The classical dogma of the central nervous system as an unchangeable entity has been refuted by the advent of direct lineage reprogramming of non-neuronal cells into neurons. During the last decade many studies including from the host laboratory have shown that brain-resident cells such as astroglia, NG2 glia, and brain pericytes, can be converted into functional induced neurons (iNs) in vitro. Transduction of glia with viruses encoding transcription factors that play key roles in neurogenesis during development were sufficient to direct astroglia towards a glutamatergic or GABAergic neuron fate. Recently, several groups, including the host lab, have succeeded in going a step further by demonstrating the feasibility of reprogramming in vivo. In fact, the host laboratory has published that Sox2 (alone or in combination with Ascl1) is able to convert NG2 glia into iNs in the injured adult mouse cerebral cortex. However, it remains unknown whether such lineage reprogramming in vivo can also be achieved in the case of human astrocytes which differ markedly from their rodent counterparts in size and complexity. To study lineage conversion of human astrocytes in vivo has been hindered by the lack of experimental systems that allow for the growth and maturation of human astrocytes within a human in vivo-like tissue context. In this project, I took advantage of the recent advent of human cerebral organoids technology. Several groups have in parallel developed the technology of cerebral organoids derived from human induced pluripotent stem cells (hiPSCs). These 3D systems enable the generation of miniature organ-like structures that mimic in many aspects human neurodevelopment. As occurs in normal brain development, in these cerebral organoids astrogliogenesis follows upon neurogenesis of deep and superficial layer neurons. In this proposal I defined the following objectives (Figure 1):
1- Generation of hiPSC lines allowing for inducible and cell-type specific expression of reprogramming factors by genome-editing
2- Characterization of astrogliogenesis in human cerebral organoids
3- Induction of human astroglia reprogramming in a human in vivo-like tissue context using cerebral organoids
