBs to direct cell differentiation was examined. Microsphere-mediated delivery of retinoic acid (RA) induced formation of uniquely cystic spheroids with a visceral endoderm layer enveloping a pseudo-stratified columnar epithelium, and with spatial localization of transcriptional profiles similar to the early primitive streak stage of mouse development. Continued differentiation of RA MS EBs in defined media conditions was assessed. Gene expression demonstrated that regular serum enhanced endoderm induction, serum-free media supported ectoderm differentiation, while mesoderm was most prominent in untreated EBs in full serum.
In summary, this work has realized a unique approach for stem cell differentiation through modification of the internal microenvironment of ESC spheroids. This novel inside-out method toward engineering EBs demonstrated that the mode of morphogen delivery significantly affected the course of differentiation. These studies provide the basis for ongoing work, which will utilize the choice of microsphere material, coating, and morphogen in order to uniquely study mechanisms of ESC differentiation and achieve unparalleled engineering of the EB microenvironment.
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Torres, Fabrício Carvalho. "Panículo adiposo interescapular de coelho da espécie Oryctolagus cuniculus como fonte de células-tronco." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/5/5132/tde-31082009-162219/.
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Nos últimos anos, as células-tronco, devido a sua capacidade de originar diversos tecidos corporais e pelo poder de auto-renovação, impulsionaram os estudos de engenharia tecidual, sobretudo em medicina regenerativa. Nesse aspecto, o tecido adiposo vem se mostrando como fonte ideal para obtenção de tais células, devido à facilidade de captação, à baixa morbidade associada ao procedimento e ao elevado rendimento celular. Com o objetivo de estabelecer um modelo experimental versátil e que satisfizesse várias áreas de interesse, propôs-se o coelho Oryctolagus cuniculus como fonte de tecido adiposo. Esse animal apresenta bolsa adiposa interescapular com peso médio de 17,2g, o que corresponde a cerca de 6,6 g/Kg em machos adultos (peso corporal médio de 2590g). A coleta do material foi por meio de lipoaspiração a seco, com cânula de 3,5mm; levou-se em média, 11 minutos para o procedimento, obtendo-se aproximadamente 10 ml de gordura. Após o processamento pela técnica enzimática, em cada mililitro de gordura encontrou-se em média 1x105 células-tronco. O estudo constatou ainda que, por meio da criopreservação em nitrogênio líquido, as células mantinham suas características citométricas após períodos de congelamento que variaram de uma semana a 13 meses. As células apresentaram características de sua indiferenciação, como a expressão dos marcadores de superfície: CD90, 80,6%; HLA-DR, 2,8% e caspase 3, 10,5%. A análise do ciclo celular com 100% de confluência mostrou que 70,8% das células encontravamse quiescentes; 22,1% apoptóticas. As células com alta capacidade replicativa, que corresponde à fase S do ciclo celular, 1,4% e 0,9% encontravam-se em replicação, mostrando que as células-tronco do tecido adiposo, em cultura, não apresentam uma proliferação descontrolada, tendendo a se estabilizar, principalmente quando atingem confluência máxima em monocamada. Todas essas vantagens fazem com que o modelo proposto possa ser facilmente reprodutível, contribuindo para o estudo das células-tronco do tecido adiposo.In the latest years, the study on tissue engineering, mainly in the area of regenerative medicine, has advanced because the medical community is highly interested in stem cells. This is due to both the potential of these cells to originate any body tissue and their power of self-renewal. Adipose tissue has been used as an ideal source of such cells, due to the simplicity of their collection, high cellular yield, and low morbidity associated with the procedure. In order to establish a versatile experimental model, which could meet the needs of researchers from various areas, the rabbit Oryctolagus cuniculus was proposed as a source of adipose tissue. This animal has an adipose pad in the interscapular region with an average weight of 17.2g, which corresponds to about 6.6g of fat material per kilogram of an adult male animal (mean body weight = 2.6kg). The material was collected by means of a liposuction procedure. Using a 3.5- mm diameter tube, a volume of nearly 10ml of fat material was obtained in a mean time of 11min. After processing the fat tissue by enzymatic technique, about 1x105 stem cells were found per milliliter of fat material. Using cryopreservation of the cells by freezing them in liquid nitrogen, it was observed that the cytometric characteristics were maintained after a period of time ranging from 1 week to 13 months. The cells presented evident characteristics of undifferentiation, such as expression of the surface markers CD90, HLA-DR, and Caspase-3 (80.6, 2.8, and 10.5 %, respectively). Analysis of the cellular cycle with 100% confluence allowed us to show that 70.8% of the cells were quiescent, 22.1% were apoptotic, 1.4% had high replication capacity (phase S of the cellular cycle) and 0.9% were already in replication (phase G2/M), indicating that stem cells from adipose tissue did not show uncontrolled proliferation, tending to stabilize, mainly when they reach maximal confluence in monolayer. These advantages make this model easily reproducible, facilitating the study of adipose tissue stem cells.
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Alt, Daniel Scott. "PREVASCULAR CELL CONDENSATIONS FOR MODULAR TISSUE ENGINEERING." Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1599521079956842.
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Kinney, Melissa. "Biophysical and biochemical control of three-dimensional embryonic stem cell differentiation and morphogenesis." Diss., Georgia Institute of Technology, 2014. http://hdl.handle.net/1853/53465.
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Stem cell differentiation is regulated by the complex interplay of multiple parameters, including adhesive intercellular interactions, cytoskeletal and extracellular matrix remodeling, and gradients of agonists and antagonists that individually and collectively vary as a function of spatial locale and temporal stages of development. Directed differentiation approaches have traditionally focused on the delivery of soluble morphogens and/or the manipulation of culture substrates in two-dimensional, monolayer cultures, with the objective of achieving large yields of homogeneously differentiated cells. However, a more complete understanding of stem cell niche complexity motivates tissue engineering approaches to inform the development of physiologically relevant, biomimetic models of stem cell differentiation. The capacity of pluripotent stem cells to simultaneously differentiate toward multiple tissue-specific cell lineages has prompted the development of new strategies to guide complex, three-dimensional morphogenesis of functional tissue structures. The objective of this project was to characterize the spatiotemporal dynamics of stem cell biophysical characteristics and morphogenesis, to inform the development of ESC culture technologies to present defined and tunable cues within the three-dimensional spheroid microenvironment. The hypothesis was that the biophysical and biochemical cues present within the 3D microenvironment are altered in conjunction with morphogenesis as a function of stem cell differentiation stage. Understanding biochemical and physical tissue morphogenesis, including the relationships between remodeling of cytoskeletal elements and intercellular adhesions, associated developmentally relevant signaling pathways, and the physical properties of the EB structure together elucidate fundamental cellular interactions governing embryonic morphogenesis and cell specification. Together, this project has established a foundation for controlling, characterizing, and systematically perturbing aspects of stem cell microenvironments in order to guide the development of complex, functional tissue structures for regenerative therapies.
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Kaur, Navdeep. "Influence of culture conditions on the molecular signature of mesenchymal stem cells." Thesis, Queensland University of Technology, 2010. https://eprints.qut.edu.au/43719/1/Navdeep_Kaur_Thesis.pdf.
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Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering.
Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary.
The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential.
Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations.
A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages.
One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations.
In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.
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Shafiee, Abbas. "Isolation and characterisation of primitive stem cell populations from placenta." Thesis, Queensland University of Technology, 2016. https://eprints.qut.edu.au/95086/1/Abbas_Shafiee_Thesis.pdf.
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Cardiovascular diseases (CVD) are the leading cause of death in developed societies. Vascular stem cells (VSCs) have been proposed as a cell population with regenerative potential for CVD. However, the in vivo ontogeny of VSCs remains controversial. In current study, we have isolated VSCs from the human term placenta and characterized in vivo cell populations which give rise to different stem cells. Furthermore, a novel hierarchy among vascular cells as well as their physiological localization were established. Collectively, this is an important finding for both developmental biology and cell-based therapy and may potentially provide an avenue for future cell therapies.
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Heathman, Thomas R. J. "Developing a process control strategy for the consistent and scalable manufacture of human mesenchymal stem cells." Thesis, Loughborough University, 2015. https://dspace.lboro.ac.uk/2134/22174.
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Human mesenchymal stem cells (hMSCs) have been identified as a promising cell-based therapy candidate to treat a number of unmet clinical indications, however, in vitro expansion will be required to increase the available number of cells and meet this demand. Scalable manufacturing processes, amenable to closed, single-use and automated technology, must therefore be developed in order to produce safe, effective and affordable hMSC therapies. To address this challenge, a controlled serum-free end-to-end microcarrier process has been developed for hMSCs, which is amenable to large-scale manufacture and therefore increasing economies of scale. Preliminary studies in monolayer culture assessed the level of variability in growth between five hMSC donors, which was found to have a variance of 25.3 % after 30 days in culture. This variance was subsequently reduced to 4.5% by the development of a serum-free monolayer culture process with the maintenance of critical hMSC characteristics and an increased number of population doublings. In order to transfer this into a scalable system, the serum and serum-free expansion processes were transferred into suspension by the addition of plastic microcarriers in 100 mL spinner flasks without control of pH or dissolved oxygen (DO). This achieved a maximum cell density of 0.08 ± 0.01 · 106 cells.mL-1 in FBS-based medium, 0.12 ± 0.01 · 106 cells.mL-1 in HPL-based medium and 0.27 ± 0.03 · 106 cells.mL-1 in serum free medium after six days. In order to drive consistency and yield into the manufacturing process, a process control system was developed for the FBS-based microcarrier expansion process in a 100 mL DASbox bioreactor platform to control DO, pH, impeller rate and temperature. Reduced impeller rates and DO concentrations were found to be beneficial, with a final cell density of 0.11 ± 0.02 · 106 cells.mL-1 and improved post-harvest outgrowth and colony-forming unit (CFU) potential compared to uncontrolled microcarrier and monolayer culture. This controlled bioreactor expansion process was then applied to the previously developed serum-free microcarrier process, eventually achieving a final cell density of 1.04 ± 0.07 · 106 cells.mL-1, whilst retaining key post-harvest hMSC characteristics. Following the controlled serum-free expansion and harvest of hMSCs, a downstream and cryopreservation process was developed to assess the impact of prolonged holding times and subsequent unit-operations on hMSC quality characteristics. This showed that hMSCs are able to maintain key characteristics throughout the entire end-to-end process, demonstrating their potential for commercial scale manufacture.
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Speccher, Alessandra. "Tissue engineering approaches for brain injury applications." Doctoral thesis, Università degli studi di Trento, 2020. http://hdl.handle.net/11572/262798.
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Due to the limited regenerative capacity of the central nervous system (CNS) upon injury, regenerative medicine and tissue engineering strategies show great promise for treatment. These aim to restore tissue functions by combining principles of cell biology and engineering, using biomaterial scaffolds which can help in recapitulating the 3D environment of the brain and improving cell survival after grafting.
Stroke and TBI are severe forms of disruptions of brain architecture, and two of the leading causes of mortality and morbidity worldwide, as no effective treatments are available. Several studies report how neural stem cells (NSCs) are able to improve functional recovery upon transplantation. However, the efficacy of these treatments is limited because of the mortality these cells are subject to after transplantation. In this context, the transplantation of mesenchymal cells (MSCs) has shown beneficial effects by secreting molecules and factors that help in the healing process.
In this study, we tested alginate-based hydrogels as candidates to support human NSCs and MSCs transplantation into the brain, in the view of exploiting the beneficial effects of both and analyzing whether their combined use could have a synergistic effect.
In the first part, we studied the suitability of alginate-based scaffolds for the three-dimensional encapsulation and culture of hNSCs and hMSCs. We analyzed their ability to support cell survival, and we evaluated whether changes in their concentration or modifications with ECM molecules could influence cell viability. We showed that the best survival conditions are found when using an RGDs-functionalized alginate scaffold at a low concentration (0.5% w/v). We then worked on the identification of the best conditions for MSCs culture and the definition of coculture conditions. Since serum is necessary for MSCs, but it is reported to induce glial differentiation of NSCs, we explored two different experimental setups. On one hand, we investigated the feasibility to exploit biomaterials to create "compartmentalized" cocultures that would at least partially retain serum. In parallel, we positively observed that MSCs can survive, proliferate and maintain their stemness even in absence of serum, supporting the hypothesis that the use of “compartmentalized” coculture systems would likely be exploitable for MSCs culture.
Finally, we tested the reported beneficial effects of MSCs in our 3D culture system, in which NSCs do not show a great viability. Encapsulated NSCs were cultured on an MSCs monolayer, and we analyzed cell survival, proliferation, differentiation and stemness retention. Gene expression analyses highlighted that NSCs maintain stemness characteristics, but we were not able to observe any improvement in NSCs survival in coculture, with respect to standard culture.
In the last part of the project we decided to test our system for tissue engineering approaches, exploiting axotomized brain organotypic slices (OSCs). We evaluated the presence of cells 7 days after transplantation, their integration in the OSCs and glial response. Preliminary results suggest that the biomaterial does not cause activation of glial cells, although stem cells do not seem to migrate out of scaffold and integrate into the brain slice.
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Fotticchia, Andrea. "Design and development of anisotropic laminate scaffolds of electrospun polycaprolactone for annulus fibrosus tissue engineering applications." Thesis, Loughborough University, 2016. https://dspace.lboro.ac.uk/2134/21407.
Full textAbstract:
In several cases, current therapies available to treat a large number of musculoskeletal system diseases are unsatisfactory as they provide only temporary or partial restoration of the damaged or degenerated site. In an attempt to maintain a high standard of life quality and minimise the economic losses due to the treatments of these frequently occurring ailments and subsequent lost working days, alternative therapies are being explored. Contrary to the current treatments, tissue engineering aims to regenerate the impaired tissue rather than repair and alleviate the symptoms; thus offering a definitive solution. The annulus fibrosus (AF) of the intervertebral disc (IVD) is a musculoskeletal system component frequently subjected to degeneration and rupture, characterised by predominance of anisotropically arranged collagen fibres. In the present thesis, electrospinning technology is used to fabricate polycaprolactone (PCL) scaffolds intended to replicate the anisotropic structure of the AF.