Thesis (Ph. D.)--Bioengineering, Georgia Institute of Technology, 2008. / Guldberg, Robert, Committee Chair ; Boyan, Barbara, Committee Member ; O'Keefe, Regis, Committee Member ; Vito, Ray, Committee Member ; Bellankonda, Ravi, Committee Member.

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.

The family with sequence similarity 65, member B (Fam65b) protein is thought to facilitate fusion of myocytes and formation of myotubes during the differentiation of human myogenic cells. Fam65b and histone deacetylase 6 (HDAC6) co-immunoprecipitate and together regulate the levels of acetylated tubulin, which might control microtubule stability in myogenic cells. In this thesis, to gain further insight on the role of Fam65b in the differentiation pathway and motility of myogenic cells, we characterized a Fam65b knock-out (KO) mouse model. Genotyping and transcriptional analysis revealed that a thirteen exons-long region of the Fam65b gene has been successfully ablated and the mRNA amplicons within the deleted segment are not transcribed. Nevertheless, mRNA products corresponding to genomic regions downstream of the deleted area are still detected. Furthermore, analysis of skeletal muscle lysates via western blot (WB) does not show a complete loss of Fam65b expression, but only reduced translation of some isoforms. Nevertheless, WBs of myogenic cells that have been directly isolated from Fam65b KO mice and expanded in vitro revealed the absence of a 120 Kd band, which putatively corresponds to the long isoform of Fam65b. Finally, our data show that Fam65b KO mice are significantly heavier than wild type (WT) mice, and that this phenotype is consistently observed across both genders during the first seven months of age. While functional and molecular analyses of the KO mouse model are still ongoing, future work might include generating a new KO model via the CRISPR-Cas9 technology to ablate all isoforms of Fam65b.

Liver injury stimulates hepatocyte proliferation, regenerating the liver through self-replication. In cases where there is severe, repetitive, parenchymal damage, as seen in human chronic liver disease, hepatocyte mediated regeneration becomes impaired. In this setting it is currently unclear whether endogenous biliary epithelial cells can repopulate the hepatocyte compartment. This thesis therefore aimed to address this point by lineage tracing the main two liver epithelia populations on a background of impaired hepatocyte regeneration. To impair regeneration, an Itgb1 transgene was specifically deleted, conditionally, from the hepatocyte epithelium. Long-term loss of β1-Integrin alone or with additional injury caused an epithelial ductular reaction of biliary origin. Alongside β1-Integrin ablation, the hepatocyte epithelium was also labelled with a heritable ROSA26LSLtdTomato reporter. Impaired hepatocyte regeneration mediated by β1- integrin ablation resulted in 25% of hepatocytes becoming tdTomato negative (non-hepatocyte derived). To verify that the non-hepatocyte mediated regeneration was originating from the biliary epithelium, anti-Itgb1 RNAi was administrated to K19CreERT LSLtdTomato mice. Resulting in tdTomato positive hepatocytes that had differentiated from the labelled tdTomato positive biliary epithelial cells. In summary, this thesis demonstrates that hepatocyte β1-Integrin ablation combined with toxic damage causes marked ductular reactions and results in a substantial regeneration of functional hepatocytes from the biliary epithelium.

Pericytes represent a population of potential mesenchymal stem cells (MSC) that reside within a perivascular niche until they are required in normal homeostasis and the response to injury. Their mesenchymal capacities for multipotent differentiation, immune modulation and release of trophic factors hold great promise for regenerative therapies. Pathological expression of these potentials has been described in disease states, while acute or chronic inflammation following injury can lead to the production of signalling molecules that ultimately drive these progenitors to a fibrotic fate. The aim of this work was to explore how fate decisions of pericytes are regulated by their niche (in the setting of osteogenesis), and in the response to acute and chronic injury (in the setting of fibrosis). It was hypothesized that interactions between pericytes and endothelial cells (EC) within their perivascular niche are responsible for regulating mesenchymal differentiation. The osteogenic, adipogenic and chondrogenic potential of pericytes following isolation from multiple human organs was confirmed. The interactions between pericytes and EC in 2D and 3D coculture and the production of basement membrane proteins in these settings were confirmed. The osteogenic differentiation of pericytes was accelerated by EC but no influence of EC on the adipogenic and chondrogenic differentiation of pericytes was detected. Furthermore, data indicated that the influence on pericyte osteogenic potential by EC may occur through wnt signaling. The activation of TGFβ (transforming growth factor beta) through αv integrins has been suggested as central mediator of fibrosis in multiple organs. We hypothesized that selective αv integrin deletions in PDGFRβ (platelet derived growth factor receptor beta) expressing pericytes identifies a targetable pathway regulating fibrosis in skeletal muscle. We report that PDGFRβ-Cre inactivates genes in murine skeletal muscle pericytes with high efficiency. Deletion of the αv integrin subunit in pericytes protected mice from chemical injury induced skeletal muscle fibrosis. Pharmacological blockade of αv integrins by a novel small molecule (CWHM 12) attenuated muscle fibrosis, even when administered after fibrosis was established.

The exogenous expression of master transcription factors (TFs) to drive cell identity changes is an exciting and powerful approach to cell and tissue engineering. Yet, the generation of desired cell types is often plagued by inefficiency and inability to produce mature cell types. Through investigations of the molecular mechanisms of induced pluripotent stem cell (iPSC) generation, I discovered that expression of constitutively active Smad2/3 (Smad2CA/3CA), together with the Yamanaka factors, could dramatically improve the efficiency of reprogramming. Mechanistically, SMAD3 interacted with both co-activators and reprogramming factors, bridging their interaction during reprogramming. Because SMAD2/3 interact with a multitude of master TFs in different cell types, I tested the conversions of B cells to macrophages, myoblasts to adipocytes, and human fibroblasts to neurons. Remarkably, each conversion system was markedly enhanced when the master TFs were co-expressed with Smad3CA. These results revealed the existence of shared molecular mechanisms underlying diverse TF-mediated cellular conversions, and demonstrated SMAD2/3 as a widely applicable cofactor that potentiates the generation of diverse cell types with profound efficiency and maturity.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement therapies may hold the potential for repair in a degenerate retina, by reinstating light sensitive cells to project and form connections with downstream retinal cells and finally the visual cortex. Patient-specific induced pluripotent stem cells (iPSc) could provide an autologous source of cells to replace lost tissue. However, the use of patient-derived iPSc would require that the disease-causing gene mutation be corrected in cells before transplantation. Ex vivo gene therapy of mouse photoreceptor precursor (PhRP) cells and subretinal transplantation of treated cells are here studied in a disease-specific animal model of RP; rhodopsin was ectopically expressed ex vivo in rod precursor cells, sourced from a transgenic model lacking the rhodopsin gene. Treated rod precursors were here transplanted in mice of the same disease model and are shown to gain expression of rhodopsin and mature to regenerate the absent outer nuclear layer (ONL) of degenerate mice. Visual function, assayed in the same animals before and after transplantation, was restored in animals which had no rod function at baseline. Delivery of the rhodopsin gene by both an adeno associated viral (AAV) vector and a non-viral minicircle DNA vector developed here for ex vivo gene delivery to rod photoreceptor precursors showed comparable efficiency and sustained expression. The non-viral minicircle method provides a novel system for efficient photoreceptor therapy and may offer a platform of genetic treatment of photoreceptor degenerations in which the gene in focus exceeds the size limit for packaging in AAV. Human embryonic stem cell (ESC) and human iPSC-derived PhRPs were also transplanted in mice with complete ONL degeneration and were able to reform the lost photoreceptor layer and mature in the host retina. Human cells developed light-sensitive outer segments, and reconnect with host neurons downstream to improve vision in previously blind mice. Efficient transplantation of ex vivo genetically treated rod precursors and human stem cell-derived PhRPs in animal models of progressive RP may provide a clinically-relevant model for the investigation of cell-replacement therapy for photoreceptor regeneration in retinal disease.

Orientador: Noeme Sousa Rocha / Resumo: O tumor venéreo transmissível (TVT) canino é uma neoplasia transplantável, considerada um alo-enxerto. Entretanto, pouco se sabe a respeito da origem e processo de cacinogênese. Atualmente, postula-se que alguns tumores originem-se de células iniciadoras tumorais, classicamente descritas nas leucemias mielóide humanas. As características intrínsecas do TVT fornecem indícios de uma possível participação de células iniciadores tumorais no processo de carcinogênese nesse tumor. Foi realizado estudo de fenotipagem do TVT canino para avaliar a marcação das proteínas CD44, CD133, CD90 e CD34, comumente associadas ao potencial iniciador tumoral. Para tanto utilizou-se a citometria de fluxo, imuno-histoquímica e RTq-PCR. Foram analisados também as frações de crescimento pelo Ki-67) e o número de células em apoptose pela caspase-3 clivada. Trinta e oito amostras de TVT foram obtidas de pacientes sem tratamento quimioterápico prévio. As amostras foram classificadas em plasmocitóide ou mistas, de acordo com o subtipo citológico; as células positivas na citometria de fluxo foram representadas em termos percentuais para os marcadores CD44, CD34, CD90 e CD133; a fração de crescimento foi representada pela técnica do H-Score; a quantidade de células apoptóticas foi representada pelo somatório de células positivas para a caspase-3 clivada; as imuno-marcações das proteínas CD44 e CD34 foram representadas por escores semi-quantitativos baseados na intensidade e percentual de células positivas;... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The canine transmissible venereal tumor (CTVT) is a transplantable neoplasia considered an allograft. Information about the origin and carcinogenesis process is scarcely known. Currently, some neoplasms are believed to arise from a tumor-initiating cell (TIC's) classically described in human myeloid leukemia. TVT intrinsic characteristics provide evidence of a possible TIC's participation in carcinogenesis process of this malignancy. Thus, a phenotyping study of CTVT was conducted to assess the immunophenotyping properties of the proteins CD44, CD133, CD90 and CD34, already known to be associated to tumor initiator potential. The use of flow cytometry and immunohistochemistry contributed to this purpose. In addition, growth fractions and cells undergoing apoptosis were examined by Ki-67 and caspase-3 cleaved, respectively. Thirty-eight samples were chosen from patients having no previous chemotherapy and cytological diagnosis of CTVT. Samples were classified into plasmacytoid or mixed according to cytological subtype. Positive cells in the flow cytometry were expressed in percentage for the markers CD44, CD34, CD90 and CD133. H-score technique helped to represent growth fractions. Apoptotic cell quantity was calculated by summing positive cells. Immunohistochemical marking of CD44 and CD34 proteins were determined by semiquantitative scores based on the intensity and percentage of positive cells. Moreover, specimens were divided into resistant and non-resistant groups and com... (Complete abstract click electronic access below) / Doutor

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), whose function is to optimise neuronal transmission and preserving axonal integrity. Oligodendrocytes are derived from a stem cell population, called oligodendrocyte progenitor cells (OPCs). Oligodendrocyte lineage cells (OLCs) have been implicated in the pathophysiology of various diseases including not only demyelinating diseases (eg. Multiple Sclerosis (MS) or Pelizaeus-Merzbacher disease (PMD)), but also psychiatric disorders (eg. schizophrenia or Rett syndrome (RTT)). Regardless of the type of disease, understanding the underlying fundamental biology of the oligodendrocyte lineage cells is pivotal to develop therapeutic strategies. In the mouse embryonic forebrain OPCs are generated in consecutive waves from distinct brain regions along a spatiotemporal gradient; with ventral OPCs emerging before dorsal OPCs. The developmentally distinct OPCs, and their progenies, persist in the brain throughout life. To investigate whether ventrally and dorsally derived OLCs fulfil different functions in the adult brain, dorsally derived OPCs were ablated in development using a \textit{Sox10}-driven diphtheria toxin fragment A (DTA) mouse model. As dorsally derived OPCs populate the cortex, locomotor coordination and cognition were investigated following dorsal OPC ablation. Mice ablated of the dorsal OPC population do not show a significant deficit in learning and attentional function. In contrast, ablated mice show an impaired locomotor coordination, while general vigilance, gait, balance and sensation are comparable to control groups. The locomotor coordination disabilities are a result of alterations of brain, not spinal cord homeostasis, as only a neglect able number of OLCs in the spinal cord are affected by the ablation model. In addition, no signs of neuronal cell death or chronic inflammatory response was detected in response to the ablation. As the oligodendrocyte numbers are similar between control and ablated animals, the locomotor coordination phenotype can also not be explained by reduced number of oligodendrocytes. However, clustering analysis following single-cell Drop-sequencing uncovered a heterogeneity of oligodendrocyte (OL) subpopulations in the motor cortex. Whilst some OL subpopulations are of mixed developmental origin, others are exclusively formed by either ventrally or dorsally derived OLs, arguing that dorsal oligodendrocyte subpopulations are crucial for homeostatic brain function. In the absence of dorsal OPCs, ventral OPCs are not capable of forming dorsal oligodendrocyte subpopulations in response to dorsal OPC ablation. In conclusion, my results indicate a functional heterogeneity of developmentally-distinct oligodendrocytes in physiological brain function.

Differentiation and cell cycle regulation in stem cell have a key function for embryonic development, organ homeostasis and tissue repair. Recent results have shown that these two mechanisms are intrinsically connected. Indeed, cell cycle machinery directly controls maintenance of pluripotency and initiation of differentiation. More precisely, the cell cycle regulator Cyclin D appears to control the transcriptional activity of Activin/Nodal signalling during progression of the cell cycle in human Embryonic Stem Cells (hESCs). As a consequence, hESCs can only differentiate into endoderm in the Early G1 phase when Cyclin Ds are expressed at low levels. These results show the mechanisms by which the cell cycle defines differentiation propensity of stem cells. However, these observations also imply the existence of interplays coordinating extra cellular signalling pathways with the epigenetic state, chromatin structure and transcriptional networks during cell cycle progression and these mechanisms remain to be fully uncovered. Here, I have utilised the FUCCI reporter system combined with ATAC-Seq to analyse chromatin dynamics during cell cycle progression in hESCs. Furthermore, I performed ChIP-Seq analyses to define the genomic location of transcriptional regulators during cell cycle progression as well as RNA-Seq to confirm variation in gene expression pattern. Integration of these data shows that the chromatin status in hESCs is highly dynamic and the core pluripotency transcription factors and epigenetic modifiers change genomic location during cell cycle progression. I also showed that hESCs in the Late G1 phase accumulate transcripts that are important for differentiation and development; therefore, indicating this phase represents a unique portion of the cell cycle for cell fate decisions. Taken together, these results uncover that transcriptional networks are unexpectedly dynamic during the progression of cell cycle in stem cells. I hypothesise that these modifications are necessary to prime hESCs for different cell fate choices allowing a diversity of differentiation that is otherwise impossible. Overall these mechanisms underline the need to study transcriptional and epigenetic mechanisms in the dynamic context of the cell cycle and have major implications for adult tissue homeostasis and disease.