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Characterization of Thy-1 expression on human hematopoietic cells

University of British Columbia

Hematopoietic stem cells have extensive proliferative capacity and the ability to produce daughter cells of all hematopoietic lineages. However, their characterization is complicated by their low frequency and the heterogeneous cellular composition of the tissues in which they are found. The ability to obtain pure populations of stem cells would be useful not only for basic studies of stem cell biology, but additionally for clinical procedures involving gene therapy or tumour cell purging. Numerous techniques and reagents have been developed towards this goal; however, it has been very difficult to obtain a biologically homogeneous population of human hematopoietic stem cells. With the initial purpose of producing new reagents for human hematopoietic stem cell purification, I helped to generate a panel of monoclonal antibodies by immunizing mice with various human hematopoietic cell lines. Several monoclonal antibodies were isolated which reacted with subpopulations of the few percent of human bone marrow cells that express CD34, a molecule that is selectively expressed on hematopoietic precursor cells including those required for long-term reconstitution of hematopoiesis in vivo. The property of one of these monoclonal antibodies, referred to as 5E10, that was raised following immunization with a human erythroleukemia cell line, HEL, was chosen for more detailed studies. 5E10 was subsequently shown to react with Thy-1, a phosphatidylinositol-anchored cell surface protein belonging to the immunoglobulin supergene family. This was established by isolation and sequencing of a cDNA cloned by immunoselection of COS cells transfected with a cDNA library derived from a 5E10⁺ cell line. 5E10 staining of hematopoietic cells from human fetal liver, cord blood, adult peripheral blood, and bone marrow samples revealed that Thy-1 expression is restricted to approximately 1-4% of fetal liver, cord blood and bone marrow cells. These consist of a small subset of lymphoid cells and approximately 25% of all CD34⁺ cells. Thy-1⁺CD34⁺ cells were further characterized as CD38lo/CD45RO⁺/CD45RA⁻/CD71lo/c-kitlo and rhodamine-123dull. When CD34⁺ cells were sorted on the basis of Thy-1 expression, the majority of clonogenic hematopoietic progenitor cells were recovered in the Thy-1⁻CD34⁺ fraction, whereas the majority of cells giving rise to clonogenic progenitors after 5-8 weeks of culture on irradiated marrow adherent feeder layers (i.e., long-term culture initiating cells or LTC-IC) were recovered in the Thy-1⁺CD34⁺ fraction. In addition to being expressed on a subset of CD34⁺ cells, Thy-1 was found on a small (<1%) number of CD3⁺CD4⁺ CD34⁻ lymphocytes in bone marrow, cord blood, and peripheral blood. The demonstration of a restricted expression of Thy-1 on primitive human hematopoietic cells confirms and extends recent observations of others indicating that Thy-1 expression can be used to obtain enriched populations of very primitive hematopoietic cells. While the function of Thy-1 is unknown, its differential expression on early stages of hematopoietic cell development suggests that it may have a specific role in very early and/or quiescent cells.

Thesis/Dissertation

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2008-08-08

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http://hdl.handle.net/2429/1319

Genetics

University of British Columbia

UBCV

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