Biology of adult human normal and leukaemia CD133+ stem cells
Recently, CD133 has been used extensively as a marker for identification of stem cells from human normal and malignant tissues. Human normal CD 133+ stem cells are capable of multilineage in vitro and in vivo differentiation providing a novel source of stem cells for regenerative therapy, whereas, malignant CD133+ stem cells are a potential therapeutic target for anti-cancer treatment. This PhD thesis investigated the neural differentiation of human adult bone marrow and foetal liver CD133+ stem cells; explored the presence of CD133 and embryonic stem cell marker Oct-4 positive stem cells in adult human normal and malignant tissues, including normal brain tissues, normal and malignant bone marrow stem cells; and evaluated the anti-acute myeloid leukaemia (AML) effect of 5 novel synthetic ajoene compounds using drug-resistant AML cell line for overcoming drug resistance in elderly AML patients. A novel serum-free culture system for inducing neural differentiation of human adult bone marrow and foetal liver CD 133+ stem cells in vitro was demonstrated. Following only two weeks' culture of selected bone marrow CD 133+ cells in serum-free medium supplemented by 50% human astrocyte conditioned medium and other cytokines, 25.5% of bone marrow CD 133+ stem cells differentiated into neural (17.8%) and glial (7.7%) cells. After three weeks' culture of selected foetal liver CD133+ cells in serum-free medium supplemented by several cytokines without astrocyte conditioned medium, only 10.8% of these stem cells differentiated into neural and glial cells. These neural differentiated cells expressed mature neural and glial markers including NF-h, NF-m, NSF and GFAP, and exhibited mature neural morphologies. The astrocyte conditioned medium was the essential ingredient in all effective serum-free culture conditions suggesting it may contain other more potent neural/glial inducing factors. The serumfree culture system is clinically relevant and provides a vehicle for generating neural cells from adult human bone marrow CD 133+ stem cells for the treatment of patients with neurodegenerative diseases. CD 1 33-isoform 2 (CD 133-2) and embryonic stem cell marker Oct-4 expression was revealed in three adult human normal and malignant tissues and cells, including normal brain (substantia nigra and striatum), normal and malignant CD34+ marrow stem cells. There was no expression of CD133-isoform I (CD133-1) in these tissues. The very small population of CD133-2 and Oct-4 positive stem cells in adult human normal substantia nigra and striatum may represent the embryonic remnants and provide a potential source of adult neural stem cells that may be induced to undergo neural diffeentiation for the treatment of neurodegenerative conditions, such as Parkinson's disease. The substantial expression of Oct-4 in adult human normal and drug-resistant myeloid leukaemia KG1a marrow stem cells demonstrates that, alongside CD133, Oct-4 is a novel cancer stem cell marker suggesting the CD 133-2 and Oct-4 positive KGIa cells are the most likely targets in disease development and are novel therapeutic targets for effective treatment of AML. Novel synthetic ajoene compounds were shown to significantly inhibit growth, induce apoptosis plus necrosis, and reduce Bcl-2 expression in human drug-resistant CD34+ AML KG 1 a cells, both alone and in combination with low dose (1 jxM) cytarabine. E/Zbenzyl was the most potent growth inhibition compound,. whereas, Z-ajoene was the most cytotoxic compound. Low dose cytarabine-induced cytotoxicity was significantly enhanced by the five novel compounds in the following order: Z-ajoene > E-ajoene > Ephthalimide> E/Z-benzyl > Z-pthalimide. The combination of Z-ajoene and low dose cytarabine provides a promising combination therapy for elderly AML patients to overcome drug resistance. This PhD thesis provides the basis for appropriate identification and clinical application of stem cells from adult human normal tissues (brain and bone marrow) and foetal liver for autologous transplantation for the treatment of neurodegenerative diseases, such as Parkinson's disease, and also the identification of cancer stem cells in human AML marrow stem cells indicating novel therapeutic targets for anti-cancer treatment. Moreover, it demonstrates significant anti-cancer effect of 5 novel synthetic ajoene compounds in drug-resistant AML cells highlighting their potential for overcoming drug resistance in elderly patients with AML.
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