Dissertations / Theses on the topic 'Biomaterials, neural stem cell'

Bioengineering
Ph.D.
Within the last decade, neurodegenerative diseases such as Alzheimer’s and Parkinson’s have emerged as one of the top 5 leading causes of death globally, and there is currently no cure. All neurodegenerative diseases lead to loss of the functional cells in the nervous system, the neurons. One therapeutic approach is to replace the damaged and lost neurons with new, healthy neurons. Unfortunately, this is a difficult endeavor since mature neurons are not capable of cell division. Instead, researchers are turning to neural stem cells, which are able to self-renew and be rapidly expanded before being differentiated into functional cell phenotypes, such as neurons, allowing for large numbers of cells to be generated in vitro. Controlled differentiation of human neural stem cells into new neurons has been of interest due to the immense potential for improving clinical outcomes. Adult neural stem cell behavior, however, is not well understood and the transplanted stem cells are at risk for tumorigenesis. The focus of this dissertation is the development of engineered biomaterials as tools to study human neural stem cell behavior and neurogenesis (differentiation). A novel cell penetrating peptide was developed to enhance intracellular delivery of retinoic acid, a bioactive lipid known to induce differentiation. A hydrogel platform fabricated from hyaluronic acid, a naturally-occurring polysaccharide found in brain extracellular space, was designed to serve as a biomimetic soft substrate with similar mechanical properties to the brain. The biological behavior of the stem cells was characterized in response to chemical and physical cues.
Temple University--Theses

Bioengineering
M.S.
In this work, a hydrogel made from hyaluronic acid is synthesized and utilized to study neural stem cell behavior within a custom tailored three dimensional microenvironment. The physical properties of the hydrogel have been optimized to create an environment conducive for neural stem cell differentiation by mimicking the native brain extracellular matrix (ECM) environment. The physical properties characterized include degree of methacrylation, swelling ratios, enzymatic degradation rates, and viscoelastic moduli. One dimensional proton nuclear magnetic resonance (1HNMR) confirms modification of the hyaluronic acid polymers, and is used to quantify the degree of methacrylation. Rheological measurements are made to quantify the viscoelastic moduli. Further post-processing by lyophilization leads to generation of large voids to facilitate re-swelling and cell infiltration. ReNcell VM (RVM), and adult human neural stem cell line derived from the ventral mesencephalon, are cultured and differentiated inside the hydrogel for up to 2 weeks. Differentiation is characterized by immunocytochemistry (ICC) and real time quantitative polymerase chain reaction (qRT-PCR).
Temple University--Theses

The design and application of bionanotechnologies aimed at the nervous system provide powerful new approaches for studying cell and molecular biology and physiology. The successful and development of bionanotechnologies designed to interact with the nervous system as research or clinical tools requires an understanding of the relevant neurophysiology and neuropathology, and an understanding of the relevant chemistry and materials science and engineering. Materials designed molecularly for regeneration of tissues are becoming of great interest in advanced medicine and improvements in the understanding of self-assembly process offer new opportunities in molecular design of biomaterials for vary applications. In this project, two classes of biomaterials were studied with the same final achievement: the application to the regeneration of nervous systems. RADA16-I (AcN-RADARADARADARADA-CNH2), representative of a class of self-assembling peptides with alternate hydrophobic and hydrophilic residues, self-assembles into β-sheet bilayer filaments. Though molecular studies for this class of peptides has been recently developed, new investigations are required to explain how RADA16-I functionalization with biological active motifs, may influence the self-assembling tendency of new functionalized peptides (FP). Since FPs recently became a promising class of biomaterials, a better understanding of the phenomenon is necessary to design new scaffolds for cell biology and nanobiomedical applications. The first part of this project was based on the investigation with computational and experimental tools about the self-assembly of different FPs showing diverse sequences and "in vitro" behaviors. For the first time spectroscopic techniques (Raman and ATR/FTIR) was applied to these class of peptides and new vibrational modes were used to describe the nanostructure. Thanks to molecular dynamic simulations it was possible increase the experimental findings. The functionalizing self-assembling peptides can strongly influence or prevent assembly into nanostructure. Moreover the designing strategies were enhanced thanks to a deep investigation about the Glicines hinge between self assembling core and biological functionalization. The study of this structural group involved a refinement of a functionalized self-assembling peptide with the direct application on neural stem cells, and a then a future in vivo application. In the second part of this project electrospun tubes, formed by micro and nanofibers, were used to regenerate a 10-mm nerve gap in rat sciatic nerve in vivo. This work provided evidence that electrospun micro- and nanofiber PCL/PLGA channels are promising bioabsorbable scaffolds for stimulating and guiding peripheral nerve regeneration in rat models of sciatic nerve transection. This nanotechnological approach shows very encouraging results in peripheral nervous system regeneration that can ameliorate with surgery shrewdness, rehabilitative training and biomaterial modification, or better a combination of both eletrospun and self-assembling fibers. Finally in this project it was shown how a deeply investigation about self-assembling process, starting from theoretical part, could be applied directly in the development of many new biomaterials for specific nanobiomedical applications with the hope of increasing of the application range.

Peripheral nerves have demonstrated the ability to bridge gaps of up to 6 mm. Peripheral Nerve System injury sites beyond this range need autograft or allograft surgery. Central Nerve System cells do not allow spontaneous regeneration due to the intrinsic environmental inhibition. Although stem cell therapy seems to be a promising approach towards nerve repair, it is essential to use the distinct three-dimensional architecture of a cell scaffold with proper biomolecule embedding in order to ensure that the local environment can be controlled well enough for growth and survival. Many approaches have been developed for the fabrication of 3D scaffolds, and more recently, fiber-based scaffolds produced via the electrospinning have been garnering increasing interest, as it offers the opportunity for control over fiber composition, as well as fiber mesh porosity using a relatively simple experimental setup. All these attributes make electrospun fibers a new class of promising scaffolds for neural tissue engineering. Therefore, the purpose of this doctoral study is to investigate the use of the novel material PGD and its derivative PGDF for obtaining fiber scaffolds using the electrospinning. The performance of these scaffolds, combined with neural lineage cells derived from ESCs, was evaluated by the dissolvability test, Raman spectroscopy, cell viability assay, real time PCR, Immunocytochemistry, extracellular electrophysiology, etc. The newly designed collector makes it possible to easily obtain fibers with adequate length and integrity. The utilization of a solvent like ethanol and water for electrospinning of fibrous scaffolds provides a potentially less toxic and more biocompatible fabrication method. Cell viability testing demonstrated that the addition of gelatin leads to significant improvement of cell proliferation on the scaffolds. Both real time PCR and Immunocytochemistry analysis indicated that motor neuron differentiation was achieved through the high motor neuron gene expression using the metabolites approach. The addition of Fumaric acid into fiber scaffolds further promoted the differentiation. Based on the results, this newly fabricated electrospun fiber scaffold, combined with neural lineage cells, provides a potential alternate strategy for nerve injury repair.

End-stage kidney disease is increasing in prevalence and is associated with high levels of morbidity and mortality. At present, the only treatment options are dialysis or renal transplantation. However, dialysis is very costly and is associated with high levels of morbidity, whereas the problem with transplantation is that there is a shortage of organ donors. For these reasons, over recent years, there has been an increasing interest in developing novel therapies in the field of regenerative medicine including stem cell based therapies and tissue engineering. Stem cells could be used in a number of ways to develop new therapies for kidney disease. Firstly, they could be administered as cell therapies to patients with kidney disease, and secondly, they could be used to generate specific types of renal cells in vitro that could be used for understanding disease mechanisms and for drug discovery programmes. The barriers to the development of novel stem cell therapies include the difficulties in expanding kidney-derived stem cells in culture without altering their phenotype, and directing their differentiation to specific types of renal cells. These issues could be addressed by developing biomaterial substrates that provide an appropriate microenvironment for the successful culture and differentiation of stem cells. Within this study we interrogated a wide range of biomaterial substrates for their capability to direct the differentiation of kidney derived progenitor / stem cells. These materials were thoroughly characterised in terms of their physicochemical properties, such as surface chemistry, nanotopography and wettability by employing a wide range of analytic techniques, including X-Ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), colorimetry and contact angle measurements. We firstly investigated a range of polyacrylates. These substrates were novel in that, they were precisely designed to mimic cell binding motifs of the extracellular matrix stereochemically by using monomeric precursors that display particular chemical functional group chemistries, namely amine, hydroxyl, carboxyl groups or aliphatic spacer groups. We found that these materials differed strongly in the presence and distribution of surface functional group chemistries and topographical features, including the distribution of surface artefacts on a macroscale. Moreover, some of these materials were able to direct the differentiation into specialised renal cell lines. Two substrates, namely ESP 003 and ESP 004, directed the differentiation of kidney derived stem cells into podocytes and two further substrates, namely ESP 007 and BTL 15, directed differentiation into functional proximal tubule cells. These four substrates stimulated cell differentiation to an extent of about 40 to 50% after only 96 h in cell culture. We were moreover able to identify surface physicochemical cues, including surface micro- and nanoscale topography and surface functional group chemistries that are important to stimulate the differentiation process. In addition, we investigated a range of plasma polymer coatings composed of allylamine and octadiene that were provided as homo-or copolymers and in form of chemical gradients, the latter one differing in the amount of nitrogen functional group chemistries across the surfaces. We found that substrates with higher allylamine content displayed a greater amount of nitrogen functional groups and therefore increased in wettability. Moreover, those plasma polymer substrates with higher amine functionality directed kidney progenitor cell differentiation into podocytes, whereas substrates with higher octadiene concentration directed cell differentiation into functional proximal tubule cells, both to an extent of 35 to 45% after only 96 h in culture. To further study cell differentiation, we then incorporated gold nanoparticles underneath these plasma coatings, either in form of homogeneous coatings or in form of a nanoparticle density gradient. We found that surface topographic gradients increased cell differentiation into podocytes 3- to 4-fold, whereas differentiation into proximal tubule cells was only dependent on surface chemistry. Our studies on plasma polymer substrates highlighted not only the great potential of plasma polymers to modify surface functionality of a wide range of surfaces, but also emphasized the great capabilities of surface gradients, whether chemical or topographical in nature, to effect cellular fate. In summary, the results of this study include the identification of biomaterial substrates that have the potential to differentiate kidney-derived progenitor/stem cells in vitro and of the cues that are necessary to assist in the differentiation process. In the future, these biomaterials could be useful for directing the differentiation of pluripotent stem cell-derived renal progenitors to specific types of renal cells that could be used for applications in regenerative medicine and drug discovery programmes.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008.
Additional advisors: Yogesh K. Vohra, Xu Feng, Jack E. Lemons, Timothy M. Wick. Description based on contents viewed July 8, 2009; title from PDF t.p. Includes bibliographical references.

Thesis (Ph. D.)--University of Oregon, 2003.
Typescript. Includes vita and abstract. Includes bibliographical references (leaves 101-117). Also available for download via the World Wide Web; free to University of Oregon users.

This thesis is focused on neural stem cell (NSC) and glioma biology. I discuss how NSCs interact with extracellular matrix (ECM) proteins in the stem cell niche, and investigate the consequences of deregulated Platelet-derived growth factor (PDGF) signaling for embryonic NSCs in transgenic mice. Furthermore I present cell cultures of human glioblastoma multiforme (GBM) that models human disease, taking into account the heterogeneity of GBM. Finally, interactions between brain tumors and mast cells are studied using the glioma cultures. In paper I, the importance of NSC interactions with the ECM in the stem cell niche during development is discussed. Contacts between NSCs and the ECM in the subventricular zone (SVZ) are emerging as important regulatory mechanisms. We show that early postnatal neural stem and progenitor cells (NSPC) attach to collagen I, and that the adhesion is explained by higher expression of collagen receptor integrins compared to adult NSPC. Further, blood vessels in the SVZ express collagen I, indicating a possible functional relationship. Growth factors, e.g. PDGF, regulate NSC proliferation and differentiation. Aberrant activation of growth factor signaling pathways also plays a role in brain tumor formation. Paper II demonstrates that transgenic mice expressing PDGF-B at high levels in embryonic NSCs displayed mild neurological defects but no hyperplasia or brain tumors. This suggests that a high level of PDGF is not sufficient to induce brain tumors from NSCs without further mutations. Paper III presents a novel panel of human glioma stem cell (GSC) lines from GBM that display NSC markers in vitro and form secondary orthotopic tumors in vivo. GBM has recently been categorized in molecular subclasses and we demonstrate, for the first time, that these subclasses can be retained in vitro by stem cell culture conditions. We have thus generated models for research and drug development aiming at a focused treatment depending on GBM subtype. Interactions with the immune system are integral parts of tumorigenesis. Mast cells are found in glioma and in paper IV we demonstrate that the grade-dependent infiltration of mast cells is in part mediated by macrophage migration inhibitory factor and phosphorylation of STAT5.

In this bachelor thesis project, the problem of imageclassification with convolutional neural networks is considered.In several fields of biology, automatized cell detection is a helpfultool for facilitating the process of cellular analysis. This reportanswers the question whether a computer program can tell if animage contains muscle stem cells or not. Analogously to the neuronsof the human brain, the creation of such a program involvestraining thousands of mathematically modeled artificial neuronsto maximize the likelihood of producing correct classifications.This report covers how such a network is implemented and showshow its performance dependens on the network’s dimensions. It isrevealed that a neural network indeed can replace and speed upthe manual process of classifying images. With an image datasetof cells, the best performing networks manage to classify imageswith an accuracy of up to 90%.

Neurogenesis can be induced by several physiological and pathological conditions, such as environmental enrichment, stroke, epilepsy or inflammatory demyelination. The aim of this project was to investigate the possible effect of prion disease on neurogenesis, i.e. proliferation and differentiation of neural stem cells with a particular focus on the hippocampus and the neurogenic area of the dentate gyrus. We studied stem cell recruitment in non-infected and prion infected wild type mice. In addition, we examined transgenic mice which express prion protein at 3 times wt level (tg37), show profound hippocampal degeneration upon scrapie infection and that can be rescued by Cre-mediated inactivation of the Prnp gene in neurons. Stem cell proliferation and differentiation was analysed by immunostaining of vibratome sections. To study stem cell proliferation, scrapie-infected and control mice received one intraperitoneal injection of BrdU (50 pg/g body weight) 30 minutes before culling. BrdU gets incorporated into proliferating cells. To study differentiation of labelled cells, animals were culled at different time points after BrdU injection (e.g. 7 and 8 days). BrdU-labelled cells were further characterised by the expression of astroglial (GFAP) and early neuronal (p-tubulin) markers using double labelling immuno-fluorescence. Also, we identified proliferating microglial cells using the marker Iba-1. Neuropathology of prion infected animals was assessed by immunostaining of paraffin-embedded brain sections. Cellular morphology was examined by haematoxylin and eosin staining. Astrogliosis and microglia activation was analyzed by immunostainings for the astroglial marker GFAP and the microglial marker Iba-1 respectively. Finally, PrPSc accumulation was verified by immunostaining with the PrP-specific antibody ICSM35. Late-stage scrapie increased cell proliferation in the hippocampus but not the dentate gyrus of wild type mice while it profoundly increased cell proliferation throughout the area of the hippocampus of tg37 transgenic mice. BrdU-labelled proliferating cells in the dentate gyrus of wt mice acquired a neuronal phenotype within 8 days, thus confirming that BrdU labelling identifies proliferating progenitor cells, and further that the cell proliferation we estimated in the dentate gyrus partly reflects proliferation of stem cells. Proliferating cells in the dentate gyrus of prion- infected tg37 mice were identified as astrocytes and microglial cells. Also there was a significant increase in BrdU-labelled cells expressing P-tubulin. Inactivation of PrP expression in neuronal cells during scrapie incubation, significantly decreased cell proliferation in the hippocampus. This decrease in part corresponded to a reduction in the number of proliferating astrocytes and microglial cells in the dentate gyrus. However, the number of proliferating cells acquiring a neuronal phenotype (P-tubulin positive) was unaffected by PrP depletion (and the subsequent reversal of clinical prion disease) at least at an early time point following PrP depletion. Depletion of neuronal PrP also led to reduced proliferation in the hippocampus of un-inoculated mice, suggesting that PrP plays a role in cell proliferation. We have demonstrated that prion disease and the resulting neuronal loss in the hippocampus increases neurogenesis (BrdU-labelled cells acquiring a neuronal phenotype) and also increases the numbers of proliferating astrocytes and microglia cells in the dentate gyrus of PrP over-expressing mice (tg37).

In the last decade, multipotent mesenchymal stromal cells (MSCs) demonstrated a significant therapeutic efficacy, particularly in cell therapy approaches aiming at tissue regeneration. MSCs exert their action via trophic support, induction of angiogenesis, immunomodulation and reduction of necrosis at affected tissues. Importantly, these regenerative and protective properties are largely associated to MSC secretome. Unfortunately, cell-based approaches not always meet the criteria for a smooth translation to the clinic. For instance, the use of stem cells in pathologies with a very short therapeutic window, such as few hours, is not compatible with the requested minimal criteria for MSC release, before administration to the patient. Notwithstanding, in the regenerative medicine field, the MSC mechanism of action paradigm was recently extended to include the action of extracellular vesicles (EVs), which are cytoplasm-containing cellular bodies secreted by a wide range of cell types. Intriguingly, many studies reported that EVs generated by MSCs are able to recapitulate the majority of the regenerative properties of parental MSCs. Starting from these premises, the objectives of the present doctoral research project were: to address EV-mediated cell-to-cell communication as novel MSC mechanism of action; to address reprogrammed MSC-EV generation; to define, for the first time in the literature, stem cell EV molecular content (e.g.: miRNome), comparing reprogrammed to non-reprogrammed MSC-EVs; to challenge stem cell-EV therapeutic potential in a model of acute tissue damage, as a proof-of-concept for feasibility and effectiveness of a stem cell-based albeit cell-free regenerative strategy. Intriguingly, EVs may be produced in a ready-to-use formulation, so that clinicians could use them as soon as a therapeutic need arises, also in the case of an urgent one. In this way, EV-shuttled MSC regenerative properties could exert beneficial effects also on pathologies currently lacking any cell therapy option. To develop this innovative therapeutic strategy, MSCs were isolated from different tissues and their biological properties were evaluated in order to choose the MSC source most suitable for the implementation of the project. Thus, both MSC transcriptome and immunophenotype were addressed. MSCs from adult sources (e.g.: bone marrow) showed senescence-related features in vitro, correlated to donor’s age in vivo. On the other hand, MSCs from perinatal tissues (e.g.: cord blood) showed a phenotype more similar to that of pericytes, which are the in vivo progenitors of MSCs. Therefore, cord blood was chosen as MSC source, also in the prospective of clinical translation, since public banking of cord blood units for clinical use already exists worldwide. Next, thanks to an extended analysis of the stromal populations present in cord blood, a MSC subpopulation showing higher proliferation properties and significantly longer telomeres was isolated. In addition, the standard cord blood MSC isolation protocol was improved, leading to an efficiency of 80%. Eventually, MSC secretome-associated anti-inflammatory and anti-apoptotic properties were observed in vitro and in vivo. In order to investigate if EVs contributed to MSC paracrine properties, MSC-EV secretion and regenerative properties were assessed. The MSC-EV therapeutic effectiveness was challenged in an in vitro model of acute tissue damage. Intriguingly, MSC-EVs could rescue damage-induced cell mortality, showing the same protective effect of parental MSCs. In spite of the use of a high proliferative cord blood MSC subpopulation, primary cultures still show a limited lifespan. In order to increase their replicative potential and to better exploit their EV production, induced cellular reprogramming was tested on MSCs as an alternative to traditional immortalization techniques. In this way, MSC-derived cell lines endowed with unlimited lifespan were generated, and permanent modification of their genome was avoided. The next step was to confirm the generation of EVs from reprogrammed MSCs, since reprogramming drastically changes cell identity. Furthermore, the EV miRNome load of reprogrammed and non-reprogrammed MSCs was addressed. Importantly, the majority of miRNAs were common between the two samples, indicating that reprogramming did not change the EV miRNA content. This result could have relevant consequences on the functional features of reprogrammed MSC-EVs, since EV-mediated miRNA transfer from donor to target cells was proposed as one of MSC mechanisms of action. In the last part of this doctoral research, stem cell (non-reprogrammed and reprogrammed MSCs)-EV therapeutic effectiveness was addressed and compared to that of parental MSCs. In order to do that, an organotypic ex vivo mouse model of brain ischemia was used. This model recapitulated the modulation of some ischemic damage-related parameters, including increased secretion of inflammatory cytokines, high tissue necrosis and the impairment of neuronal and astrocytic cell populations. Therefore, this model mimicked early phase events of brain ischemia, whose thrombolytic clinical treatment must be administered within 3-6 hours of first signs of ischemia. Notably, stem cell-EVs were tested for the first time in this pathological context to verify their potential role in tissue regeneration. Strikingly, stem cell-EV administration to affected tissues showed significant neuroprotective properties, which were comparable to those of parental MSCs. Importantly, the ischemic damage-related parameters previously described were rescued. In particular, inflammatory-associated parameters underwent the most statistically significant decrease, showing levels similar to or better than those of the uninjured brain tissue. This is of uttermost importance, considering that chronic inflammation is detrimental to tissue regeneration. To conclude, the results of the present PhD thesis confirmed the feasibility of stem cell EV-based therapies in regenerative medicine approaches. In the future, this innovative EV therapy may be applied to pathological contexts currently without a cell therapy option. In the framework of advanced therapy medicinal products, the new drug would be the EVs, rather than the parental stem cells. Finally, EVs could play the role of ready-to-use anti-inflammatory molecule carriers, in order to guarantee a rapid therapeutic action for the regeneration of injured tissues.
Negli ultimi anni, le cellule stromali mesenchimali multipotenti umane (CSM) hanno mostrato una grande efficacia terapeutica, soprattutto in approcci di terapia cellulare aventi come obiettivo la rigenerazione tissutale. L’azione delle CSM avviene attraverso supporto trofico, induzione di angiogenesi, modulazione della risposta immunitaria e diminuzione della necrosi a livello dei tessuti colpiti. Inoltre, recente letteratura ha dimostrato che queste capacità rigenerative e protettive sono in larga parte associate al secretoma delle CSM. Purtroppo, gli approcci di terapia cellulare non sono sempre traslabili alla clinica. Ad esempio, l’utilizzo di cellule staminali in patologie caratterizzate da una finestra terapeutica molto stretta, dell’ordine di poche ore, non è compatibile con la necessità di scongelare e valutare i minimi standard di qualità delle CSM prima della somministrazione al paziente. Nonostante ciò, il paradigma del meccanismo d’azione delle CSM nel campo della medicina rigenerativa si è ulteriormente arricchito. Infatti, molti recenti studi hanno dimostrato che le vescicole extracellulari, ossia porzioni di citoplasma delimitate da membrana cellulare secrete dalle CSM, sono in grado di riprodurre la maggior parte delle proprietà rigenerative delle CSM stesse. Date queste premesse, gli obiettivi del progetto di ricerca del presente Dottorato sono stati i seguenti: indagare la comunicazione intercellulare tramite vescicole extracellulari quale innovativo meccanismo d’azione delle CSM; studiare la produzione di vescicole extracellulari da parte di CSM riprogrammate, e, per la prima volta in letteratura, definirne il contenuto molecolare (es.: miRNoma), a confronto con le CSM d’origine; testare il potenziale terapeutico di vescicole extracellulari da cellule staminali in un modello di danno tissutale acuto, come proof-of-concept della funzionalità di una strategia terapeutica cell-free. Infatti, le vescicole extracellulari potrebbero essere prodotte in formulazioni pronte all’uso, a immediata disposizione per ogni richiesta clinica, anche urgente. In questo modo le proprietà rigenerative delle CSM potrebbero essere veicolate dalle vescicole extracellulari anche in contesti patologici attualmente senza alcuna opzione di terapia cellulare. Per lo sviluppo di questa innovativa strategia terapeutica, CSM isolate da vari tessuti sono state caratterizzate e confrontate in base al loro trascrittoma e al loro immunofenotipo, allo scopo di valutarne le proprietà biologiche e quindi scegliere le CSM più adatte all’implementazione del progetto di Dottorato. Le CSM da tessuti adulti (e.g.: midollo osseo) hanno mostrato in vitro caratteristiche di senescenza correlate all’età del donatore in vivo. Al contrario, le CSM da tessuti perinatali (e.g.: sangue di cordone ombelicale) hanno mostrato un fenotipo più simile a quello dei periciti, ossia i progenitori delle CSM in vivo. Quindi, tenuto conto anche della traslabilità clinica, il sangue di cordone ombelicale è stato scelto come fonte di CSM, visto che la raccolta e la crioconservazione di unità di sangue placentare a fini terapeutici è già una realtà clinica. In seguito, un’analisi estesa delle popolazioni stromali presenti nel sangue di cordone ombelicale ha portato alla definizione di una sottopopolazione di CSM dotata di maggiori capacità proliferative e con una lunghezza del telomero significativamente più alta. Inoltre il protocollo standard di isolamento delle CSM da sangue di cordone ombelicale è stato migliorato, arrivando ad un’efficienza di circa 80%. Infine, le proprietà anti-infiammatorie e anti-apoptotiche del secretoma delle CSM sono state studiate sia in vitro che in vivo. Al fine di verificare se le vescicole extracellulari contribuissero alle proprietà paracrine delle CSM, se ne è caratterizzata la secrezione e se ne sono indagate le proprietà rigenerative. Una chiara efficacia terapeutica da parte delle vescicole extracellulari di CSM è stata dimostrata in un modello in vitro di danno tissutale acuto, in cui le vescicole extracellulari hanno eguagliato i risultati ottenuti con le CSM stesse. Nonostante l’utilizzo di CSM dalle elevate proprietà proliferative, la loro lifespan in coltura, in quanto cellule primarie, rimane limitata. Allo scopo di aumentarne il potenziale replicativo e di sfruttarne al meglio così la produzione di vescicole extracellulari, le CSM sono state sottoposte alla riprogrammazione cellulare indotta. In questo modo sono state generate linee cellulari derivate da CSM dal potenziale di crescita illimitato, evitando però di modificarne il genoma come nelle tradizionali tecniche di immortalizzazione. Siccome la riprogrammazione implica una modificazione radicale dell’identità della cellula d’origine, il passo successivo è stato quello di confermare la capacità di questa nuova popolazione di generare vescicole extracellulari, poi opportunamente caratterizzate. In particolare, il miRNoma delle vescicole extracellulari da cellule riprogrammate è stato oggetto di studio e di confronto con quello delle vescicole extracellulari delle CSM d’origine. Si è così potuto dimostrare che la maggior parte dei miRNA era presente nelle vescicole extracellulari sia prima che dopo la riprogrammazione. Ciò indica che il processo di riprogrammazione non ne ha alterato in modo sostanziale il contenuto. Questo potrebbe avere importanti ricadute sugli aspetti funzionali delle vescicole extracellulari da CSM riprogrammate. Infatti è stato ipotizzato che il trasferimento di miRNA specifici da cellule donatrici a cellule target mediato dalle vescicole extracellulari sia uno dei meccanismi d’azione delle CSM. Nell’ultima parte di questo progetto di Dottorato, l’utilità terapeutica delle vescicole extracellulari da cellule staminali (CSM e CSM riprogrammate) è stata confrontata con quella delle CSM d’origine. A tale scopo è stato utilizzato un modello ex vivo di ischemia cerebrale, in cui è stato osservato il movimento di alcuni parametri di danno ischemico acuto, tra cui un picco di produzione di citochine infiammatorie, una forte necrosi tissutale e una riduzione delle popolazioni cellulari neuronali e astrocitiche. Questo particolare modello mima infatti la fase acuta di questa condizione patologica, il cui trattamento a base di agenti trombolitici deve avvenire entro 3-6 ore dall’insorgenza dei primi sintomi. Quindi le vescicole extracellulari prodotte dalle CSM riprogrammate sono state testate per la prima volta in questo contesto patologico per verificare se potessero esercitare una funzione rigenerativa. La loro somministrazione al tessuto colpito dal danno ischemico ha generato uno spiccato effetto neuroprotettivo, pari a quello delle CSM d’origine, che ha riportato a valori simili a quelli del tessuto cerebrale non danneggiato i parametri di danno sopra descritti. Il risultato più interessante e statisticamente significativo è stato soprattutto a carico di quei parametri legati ai processi infiammatori, i quali sfavoriscono il recupero del danno tissutale. In conclusione, i risultati presentati in questa tesi di Dottorato confermano la possibilità di utilizzo di vescicole extracellulari secrete da cellule staminali in strategie di medicina rigenerativa. Questa innovativa extracellular vesicle therapy potrebbe in futuro essere applicata in contesti patologici per i quali ad oggi non è praticabile una terapia cellulare. A questo punto, nel quadro dei prodotti medicinali per le terapie avanzate, il “farmaco” non sarebbe più la cellula staminale, ma le rispettive vescicole extracellulari. Queste acquisirebbero così il ruolo di carrier di molecole antinfiammatorie, pronte all’uso e capaci di garantire un’azione terapeutica tempestiva per la rigenerazione di tessuti danneggiati.

Cell instructive biointerfaces are versatile tools to mimic a natural cellular environment and control cell fate in vitro. The particular interest lies in combining information gained from surface and interface analysis tools with biological analysis to explore and understand fundamental processes such as neuronal stem cell differentiation at the biointerface. A major challenge in biointerface design is to mimic and study the complex interactions of the natural processes in the extracellular matrix (ECM) with artificially designed surfaces and interfaces. In the past, peptide surfaces have been used as ECM mimics, however, more research is required in this field to tune the properties of peptide surface to modulate the outcome of stem cell fate. The present work aims to address this challenge by designing new synthetic peptide surfaces with well controlled composition and functionality able to impart have control over the differentiation of neuronal stem cells with the ultimate goal to relate surface properties and stem cell response to understand and control how neuronal networks function. Compositionally well-defined surfaces of two short laminin peptide sequences, Arg-Gly-Asp (RGD) and Ile-Lys-Val-Ala-Val (IKVAV) were prepared of various ratios via the “grafting from” stepwise approach. The surface modification was confirmed with surface analysis techniques to indicate successful peptide functionalization. The neural progenitor stem cells (NPSCs) were set up from embryonic rat hippocampi (E18). Immunocytochemistry (ICC) observed cell viability and differentiation to specific NPSCs lineages for βIII-Tubulin and GFAP. The surface characterizing techniques of WCA, AFM, ToF-SIMS, and XPS validated the successful orthogonal functionalization of controllable peptides composition surfaces with the increase of RGD composition a relative decrease in the IKVAV composition was observed. The increase in the normalized total ion fragments of RGD in the ToF-SIMS measurements can be related linearly to the % area coverage of neurons versus astrocytes observed on the controllable peptide composition surfaces. Well-defined peptide surfaces were designed and successfully demonstrated that the amount of RGD peptide composition present on the surface influences cell adhesion, proliferation and differentiation to a desirable cell fate or controllable cell population (i.e. neurons and astrocytes). Recent technological developments demonstrate a shift from two-dimensional (2D) surfaces to three-dimensional (3D) surfaces, so we used the designed versatile 2D surfaces as the template to design comparable 3D surfaces to examine the biological response of NPSCs to both microenvironments. This study proposes the design of novel 3D nanoparticles (NPs) made of gold and surface-conjugated with differentiation-inducing peptides. The NPs-peptides will guide stem cell differentiation to neurons that self-aggregate around the NPs by cell-cell contacts to form neurospheres. The neural stem cells will establish 3D structures biomimicking the cytoarchitecture of the brain. Successively, they could be used as an alternative 3D in vitro model for neurotoxicity testing of drugs and chemicals. Size-controllable NPs will be surface-conjugated with RGDC and IKVAVC(19) peptides via the SPPS “grafting to” approach. Two different sizes of NPs were characterized as AuNPs and SiO2@AuNPs characterization and validated by TEM, DLS, -potential, EDX; and the surface functionalization on NPs was successfully confirmed by UV/vis spectroscopy and -potential. The NPSCs were set up from E18 rat hippocampi and cell viability and differentiation to specific NSPCs lineages was stained for βIII-Tubulin and GFAP. AuNPs and SiO2@AuNPs peptide immobilized surfaces supported cell adhesion, proliferation, and survival. The confocal light scanning microscope (CLSM) images indicate that the RGDC functionalized AuNPs and SiO2@AuNPs surfaces induced a preferential differentiation towards a neurons cell fate and the IKVAVC(19) functionalized surfaces of AuNPs and SiO2@AuNPs favored an astrocytes cell. The cellular uptake of functionalized AuNPs by the cells in the neurospheres was observed via TEM micrographs, whilst the micrographs of functionalized SiO2@AuNPs surfaces suggest that they were not taken-up into the cells. Hence, indicating that there is a difference in the cellular uptake mechanism for the functionalized AuNPs and SiO2@AuNPs, which might be due to the agglomeration of the nanoparticles than the individual size of the nanoparticles. In conclusion, the relation between the 2D and 3D surfaces may provide new insight in understanding how surface properties affect the regulation of physiological relevance in directing neural cell differentiation, which will be essential to understand how neural networks function.

Neural stem cells showed strong electrotaxis, evidenced by highly directed migration towards the cathode.  Optimal electrotaxis was found to require growth factors and phosphoinositide 3-kinase (PI3-K) signalling, although reduced electrotaxis could be obtained without growth factors at the highest EFs used.  After EF exposure, neural stem cell trajectories became much more linear, and a reduction in the number of cell protrusions oriented towards the anode was observed.  Also, protrusions initially orienting towards the cathode retracted after the polarity of the EF was reversed, suggesting that EFs could inhibit the extension of anodal protrusions.  A simple model of neural stem cell migration was built with only two key parameters, which reproduced accurately neural stem cell migration patterns, and predicted that PI3-K functions in electrotaxis mainly by controlling cell orientation.  Finally, wild-type and Pax6^-/- embryonic neural stem cells were exposed simultaneously to EFs and contact guidance cues in conflicting orientations.  Only wild-type neural stem cells showed significant integrative migratory responses, suggesting that Pax6 is important for integration of diverse guidance cues during cell migration. The results obtained in this thesis show that neural stem cells display strong electrotaxis in vitro, which is accompanied by a qualitative change in the pattern of migration.  The results also identify the control of protrusion orientation by EFs as an important element in neural stem cell electrotaxis, contributing insight into the mechanisms of electrotaxis.  Finally, these results warrant further studies to assess the possibility of using EFs in brain repair therapies.

Ischaemic stroke is a major cause of mortality and chronic disability for which there is no effective treatment. The subventricular zone (SVZ) is an adult neurogenic niche which mediates limited endogenous repair following stroke. To harness this phenomenon for therapy, it is important to understand how the SVZ niche is altered in stroke, and the processes that recruit neural precursors to the site of injury, which becomes a de facto neurogenic niche. Galectin-3 (Gal-3) is a β-galactoside binding protein involved in cellular adhesion, inflammation and tumour metastasis. Gal-3 is specifically expressed in the SVZ and maintains neuroblast migration to the olfactory bulb, although its role in post-stroke neurogenesis is not well-understood. Therefore, this project aimed to (1) characterise the cytoarchitecture of the SVZ in response to stroke, and (2) examine the role of Gal-3 in stroke outcome and tissue remodelling, and test the hypothesis that Gal-3 is required for neuroblast ectopic migration into the ischaemic striatum. Using the intraluminal filament model of middle cerebral artery occlusion (MCAO) in mice, and whole mounts of the lateral ventricular wall, significant SVZ reactive astrocytosis and increased vascular branching were observed, thereby disrupting the neuroblast migratory scaffold. Stroke increased SVZ cell proliferation without increase in cell death. Post-stroke ependymal cells were enlarged and non-proliferative, and assumed a reactive astroglial phenotype, expressing de novo high levels of glial fibrillary acidic protein. This was associated with focal planar cell polarity misalignment, and turbulent and decreased rate of cerebrospinal fluid flow. These findings demonstrate significant changes in multiple SVZ cell types which are positioned to influence post-stroke neurogenesis and regulation of the neural stem cell niche Gal-3 was up-regulated in the ischaemic brain and ipsilateral SVZ. To elucidate the role of Gal-3 after stroke, MCAO was performed in wildtype and Gal-3 null (Gal-3^-/-) mice, and parameters of stroke outcome and post-stroke neurogenesis compared. The deletion of Gal-3 did not affect infarct volumes or neurological outcomes, although neuroblast migration into the ischaemic striatum was increased in Gal-3^-/- brains. Gal-3^-/- mice failed to mount an angiogenic response in the ischaemic striatum, and this was associated with lower levels of vascular endothelial growth factor (VEGF) and increased anti-angiogenic protein levels. Loss of Gal-3 further disrupted the pro-proliferative neural-vascular interaction at the basement membrane. The current data indicate that Gal-3 is a pleiotropic molecule which has distinct roles in both the SVZ and the post-stroke striatum as niches of adult neurogenesis.

Thesis (Ph.D.)--University of California, San Francisco, 2008.
Source: Dissertation Abstracts International, Volume: 69-06, Section: B, page: 3358. Adviser: Arturo Alvarez-Buylla. Includes supplementary digital materials.

Despite the remarkable beneficial effects of disease-modifying agents in relapsing-remitting multiple sclerosis (MS) patients, progressive forms of (P)MS still lack effective treatments. This stark contrast is partially dependent on the difficulties researchers have found in tackling the complex pathophysiology of this phase of disease, in which chronic inflammation within the central nervous system (CNS) is coupled by ongoing neurodegeneration and demyelination. Cell transplantation is among the most promising therapeutic approaches in regenerative medicine, combining tissue trophic and immunomodulatory effects of the graft with its intrinsic potential for cellreplacement. These are all attributes that can be harnessed to treated patients with PMS. As such, within this thesis, I have focused my attention on investigating how cellular therapies could be used to (i) prevent neuronal damage, (ii) modulate the chronic activation of the immune system and (iii) replace the damaged myelin in PMS. Olfactory Ensheathing Cells (OECs) are a special population of glial cells known to exert neuroprotective mechanisms and capable of promoting neuroprotection. Using in vitro models of neuron-like cells, I have demonstrated that OECs exert their neuroprotective effect by reducing Cx43-mediated cell-to-cell and cell-toextracellular environment communications. Despite this important finding, the immunomodulatory and remyelinating potential of OECs is still limited. As such, I decided to study a complementary stem cell approach that conjugates these attributes with ease in clinical applicability. Induced Neural Stem Cells (iNSCs) are a source of autologous, stably expandable, tissue specific and easily accessible stem cells, which have the potential to differentiate into the three main neural lineages. Mouse iNSCs were characterized in vitro and in vivo and their immunomodulatory potential was initially studied. This work uncovered a novel mechanism that underpins the potential of iNSCs to interact with the chronic CNS compartmentalised activation of the innate immune system. Specifically, I found that iNSCs are able to sense extracellular metabolites, which accumulate in the chronically inflamed CNS, and to ameliorate neuroinflammation via succinate-SUCNR1-dependend mechanisms. To characterize the potential for tissue replacement and remyelination of such a promising cell line, I have also analysed how iNSCs grafts differentiate in an experimental model of focal demyelination. I found that iNSCs are able to integrate and differentiate into remyelinating oligodendrocytes (OLs) in chronic demyelinated CNS. These data suggest that iNSCs are indeed an effective source of stem cell transplantation, being able to modulate inflammation and to effectively replace lost tissue in mouse models of PMS. Altogether the evidences gathered in this thesis are important new steps in the field of cell transplantation, which will be pivotal in the march forward for future clinical applications in chronic demyelinating CNS disorders.

Despite advances in treatment, heart failure remains one of the top killers in Canada. This recognition motivates a new research focus to harness the fundamental repair properties of the human heart, with human cardiac stem cells (CSCs) emerging as a promising cell candidate to regenerate damaged myocardium. The rationale of this approach is simple with ex vivo amplification of CSCs from clinical grade biopsies, followed by delivery to areas of injury, where they engraft and regenerate the heart. Currently, outcomes are limited by modest engraftment and poor long-term survival of the injected CSCs due to on-going cell loss during transplantation. As such, we explored the effect of cell encapsulation to increase CSC engraftment and survival after myocardial injection. Transcript and protein profiling of human atrial appendage sourced CSCs revealed strong expression the pro-survival integrin dimers αVβ3 and α5β1- thus rationalizing the integration of fibronectin and fibrinogen into a supportive intra-capsular matrix. Encapsulation maintained CSC viability and expression of pro-survival transcripts when compared to standard suspended CSCs. Media conditioned by encapsulated CSCs demonstrated superior production of pro-angiogenic/ cardioprotective cytokines, angiogenesis and recruitment of circulating angiogenic cells. Intra-myocardial injection of encapsulated CSCs after experimental myocardial infarction favorably affected long-term retention of CSCs, reduced scar burden and improved overall cardiac function. Taken together, cell encapsulation of CSCs prevents detachment induced cell death while boosting the mechanical retention of CSCs to enhance repair of damaged myocardium.

Biomaterials are becoming the most commonly used therapeutic method for treatment of lost or damaged tissue in the body. Metallic materials are chosen for high strength orthopaedic and dental applications. Titanium (Ti) implants are highly successful in young, healthy patients with the ability to fully integrate to surrounding tissue. However the main population requiring these corrective treatments will not be healthy or young, therefore further research into material modifications have been started to improve outcomes in compromised patients. The body’s immune system will generate a response to any implanted material, and control the final outcome. Among the first and most influential, cells to interact with the implant will be macrophages. Throughout this study we have 1) established the ability of macrophages to recognize and differentially activate in response to material surface properties, 2) investigated the role of integrin binding in macrophage activation to material properties, and 3) confirmed the importance of macrophage activation in vivo following Ti implant placement. The generation of a hydrophilic implant surface promoted the greatest anti-inflammatory and pro-regenerative macrophage activation. Surface wettability will control protein adsorption which can activated different integrin binding on macrophages and may be responsible for changes in activation. When integrin β3 subunit binding was prevented hydrophilic surfaces no longer promoted an anti-inflammatory macrophage activation. Additionally, when macrophage levels were reduced using two separate ablation models, MaFIA mice and clodronate liposomes, hydrophilic surfaces no longer promoted anti-inflammatory T-cell populations and cytokine profiles. There were also fewer stem cells adhered to implant surfaces at 1, 3, and 7 days when macrophage populations were compromised.

I biomateriali per applicazioni di ingegneria dei tessuti devono soddisfare diversi requisiti, come sicurezza, biocompatibilità e caratteristiche meccaniche appropriate. Il processo di sviluppo di questi biomateriali comprende diversi approcci scientifici, che vanno dall' in-silico all'in-vivo. L'ottimizzazione in silico delle caratteristiche dei biomateriali sta attirando un'attenzione sempre maggiore. Infatti, il miglioramento di questo approccio consentirà di ridurre i costi aggiuntivi nel processo di sviluppo dei biomateriali, a causa di caratterizzazioni sperimentali non necessarie. Secondo questo punto di vista, in questa tesi viene presentato un approccio di dinamica molecolare per la caratterizzazione dei biomateriali. Più in dettaglio, gli scaffold degli idrogel di peptidi autoassemblanti (SAP) sono stati studiati su nanoscala e microscala, al fine di chiarire le loro relazioni intrinseche struttura-proprietà-funzione. Le dinamiche molecolari atomistiche a grana grossa (CG-MD) sono state utilizzate per studiare i meccanismi di auto-assemblamento che portano alla formazione di scaffold peptidici. A causa della mancanza di informazioni strutturali cruciali nelle simulazioni CG-MD, l'innovativa suite software, denominata Morphoscanner, è stata impiegata per la classificazione dei modelli di aggregazione conformazionale dei SAP. Quindi, le proprietà meccaniche e i meccanismi di rottura delle nanostrutture SAP sono stati studiati attraverso le simulazioni MD vincolate. Queste evidenze hanno portato allo sviluppo di un approccio CG-MD che mira a chiarire la complessa interazione tra membrane cellulari e nanofibrille SAP. In particolare, le simulazioni MARTINI CG-MD sono state utilizzate per comprendere gli effetti della nanofibrille peptidiche sulla dinamica dei domini lipidici nelle membrane neurali. Tali risultati aprono nuove dimensioni nel campo della biomateriomica, consentendo di comprendere ed eventualmente controllare i complessi fenomeni che influenzano le proprietà meccaniche e la biocompatibilità dei biomateriali a base peptidica per applicazioni di ingegneria tissutale.
Biomaterials for tissue engineering applications have to comply with several requirements, such as safety, biocompatibility and appropriate mechanical features. The development process of these biomaterials encompasses several scientific approaches, ranging from in-silico to in-vivo. The in-silico optimization of biomaterials features is attracting even larger attention. Indeed, the improvement of this approach will allow to reduce additional costs int the biomaterials development process, due to unnecessary experimental characterizations. According to this point-of-view, in this thesis is presented a molecular dynamics approach for biomaterial characterization. More in details, self-assembling peptides (SAPs) hydrogels scaffolds have been investigated at the nano-scale and micro-scale, to elucidate their intrinsic structure-property-function relationships. The atomistic and coarse-grained molecular dynamics (CG-MD) have been used for the elucidation of self-assembling pathways of peptide-based scaffolds. Due to the lack of crucial structural information in CG-MD simulations, the innovative software suite, dubbed Morphoscanner, has been employed for the elucidation of conformational aggregation patterns of SAPs. Then, the mechanical properties and failure mechanisms of SAPs nanostructures have been investigated through the steered MD simulations. These evidences led the development of a CG-MD approach aiming to elucidate the complex interplay between cell membranes and SAPs nanofibrils. In particular, MARTINI CG-MD simulations have been used for understanding the effects of SAPS nanofibril on dynamics of lipid domains in neural membranes. Such achievements open up new dimensions in the field of biomateriomics, allowing to understand and eventually orchestrate the complex phenomena which affect the mechanical properties and biocompatibility of SAPs biomaterials for tissue engineering applications.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
A engenharia de tecidos combina células humanos, materiais e engenharia de modo a induzir respostas biológicas com o objetivo de proporcionar uma regeneração rápida e correta do tecido danificado. O uso de matrizes tridimensionais (3D) para suportar o crescimento celular, ao contrário dos convencionais materiais 2D, é de grande importância para a simulação da organização estrutural de tecidos biológicos. Outros aspetos da matriz extracelular (ECM), para além da sua arquitetura são conhecidos por afetar a resposta celular. Fatores biomecânicos apresentados às células através das proteínas da ECM influenciam a adesão celular e fenómenos tais como manutenção do fenótipo, diferenciação celular e proliferação. Estudos in vitro muitas vezes falham na apresentação de fatores fisiológicos que incluem dinâmica de fluidos, o qual pode levar a uma correta oxigenação do biomaterial com células incorporadas, bem como a fenómenos de mecanotransdução. Neste trabalho, propomos um sistema que revela o efeito de 32 combinações de proteínas da ECM na adesão e expressão da alcalina fosfatasse (ALP) em células estaminais derivadas da coluna óssea (MSCs), tanto em ambiente estático como dinâmico. Um bioreator foi desenhado de modo a permitir um estudo high-throughput, para que fossem analisadas 32 combinações biomaterial-célula simultaneamente. Este bioreactor foi construído a partir de material de laboratório comum e de baixo custo (incluindo tubos e seringas descartáveis). As MSCs foram semeadas em scaffolds de quitosano poroso, modificado covalentemente com proteínas da ECM do osso, assim como proteínas responsáveis por contacto célula-célula e componentes do esmalte. Uma análise fatorial permitiu correlacionar a presença das várias combinações proteicas com melhor adesão celular ao biomaterial, assim como uma expressão de ALP após 24 horas e 5 dias de cultura. Os dados foram analisados tanto para ambiente estático, como dinâmico na presença de um pequeno fluxo, previamente comprovado como potenciador da diferenciação osteogénica de MSCs. O sistema desenvolvido foi útil na interpretação da grande complexidade das interações célula-ECM, e poderá ter possível aplicação no desenvolvimento de biomateriais para regeneração óssea, bem como em futuras aplicações como modelos de doença.
Tissue engineering combines human cells, materials and engineering to induce biological responses seeking the rapid and accurate healing of damaged tissues. The use of three-dimensional (3D) matrices to support cellular growth, in opposition to traditionally used two dimensional (2D) materials, are of utmost importance to emulate the structural organization of biological tissues. Other aspects of the extracellular matrix (ECM) beyond its architecture are known to affect cell response. The biochemical cues presented to cells by ECM proteins influence cell adhesion and phenomena as cell phenotype maintenance, cell differentiation and proliferation. In vitro studies often lack physiological-like cues that include slow fluid dynamics, which may impair the correct oxygenation of the biomaterial-cells construct. Here, we engineered a system to disclose the effect of 32 different ECM protein combinations on the adhesion and alkaline phosphatase (ALP) expression of bone marrow-derived mesenchymal stem cells (MSCs), both under static and flow perfusion conditions. A novel bioreactor was designed to enable a high-throughput study, that allowed to withdraw data from 32 biomaterial-cell combinations in one single test. The bioreactor was assembled from widely available affordable labware (including plastic tubes and disposable syringes). MSCs were seeded on chitosan porous scaffolds covalently modified with bone ECM proteins, as well as cell-cell contact proteins and enamel components. A factorial analysis study allowed correlating the presence of single and combinations of proteins with improved cell adhesion to biomaterials, as well as improved ALP quantification after 24 hours and 5 days of culture. The data was analyzed both for static culture conditions, as well as in the presence of a slow perfusion rate, previously shown to potentiate MSCs osteogenic differentiation. The developed system has proven to be useful in the interpretation of the wide complexity of cells-ECM interactions, and may find application in the development of biomaterials for tissue regeneration or as disease model platforms.

Thesis (Ph. D.)--University of Missouri-Columbia, 2008.
The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 3, 2009) Vita. Includes bibliographical references.

The main goal of this PhD thesis was to understand the mechanisms underlying GBM tumor formation and progression, with a special emphasis on the study of factors that have a role in normal NSC biology, which might be providing cancer cells with the stem-like properties required to acquire tumorigenic potential. With this purpose, this PhD thesis was divided into two different projects. 1. Transcriptional profiling of hypoxic NSC identifies calcineurin-NFATc4 signaling as a major regulator of NSC biology. One of the main goals of our laboratory is to get a deeper understanding of NSC biology as a first step prior to applying this knowledge to the study of gliomas. Therefore, in the first project of this PhD thesis, we set out to identify novel factors that control NSC biology (whose role in NSC was previously unknown). The specific objectives of this project have been: - Characterization of the effect of physiologic oxygen concentrations on NSC biology. - Identification of novel signaling pathways that are operative in NSC cultured under physiologic oxygen concentrations. - Identification of novel TF that orchestrate the NSC response to physiological oxygen concentrations - Functional validation of the role of NFATc4, one of the most promising candidate TF identified above on NSC properties under physiologic oxygen concentrations. 2. GPR56 is a NSC factor that restricts the proneural to mesenchymal transition in GBM by inhibiting the NF-kB pathway. The second project was focused on the identification of factors that are involved in GBM progression from a PN to a MES subtype (PN-to-MES transition). In a candidate-driven approach, we selected GPR56 as the focus of our studies based on its high enrichment in normal NSC as well as its function as an adhesion receptor. The main objectives of this project are listed below: - Characterization of GPR56 expression in the adult mouse brain and during ESC differentiation into the neural lineage. - Evaluation of GPR56 expression in the different GBM subtypes and correlation with GBM subtype markers. - Functional characterization of the impact of GPR56 knockdown in GIC in vitro and in vivo. - Characterization of the role of GPR56 as an adhesion molecule in GBM. - Study of the transcriptional regulation and signaling mediators of the GPR56 pathway. - Clinical association studies between GPR56 and the GPR56-associated signature and GBM clinical features, including patient survival and MRI characteristics. - Evaluation of the expression of GPR56 and GPR56-associated signature in other tumor types beyond GBM.

Stem cells have the capacity for both self renewal, and to form all cell types in the body. Interestingly, so called neural stem cells (NSCs) are found in the adult human brain, which is of significance both out of a developmental perspective and from a clinical point of view. At the present moment, the regulation of neural stem cell (NSC) proliferation and fate determination is not completely understood.

The overall aim of this thesis was to study the mechanisms that regulate NSC proliferation and fate determination in vitro and in vivo. In particular, the roles of the female sex hormone estrogen and the testosterone analogue nandrolone, as well as the melanocortin α-melanocyte stimulating hormone (α-MSH), were analyzed in this context. Also, the breast cancer susceptibility gene one (BRCA-1), was studied in the brain with emphasis on regions containing NSCs.

Our findings show that estrogen and nandrolone have similar effects on NSCs; both decreased NSC proliferation and increased neurogenesis. Estrogen's ability to reduce proliferation was due to increased levels of p21, an inhibitor of cyclin dependent kinases. In contrast, no change in p21 was observed in the case of nandrolone, indicating differential regulation. Adult rats subjected to nandrolone injections had 30% reduced NSC proliferation in the dentate gyrus, indicating profound effects on NSCs in vivo.

The melanocortin α-MSH acted as a mitogen by increasing levels of cyclinD1 and retinoblastoma protein; as a result NSC proliferation was doubled.

Finally, BRCA-1 is expressed while NSCs proliferate, but is drastically down regulated upon differentiation, indicating that BRCA-1 could be used as a possible NSC marker.

In summary, in this thesis estrogen and nandrolone were identified as NSC regulators which decrease proliferation and positively influence neurogenesis. Also, we have identified the hormone α-MSH as a NSC mitogen, and BRCA-1 as a possible NSC marker.

In order to coordinate brain development with the growth of the organism, neurogenesis is highly dependent on the organism’s nutritional intake. The transition between neural stem cell (NSC) quiescence and reactivation is a key point of regulation during neurogenesis, and in Drosophila, this process is tightly coupled to nutrient availability. Post-embryonic NSCs in Drosophila only exit quiescence when larvae are fed a diet containing essential amino acids. The fat body, a functional homologue of the liver and adipose tissue, acts as a systemic nutrient sensor. Existing evidence from both in vivo and in vitro studies suggest that upon sensing dietary amino acids, the fat body secretes unknown factor(s) that induce glial secretion of insulin/IGF-peptides, which are sensed by underlying NSCs to trigger their reactivation. Despite recent work on nutritional regulation of NSC reactivation, key questions still remain: what are the signal(s) from the fat body, and how do they interact with the glial cells to induce glial production of DIlps? In addition, a number of glial subtypes are closely associated with the NSCs, but each subtype’s individual contribution to NSC reactivation remains elusive. In order to search for the unknown fat body factor(s) and investigate how they interact with the glial cells, I compared the transcriptome of the fat body under fed and starvation conditions during the time window of NSC reactivation. I identified an extracellular matrix protein (ECM), Collagen IV, as a secreted fat body signal whose deposition on the CNS is required for NSC reactivation. Collagen IV recruits glial-derived Perlecan, an- other ECM protein and a known requirement for NSC reactivation, to the vicinity of NSCs. Both ECM proteins are indispensable for the induction of glial insulin/IGF signalling. NSCs are separated from the hemolymph by a blood brain barrier (BBB). In collaboration with Pauline Speder and Jessie Van Buggenum (Andrea Brand lab), we confirmed crucial roles of the two BBB glial populations as a nutrient-sensitive NSC niche and identified each subpopulation’s contribution to NSC reactivation. Transcriptional profiling revealed that both BBB glial populations transcribe Dilp6 and perineurial glia is the source of Perlecan. Together, Collagen IV and Perlecan trigger NSC reaction via binding to their integrin receptors and subsequent induction of insulin signalling from the BBB glia.

Transplantation of genetically engineered neural stem cells (NSCs) into sites of central nervous system (CNS) disease/injury is a promising strategy to promote repair of damaged tissue. However, translating this strategy into the clinic requires several challenges to be overcome including facilitating ‘combinatorial therapy’ (achieving multiple therapeutic goals – essential in CNS injury/disease). Nanotechnologies are emerging as multifunctional platforms capable of meeting this requirement. For example, magnetic particles (MPs) and implantable hydrogels offer several biomedical advantages for transplant populations, including: safe genetic manipulation; non-invasive cell tracking, via MRI; and safe and efficient accumulation of cells at sites of injury. However, the use of these nanotechnologies remains to be explored in detail for NSC transplantation therapies. In this thesis, it is shown that MPs can mediate gene delivery to NSCs grown as neurospheres and monolayers with the most efficient transfection efficiencies achieved using oscillating magnetofection protocols (9.4% and 32.2% respectively). In both culture systems, developed protocols had no effect on key regenerative properties of NSCs such as cell viability, proliferation, stemness and differentiation. Further, ‘magnetofected’ monolayer NSCs were shown to have survived and differentiated in a cerebellum slice model acting as host tissue, indicating safety of the procedures. It was also shown that assessing procedural safety and extent of transfection of magnetofection protocols may be feasible by employing mass spectrometry and proteomics analysis. It was also found that tailored enhancement of particle magnetite content offers a means to efficiently label NSCs, up to a maximum of 95.8%. Labelling procedures had no effect on cell viability, proliferation, stemness or differentiation. In addition, labelled cells could survive and differentiate in a slice model of spinal cord injury indicating safety of the labelling procedures. Functional labelling was also demonstrated by magnetic capture of labelled cells in an in vitro flow system. Hydrogels offer major advantages for delivery of transplant populations into injury sites. Here it was shown that an intraconstruct genetic engineering approach was feasible for NSCs cultured with a clinically translatable, collagen hydrogel system. Magnetofection protocols safely increased MP mediated transfection of NSCs grown in ‘2-D’ and ‘3-D’ hydrogel cultures.

NG2-glia, identified by their expression of the NG2 chondroitin sulphate proteoglycan (CSPG), represent a fifth glial cell population in the central nervous system (CNS). NG2-glia were originally considered to be oligodendrocyte progenitor cells (OPCs), but they have properties and distribution in the adult that is distinct from OPCs. NG2-glia have been shown to form contacts with axons at nodes of Ranvier in white matter and to respond to synaptically released glutamate in grey matter. Furthermore, NG2-glia are known to give rise to oligodendrocytes after demyelination in vivo and to form neurons and astrocytes in vitro. However, the function of NG2-glia remains largely undefined. Therefore, the aim of this thesis was to define the interrelations of NG2-glia with other elements in the CNS and test the hypothesis that they are multipotent neural stem cells (NSCs).

The research reported in this thesis focused on the potential of neural precursor cells to provide a suitable source of neurones which can be used in cell replacement strategies for Huntington's disease. Specifically, the parameters affecting the differentiation of these cells into neuronal phenotypes were addressed and increasing the survival of proliferating and differentiating neurones was attempted. In vivo characteristics and the fibre projections of primary and 10 day expanded ENPs was also assessed. The limitations of xenografts in this thesis led to the search for an alternative model system for such experiments. Chapter Three involved an extensive study investigating the effects of a range of concentrations of FGF-2 and EGF on the proliferation and more importantly the neuronal differentiation of murine ENPs over 6 passages in culture, and it was found that the concentration had an effect on the neuronal proportion as well as the neuronal yield of these cultures. Chapter 4 examined the turnover of neuronal precursors in the ENP population cultured in the presence of FGF2 and EGF, using BrdU. The ongoing proliferation of neuronal precursors within ENP cultures was observed and the addition of the growth factors: CNTF, BDNF, HGF and NGF, to enhance the survival of these neurons on differentiation had no effect. Chapter 5 examined the potential of 10 day expanded human striatal ENPs to maintain a striatal like phenotype both in vitro and in vivo in comparison to primary foetal tissue. In vitro after 10 days expansion ENPs differentiated into DARPP-32 positive neurons and this characteristic was maintained in vivo, in a lesion model of HD, albeit to a much lesser extent. This study was limited by the need for ongoing immunosuppression which reduced the life span of the host animal. Chapter 6 investigates further the potential of ENPs. The ability for these cells to send long projections in the host brain and therefore repairing the circuitry lost or damaged as a result of the disease. A four way analysis was carried out examining both alio- and xenograft environments with both primary and 10 day expanded ENPs. Mouse grafts were used to address the allograft environment given that such an experiment is not possible with human tissue and both human and mouse tissue addressed the xenograft environment. To overcome the issues associated with labelling the grafted tissue in the host brain, several techniques were employed, including the use of the GFP transgenic mouse, lentiviral labelling of the cells with the LacZ gene and iontophoretic labelling of the graft with anterograde tracers. ENP grafts were shown to send out longer projections than that of primary tissue although this may be due to migration of the grafted cells. Chapter 7 addresses the issue of immunosuppression of xenografted animals. An alternative model system was explored with the hypothesis being that it would be possible to tolerise the animal in the neonatal period to the xenograft tissue that would subsequently be used for intrastriatal grafting in the adult. Indeed, tolerising the animal resulted in healthy surviving grafts in the adult without the need for daily immunosuppression. Work presented in this thesis contributes some understanding to the biology of neural stem cells and neural xenografts that may ultimately be used for neural transplantation therapies in HD.

Le développement du système nerveux s'appuie sur un réseau complexe de régulation qui contrôle l'acquisition des destins neuronaux et gliaux. Le facteur de transcription (TF) Glial cells missing (Gcm) est connu pour être le facteur de la détermination gliale dans le système nerveux , embryonnaire de la drosophile. Lorsqu'on surexprime Gcm dans les tissus neuronaux, les cellules souches produisant normalement des neurones produisent des cellules gliales. Cela montre qu'elles changent du destin entrainant une reprogrammation épigénomique qui exige la répression des gènes neuronaux et l'activation des gènes gliogeniques. Nous avons montré que le changement de destin des lignées neuronales par Gcm nécessite des changements globaux au niveau de la chromatine et que le histone acétyl-transférase dCBP joue un rôle important dans la plasticité cellulaire. La quantité de dCBP est globalement diminuée dans · les cellules gliales ectopiques pour qu'elles s'adaptent à un nouveau statut d'acétylation. De plus, on a montré que dans les cellules gliales, dCBP agit avec Gcm pour activer les gènes gliogeniques · d'une manière différente par rapport à son action globale. Il est fondamental de définir plus en détail les mécanismes moléculaires de cet événement pour pouvoir comprendre l'interconversion des cellules et aussi pour améliorer notre capacité de les reprogrammer
Development of the nervous system relies on a complex network where acquisition of , neuronal and glial fates are tightly regulated. The transcription factor (TF) Glial cells missing (Gcm) is known to be the glial determinant Drosophila embryonic nervous system. When Gcm is overexpressed in the nervous system, neural stem cells that produce normally neurons start to produce glia. This causes a cell fate change that leads to a whole epigenomic reprogramming that requires repression of neuronal genes and activation of glial genes. We have shown that the change of fate of a whole neuronallineage by Gcm requires global changes at the chromatin level and that histone acetyl-transferase dCBP plays an , important role in cellular plasticity. DCBP levels are decreased globally in ectopic glia, for them · to adapt a new acetylation status. Furthermore, we have shown that dCBP in endogenous glia may act together with Gcm to activate late glial genes, exhibiting a different mode of action compared to its role during reprogramming. Defining further the molecular mechanisms of this event is fundamental for understanding the developmental biology of inter-converting cells and also for improving our ability to reprogram cells

The neural crest is a developmental tissue that arises in the vertebrate ectoderm and is unique for its capacity to differentiate into a wide range of cell types and ability to migrate throughout the developing embryo. Due to the early stage at which it arises during embryonic development and the ethical issues with experimenting on human embryos little is directly known about human neural crest and instead much of what we understand has been gleaned from animal models of vertebrate development. Human pluripotent stem (hPS) cells offer us a tool that we can use to support the knowledge derived from these models by applying it to human cells, but to date the differentiation of hPS cells into neural crest has only been achieved in ill-defined conditions that compromise the efficacy of the model. The work presented here describes the development of a fully defined system for generating neural crest from hPS cells that can be manipulated in a biologically relevant way to produce other ectodermal phenotypes. As such it represents the first fully defined system for pan- ectodermal differentiation of hPS cells that has been described. The response of the cells undergoing this process to known ectodermal morphogens, particularly BMP, is assessed in depth and the neural crest cells are subjected to rigorous transcriptional and functional analysis. In the future this system could be used to investigate multiple aspects of neural crest and ectodermal biology in the human in a way that has not yet been possible and offers support for the knowledge that has already been collected with classic developmental biology approaches.

This thesis investigated how human neural stem cells are regulated, focusing specifically on heparan sulfate proteoglycans, the key proteins of the extracellular space. The findings of this study identified central roles for proteoglycans in mediating neural stem cell events, including self-renewal and differentiation. This research has improved our understanding of human stem cell and human neurogenesis biology and provided novel approaches for the development of improved neural stem cell applications, including using these cells for brain damage therapy.

Multiple sclerosis (MS) is a leading cause of neurological disability in young adults. Although current treatments can reduce symptomology and relapse rate, they are unable to prevent the chronic neurodegeneration that occurs at later stages. MS pathology is mediated by complex interactions between invading immune cells, neurons, glia, and endogenous stores of neural progenitor cells (NPCs). Factors critical to NPC/immune cell communication as well as the survival, differentiation, and proliferation of NPCs are not well defined. Elucidation of these factors will allow for the advancement of NPC transplantation therapies as well as the identification of novel pharmacological targets. Fas – a member of the tumor necrosis superfamily of death receptors – has diverse, cell-specific functions and is a major modulator of autoregulation within the immune system. Although Fas is expressed by NPCs, its exact role in this cell type was previously unknown. To contribute to this body of knowledge, the experiments in this dissertation examined the role of the Fas receptor (Fas) and Fas ligand (FasL) in NPC survival, differentiation, and T-cell cross-talk in vitro and in vivo in experimental autoimmune encephalomyelitis (EAE; a well-established animal model of MS). Activation of Fas via FasL increased NPC survival by decreasing apoptosis (as opposed to increasing proliferation) in vitro. This decreased apoptosis correlates with upregulation of the inhibitor of apoptosis protein (IAP) Birc3. Further investigation into the importance of Fas in NPCs was accomplished by comparing wild-type and Fas-deficient (lpr) NPCs. Lpr NPCs exhibited decreased apoptosis, decreased proliferation, and increased differentiation to oligoprogenitor and neuronal lineages. These studies suggest the Fas system plays multifaceted roles in NPCs and that its exact functions are dependent on both functional Fas expression and presence or absence of FasL. To determine the role of Fas/FasL in neuroimmune cross-talk, co-cultures of wild-type or lpr NPCs with different T-cell subtypes (Th1, Th2, and Th17 cells) were performed. Th1 cells were the only subtype capable of inducing NPC apoptosis. Th1-mediated death was dose-dependent and was not mediated via Fas. On the other hand, NPCs were able to induce significant apoptosis in pro-inflammatory Th1 and Th17 cells without affecting anti-inflammatory Th2 cells. NPC-induced Th17 cell death was mediated via Fas. These data suggest NPCs can specifically target pro-inflammatory T-cells and can promote neuroprotection by inducing death of these proencephalogenic cells. Finally, intravenous injection of wild-type or lpr NPCs into EAE mice reduced clinical symptoms and CNS immune infiltrate to the same extent. Few NPCs enter the CNS, where they remain undifferentiated. This suggests the main mechanism through which NPCs produce beneficial results in EAE is via peripheral immunoregulation, which is not dependent on Fas expression. Overall, this dissertation elucidates the Fas system as an important modulator of NPC cell-fate and immunoregulatory capacity.

La thérapie cellulaire se positionne comme une stratégie prometteuse pour inciter le cœur infarci à se régénérer. A cet effet, des études récentes placent des espoirs considérables dans l’utilisation des cellules souches embryonnaires et notre laboratoire a déjà démontré comment les différencier en progéniteurs cardiovasculaires, un type de précurseurs cellulaires qui ne peut aboutir qu’à la formation de cardiomyocytes, de cellules endothéliales ou de cellules de muscles lisses. Cet engagement précoce réduit leur capacité de prolifération anarchique et en même temps leur permet de rester suffisamment plastiques pour éventuellement s’intégrer plus facilement avec le tissue hôte. Cependant, les études précliniques et cliniques d’injection de ces cellules s’avérèrent décevantes. Malgré de légères améliorations de la fonction cardiaque, on observa une trop faible survie cellulaire ainsi qu’un taux de rétention des cellules dans le myocarde remarquablement bas. Afin d’étudier ce problème, mes travaux de thèse ont porté non seulement sur la conception de nouveaux biomatériaux pouvant servir de moyen de transport et d’intégration des cellules dans la zone infarcie, mais aussi sur la conception de biomatériaux permettant de contrôler précisément l’environnement cellulaire au cours du processus de différenciation de cellules souches pluripotentes humaines en cardiomyocytes. Grâce aux importantes interactions entre nos laboratoires de recherche fondamentale et de recherche clinique, nous avons tout d’abord développé de nouvelles techniques de fabrication et de caractérisation de patches de fibrine cellularisés qui sont récemment entrés dans un essai clinique de phase I. A partir de cette formulation clinique approuvée par les autorités de régulation, nous avons élaboré toute une gamme de matériaux composites uniquement à base de matières premières pertinentes dans ce cadre clinique, dans le but d’améliorer la maturation des progéniteurs cardiovasculaires une fois greffés sur le cœur défaillant. Dans cette optique, nous avons également développé un modèle in vitro permettant d’étudier précisément l’influence combinée de la rigidité du substrat et du confinement spatial sur la différenciation des cellules souches en cardiomyocytes. Grâce à des techniques de microfabrication sur substrat mou, il a été possible de positionner précisément les cellules souches pluripotentes dans des espaces restreints d’élasticité variable. Ainsi, nous avons pu observer que même en utilisant des protocoles chimiques éprouvés basés sur la modulation de cascades de signalisation impliquées dans le développement cardiaque, une très forte hétérogénéité pouvait apparaître en fonction de l’environnement physique des cellules. Nous avons ainsi pu extraire les caractéristiques principales permettant une différenciation cardiaque efficace, reproductible et standardisée et les avons appliquées à la fabrication d’une nouvelle génération de patches composés de matériaux cliniques et de couches multiples de bandes synchrones de cardiomyocytes. De fait, ces travaux ouvrent de nouvelles voies dans l’utilisation de biomatériaux pour la production industrielle de cardiomyocytes et pour la fabrication de patches cliniques, cellularisés ou non, dans le traitement de l’insuffisance cardiaque
Cell therapy is a promising strategy to help regenerate the damaged heart. Recent studies have placed a lot of hopes in embryonic stem cells and our lab had previously found a way to differentiate them into cardiac progenitors, cells that can only differentiate into cardiomyocyte, endothelial cells or smooth muscle cells. This early commitment decreases their proliferative capabilities, yet maintains their plasticity for better integration inside the host tissue. However, clinical and pre-clinical injection studies did not really meet the expectations. Even though slight improvements in cardiac function were demonstrated, very low cell viability has been observed, as well as a very low retention of the cells inside the myocardium. To address this problem, my PhD projects not only focus on the design of new biomaterials to act as a vehicle for cell delivery and retention in the infarcted area, but also on the design of biomaterials that control the cellular environment during the differentiation of pluripotent stem cells into cardiomyocytes. Going back and forth between the labs and the clinics, we first developed new techniques for the fabrication and the characterization of a cell-laden fibrin patch that is now undergoing phase I clinical trial. From the approved clinical formulation, we then propose new blends of clinical materials that will eventually improve the maturation of the cardiac progenitors once grafted onto the failing heart. In this perspective, we developed an in vitro model to investigate the combined influence of matrix elasticity and topographical confinement on stem cell differentiation into cardiomyocytes. By using microfabrication techniques to pattern pluripotent stem cells on substrates of controlled stiffness, we demonstrate that even using a widely recognized chemical-based protocol to modulate signaling cascades during differentiation, much heterogeneity emerges depending on the cellular physical environment. We thus extracted the main features that led to controlled and reproducible cardiac differentiation and applied it to the fabrication of next generation of multi-layered anisotropic cardiac patches in compliances with clinical requirements. This work opens new routes to high-scale production of cardiomyocytes and the fabrication of cell-laden or cell-free clinical patches

Transplantation of neural progenitor cells (NPC) constitutes a putative therapeutic maneuver for use in treatment of neurodegenerative diseases. At present, effects of NPC transplantation in the Alzheimer’s disease (AD) brain are largely unknown and a primary objective of this work is to demonstrate possible efficacy of NPC administration in an AD animal model. The benefits of transplantation could involve a spectrum of effects including replacement of endogenous neurons, conferring neuroprotection with enhancement of neurotrophic factors, and diminishing levels of neurotoxic agents. Additionally, since chronic inflammation is a characteristic property of the AD brain, I considered NPC transplantation could have a particular utility in inhibiting ongoing inflammatory reactivity. Accordingly, intra-hippocampal transplantation of NPC has been examined for efficacy in attenuating inflammatory responses and conferring neuroprotection in the hippocampus. These findings indicate efficacy for NPC transplantation with effects consistent with cellular actions to attenuate inflammatory reactivity. Synaptic plasticity, such as long-term potentiation (LTP), is thought to play a critical role in modification of neuronal circuitry in learning and memory, but the role in neurogenesis is not well known. A critical aspect of my study was to examine potential roles of N-methyl-D-aspartate receptor (NMDAR)-dependent LTP in promoting neurogenesis by facilitating proliferation/survival and neuronal differentiation of endogenous NPCs in the dentate gyrus (DG) and exogenously transplanted neural stem cells (NSCs) in the CA1. I found that LTP induction significantly facilitates proliferation/survival and neuronal differentiation of endogenous NPCs and exogenously transplanted NSCs in the hippocampus. These effects were eliminated by a NMDAR competitive antagonist, CPP. Accordingly, chemical LTP stimulation reproduced enhanced proliferation/survival and neuronal differentiation of NSCs when co-cultured with hippocampal neurons. These effects were eliminated by a NMDAR competitive antagonist, D-APV and inhibited by the tyrosine kinase inhibitor, K252a. ELISA and biotinylation results revealed that NMDAR-mediated LTP facilitates the release of a neurotrophic factor, BDNF. The conditioned media from cLTP-induced hippocampal neurons were sufficient to activate the BDNF receptor, TrkB. Overall, my results suggest that NMDAR-dependent LTP plays a critical role in neurogenesis and may contribute to the utility of NSC transplantation as an effective cell therapy for a variety of neurodegenerative diseases.