Small molecule to enhance the haematopoietic differentiation capability in mouse induced pluripotent stem cells

Haematopoiesis is the process of blood and immune cell production that, for life, is sustained by a rare population of bone marrow resident haematopoietic stem and progenitor cells (HSPCs). Over the last 50 years, the ability of HPSCs to rescue haematopoiesis has been used therapeutically in bone marrow transplantation following myeloablative therapy for haematological malignancies/disorders. Of further biological and clinical relevance, HSPC function must be strictly regulated in order to prevent the accumulation of genetic/epigenetic mutations that generate leukaemia stem cells (LSCs) which cause leukaemia. In this thesis, I have built on previous work in the laboratory where a multi-species stem cell based forward screen was conducted on a chemical library with the objective to (i) target HSPC function in vitro and in vivo to improve the practice of bone marrow transplantation and (ii) therapeutically target LSCs, which are the basis for therapy resistance and relapse in a sub-type of leukaemia called acute myeloid leukaemia (AML). Three lead small molecules were identified in this screen: Phthalylsulfathiazole, a sulphonamide, Yohimbine, an alpha-2 adrenergic receptor antagonist, and, Cis-2-Methyl-5-trimethylammonium methyl-1, 3-oxathiolane iodide (Oxa-22), M3 muscarinic acetylcholine receptor agonist. Here, I used induced pluripotent stem cells (iPSCs), a type of stem cell produced in the laboratory by genetic reprogramming of somatic cells, to assess the ability of Phthalylsulfathiazole and Yohimbine to potentiate the hematopoietic differentiation programme of iPSCs, which could eventually be used in bone marrow transplantation or transfusion medicine. As proof-of-principle for clinical application, we initially established a mouse iPSC culture and haematopoietic differentiation system in the laboratory. Gene expression analysis revealed that Phthalylsulfathiazole exposure appeared to enhance haematopoietic gene expression following haematopoietic commitment of iPSCs, while Yohimbine increased the haematopoietic gene expression programme during both haematopoietic specification from iPSCs and post-haematopoietic commitment. In functional experiments, iPSCs that were exposed to Yohimbine demonstrated improved granulocyte-macrophage potential in colony-forming cell assays. These data suggest that both Phthalylsulfathiazole and Yohimbine are promising agents to enhance haematopoietic differentiation from iPSCs in vitro. As Yohimbine and Oxa-22 has been shown to enhance HSPC function in vivo after transplantation, in the short-term, I also sought to evaluate whether these small molecules affected long-term functioning of HSCs, including haematopoietic differentiation potential. Yohimbine and Oxa-22 did not affect long-term HSC function or differentiation. Preliminary evidence, in contrast, suggests that Phthalylsulfathiazole administration in vivo may be detrimental to long-term HSC functioning. Finally, I assessed the impact of the small molecules to target human AML cell lines. In THP-1 cells, Yohimbine and Oxa-22, but not Phthalylsulfathiazole, alleviated the differentiation block normally observed in AML cells, as assessed by immunophenotyping for myeloid markers, but without impacting either cell cycling status or cell survival. Collectively, the data presented in this thesis suggest that both Yohimbine and Oxa-22 have the conserved ability to potentiate haematopoietic differentiation from both iPSCs and AML cells in vitro and they do not adversely impact HSPC function in vivo. Further experimentation is required to elucidate the molecular mechanisms underscoring Yohimbine and Oxa-22 mediated normal and leukaemic function in vitro and in vivo with a view that these investigations could facilitate their application in clinical haematology settings