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Pulmonary arterial hypertension (PAH) is a devastating lung disease characterized by arterial pruning, occlusive vascular remodeling, and inflammation contributing to increased pulmonary vascular resistance with resultant right heart failure. Endothelial cell (EC) injury and apoptosis are commonly considered triggers for PAH, the mechanisms leading from injury to complex arterial remodeling are incompletely understood. While current therapies can improving symptoms, with the exception of parenteral prostacyclin, they do not significantly prolong transplant free survival. As well, there are no therapies that can regenerate the damaged lung short of transplantation. In this project, I sought to both advance the understanding of disease pathogenesis and explore regenerative therapeutic options for PAH. To this end, I first employed single cell RNA sequencing (scRNA-seq) at multiple time points during the Sugen 5416 (SU) – chronic hypoxia (CH) model of PAH, to provide new insights into PAH pathogenesis both during onset and progression of disease. We also employed microCT analysis to visualize and quantify the arterial pruning associated with PH and found significant loss up to 65% of the healthy arteriolar volume in this model. Through scRNA-seq analysis performed at four timepoints spanning the onset and progression of disease, two disease-specific EC cell types emerged as key drivers of PAH pathogenesis. The first was the emergence of capillary ECs with a de-differentiated gene expression profile, which we termed dedifferentiated capillary (dCap) ECs, with enrichment for the Cd74 gene. Interestingly, RNA velocity analysis suggested that these cells may be undergoing endothelial to mesenchymal transition during PAH development. At later times, a second arterial EC population became apparent, which we termed activated arterial ECs (aAECs), since it uniquely exhibited persistently elevated levels of differential gene expression consistent with a migratory, invasive and proliferative state. Interestingly, the aAECs together with the smooth muscle (SM)-like pericytes, a population which was also greatly expanded in PAH, expressed Tm4sf1, a gene previously associated with a number of cancers and abnormal cell growth. Furthermore, by immunostaining, TM4SF1 was found to be spatially localized to sites of complex and occlusive arterial remodeling, associated with both endothelial cells and pericytes in these lesions, suggesting an important role for the aAECs and SM-like pericytes in arterial remodeling and PH progression. Together, these findings suggest that aAECs, dCap ECs, and SM-like pericytes are emerging cell populations responsible for lung arterial remodeling in PAH, which drives disease progression, and that TM4SF1 may be a novel therapeutic target for this disease. As a first step in trying to develop approaches to regenerate lung arterial bed that is lost in PAH, we investigated the potential role of endothelial colony forming cells (ECFCs) and mesenchymal stromal cell (MSC) derived extracellular vesicles (EVs) as novel therapeutics, on the premise that these stem/progenitor cells would stimulate lung regeneration by mainly paracrine mechanisms. Additionally, we used biomaterials to microencapsulate cells and EVs to improve their local delivery and retention. While ECFCs were found to be ineffective in treating the monocrotaline model on their own, they were poorly retained in the lung and microencapsulation of ECFCs led to enhanced lung delivery within the first 72 hours, with resultant hemodynamic improvements in this model of PAH. MSCs are well known to be immunomodulatory and proangiogenic, largely acting through paracrine mechanisms, including by the release of EVs. Yet, following intravenous administration, nano sized EVs are rapidly cleared from circulation, potentially limiting their therapeutic potential. I adapted our microencapsulation strategy for EVs, and demonstrated significantly greater retention of microgel-loaded EVs were within the lung, resulting in enhanced local cell uptake. Interestingly, the hydrogel used for microencapsulation induced a local immune response which made it unsuitable for testing any potential therapeutic benefits of MSC-EVs in this study. Nonetheless, this work demonstrated proof-of-principle for the utility of microencapsulation as a strategy to enhance EV lung delivery. Overall, this work has identified novel lung cell populations (aAECs, dCap ECs, SM-like pericytes) driving arterial remodeling associated with PH progression, demonstrated the potential of ECFCs as a regenerative cell for the treatment of PAH, and illustrated the utility of microencapsulation as a tool to enhance lung targeting of both cells and EVs.

<p>Immunodeficient-causing diseases such as HIV and leukemia have no cures, often require meticulous treatments and result in high morbidity or mortality. Although bone marrow transplants are an option for a subset of leukemia patients, the shortage of donors and the requirement for donor matching restricts the efficacy of this treatment option. Therefore there is a prominent clinical need for alternative sources of hematopoietic stem/progentior cells with lymphopoietic potential. Recently we described the direct conversion of human dermal fibroblasts to multilineage hematopoietic progenitors by ectopic expression of OCT4. This direct conversion method was used to assess whether OCT4-transduced fibroblasts had the capacity to derive cells of the lymphoid lineage. This work shows the transient co-expression of CD34 and CD45 of fibroblasts within 7 days of OCT4 transduction followed by stable expression of CD45 on fibroblasts by day 15. The acquisition of hematopoietic markers, however, did not coincide with colony formation as previously described. Furthermore, CD45+ cells that were enriched and cultured in hematopoietic conducive conditions did not acquire co-expression of CD34 as previously shown. Interestingly, CD34 expression was shown to be inversely correlated with OCT4 expression. Therefore the constitutive expression of OCT4 may have (1) inhibited the acquisition of CD34 expression on CD45+ cells (2) downregulated the expression of CD34 on the day 7 CD34+CD45+ fibroblasts, thereby resulting in the transient expression of these markers. Furthermore, this work shows that expression of CD45 on OCT4-transduced fibroblasts is required for survival on the MS5 stromal cell line used to support hematopoietic progenitors with lymphopoietic potential, while supplementation of CD45+ fibroblasts with hematopoietic progenitor supportive conditions resulting in co-expression of CD34 and CD45 is required for acquisition of CD19, a pan-B cell marker on CD45+ fibroblasts. These findings suggest OCT4-transduced fibroblasts have lymphopoietic potential.</p> / Master of Science (MSc)

<p><strong>Background:</strong> Heart failure (HF) constitutes an important growing medical and economic problem with high prevalence and mortality. Prognosis assessment remains a challenge because of the dynamic nature of HF and the existence of some unexplained variation in outcomes. Our objective was to refine the process of prognostic assessment in current HF patients.</p> <p><strong>Methods:</strong> We conducted a systematic review to identify existing risk prediction models in ambulatory HF patients, a meta-analysis to identify mortality predictors in HF patients treated with an implantable cardioverter defibrillator (ICD), a retrospective cohort study to validate a new model, the HF Meta-Score, derived from the results of the meta-analysis and a cross-sectional and prospective cohort study to evaluate whether circulating progenitor cells (CPCs) are associated with functional capacity and mortality in ambulatory HF patients.</p> <p><strong>Results:</strong> We identified 20 risk prediction models in ambulatory HF patients; only five were externally validated showing limited discrimination and calibration. The two most validated models were derived from HF cohorts from the 1990s and reported limited performance in ICD patients. In a meta-analysis, we identified that age, baseline renal function, history of heart failure, chronic obstructive pulmonary disease, diabetes, peripheral vascular disease, left ventricular ejection fraction, NYHA class, atrial fibrillation, wide QRS and the occurrence of appropriate or inappropriate ICD shocks were independent mortality predictors. Some of these predictors were omitted in previously identified models. From the results of the meta-analysis, we developed the HF Meta-Score that showed better performance that an existing model. We observed that CPCs were independently associated with functional capacity and outcomes in ambulatory HF patients.</p> <p><strong>Conclusions:</strong> These results open many pathways to further refine the prognostic assessment in ambulatory HF patients. The HF Meta-Score is a promising score. The clinical utility of the HF Meta-Score and of the incorporation of new predictive factors, such as CPCs, needs to be tested.</p> / Doctor of Philosophy (PhD)

Atopic dermatitis (AD) is a common skin disease that is characterized by chronic, relapsing skin inflammation and eczematous, itchy lesions. In AD, systemic and local eosinophilia and basophilia is thought to contribute to disease progression in both acute and chronic lesions. It has been previously shown that in chronic allergic inflammatory diseases, tissue eosinophilia and basophilia may in part result from eosinophil/basophil (Eo/B) progenitors trafficking from the bone marrow and maturing in tissue in response to type 2 cytokines including IL-5 and IL-3. We therefore proposed that a similar mechanism could be contributing to the pathogenesis of AD. First, we compared lesional and non-lesional AD tissue, and found approximately 10-fold higher levels of Eo/B progenitors in the lesional tissue (p<0.05). As previous research has shown an increase in Eo/B progenitors in the airways of allergic asthmatics post inhaled allergen challenge, we next examined whether Eo/B progenitors increased locally in the acute phase of AD using the intradermal allergen challenge model. Compared to intradermal diluent challenge there was an increase in Eo/B progenitors (5.5-fold), eosinophils (18-fold) and basophils (2.5-fold) 24 hours post intradermal allergen challenge (all p<0.05). These increases were consistent with findings in allergic airways. Lastly, we examined the relationship between disease severity and Eo/B progenitors in inflamed lesional (chronic) and allergen-challenged (acute) tissue. We found that Eo/B progenitors in lesional tissue positively correlated with disease severity (EASI R=0.71, p<0.05 and SCORAD R=0.65, p<0.05), while in allergen-challenged tissue a trend was seen for a positive correlation between Eo/B progenitors and disease severity (EASI R=0.48, p=0.07 and SCORAD R=0.46, p=0.09). These results highlight the potential involvement of Eo/B progenitors in the disease pathogenesis of AD. / Thesis / Master of Science (MSc) / Atopic dermatitis is a common skin disease that is characterized by chronic, relapsing skin inflammation and eczematous, itchy lesions. In other allergic diseases, a cell called the “eosinophil/basophil progenitor” contributes to the accumulation of inflammatory cells in the diseased organ. We proposed that eosinophil/basophil progenitors found in the skin may be contributing to the development of local allergic inflammation. In patients with moderate-to-severe atopic dermatitis we compared acute responses to intradermal allergen and chronic skin lesions to diluent-challenged and un-affected skin, respectively. Allergen-challenged skin had more eosinophil/basophil progenitors, mature eosinophils and basophils 24 hours’ post-challenge compared to unchallenged skin (p<0.05). Chronic skin lesions had more eosinophil/basophil progenitors than un-affected skin (p<0.05). The number of eosinophil/basophil progenitors positively correlated to disease severity as determined by EASI and SCORAD. Our results suggest that accumulation of eosinophil/basophil progenitors in skin of atopic dermatitis patients could support allergic inflammation and contribute to disease severity.

No / A neurological disorder is any disorder or abnormality in the nervous system. Among different neurological disorders, Alzheimer’s disease (AD) is recognized as the sixth leading cause of death globally. Considerable research has been conducted to find pioneer treatments for this devastating disorder among which cell therapy has attracted remarkable attentions over the last decade. Up to now, targeted differentiation into specific desirable cell types has remained a major obstacle to clinical application of cell therapy. Also, potential risks including uncontrolled growth of stem cells could be disastrous. In our novel protocol, we used basal forebrain cholinergic progenitor cells (BFCN) derived from human chorion-derived mesenchymal stem cells (hC-MSCs) which made it possible to obtain high-quality population of cholinergic neurons and in vivo in much shorter time period than previous established methods. Remarkably, the transplanted progenitors fully differentiated to cholinergic neurons which in turn integrated in higher cortical networks of host brains, resulting in significant improvement in cognitive assessments. This method may have profound implications in cell therapies for any other neurodegenerative disorders. / This work was carried outwithin the framework of a collaborative project (Project Grant No. 94-02-30-25922) by the School of Medicine, Iran University of Medical Sciences, (Project Grant No. REP209) council for stem cell sciences and technologies (Presidency of the Islamic Republic of Iran, vice-presidency for science and technology), and Iran National Science Foundation (INSF).

No / Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.