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.

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

No / There is growing interest in the use of mesenchymal stem cells (MSC) for immune therapy. Clinical trials that use MSC for treatment of therapy resistant graft versus host disease, Crohn's disease and organ transplantation have initiated. Nevertheless, the immunomodulatory effects of MSC are only partly understood. Clinical trials that are supported by basic research will lead to better understanding of the potential of MSC for immunomodulatory applications and to optimization of such therapies. In this manuscript we review some recent literature on the mechanisms of immunomodulation by MSC in vitro and animal models, present new data on the secretion of pro-inflammatory and anti-inflammatory cytokines, chemokines and prostaglandins by MSC under resting and inflammatory conditions and discuss the hopes and expectations of MSC-based immune therapy.

Yes / Glioblastoma multiforme (GBM) is a grade IV brain tumor characterized by a heterogeneous population of cells that are highly infiltrative, angiogenic and resistant to chemotherapy. The current standard of care, comprised of surgical resection followed by radiation and the chemotherapeutic agent temozolomide, only provides patients with a 12–14 month survival period post-diagnosis. Long-term survival for GBM patients remains uncommon as cells with intrinsic or acquired resistance to treatment repopulate the tumor. In this review we will describe the mechanisms of resistance, and how they may be overcome to improve the survival of GBM patients by implementing novel chemotherapy drugs, new drug combinations and new approaches relating to DNA damage, angiogenesis and autophagy.

No / Tissue stem cells and germ line or embryonic stem cells were shown to have reduced oxidative metabolism, which was proposed to be an adaptive mechanism to reduce damage accumulation caused by reactive oxygen species. However, an alternate explanation is that stem cells are less dependent on specialized cytoplasmic functions compared with differentiated cells, therefore, having a high nuclear-to-cytoplasmic volume ratio and consequently a low mitochondrial content. To determine whether stem cells rely or not on mitochondrial respiration, we selectively ablated the electron transport chain in the basal layer of the epidermis, which includes the epidermal progenitor/stem cells (EPSCs). This was achieved using a loxP-flanked mitochondrial transcription factor A (Tfam) allele in conjunction with a keratin 14 Cre transgene. The epidermis of these animals (TfamEKO) showed a profound depletion of mitochondrial DNA and complete absence of respiratory chain complexes. However, despite a short lifespan due to malnutrition, epidermal development and skin barrier function were not impaired. Differentiation of epidermal layers was normal and no proliferation defect or major increase of apoptosis could be observed. In contrast, mice with an epidermal ablation of prohibitin-2, a scaffold protein in the inner mitochondrial membrane, displayed a dramatic phenotype observable already in utero, with severely impaired skin architecture and barrier function, ultimately causing death from dehydration shortly after birth. In conclusion, we here provide unequivocal evidence that EPSCs, and probably tissue stem cells in general, are independent of the mitochondrial respiratory chain, but still require a functional dynamic mitochondrial compartment.

Yes / Humans are social animals that communicate disproportionately via potent genetic signals imbued in the skin and hair, including racial, ethnic, health, gender, and age status. For the vast majority of us, age-related hair pigment loss becomes the inescapable signal of our disappearing youth. The hair follicle (HF) pigmentary unit is a wonderful tissue for studying mechanisms generally regulating aging, often before this becomes evident elsewhere in the body. Given that follicular melanocytes (unlike those in the epidermis) are regulated by the hair growth cycle, this cycle is likely to impact the process of aging in the HF pigmentary unit. The formal identification of melanocyte stem cells in the mouse skin has spurred a flurry of reports on the potential involvement of melanocyte stem cell depletion in hair graying (i.e., canities). Caution is recommended, however, against simple extrapolation of murine data to humans. Regardless, hair graying in both species is likely to involve an age-related imbalance in the tissue's oxidative stress handling that will impact not only melanogenesis but also melanocyte stem cell and melanocyte homeostasis and survival. There is some emerging evidence that the HF pigmentary unit may have regenerative potential, even after it has begun to produce white hair fibers. It may therefore be feasible to develop strategies to modulate some aging-associated changes to maintain melanin production for longer.

Yes / The spermatogonial stem cells (SSCs) are the only germline stem cells in adults that are responsible for the transmission of genetic information from mammals to the next generation. SSCs play a very important role in the maintenance of progression of spermatogenesis and help provide an understanding of the reproductive biology of future gametes and a strategy for diagnosis and treatment of infertility and male reproductive toxicity. Androgens/oestrogens are very important for the suitable maintenance of male germ cells. There is also evidence confirming the damaging effects of oestrogen-like compounds on male reproductive health. We investigated the effects in vitro, of diethylstilbestrol (DES) on mouse spermatogonial stem cells separated using Staput unit-gravity velocity sedimentation, evaluating any DNA damage using the Comet assay and apoptotic cells in the TUNEL assay. Immunocytochemistry assays showed that the purity of isolated mouse spermatogonial cells was 90%, and the viability of these isolated cells was over 96%. Intracellular superoxide anion production (O2−) in SSCs was detected using p-Nitro Blue Tetrazolium (NBT) assay. The viability of cells after DES treatment was examined in the CCK8 (cell counting kit-8) cytotoxicity assay. The results showed that DES-induced DNA damage causes an increase in intracellular superoxide anions which are reduced by the flavonoid, quercetin. Investigating the molecular mechanisms and biology of SSCs provides a better understanding of spermatogonial stem cell regulation in the testis.

Yes / Hypothalamic tanycytes in median eminence (ME) are emerging as a crucial cell population that regulates endocrine output, energy balance and the diffusion of blood-born molecules. Tanycytes have recently been considered as potential somatic stem cells in the adult mammalian brain, but their regenerative and tumorigenic capacities are largely unknown. Here we found that Rax+ tanycytes in ME of mice are largely quiescent but quickly enter the cell cycle upon neural injury for self-renewal and regeneration. Mechanistically, Igf1r signaling in tanycytes is required for tissue repair under injury conditions. Furthermore, Braf oncogenic activation is sufficient to transform Rax+ tanycytes into actively dividing tumor cells that eventually develop into a papillary craniopharyngioma-like tumor. Together, these findings uncover the regenerative and tumorigenic potential of tanycytes. Our study offers insights into the properties of tanycytes, which may help to manipulate tanycyte biology for regulating hypothalamic function and investigate the pathogenesis of clinically relevant tumors.

Mouse embryonic stem cells (ESCs) are capable of differentiating into any somatic cell type and are known to display fragile immune privilege in vivo and in vitro. The extent to which the depletion of essential amino acids (EAAs) by ESCs contributes to this phenomenon was investigated. ESCs were found to express various enzymes capable of catabolising EAAs within the culture medium. In particular, depletion of threonine, valine and lysine was found to have significant impact on T cell proliferation and differentiation, biasing their polarisation towards a FoxP3⁺ T regulatory (T_reg) phenotype. Supplementing ESC conditioned medium with these three EAAs alone rescued normal T cell proliferation, whereas artificially limiting their availability was sufficient to induce T_reg cell differentiation to a level equivalent to general EAA depletion. The pattern of EAA catabolism by mouse ESC was shared by induced pluripotent stem cells, while mouse melanoma cell lines and human ESCs displayed distinct patterns of EAA depletion. The cytosolic branched chain aminotransferase enzyme, Bcat1, catalyses the first step of branched chain amino acid catabolism (isoleucine, leucine and valine), and is highly expressed by both mouse and human ESCs. The contribution of this enzyme to the establishment of acquired immune privilege by ESC-derived tissues was, therefore, investigated. ESC lines were derived from mice lacking Bcat1 activity and were characterised. Bcat1^−/− ESC lines displayed no difference to their wildtype counterparts (Bcat1^LoxP) in terms of in vitro proliferation and their capacity to form teratomas in vivo. Furthermore, the loss of Bcat1 function had little impact on the inhibition of T cell proliferation in culture, ability to induce T_reg cell commitment or their ability to prevent rejection by T cell receptor transgenic recipients, suggesting the minimal contribution of Bcat1 to the depletion of EAAs by ESCs. In conclusion, EAA depletion by mouse ESC may provide a mechanistic explanation for the previously described immune-suppressive capacity of ESC.

Multipotent haematopoietic stem cells (HSCs) originate in the dorsal aorta (DA) during vertebrate embryogenesis, and after migrating to a permanent niche, give rise to a continuous supply of mature blood cells of all lineages throughout adult life. Previous cell tracing experiments have shown that the cells of the DA migrate here from an early collection of haemangioblasts (bipotential precursors of blood and endothelial cells) which reside in the dorsolateral plate (DLP) mesoderm. Development of HSCs is tightly regulated by a number of key signalling pathways in both the DLP and the DA. In particular, notch signalling is considered an important factor in vascular, arterial and HSC development. Here, the relatively slow development and the spatial separation of definitive haematopoiesis from primitive haematopoiesis in Xenopus laevis has been exploited to reveal the first defect of reduced notch signalling in the Xenopus DA. Two notch inputs to HSC programming have been identified in Xenopus: notch4 and its target genes, esr7 and esr10, are expressed from stage 31, immediately after migrating haemangioblast cells reach the midline of the embryo to form the DA, whilst notch1 is expressed slightly later, from stage 34, and controls expression of two further notch target genes, esr1 and hesr1. Using both morpholino knockdown of these six genes, and chemical inhibition of notch signalling using a specific γ-secretase inhibitor, notch signalling has been demonstrated to be essential for HSC programming but dispensable for earlier haemangioblast and arterial programming. Furthermore, esr1, downstream of both notch1 and notch4, is shown to be responsible for repression of endothelial genes in the DA. Taken together, this demonstrates that a cascade of notch and notch effector genes are essential for the programming of Xenopus HSCs.