The selective degeneration of motoneurons in the spinal cord, the brainstem and the brain cortex is the core pathology of amyotrophic lateral sclerosis (ALS), but evidences suggest that the neighbouring non-neuronal cells are also involved in the disease progression. Beside Riluzole, only drug approved to treat this fatal neurodegenerative disease, new pharmaceutical agents or novel strategies including stem cell therapy are currently under development and evaluated preclinically in front line on mutant SOD1 rodents mimicking all hallmarks of the human disease. Current intravenously delivered drugs tested in ALS therapy assume an intact blood-brain barrier and suppose the passage across the endothelium to hit their targets in the CNS parenchyma. If BBB impairment occurs in ALS, it may lead to revision of planned pharmaceutical treatment. In the first part of the work, we have validated the mutant SOD1 rat model of ALS and we characterized properties and integrity of its BBB. We observed a significant BBB disruption at symptomatic phase of ALS, evidenced by blood protein leakage, IgG accumulation and microhemorrhage. To look for the mechanism of BBB opening, we demonstrated that the expression of key genes involved in the BBB integrity was decreased. At the ultrastructure, the morphology of endothelial cells and vascular astrocyte end-feet was altered. Our results suggest that BBB disruption is a late event in ALS disease course and appears like a consequence of the local degenerative process or neuroinflammation rather than a cause. Since a lot of extracellular oedema and swollen astrocyte end-feet were found in mutant SOD1 rats, we also looked at the expression and localization of aquaporin-4, a key protein involved in CNS water movement. We found that its expression was highly increased in the symptomatic phase of ALS course and we hypothesize that this overexpression might be related to the resolution of oedema after BBB opening. In the second part of the work, we considered an original, easy, non-invasive and safe therapeutical approach of stem cell delivery in ALS rats. Since ALS affects the motoneurons throughout the CNS, we decided to use the bloodstream to deliver neural stem cells. We studied cell homing, survival, proliferation, integration and differentiation. Interestingly, the highest efficiency of cell delivery to the CNS was found in symptomatic ALS and the lowest in healthy animals. Neural stem cells injected into ALS animals preferentially colonized the motor cortex, hippocampus and spinal cord. We detected their successful differentiation into neural lineages by the appearance of MAP2-, GFAP-positive cells and the decrease of nestin expression. One of the realistic near-term clinical goals for ALS is the transplantation of stem cells that counteract the loss of motoneurons by secreting neuroprotective factors. Accordingly, we evaluated in vitro the expression of neurotrophic factors released by stem cells after stimulation with tissue extracts from ALS rats. The aim of this paradigm was to determine whether the ALS environment triggers neuroprotective factors release from stem cells. Mesenchymal stem cells and neural stem cells were able to express a wider range of growth factors than fibroblasts. According to the stem cell population stimulated, we obtained differential expression pattern, raising the choice of cell population for appropriate clinical applications in ALS.

Stem cells are cells that have a unique ability to divide for an indefinite period. Additionally, they can give rise to a plethora of specialized cell types. The advent of high-throughput technologies made it possible to investigate gene expression on a large scale. This enabled scientists to perform comprehensive gene profiling studies of stem cells. Several authors have suggested that there might be a common set of genes that control the stemness of stem cells. In this study, we suggest that ”stemness” genes that are related to ”stemness” characteristics show a statistically significant down-regulation between undifferentiated and differentiated cells. For this we have analyzed microarray data from five different cell lines and compared their global expression profiles. Common down-regulated transcripts among those data sets were de- rived by using a well-established gene expression analysis procedure called Significance Analysis of Microarrays. Since all three data sets were provided by Cellartis AB, the derived list of common transcripts was subsequently compared with an external study. Moreover, we also performed a comparison with down-regulated genes derived from mouse embryonic stem cells. This was done to determine if there is a common set of stemness genes even across distinct species. Re- sults were further evaluated using a comprehensive data-set from a study by Skottman et al. (2005). All results where compared uti- lizing using a range of false discovery rate threshold values and the results were subsequently used for gene ontology term enrichment. GO terms where utilized to functionally annotate and classify those embryonic stem cell transcripts, that were found to be common in all data-sets and identify over-represented biological processes related to those genes.

<p>Stem cells are cells that have a unique ability to divide for an indefinite period. Additionally, they can give rise to a plethora of specialized cell types. The advent of high-throughput technologies made it possible to investigate gene expression on a large scale. This enabled scientists to perform comprehensive gene profiling studies of stem cells. Several authors have suggested that there might be a common set of genes that control the stemness of stem cells. In this study, we suggest that ”stemness” genes that are related to ”stemness” characteristics show a statistically significant down-regulation between undifferentiated and differentiated cells. For this we have analyzed microarray data from five different cell lines and compared their global expression profiles. Common down-regulated transcripts among those data sets were de- rived by using a well-established gene expression analysis procedure called Significance Analysis of Microarrays. Since all three data sets were provided by Cellartis AB, the derived list of common transcripts was subsequently compared with an external study. Moreover, we also performed a comparison with down-regulated genes derived from mouse embryonic stem cells. This was done to determine if there is a common set of stemness genes even across distinct species. Re- sults were further evaluated using a comprehensive data-set from a study by Skottman et al. (2005). All results where compared uti- lizing using a range of false discovery rate threshold values and the results were subsequently used for gene ontology term enrichment. GO terms where utilized to functionally annotate and classify those embryonic stem cell transcripts, that were found to be common in all data-sets and identify over-represented biological processes related to those genes.</p>

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: Hematopoietic stem cells; Umbilical cord blood. Includes bibliographical references.

Stem cell therapy is a promising approach for tissue regeneration but there exists a problem of low engraftment rate to the injury site. Our laboratory has shown that hMSCs that were capable to penetrate through collagen barrier have higher migration capacity and engraftment efficiency than those remained inside the collagen matrix and those in traditional 2D culture. These cells capable of penetrating through collagen barrier might be hopeful candidate for improving engraftment efficacy. Major processes of engraftment, such as transmigration through basement membrane and invasion to the site of injury, involve cell-extracellular matrix-interacting proteins. As matrix metalloproteinases (MMP) and integrins are the key players in these processes, MMP and integrin profiles of the hMSCs were studied In this study, we demonstrated that hMSCs that were capable of penetrating through the collagen barrier have distinctive MMP profile to traditional 2D culture. These cells secrete significantly higher amount of MMP-1 than 2D culture, but the amount of MMP-2 secreted is comparable to traditional 2D culture. On the other hand, MMP-9 and MMP-13 were below detection limit by ELISA in both groups. Moreover, we have investigated the subcellular localization of MMPs and integrins. The cells were seeded on dishes with or without ECM coatings. It was demonstrated that hMSCs capable of penetrating through collagen barrier exhibit higher amount of subcellular MT1-MMP and integrin 6271 on ECM coated dish. Moreover, these cells exhibit a prominent feature of perinuclear localization of MT1-MMP., whereas the level of subcellular MMP-2, integrin 65 and 6v73 is comparable to that in 2D culture. We have also investigated the stem properties of hMSCs penetrated through collagen barrier. These properties include proliferation capacity, self-renewal capacity and differentiation potential towards chondrogenic, osteogenic and adipogenic lineages. It has been demonstrated that these properties are not compromised for superior migratory activities. / published_or_final_version / Mechanical Engineering / Master / Master of Philosophy

A Disintegrin-like And Metalloproteinase with Thrombospondin (TSP)-Type I sequence motifs 9 (ADAMTS9) is widely expressed in mouse and human fetal tissues. ADAMTS9 null mice cannot survive beyond E7.5 and its haploinsufficiency is associated with cardiac and aortic anomalies. This project hypothesized that ADAMTS-9 plays an important role during early embryogenesis. By using human embryonic stem cells (hESCs) as a model, the objectives of this study were to study the expression of ADAMTS9 and to determine the effects of ADAMTS9 perturbation on hESC differentiations. The expression of ADAMTS9 was compared between undifferentiated and differentiated hESCs. Its mRNA was maintained at similar levels in different passages of normal hESC lines. Interestingly, significantly lower expression was detected in karyotypically abnormal VAL4A when compared to the normal cells (H9 and VAL3). ADAMTS9 immunoreactivity was detected in cells located at the boundary of hESC colonies. The expression of ADAMTS9 was then studied during differentiation of hESC. ADAMTS9 mRNA and protein expression increased time-dependently during the first 24 days? of embryoid body (EB) formation. The expression pattern was similar to that of mesoderm and endoderm markers. Upon more specific lineage differentiation induced by retinoic acid and bone morphogenesis protein 4, ADAMTS9 mRNA expression was significantly increased. The positive correlation of ADAMTS9 with ESC differentiation was also found in mouse system, in which ADAMTS9 was increased time-dependently during mouse EB formation and down-regulated during reprogramming from somatic cells into induced pluripotent cells. Previous studies have shown that down-regulation of ADAMTS9 in several tumor tissues was attributed to ADAMTS9 hypomethylation. However, the present study demonstrated that the expression of ADAMTS9 was reduced in hESC after treatments with inhibitors of DNA methylation (5-aza-2?deoxycytidine) and histone deacetylase (VPA). The cellular localization of ADAMTS9 during hESC differentiation was further studied by co-localization of ADAMTS9 with several lineage specific markers. It was found that ADAMTS9 co-localized with mesoderm and endoderm markers. The functional role of ADAMTS9 in hESCs was then studied by transient ADAMTS9 knockdown. ADAMTS9 siRNA significantly decreased the expression level of mesoderm marker, REN. Thus, the role of ADAMTS9 during mesoderm differentiation was followed. ADAMTS9 was found to be dramatically increased after mesoderm differentiation of hESCs. In mesodermal cells, ADAMTS9 was co-expressed with vascular endothelial markers, VEGF and CD31, but not with pericyte markers, alpha muscle actin. Lentiviral vector encoding ADMATS9 shRNA was used for long term knockdown of ADAMTS9. ADAMTS9 down-regulation had no effect on the proliferation of hESCs. In agreement with the siRNA study, ADAMTS9 shRNA also significantly reduced the expression of REN. Upon mesoderm differentiation, ADAMTS9 knockdown resulted in a decreasing trend of mesoderm marker, CD34. In conclusion, the present study demonstrated a positive association of ADAMTS9 expression with hESC differentiation. ADAMTS9 was dramatically induced during mesoderm differentiation and its knockdown led to down-regulation of mesoderm markers. Together with the fact that ADAMTS9 expression was associated with endothelial cell markers suggested its possible role during endothelial cells formation. The roles of ADAMTS9 during hESC differentiation and early embryo development warrant further investigation. / published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

Background Mesenchymal stem cells (MSC) were first discovered in the 1970s by scientist A.J. Friedenstein and his colleagues. Friedenstein isolated the first mesenchymal stem cells and was credited for discovering its multilineage differentiation potential. To this day, an extensive amount of research has been conducted on the use of these cells in the treatment of degenerative diseases and various autoimmune disorders. Its migratory ability and immunosuppressive characteristics make MSCs advantageous in an inflammatory environment. Recently, MSCs were also found to have the ability to phagocytose apoptotic bodies generated from T-cells and B-cells. Objectives In this study, we sought to investigate the phagocytic capability of MSCs further in a cancer setting and observe whether or not MSCs could become immunostimulatory cells after phagocytosis of apoptotic cancer cells. Methods To conduct this study, we first used UV-irradiation to generate apoptotic cells from 3 neuroblastoma (NB) cell lines. After apoptotic NB cells were generated, they were then co-cultured with MSCs for phagocytosis to occur. To detect phagocytosis, we stained the apoptotic NB cells with a red fluorescent dye PKH-26 and MSCs with CFSE, a green fluorescent dye. Then, we used flow cytometry to detect the percentage of phagocytosis. After phagocytosis, we collected the supernatants from the MSCs treated with the apoptotic NB cells and observed how the IL-6 and IL-8 cytokine levels changed compared to the levels from the supernatant of the MSCs only. Results and Conclusions After conducting this experiment, our results showed that in a cancer environment MSCs were able to phagocytose apoptotic NB cells. Furthermore, after phagocytosis the IL-6 and IL-8 cytokine levels increased significantly in the MSCs treated with apoptotic NB cells compared to the control group with MSCs only. Since IL-6 and IL-8 are both considered pro-inflammatory cytokines, we can conclude that after phagocytosis of apoptotic NB cells, MSCs can become immunostimulatory cells. To further confirm our findings, various other cytokines should be tested and more experiments should be done. This way, a more complete picture can be generated describing how MSC cytokine secretion activity changes after phagocytosis of apoptotic neuroblastoma cells. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Medical Sciences

Neural stem cell (NSC) transplantation strategy offers great potential to treat spinal cord injury (SCI). NSCs may replace lost neurons or oligodendrocytes, and act as a source of neurotrophic factors to support the survival of remaining cells. Their efficiency was limited by poor survival after transplantation, and they had more tendencies to differentiate into astrocytes, but not neurons and oligodendrocytes. This project investigated whether activated microglia is a factor that contributes to this phenomenon, and studied the potential role of minocycline, a widely used antibiotic drug, to modify the negative effects of microglia on NSCs. In the first part of this study, we used organotypic spinal cord slice (SCS) culture to mimic in vivo local environment after SCI, and NSCs were grafted on their surface or shared culture medium with them. After specific depletion of microglia with clodronate loaded liposome, more grafted NSCs survived, and in the co-culture system, the NSC neuronal differentiation rate increased while glial differentiation rate decreased, the apoptosis rate also decreased. This suggested that activated microglia may impair NSC survival, and neuronal differentiation, but improve glial differentiation. In the second part of this study, we first tested the direct effects of minocycline on NSC apoptosis, proliferation and differentiation in vitro, to test whether minocycline has any side effect on NSCs. The results showed that at the concentration 10μg/ml or lower, minocycline did not affect NSC survival and proliferation, but impaired neuronal differentiation. Then we treated primary microglia culture with LPS or LPS plus minocycline, and collected the conditioned mediums (CM-LPS and CM-LPSMC) to test their effects on NSC　apoptosis and differentiation. The results showed that compared with CM-LPS, CM-LPSMC resulted in a significantly lower apoptotic rate of NSCs, also allowed NSC neuronal differentiation. This suggested that minocycline may impair the pro-apoptotic effect of activated microglia on NSCs. In conclusion, our study showed that activated microglia may impair NSC survival and neuronal differentiation. This indicated that in NSC transplantation strategy for SCI, microglia would be a target to be manipulated to improve graft survival and neuronal differentiation. Although minocycline may suppress NSC differentiation towards neurons, it has the potential to protect NSCs from the toxic effects of activated microglia. This showed the therapeutic potential of minocycline in NSC transplantation strategies for SCI. / published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Functional mesenchymal stem cells (MSCs) derived from human induced pluripotent stem cells (iPSCs) may represent an unlimited cell source with superior therapeutic benefits for tissue regeneration to somatic tissue, such as bone marrow (BM)-derived MSC. In the first part of this project, I investigated whether the differential expression of ion channels in iPSC-MSCs was responsible for their higher proliferation capacity than that of BM-MSCs. The expression of ion channels for K+, Na+, Ca2+ and Cl- currents was assessed by reverse transcription-polymerase chain reaction (RT-PCR). The functional role of these ion channels were then verified by patch clamp experiments to compare the electrophysiological properties of iPSC-MSCs versus BM-MSCs. I detected significant mRNA expression of ion channel genes including KCa1.1, KCa3.1, KCNH1, Kir2.1, SCN9A, CACNA1C and Clcn3 in both human iPSC-MSCs and BM-MSCs; while Kir2.2 and Kir2.3 were only observed in human iPSC-MSCs. Furthermore, I identified five types of currents (BKCa, IKDR, IKir, IKCa and ICl) in iPSC-MSCs, while only four of them (BKCa, IKDR, IKir and IKCa) were observed in BM-MSCs. The rate of cell proliferation was 1.4 fold faster in iPSC-MSCs as compared to BM-MSCs. Interestingly, the proliferation rate of human iPSCMSCs was significantly reduced when inhibiting IKDR with shRNA and hEAG1 channel blockers, 4-AP and astemizole. Though to a lesser extent, the proliferation rate of human BM-MSCs also decreased by IKDR blockage. These results demonstrated that hEAG1 channel plays a crucial role in controlling the proliferation rate of human iPSC-MSCs but to a lesser extent in BM-MSCs. Next, I examined whether forced expression of a transcription factor- myocardin in iPSC-MSC using viral vectors (adenovirus or lentivirus) can further enhance their trans-differentiation to cardiomyocytes and improve their electrophysiological properties for cardiac regeneration. My results on RT-PCR and immunofluorescent staining revealed that myocardin induced the expression of several cardiac and smooth muscle cell markers, including α-MHC, cTnT, GATA4, α-actinin, and cardiac MHC, smooth muscle cell markers MYH11, calponin, and SM α-actin, but not the more mature cardiac markers such as β-MHC and MLC2v in iPSC-MSCs. These findings indicate that forced expression of myocardin in iPSC-MSC resulted in partial trans-differentiation into cardiomyocytes phenotype. Furthermore, I also discovered that myocardin altered the electrophysiological properties of iPSC-MSCs when examined by RT-PCR and patch clamp experiments. Forced expression of myocardin in iPSC-MSC enhanced the expression of Kv4.3, SCN9A and CACNA1C, but reduced that of KCa3.1 and Kir 2.2 in iPSC-MSCs. Moreover, BKCa, IKir, ICl, Ito and INa.TTX were detected in iPSC-MSC with ectopic expression of myocardin; while only BKCa, IKir, ICl, IKDR and IKCa were noted in iPSC-MSC transfected with green florescence protein. Furthermore, as measured by multi-electrode arrays recording plate, the conduction velocity of the neonatal rat ventricular cardiomyocytes cocultured iPSC-MSC monolayer was significantly increased after ectopic expression of myocardin. Taken together, I have demonstrated that hEAG1 channel is important in the regulation of iPSC-MSC proliferation and forced expression of myocardin in iPSC-MSC resulted in their partial transdifferentiation into cardiomyocytes phenotype and improved the electrical conduction during integration with mature cardiomyocytes. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

The retina is an internal photosensitive neural tunic which absorbs light and prevents it from reflecting back. The light receptors and neurons of the retina are initial processor of visual information. Various anomalies of the retina such as retinitis pigmentosa, cone-rod dystrophy to retinal degenerative diseases cause severe loss of vision since they affect photoreceptors directly or indirectly. Conventional therapies have never been fully successful in restoring vision in such diseases. However current research in stem cell therapies has shown remarkable potential. In this project, induced pluripotent stem cells from mouse were coxed into photoreceptor fate in presence and absence of Dorsomorphin using specific media in a stepwise differentiation process. Dorsomorphin is an inhibitor of Bone Morphogenic Protein (BMP) whose suppression may influence neural differentiation. Studies were done using conventional inverted microscopy and fluorescent microscopy on mouse induced pluripotent stem cells (miPS cells). Immunolabelling techniques involving Pax6, Crx, RPE65, Rhodopsin and Opsin were used to evaluate the advantage of these as markers for stem cells differentiation. Reverse Transcriptase PCR was done to confirm the gene expression on the differentiated cells. Human iPS derived Mesenchymal stem cells were cultured and the effect of different concentrations of Retinoic Acid such as 0mM, 0.1mM and 0.5mM on cell proliferation was tested in both presence and absence of Dorsomorphin. The results revealed both control and Dorsomorphin treated miPS cells successfully differentiated into photoreceptors-like cells as detected by positive staining of Rhodopsin and Opsin. The cells were however negative for Pax6, and very weak staining for RPE and Crx. The presence or absence of Dorsomorphin did not make any difference on miPS differentiation. The same observation was made on differentiating human iPS-MSC where Dorsomorphin did not reveal much effect. However highest cell count of proliferating cells was observed in the subgroups containing 0.1mM Retinoic Acid on Day7 groups as control had an average of 590 ±±317.23 and treatment 1206 .33 ±±114.99 cells, with statistical significance of P<0.05. It appears that the presence of Retinoic Acid facilitated the proliferation of human iPS-MSC. In conclusion, the study reveals that iPS can be another potential stem cell source for therapies of retinal diseases involving photoreceptors where the question of ethical issue is not a problem unlike embryonic stem cells. Also it reveals the concentration of Retinoic Acid most suited for human iPS-MSC cell proliferation. Dorsomorphin did not seem to have much effect on either type of stem cells in terms of promoting photoreceptor differentiation. / published_or_final_version / Medicine / Master / Master of Medical Sciences