Genetic regulatory networks in avian B cells

Biology is turning into an information science. The science of systems biology seeks to understand the genetic networks that govern organism development and functions. In this study the chicken was used as a model organism in the study of B cell regulatory factors. These studies open new avenues for plasma cell research by connecting the down regulation of the B cell gene expression program directly to the initiation of plasma cell differentiation. The unique advantages of the DT40 avian B cell model system, specifically its high homologous recombination rate, were utilized to study gene regulation in Pax5 knock out cell lines and to gain new insights into the B cell to plasma cell transitions that underlie the secretion of antibodies as part of the adaptive immune response. The Pax5 transcription factor is central to the commitment, development and maintenance of the B cell phenotype. Mice lacking the Pax5 gene have an arrest in development at the pro-B lymphocyte stage while DT40 cells have been derived from cells at a more mature stage of development. The DT40 Pax5-/- cells exhibited gene expression similarities with primary chicken plasma cells. The expression of the plasma cell transcription factors Blimp-1 and XBP-1 were significantly upregulated while the expression of the germinal centre factor BCL6 was diminished in Pax5-/- cells, and this alteration was normalized by Pax5 re-introduction. The Pax5-deficient cells further manifested substantially elevated secretion of IgM into the supernatant, another characteristic of plasma cells. These results for the first time indicated that the downregulation of the Pax5 gene in B cells promotes plasma cell differentiation. Cross-species meta-analysis of chicken and mouse Pax5 gene knockout studies uncovers genes and pathways whose regulatory relationship to Pax5 has remained unchanged for over 300 million years. Restriction of the hematopoietic stem cell fate to produce T, B and NK cell lineages is dependent on the Ikaros and its molecular partners, the closely related Helios and Aiolos. Ikaros family members are zinc finger proteins which act as transcriptional repressors while helping to activate lymphoid genes. Helios in mice is expressed from the hematopoietic stem cell level onwards, although later in development its expression seems to predominate in the T cell lineage. This study establishes the emergence and sequence of the chicken Ikaros family members. Helios expression in the bursa of Fabricius, germinal centres and B cell lines suggested a role for Helios in the avian B-cell lineage, too. Phylogenetic studies of the Ikaros family connect the expansion of the Ikaros family, and thus possibly the emergence of the adaptive immune system, with the second round of genome duplications originally proposed by Ohno. Paralogs that have arisen as a result of genome-wide duplications are sometimes termed ohnologs – Ikaros family proteins appear to fit that definition. This study highlighted the opportunities afforded by the genome sequencing efforts and somatic cell reverse genetics approaches using the DT40 cell line. The DT40 cell line and the avian model system promise to remain a fruitful model for mechanistic insight in the post-genomic era as well.

Effects of Silica Based Biomaterials on Bone Marrow Derived Cells - Material Aspects of Bone Regeneration

Silica based biomaterials, such as melt-derived bioactive glasses and sol-gel glasses, have been used for a long time in bone healing applications because of their ability to form hydroxyapatite and to stimulate stem cell proliferation and differentiation. In this study, bone marrow derived cells were cultured with bioactive glass and sol-gel silica, and seeded into porous polymer composite scaffolds that were then implanted femorally and subcutaneously in rats to monitor their migration inside host tissue. Bone marrow derived cells were also injected intraperitoneally. Transplanted cells migrated to various tissues inside the host, including the lung, liver spleen, thymus and bone marrow. The method of transplantation affected the time frame of cell migration, with intraperitoneal injection being the fastest and femoral implantation the slowest, but not the target tissues of migration. Transplanted donor cells had a limited lifetime in the host and were later eliminated from all tested tissues. Bioactive glass, however, affected the implanted cells negatively. When it was present in the scaffold no donor cells were found in any of the tested host tissues. Bioactive glass S53P4 was found to support both osteoblastic and osteoclastic phenotype of bone marrow derived cells, but it was resistant to the resorbing effect of osteoclastic bone marrow derived cells, showing that bioactive glass is rather dissolved through physicochemical reactions than resorbed by cells. Fast-dissolving silica sol gel in microparticulate form was found to increase collagen formation by bone marrow derived cells, while slow dissolving silica microparticles enhanced their proliferation, suggesting that the dissolution rate of silica controls the response of bone marrow derived cells.

Pluripotency and genetic stability of human pluripotent stem cells

Pluripotent cells have the potential to differentiate into all somatic cell types. As the adult human body is unable to regenerate various tissues, pluripotent cells provide an attractive source for regenerative medicine. Human embryonic stem cells (hESCs) can be isolated from blastocyst stage embryos and cultured in the laboratory environment. However, their use in regenerative medicine is restricted due to problems with immunosuppression by the host and ethical legislation. Recently, a new source of pluripotent cells was established via the direct reprogramming of somatic cells. These human induced pluripotent stem cells (hiPSCs) enable the production of patient specific cell types. However, numerous challenges, such as efficient reprogramming, optimal culture, directed differentiation, genetic stability and tumor risk need to be solved before the launch of therapeutic applications. The main objective of this thesis was to understand the unique properties of human pluripotent stem cells. The specific aims were to identify novel factors involved in maintaining pluripotency, characterize the effects of low oxygen culture on hESCs, and determine the high resolution changes in hESCs and hiPSCs during culture and reprogramming. As a result, the previously uncharacterized protein L1TD1 was determined to be specific for pluripotent cells and essential for the maintenance of pluripotency. The low oxygen culture supported undifferentiated growth and affected expression of stem cell associated transcripts. High resolution screening of hESCs identified a number of culture induced copy number variations and loss of heterozygosity changes. Further, screening of hiPSCs revealed that reprogramming induces high resolution alterations. The results obtained in this thesis have important implications for stem cell and cancer biology and the therapeutic potential of pluripotent cells.