Directed differentiation of human embryonic stem cells to microglial-like cells

Protein aggregations of β-Amyloid (Aβ) and Tau are alone not sufficient to account for all the symptoms and progression of Alzheimer’s Disease (AD), as such there is much emphasis upon the immune component of the disease. The cells of the brain that are capable of initiating an immune response are microglia; these are derivatives of hematopoietic cells and they have the capacity to phagocytose and clear Aβ aggregates or release cytokines such as TNF-a, in a neurotoxic role, promoting apoptosis of the surrounding neurons and hence aiding disease progression. To uncover the precise role of microglia in terms of AD we propose a protocol to enable differentiation of microglia from human embryonic stem cells (hESCs). The protocol we propose is a two-step differentiation procedure i) hESCs to monocytes followed by ii) ES-derived monocytes to microglia. Chapter 3: Exogenous over-expression of PU.1, a transcription factor vital in both the onset of haematopoiesis and the terminal differentiation of monocytes and microglia, was revealed to enable differentiation to a hematopoietic fate. Chapter 4: hESCS were differentiated to monocyte-like cells (CD45+/CD11b+) through culture in the presence of the hematopoietic growth factors; M-CSF and IL-3. Chapter 5: focuses on the differentiation of ES-derived monocytes to microglia. It shows that monocytes cultured in astrocyte conditioned medium give rise to cells that are IBA-1+/Glut5+/CD45low/NG2low/CD80+/CD11c+ and have a ramified microglial phenotype, which upon stimulation with Aβ(1-42) can become activated to the amoeboid phenotype. The development of the protocol for the generation of microglia holds great importance in terms of creating in vitro models for AD research as a whole and can be extended to the differentiation of patient IPSCs that contain mutations in genes associated with innate immunity or SNPs associated with AD risk, disease onset and progression