Neural stem cells (NSCs) have been recently identified in the inferior colliculus (IC). These cells are of particular interest, as no casual therapeutic options for impaired neural structures exist. This research project aims to evaluate the neurogenic potential in the rat IC from early postnatal days until adulthood. The IC of rats from postnatal day 6 up to 48 was examined by neurosphere assays and histological sections. In free-floating IC cell cultures, neurospheres formed from animals from early postnatal to adulthood. The amount of generated neurospheres decreased in older ages and increased with the number of cell line passages. Cells in the neurospheres and the histological sections stained positively with NSC markers (Doublecortin, Sox-2, Musashi-1, Nestin, and Atoh1). Dissociated single cells from the neurospheres differentiated and were stained positively for the neural lineage markers β-III-tubulin, glial fibrillary acidic protein, and myelin basic protein. In addition, NSC markers (Doublecortin, Sox-2, CDK5R1, and Ascl-1) were investigated by qRT-PCR. In conclusion, a neurogenic potential in the rat IC was detected and evaluated from early postnatal days until adulthood. The identification of NSCs in the rat IC and their age-specific characteristics contribute to a better understanding of the development and the plasticity of the auditory pathway and might be activated for therapeutic use. / Neuronale Stammzellen wurden kürzlich im unteren Colliculus inferior (CI) identifiziert. Diese Zellen sind von besonderem Interesse, da es keine therapeutischen Optionen für geschädigte neuronale Strukturen gibt. Ziel dieses Forschungsprojekts ist es, das neurogene Potenzial im CI der Ratte von den ersten postnatalen Tagen bis zum Erwachsenenalter zu untersuchen. Der CI von Ratten vom 6. bis zum 48. postnatalen Tag wurde mit Neurosphären-Assays und histologischen Schnitten untersucht. In frei schwimmenden CI-Zellkulturen bildeten sich Neurosphären bei Tieren vom frühen postnatalen Alter bis zum Erwachsenenalter. Die Menge der gebildeten Neurosphären nahm im höheren Alter ab und stieg mit der Anzahl der Zelllinienpassagen. Die Zellen in den Neurosphären und die histologischen Schnitte zeigten eine positive Färbung mit neuronalen Stammzell-Markern (Doublecortin, Sox-2, Musashi-1, Nestin und Atoh1). Dissoziierte Einzelzellen aus den Neurosphären differenzierten und wurden positiv für die neuralen Abstammungsmarker β-III-Tubulin, GFAP und MBP angefärbt. Darüber hinaus wurden neuronalen Stammzell-Marker (Doublecortin, Sox-2, CDK5R1 und Ascl-1) mittels qRT-PCR untersucht. Zusammenfassend lässt sich sagen, dass ein neurogenes Potenzial im CI der Ratte von den frühen postnatalen Tagen bis zum Erwachsenenalter nachgewiesen und bewertet wurde. Die Identifizierung von neuronalen Stammzellen im CI der Ratte und ihre altersspezifischen Merkmale tragen zu einem besseren Verständnis der Entwicklung und der Plastizität der Hörbahn bei und könnten für eine therapeutische Nutzung aktiviert werden.

This study focuses on ETV7, a transcriptional repressor, known to be up-regulated in breast cancer (BC). Firstly, we demonstrated a new role of ETV7 in promoting breast cancer stem-like cell (BCSC) plasticity and resistance to chemotherapy in BC cells. We observed that ETV7 repressed a large panel of interferon response genes and increased BCSC cell plasticity, leading to resistance to 5-fluorouracil. Then, we investigated the role of ETV7 in inflammatory and immune responses in BC cells. We identified TNFRSF1A, encoding for TNFR1, as one of the genes repressed by ETV7. We demonstrated that ETV7 directly bound to the intron I of this gene, and we showed that the ETV7-mediated down-regulation of TNFRSF1A reduced the activation of NF-κB signaling. These results suggest that ETV7 can reduce the inflammatory responses in BC cells by repressing the TNFR1/NF-κB axis. Moreover, we analyzed the role of ETV7 in the regulation of antigen presentation and confirmed that ETV7 downregulated genes involved in the antigen-presenting pathway, potentially leading to cancer immune evasion We also analyzed if the silencing of ETV7 affected the viability of cancer cells and observed that knock-down of ETV7 can induce p53-mediated apoptosis in cancer cells. Lastly, we analyzed the pro-tumorigenic potential of ETV7 using in vivo model and we observed that mammary gland tumor cells overexpressing ETV7 formed bigger tumors with higher proliferation potential. Taken collectively, the data acquired during this project confirm the role of ETV7 as an important regulator of BC aggressiveness both in vitro and in vivo and propose ETV7 as a novel player in BC immunity, opening a new research direction and giving useful insights for more effective therapeutic strategies.

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The study of cardiac regeneration can have many forms in which it is defined. It can not only be the ability to add new myocardium to dead or dying tissues, but also include the prevention of cardiac tissue degeneration, reversal of tissue remodeling, and the maintenance of systolic and diastolic function in the incidence of tissue damage, which can lead to subsequent heart failure progression. The use of stem cells for cardiac regeneration represents a growing field of new therapies for patients with end stage cardiac disease. Various studies have noted promising results in the recovery and reparation of these tissues. Cumulatively, their goals have become the identification of the most suitable cell type, as well as how to maximize functional efficiency and cost effectiveness for practical application. Many protocols simply do not ensure adequate cell engraftment, viability, and ultimately the return of normal tissue function. Investigators seek to determine how these processes can be enhanced or manipulated to promote cardiac regeneration in hopes of eventually making their clinical use a standard practice. / Physiology

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Out of the four molecular subgroups (WNT, SHH, Group 3 and Group 4), Group 3 patients face the highest incidence of leptomeningeal spread and overall patient survival of less than 50%. Current clinical trials for recurrent MB patients based on genomic profiles of primary, treatment-naïve tumors, provide limited clinical benefit since recurrent metastatic MBs are highly genetically divergent from their primary tumors. The paucity of patient matched primary and recurrent MB samples has contributed to the lack of molecular targets specific to medulloblastoma recurrence, limiting relapsing MB patients to palliation. Our previous in silico analyses revealed enriched expression of many stem cell self-renewal regulatory genes in Group 3 MB. In this work, I have set out to investigate whether by identifying genes contributing to self-renewal of Group 3 MB cells, we can characterize a population of cells responsible for therapy evasion and subsequent tumor relapse. Initially, we have adapted the existing COG (Children’s Oncology Group) protocol for children with newly diagnosed high-risk MB for treatment of immuno-deficient mice intracranially xenografted with human MB cells. Cell populations recovered separately from brains and spines mice during the course of tumor development and therapy were comprehensively profiled for gene expression analysis, stem cell and molecular features to generate a global, comparative profile of MB cells through therapy. Additionally, we have investigated therapeutic potential of small molecules targeting BMI1, a known self-renewal regulating gene. In the setting of recurrent Group 3 MB, pharmacological inhibition of BMI1, led to a remarkable decrease in cell proliferation and self-renewal in vitro as well as reduction of local and spinal metastatic disease in vivo. Finally, by combining the established therapy-adapted patient-derived xenograft mouse model and BMI1 inhibitor, PTC-596, we have demonstrated an additive effect of two modalities and provided the pre-clinical data for the upcoming Phase I trial. Biological investigations into the drivers of MB recurrence will lead to development of new therapeutic options for children who are frequently limited to palliation. Clinically relevant mouse models of MB recurrence can serve as platforms for pre-clinical testing and validation of new treatments aimed to provide therapeutic intervention rather than palliation. / Thesis / Doctor of Philosophy (PhD) / Medulloblastoma is the most common type of brain cancer that affects children. Out of the four main subgroups of medulloblastoma, tumors in Groups 3 and 4 are the most aggressive and are associated with a low overall survival in children diagnosed with this type of brain cancer. These two subtypes of medulloblastoma also account for the largest number of patients in which gold standard therapies fail and no additional therapies are available. Several studies have shown the existence of few cells within the tumor that alone can drive tumor growth. The aggressive behavior of these cells has in part been attributed to dysregulation of genes involved in cell replication and division. Further studies that will focus on understanding the significance of genes that regulate cell growth and replication can help discover a population of cells that is capable of evading therapy and contribute to tumor relapse. The identification and characterization of such population can lead to development of novel treatments for the children affected with aggressive medulloblastoma. In my thesis, I have developed a mouse model that replicates the aggressive therapy given to the medulloblastoma patients in order to study cells capable of escaping the harsh treatment and drive tumor comeback. Next, by profiling the gene expression and functional attributes of those cells, we identified genes that contribute to regulation of cell division and growth. The effects of both increasing and decreasing the activity of those genes were then tested in cells grown in the dish. Subsequently, the most promising results were verified in the established mouse models. The main objective of my thesis was to discover new opportunities in treatments the most aggressive type of brain cancer affecting children, and thus not only improve the quality of treatment but also the overall survival of patients with medulloblastoma.

Objectives: The effects of estrogens on immune system formation and function are well documented. Diethylstilbestrol (DES), a synthetic estrogen, has been linked to neoplasia and immune cell dysfunction in humans and animals exposed in-utero. In-vitro effects of DES exposure of murine embryonic stem (ES) cells on the early embryonic immune system development and the expression of cellular surface markers associated with common hemangioblastic and hematopoietic precursors of the endothelial, lymphoid & myeloid lineages were investigated. Hypothesis: Early ES cell expression of CD45 a marker common to lymphoid lineage hematopoietic stem cells and differentiation of lymphoid lineage precursors are affected by in-vitro exposure to DES. Methods: Murine ES cells were cultured using a variety of techniques: an OP9 co-culture system, and formation of embryoid bodies (EBs) in a liquid medium and hanging drop system. The OP9 co-culture system did not appear to give rise to well differentiated lymphoid lineage cells during 12 days of differentiation. The hanging drop EB culture system, previously shown to promote differentiation of endothelial and lymphoid precursor cells, was chosen for further studies of ES cell differentiation. ES cells were harvested at five time points: undifferentiated (day 0), and differentiated (days 3, 8, 12 and 16). Differentiating ES cells were treated with DES beginning on day 3. The synthetic estrogen, DES, was chosen as a treatment because of its similar potency to 17β estradiol and documented association with neoplasia in women exposed in-utero. Surface marker expression, measured by real-time RT-PCR amplification, was recorded using fluorogenic TaqMan(R) probes designed specifically for the surface proteins of interest: oct4, c-Kit, Flk1, ERα, ERβ, CD45, Flt1, & VE-cadherin. Analysis & Results: Changes in surface marker gene expression between day 0 and day 16 of differentiation were analyzed using the RT-PCR threshold counts (CT) and the comparative threshhold cycle method. The expression of each target mRNA was normalized internally to a housekeeping gene (18s rRNA) and calculated relative to day 0. ANOVA (Type 3 fixed-effects analysis, SAS) was performed using the unexponentiated ΔΔCT values. The effects of DES, time, and the interaction between DES and time were evaluated for days 8, 12 and 16. Additionally, the effects of DES on the expression of each marker were evaluated for day 16. Expression of estrogen receptor receptor α & β (ERα & β) in the EBs was established, and did not appear to be affected at any time by treatment with DES. ERα was expressed in significant levels on day 16, while ERβ was expressed in low levels throughout the period of differentiation. The expression of the cell surface marker, c-Kit was significantly (P<0.0001) altered by the presence of DES between the three time points sampled. The expression of the VEGF receptor, Flt1, and the adhesion molecule, VE-cadherin, markers of endothelial cells, were also significantly (P<0.026) altered by treatment with DES on day 16 of differentiation. Treatment with DES appeared to have no effect on the expression of CD45, a marker common to lymphoid precursor cells. Conclusions: These results indicate the presence of estrogen receptors in differentiating ES cells as early as day three in-vitro (ERβ) until day 16 (ERα). DES alters expression of common hemangioblastic and hematopoietic precursor, as well as endothelial lineage markers, but has no effect on expression of the marker of lymphoid lineage development before day 16. These effects coincided with the expression of ERα. The enduring effects of DES exposure in-utero may not be manifest in this ES model, or may occur at later stages of differentiation or in selected subpopulations of CD45+ cells. / Master of Science

Healthy stem cells are rare, long-lived cells with unlimited self-renewal potential, enabling organ regeneration. Similarly, cancer stem cells (CSCs) contribute to cancer initiation, post-chemotherapy regeneration and development of drug resistance through their stemness properties of self-renewal highlighting them as a key therapeutic target in relapse prevention. Relapse and drug resistance represent the two biggest clinical hurdles to improving patient outcomes. Since CSCs are rare and governed by different cellular pathways than the bulk of the tumour they require unique ways of targeting. However, CSCs are difficult to isolate and study. CSCs, like all stem cells, are only defined by functional (aka phenotypic) assays that measure self-renewal and differentiation. My research aims to utilize phenotypic measures of stemness to address these clinical hurdles in therapy. First, I conduct a meta-analysis to highlight the importance of phenotypic assays in drug discovery compared to targeted base approaches. This meta-analysis reveals that drugs discovered using phenotypic assays were more likely to be approved for clinical use and less likely to fail due to lack of efficacy. Based on these results I proceed in two directions: using phenotypic measures of stemness for new drug discovery and using it for personalized medicine to improve already clinically approved therapies. First, using a phenotypic chemical genomics approach I uncover a novel vulnerability of CSCs through the targeting of SARNP and the transcription and export (TREX) pathway. This work uncovers a novel role for SARNP in R-loop homeostasis and importantly identified TREX as a targetable pathway for cancer stem cell drug discovery. Secondly, I develop a novel personalized medicine platform that integrates functional measurements of stemness, quantitative drug profiling and genetics which clinicians can use in real-time to improve therapy choices. Importantly, this platform can predict patient response to chemotherapy with 90% accuracy and identify clonal populations that are drug-resistant and relapse-driving leading to refractory disease. Overall, my work highlights the importance of phenotypic approaches to stem cell biology for personalized medicine and cancer drug discovery. / Dissertation / Doctor of Philosophy (PhD) / Healthy stem cells are rare cells that can divide indefinitely and turn into other types of cells through a process called differentiation. This process is crucial for the regeneration of our tissues throughout our lives. However, when they acquire mutations healthy stem cells turn into cancer stem cells which are responsible for the initiation and regeneration of cancer leading to relapse. It is believed that release can be prevented if cancer stem cells are irradicated. Cancer stem cells are challenging to target because they are rare, distinct from the bulk of the tumor, and share many characteristics with healthy stem cells. Effective therapies must specifically eliminate CSCs without harming healthy stem cells, which are vital for tissue regeneration. Unfortunately, the development of such therapies is hindered by our limited understanding of the key differences between CSCs and healthy stem cells. To address this, my thesis uses advanced models of stem cells to identify critical distinctions between cancerous and healthy stem cells. By uncovering these differences, my goal is to develop drugs that can precisely target CSCs, improving cancer treatments while preserving healthy tissues.

Efficient in vitro generation of skeletal muscle tissue from cell lines is pivotal for the advancement of cultivated meat production. Here, we introduce a novel system employing a synthetic temperature-sensitive promoter (HSP70) carrying multiple heat shock elements (HSE), whose activity can be modulated by changes in temperature, enabling precise control of the expression of an exogenous master gene of myogenesis (MyoD) and a chromatin modifier (SMARCD3). To establish the optimal conditions, we screened different versions of temperature-inducible promoters (with 5, 15, or 45 HSE repetitions) in mammalian cell lines. Additionally, we explored the concurrent overexpression of exogenous heat shock factor 1 (HSF1), a multifaceted factor that enhances Hsp70 transcription, regulates the localization of binding partners, and modulates translation under stress. Our results demonstrate efficient induction of exogenous pro-myogenic factors with a 2-hour heat shock at 43°C. In the presence of exogenous HSF1, we observed a rapid initiation of the myogenic differentiation process and a significant acceleration in the expression of late myogenic markers (myogenin and sarcomeric myosin heavy chain), as well as the formation of skeletal muscle fibers in cells with intrinsic myogenic potential, such as L6 myoblasts. This approach allows for precise regulation of gene expression and cellular responses, leading to rapid and efficient production of mature muscle fibers without the need for expensive media formulations or genetic-based drug-inducible systems. This temperature-dependent cellular engineering method offers significant advantages, including accurate spatial and temporal control over cell myogenic maturation and scalability for large-scale production of differentiated skeletal muscle tissue for cultivated meat production.