Bright/ARID3A was initially discovered for its role in immunoglobulin heavy chain transcription in the mouse. Bright has also been implicated as a target of p53 and as an E2F binding partner. We have previously shown that Bright is necessary for hematopoietic stem cell development in the embryo. In this work, we show that Bright has a much broader role in development than previously appreciated. Loss of Bright in mice usually results in embryonic lethality due to lack of hematopoietic stem cells. Rare survivor mice initially appear smaller in size than either wildtype or heterozygous littermates, but as they age, these differences diminish. We show that adult Bright null mice have age-dependent kidney defects. Previous work in the adult mouse has not indicated a role for Bright in kidney function. We observed an increase in cellular proliferation in Bright null kidneys, indicating a possible mechanism behind our observation. Loss of Bright has recently been implicated in causing developmental plasticity in somatic cells. Our data indicate that loss of Bright is sufficient to fully reprogram mouse embryonic fibroblasts (MEFs) back to a pluripotent state. We term these cells Bright repression induced pluripotent stem cells (BriPS). BriPS derived from Bright knockout MEFs can be stably maintained in standard embryonic stem cell culture conditions: they express pluripotency markers and can form teratomas in vivo. We further viii show that Bright is active during embryonic stem cell differentiation. Bright represses key pluripotency genes, suggesting the mechanism of reprogramming may be Bright’s direct repression of key pluripotency factors in somatic cells. Recent advances in inducing pluripotency in somatic cells (iPS cells) have created a new field of disease modeling, increased our knowledge of how pluripotency is regulated, and introduced the hope that they can be adapted to treat disease. However, current methods for producing iPS involve overexpression of potentially oncogenic transcription factors, leaving a large gap between the lab and the clinic. Our results mark the first demonstration of an alternative method to reprograming somatic cells that is not mediated by overexpression of pluripotency factors. / text

This thesis shows that p53-deficient mouse glioma brain tumour stem cells (BTSCs), which fail to express pluripotency factors, can be reprogrammed with specific transcription factors to generate iPS cell lines (GNS-iPS) expressing endogenous pluripotency factors (Nanog, Oct4, and Rex1). GNS-iPS cell lines formed embryoid bodies (EBs) in vitro and undifferentiated growths in vivo that phenotypically did not resemble tumours derived from non-reprogrammed BTSCs. EBs formed from one GNS-iPS cell line expressed markers of mesoderm, endoderm, and ectoderm. Tumours produced from GNS-iPS cells had reduced astrocytic marker (GFAP) expression compared to those generated from control iPS cell lines or non-reprogrammed BTSCs. Preliminary results suggest that the reprogrammed cells can be re-differentiated into cells that show neural precursor phenotype. These findings suggest that BTSCs can acquire aspects of the pluripotent state with a defined set of transcription factors, opening the door for further exploration of reprogramming strategies to attenuate the cancer phenotype.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.

Somatic cells can be reprogrammed to induced pluripotent stem (iPS) cells by expression of defined embryonic factors. My thesis is focused on exploring the mechanisms underlying reprogramming using a secondary mouse embryonic fibroblast model that forms iPS cells with high efficiency upon inducible expression of Oct4, Klf4, c-Myc and Sox2. My analyses of the temporal changes in gene expression reveal that reprogramming is a multi-step process characterized by initiation, maturation and stabilization phases. Using functional RNAi screening, I discovered a key role for BMP signaling and the induction of mesenchymal-to-epithelial transition (MET) during the initiation phase. I showed that MET induction was linked to BMP-dependent induction of miR-205 and the miR-200 family of microRNAs. These studies thus defined a multi-step mechanism that incorporates a BMP-miRNA-MET axis during somatic cell reprogramming. Next I focused on the two later phases of reprogramming, maturation and stabilization. I showed the stabilization phase and acquisition of pluripotency is dependent on removal of transgene expression late in the maturation phase. Clonal analysis of reprogramming cells revealed subsets of stabilization competent (SC) versus stabilization incompetent (SI) cells. SC clones robustly entered the pluripotent state upon transgene withdrawal in the late, but not early maturation phase, whereas SI clones failed to reprogram at either stage. Transcriptome profiling by RNA-Seq revealed that SC clones acquire a competency gene expression signature late in the maturation phase. Functional RNAi screening of SC signature genes further identified regulators of transition to the stabilization phase, while screening of the same signature in iPS cells revealed a distinct subset of genes required for maintenance of pluripotency. These studies reveal that the acquisition and subsequent maintenance of pluripotency are controlled by distinct molecular networks and uncover a novel regulatory program that is required for transition to transgene independence.

This thesis shows that p53-deficient mouse glioma brain tumour stem cells (BTSCs), which fail to express pluripotency factors, can be reprogrammed with specific transcription factors to generate iPS cell lines (GNS-iPS) expressing endogenous pluripotency factors (Nanog, Oct4, and Rex1). GNS-iPS cell lines formed embryoid bodies (EBs) in vitro and undifferentiated growths in vivo that phenotypically did not resemble tumours derived from non-reprogrammed BTSCs. EBs formed from one GNS-iPS cell line expressed markers of mesoderm, endoderm, and ectoderm. Tumours produced from GNS-iPS cells had reduced astrocytic marker (GFAP) expression compared to those generated from control iPS cell lines or non-reprogrammed BTSCs. Preliminary results suggest that the reprogrammed cells can be re-differentiated into cells that show neural precursor phenotype. These findings suggest that BTSCs can acquire aspects of the pluripotent state with a defined set of transcription factors, opening the door for further exploration of reprogramming strategies to attenuate the cancer phenotype.

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.

Cloning through somatic cell nuclear transfer (SCNT) remains highly inefficient twenty years after the first demonstration of the technology with the birth of Dolly. By increasing efficiency by selecting the embryos early in development that are most likely to succeed following transfer into a surrogate mother, the technology could be more routinely utilized to enhance animal agriculture production. SCNT is believed to be highly inefficient as a result of incorrect DNA methylation and gene expression that are accumulated because of the SCNT technique. We proposed the use of a non-toxic, non-invasive detector of cell death, to quantitatively assess embryo competency prior to embryo transfer. We believed we could use SR-FLICA to identify the embryos with low levels of cell death as a result of proper DNA methylation and gene expression. By analyzing the whole embryo, differences in gene expression and DNA methylation were identified in embryos with high and low levels of cell death. However, the level of cell death did not prove to be a reliable indicator of embryo quality in predicting pregnancy outcome. This data supports the commonly held hypothesis that DNA methylation and gene expression after SCNT have random defects as a result of the random nature of resetting the DNA for embryo development. More research is required to identify the embryos which will prove to be successful following SCNT and embryo transfer.

No description available.

Bronchopulmonary dysplasia (BPD), the chronic lung disease that develops in premature babies following mechanical ventilation and oxygen exposure, is the most common complication of extreme prematurity. Currently, there is no cure for BPD. Increasing evidence indicates early-onset emphysema and pulmonary vascular disease in survivors with BPD (Aukland et al., 2006; Wong et al., 2008), suggesting an irreversible arrest in lung growth and/or premature lung aging resulting in life-long health problems (J. Sucre et al., 2021). Transient in vivo cellular reprogramming through the activation of the Yamanaka reprogramming factors Oct4, Sox2, Klf4, c-Myc (OSKM), ameliorate cellular and physiological hallmarks of aging and to promote tissue regeneration and improve organ function after injury. (Chen et al., 2021a; Hishida et al., 2022b; Lu et al., 2020) This thesis focuses on determining if transient in vivo cellular reprogramming can regenerate an established lung injury in a BPD mouse model. Two strategies, (a) Adeno-Associated virus (AAV) mediated transient overexpression of the OSK factors and (b) using a transgenic reprogrammable mouse line to overexpress the OSKM factors were employed to test the efficiency of in vivo cellular reprogramming in regenerating the lungs. Both the strategies, under the conditions tested, did not regenerate established lung injury in a BPD mouse model but the feasibility of both these strategies was established here laying a foundation for the next phase of the study.

No description available.