Generation of Self-Assembling 3D Neural Micro-Organoids using Human iPSC-Derived Neuronal Progenitors and Microglia

INTRODUCTION: Recent studies showed that microglia (MG) play critical roles in neurodegeneration, and mutations of specific genes in MG increase the risk of neurodegenerative diseases, including Alzheimer’s disease (AD). Although murine models have started to shed light on the role of MG in AD, the lack of human models hampers the study of MG-related neurodegeneration. With the advent of induced pluripotent stem cell (iPSC) technology, and knowing that in-vitro 3D co-culture better approximates the complex in-vivo cellular organization than 2D monolayer co-culture, we set out to generate self-assembling 3D neural micro-organoids of human origin as potential in-vitro 3D cellular models for unravelling neuronal-MG crosstalk relevant to neurodegeneration. MATERIALS AND METHODS: To facilitate lineage tracing, human iPSCs were genetically engineered to constitutively express either TdTomato or GFP fluorescent protein from the safe harbor AAVS1 locus. Using in-house protocols, the cells were differentiated into human neuronal progenitor cells (hNPCs-TdTomato) (Garcia Leon, et al., Alzheimer’s & Dementia, 2018) or human microglia-like cells (hMGs-GFP) (Claes et al., Alzheimer’s & Dementia, in press) respectively. At a hNPCs:hMGs ratio of 2:1, the cells were mixed and seeded onto agarose substrates imprinted with 121 microwells (Ф500 µm x H500 µm). The cells were cultured in neural maintenance medium to allow self-assembling of neural micro-organoids. Micro-organoid formation was monitored by brightfield and fluorescent microscopy. Differentiation and maturation of hNPCs and hMGs was analyzed by qRT-PCR. As an alternative approach, cerebral brain organoids were generated via a well-accepted published protocol, followed by seeding of hMGs onto the organoids. RESULTS AND DISCUSSION: hiPSC-derived hNPC:hMG mixtures started to self-assemble into micro-organoids after 9 days of culture (Fig. 1a). The micro-organoids consisted of hNPCs-dTomato (red) and hMGs-GFP cells, and discrete hNPCs and hMGs submicro-organoids with distinctive spheroid morphologies (Fig. 1b). Alternatively, hiPSCs were differentiated towards cerebral brain organoids, and the neuronal marker, Nestin, was detected after 21 days (Fig. 2a & 2f). Interestingly, after seeding hMGs (Fig. 2b) on the organoids, clear morphological changes were observed in the co-culture (Fig. 2c–2e). CONCLUSION: By harnessing the self-assembling property of hiPSC-derived hNPCs and hMGs, and assisted by robust differentiation protocols, neural micro-organoids or cerebral brain organoids were generated. These organoids represent unprecedented in-vitro 3D models essential for unravelling neuron-microglia crosstalk linked to neurodegeneration. ACKNOWLEDGEMENT: This research project is supported by the IWT_SBO-iPSCAF (IWT 150031 iPSC), KU Leuven C1-3DMuSyC grant (C14/17/111), and IWT Aspirant grant (SC/1S1071N).status: publishe