Human neurodegenerative disorders are among the most difficult to study. In particular, the inability to readily obtain the faulty cell types most relevant to these diseases has impeded progress for decades. Amyotrophic lateral sclerosis is a late onset neurodegenerative disease in which the upper and lower motor neurons degenerate, leading to paralysis and eventually death. Recent advances in pluripotent stem cell technology now grant access to significant quantities of disease pertinent neurons both with and without predisposing mutations. The two studies described in this thesis demonstrate the feasibility of using MNs, generated from pluripotent stem cell lines harboring known ALS mutations, to establish in-vitro models of the disease. Specifically, we first used gene targeting to establish genetically controlled systems, able to identify causal relationships between a familial ALS mutation and in vitro phenotypes. Next, using transcriptional profiling, we identified novel pathways altered by the mutation and demonstrated functional consequences of these pathways' misregulation. Furthermore, by monitoring the physiology of the pluripotent stem cell derived MNs, we discovered an increased firing rate in the mutant MNs, and identified an FDA-approved drug, retigabine, capable of rescuing this defect. Lastly, to aid in the discovery of additional therapeutic compounds, we combined gene targeting, transcriptional profiling, and a fluorescent reporter human embryonic stem cell line to establish a well-controlled in vitro system capable of identifying genetic modifiers of the phenotypes described herein.

Peripheral nerve injury (PNI) is common and despite modern microsurgical techniques of repair, functional restoration is always incomplete. This results in impaired sensation and reduced motor function alongside pain and cold intolerance. Traumatic PNI are often associated with loss of nerve tissue, creating a gap, and direct repair of the two damaged nerve stumps is not possible. These types of injuries are reconstructed using autologous nerve grafts but this is far from ideal since it necessitates the sacrifice of a functional nerve from elsewhere in the body. Chronic muscle atrophy because of the prolonged delay in nerve regeneration across gaps is a significant impediment to an optimal functional recovery.   Tissue engineering and regenerative medicine approaches to nerve repair might one day replace the need for autologous nerve grafts. This thesis investigates the effects of adipose derived stem cells (ASC) on nerve regeneration and muscle recovery by using the stem cells for intramuscular injection and combined with a biomaterial, poly-3-hydroxybutyrate (PHB), to create a bioengineered artificial nerve repair construct.  The mechanisms of interaction between the stem cells and neuromuscular system cells were investigated and with a view to translating this work into clinical practice, an optimal source of cells was investigated from human donors.   It was hypothesized that injecting regenerative cells into muscle would reduce nerve injury induced muscle atrophy. A rat sciatic nerve lesion was performed and three different types of cells were injected into the denervated gastrocnemius muscle; either (1) undifferentiated ASC, (2) ASC induced to a ‘Schwann cell-like’ phenotype (dASC) or (3) primary Schwann cells. Nerves were either repaired by direct end-end suture or capped to prevent muscle reinnervation. One month later, functionality was measured using a walking track test, and muscle atrophy was assessed by examining muscle weight and histology. The Schwann cells and dASC groups showed significantly better scores on functional tests when compared with control injections of growth medium alone. Muscle weight and histology were also significantly improved in the cell groups in comparison with the control group.   PHB strips seeded with either primary Schwann cells or dASC suspended in a fibrin glue matrix were used to bridge a 10mm rat sciatic nerve gap. After 12 weeks, functional and morphological analysis (walking track test, electromyography, muscle weight and muscle and nerve histology) was performed. The results showed significantly better functional results for the PHB strips seeded with cells versus the control group with fibrin matrix only. This correlated with less muscle atrophy and greater distal axon myelination in the cell groups.   To further optimize the nerve regeneration and muscle recovery, the nerve gap lesion was repaired by treatment with the bioengineered constructs seeded with dASC or nerve autograft in combination with stem cell injection in the muscle. After 6 weeks, the best results were obtained in the nerve graft group combined with intramuscular dASC injection which showed significantly less atrophy than the other groups. The results also showed that using the stem cells in a matrix on a PHB strip in combination with intramuscular injections could significantly reduce muscle atrophy.   In vitro experiments showed that dASC expressed a wide range of neurotrophic and myogenic factors including BDNF, VEGF-A, IGF-1 and HGF. Stem cell conditioned medium enhanced the proliferation of myoblast cell lines and primary Schwann cells. Various signaling pathways (PKA, MAP kinase) were involved in these effects dependent on the cell type investigated. Furthermore, in direct co-culture with myoblast cells, a small population of the cells fused together to form myotube-like structures and expressed myogenic markers.   Human ASC were isolated from the deep and superficial layers of abdominal fat tissue obtained during abdominoplasty procedures.  Cells from the superficial layer proliferated significantly faster than those from the deep layer. Superficial layer ASC induced significantly enhanced neurite outgrowth from neuronal cell lines when compared with the deep layer cells.  However, RT-PCR and ELISA analysis showed that ASC isolated from both layers expressed similar levels of the neurotrophic factors NGF, BDNF and GDNF.   In summary, these results show that stem cell therapy at both levels (the nerve lesion site and in the target denervated muscle) offers a promising approach for clinical application for treatment of peripheral nerve lesions. The bioengineered artificial nerve construct, combining PHB strip with cells, also provides a beneficial environment for nerve regeneration. Many of the benefits of the ASC are likely to be mediated through their secretome, a rich source of neurotrophic and myogenic factors. Thus adipose tissue contains a pool of regenerative stem cells which have significant potential application to tissue engineering and regenerative medicine for nerve repair.

Pluripotent stem cells (PSCs) are specialized cells, which have remarkable ability to maintain in an undifferentiated state and are capable of undergoing differentiation to three germ-layer lineage cell types, under differentiation-enabling conditions. PSCs include embryonic stem (ES)-cells, embryonal carcinoma (EC)-cells and embryonic germ (EG)-cells. ES-cells are derived from the inner cell mass (ICM) of day 3.5 blastocysts (mouse). On the other hand, EC- and EG-cells have different source of origin and exhibit some differences in terms of their differentiation abilities and culture requirements. These PSCs act as an ideal in-vitro model system to study early mammalian development and cell differentiation and, they could potentially be used for experimental cell-based therapy for a number of diseases. However, one of the problems encountered is the immune rejection of transplanted cells. For this, immune-matched induced pluripotent stem (iPS)-cells have been derived from somatic cells, by forced expression of a few stemness genes. Although, human PSCs lines are being experimented, their cell-therapeutic potential is still far from being thoroughly tested due to lack of our understanding regarding lineage-specific differentiation, homing and structural-functional integration of differentiated cell types in the host environment. To understand these mechanisms, it is desirable to have fluorescently-marked PSCs and their differentiated cell-types, which could facilitate experimental cell transplantation studies. In this regard, our laboratory has earlier generated enhanced green fluorescent protein (EGFP)-expressing FVB/N transgenic ‘green’ mouse: GU-3 line (Devgan et al., 2003). This transgenic mouse has been an excellent source of intrinsically green fluorescent cell types. Recently, we have derived a ‘GS-2’ ES-cell line from the GU-3 mouse line (Singh et al., 2012). Additionally, we envisaged the need for developing an iPS-cell line from the GU-3 mouse and then use them for studying cell differentiation. Thus, aims of the study described in the thesis are to: (1) develop an experimental system to derive EGFP-expressing fluorescently-marked iPS-cell line from a genetically non-permissive FVB/N mouse strain, characterize the established iPS-cell line and achieve differentiation of various cell types from EGFP-expressing iPS-cell line; (2) to study differentiation phenomenon, in particular to cardiac lineage, using select-cardiogenesis modulators and (3) to assess the gene-expression profiles and signaling system associated with cardiomyocyte differentiation of PSCs. This thesis is divided into four chapters with the 1st chapter being a review of literature followed by three data chapters. In the chapter I of the thesis, a comprehensive up-to¬date review of literature is provided pertaining to PSCs, their classification, derivation strategies especially for reprogramming of somatic cells for iPSC generation, their differentiation potential and characterization, particularly to cardiac lineage. Various molecular regulators involved in cardiac differentiation of PSCs with emphasis on epigenetic regulation involving DNA methylation and signaling pathways involved are described in detail. Subsequently, various approaches used for enhanced cardiac differentiation of PSCs and the therapeutic potential of PSC-derived differentiated cell types to treat disease(s) are discussed. Chapter-II describes the successful establishment of a permanent iPS-cell line (named ‘N9’ iPS-cell line) from the non-permissive FVB/N EGFP-transgenic GU-3 ‘green’ mouse. This chapter provides results pertaining to detailed derivation strategy and characterization of the ‘N9’ iPS-cell line which includes colony morphology, expansion (proliferation) efficiency, alkaline phosphatase staining, pluripotent markers’ expression analysis by qPCR and immunostaining approaches and karyotyping analysis. Further, in order to thoroughly assess the differentiation competence of the ‘N9’ iPS¬cell line, assessment of in-vitro and in-vivo differentiation potential of the ‘N9’ iPS-cell line by embryoid body (EB) formation and teratoma formation in nude mice and its detailed histological analysis showing three germ layer cell types and their derivatives were performed, followed by the generation of chimeric blastocysts by aggregation method. This established N9 iPS-cell line could potentially offer a suitable model system to study cardiac differentiation along with other established PSC lines such as the GS-2 and D3 ES-cell lines and the P19 EC-cell line. Following the establishment of the system to study cardiac differentiation of PSC lines, efforts were made to understand the biology of cardiac differentiation of PSCs (wild¬type and EGFP-transgenic PSC lines and P19 EC-cell line) using small molecules as modulators. Data pertaining to this is described in Chapter-III. The possible involvement of epigenetic regulation of cardiogenesis for example, DNA methylation changes in cardiogenesis-associated genes is studied using 5-aza cytidine as one of the chromatin modifiers. In order to understand the cardiac differentiation phenomenon, as a consequence of using 5-aza cytidine in cell culture, it was important to investigate its ability to induce/mediate cardiac differentiation. This involved an assessment by quantitating the cardiac beating phenotype and correlating this with enhanced cardiac-gene expression profiles. Further, DNA methylation regulation of cardiogenesis¬associated genes is described using various DNA methylation analysis techniques. Moreover, the possible involvement of other signaling members in mediating the cardiac differentiation is also studied using the P19 EC-cells. Results pertaining to the above findings are described in detail in the Chapter-III. Chapter-IV is focused on various efforts made towards investigating the ability of ascorbic acid to enhance cardiac differentiation of mouse ES-cells (GS-2 and D3 lines). Ascorbic acid has been implicated to be influencing cardiogenesis and it is reported to enhance differentiation of various cell types under certain culture conditions. Results pertaining to enhancement of cardiac differentiation of PSCs using ascorbic acid are presented in this chapter. This included assessment by quantitating cardiac beating phenotype and its correlation with enhanced cardiogenesis-associated gene expression profiles. Besides, estimation on the sorted cardiomyocyte population, derived from PSCs was also made using mature-cardiac marker. The possible underlying signaling mechanism involved was also studied in detail, using specific inhibitors for pERK (U0126), integrin signaling (pFAK; PP2) and collagen synthesis (DHP), in order to ascertain their involvement in ascorbic acid-mediated cardiac differentiation of mouse ES-cells. Subsequent to the three data chapters (II-IV), separate sections are provided for ‘Summary and Conclusion’ and for ‘Bibliography’, cited in the thesis. The overall scope of the study has been to understand the basic biology of cardiac differentiation from PSCs (EC-cells, iPS-cells and transgenic and wild-type ES-cells) and to assess, by using certain small molecules, whether PSCs could be coaxed to enhance the differentiation to a particular cell type (cardiac). The data contained in this thesis addresses the above theme.

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

Οι βάσεις δεδομένων αποτελούν πλέον επιτακτική ανάγκη στην οργάνωση, αποθήκευση, γρήγορη ανάκτηση δεδομένων, αλλά και στην εξαγωγή συμπερασμάτων μέσα από διαδικασίες στατιστικής επεξεργασίας, στα πλαίσια της αξιοποίησης και επεξεργασίας του τεράστιου όγκου πληροφορίας που ήδη υπάρχει αλλά και εξακολουθεί να παράγεται με εξαιρετικά γρήγορους ρυθμούς, μεταξύ άλλων στην ιατροβιολογική έρευνα και εν προκειμένω στο πεδίο των λήψεων και των μεταμοσχεύσεων αρχέγονων αιμοποιητικών κυττάρων. Στη βάση δεδομένων που σχεδιάστηκε και αναπτύχθηκε σύμφωνα με το σχεσιακό μοντέλο βάσεων, χρησιμοποιήθηκε λειτουργικό σύστημα Ubuntu 8.04 LTS Server Edition, ο MySQL server 5.0.51b ως το Σύστημα Διαχείρισης της Βάσης (RDBMS), Apache HTTP server edition 2.2.9 ως εξυπηρετητής φιλοξενίας της βάσης για τις ανάγκες πρόσβασης της από το δίκτυο και η εφαρμογή phpMyAdmin και συγκεκριμένα η έκδοση 2.11.7.1, ως το τελικό εργαλείο διαχείρισης της βάσης. Η γλώσσα προγραμματισμού Python επίσης αποτέλεσε σημαντικό εργαλείο για την κατασκευή scripts που χρειάστηκαν, προκειμένου να καταστούν συμβατά και να εισαχθούν στη βάση, τα αρχεία που υπήρχαν καταγεγραμμένα από τις μονάδες λήψεων και μεταμοσχεύσεων αρχέγονων αιμοποιητικών κυττάρων του Πανεπιστημιακού Νοσοκομείου του Ρίου Πατρών, αρχεία που αποτέλεσαν το πρωτογενές υλικό δοκιμών της σωστής λειτουργίας της βάσης αλλά και τα δεδομένα για την εξαγωγή στατιστικών συμπερασμάτων με απώτερο σκοπό την βελτιστοποίηση των διαδικασιών φαρμακευτικής κινητοποίησης για την συλλογή των κυττάρων, συλλογής, επεξεργασίας του προϊόντος και μεταμόσχευσης. / The databases constitute nowadays imperative tool for the organization, storage, rapid data recovery and statistical analysis in the field of the modern managing and exploitation of the huge volume of information that already exists and continues to be produced extremely fast. The databases are extremely useful in the management of bioinformation of medicine and biology both in daily diagnostics as well as research. The present study is concentrated in the application of databases in the blood stem cells collections and transplantations. The database was designed and developed according to the relational model databases, the Ubuntu 8.04 LTS Server Edition used as operating system, the MySQL server 5.0.51b as the DataBase Management System (RDBMS), Apache HTTP server edition 2.2.9 as server, hosting the basic access needs through the network and phpMyAdmin version 2.11.7.1, as the final windowed environment, database management tool. The Python programming language was also an important tool for the construction of scripts needed to convert, optimize and import in the database the files that were recorded by blood stem cells collection and transplantation units of the University Hospital of Patras in Rio, records that firstly constituted the raw material for testing the proper functioning of the database and on the other hand, the data for statistical conclusions with a view to the optimization of donor’s pharmaceutical mobilization, the cell collection procedure and the process of the transplantation.

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