Human hematopoietic microenvironments, in vivo, in vitro and on chip
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AbstractCells are directed by signals in their microenvironment. Knowledge about this communication is necessary to understand both normal processes and cancer. This PhD thesis focuses on the human bone marrow microenvironment that governs the generation of B-cells (early B-lymphopoiesis). Multiple environmental elements affecting the maturation process were explored by use of both classical and new technologies. Immunohistochemical investigation of bone marrow biopsies revealed that all stages of differentiating B-cells harbor a niche formed by slender CD10+ stromal cells, suggesting an essential supporting role. A cDNA cloning strategy (signal sequence trap, SST-REX) identified several B-cell surface proteins that may contribute in the crosstalk with the microenvironment. One unknown protein was characterised (TMEM-9), but turned out to have no obvious role in cell communication. Next, classical and new cultivation platforms were developed for assaying the impact of microenvironmental factors on early B-lymphopoiesis. Conventional cultivation based on a feeder layer of stromal cells showed that the morphogen Wnt3a reduced both proliferation of B-cell progenitors and the production of B-cells from hematopoietic stem cells. The existence of an operational Wnt-pathway in B-cell progenitors was verified by detection of central pathway molecules both at protein level (Western blot) and at expression level (RT-PCR). For the purpose of obtaining more advanced models of hematopoietic environments, a microfluidic system (chip) was fabricated by soft lithography. The system was compatible with long-term cultivation and stem cell differentiation, as shown by extensive adipogenic and osteogenic differentiation of mesenchymal stem cells (MSCs), which are reported to provide essential hematopoietic support. Exploitation of the microscale properties of this system can be used for housing hematopoietic cells in more realistic environments than those enabled by existing methods.
List of papers
|Paper I Kveine M, Tenstad E, Dosen G, Funderud S, Rian E.: ”Characterization of the novel human transmembrane protein 9 (TMEM9) that localizes to lysosomes and late endosomes.” Biochemical and Biophysical Research Communications 2002; 297 (4): 912-917. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1016/S0006-291X(02)02228-3|
|Paper II Torlakovic E*, Tenstad E*, Funderud S, Rian E.: “CD10+ stromal cells form B-lymphocyte maturation niches in the human bone marrow.” Journal of Pathology 2005; 205 (3): 311-317. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1002/path.1705|
|Paper III Dosen, G, Tenstad E, Nygren MK, Stubberud H, Funderud S, Rian E.: “Wnt expression and canonical Wnt signaling in human bone marrow B lymphopoiesis.” BMC Immunology 2006 (7), 13. Published under a Creative Commons Attribution License. The published version of this paper is available at: https://doi.org/10.1186/1471-2172-7-13|
|Paper IV Tenstad E, Myklebost O, Rian E.: “Extensive adipogenic and osteogenic differentiation of patterned human mesenchymal stem cells in a microfluidic device.” Lab on a Chip 2010, 10, 1401-1409. The paper is removed from the thesis in DUO due to publisher restrictions. The published version is available at: https://doi.org/10.1039/b926738g|