A 3D human co-culture to model neuron-astrocyte interactions in tauopathies

Abstract

Background Intraneuronal tau aggregation is the major pathological hallmark of neurodegenerative tauopathies. It is now generally acknowledged that tau aggregation also affects astrocytes in a cell non-autonomous manner. However, mechanisms involved are unclear, partly because of the lack of models that reflect the situation in the human tauopathy brain. To accurately model neuron-astrocyte interaction in tauopathies, there is a need for a model that contains both human neurons and human astrocytes, intraneuronal tau pathology and mimics the three-dimensional architecture of the brain. Results Here we established a novel 100–200 µm thick 3D human neuron/astrocyte co-culture model of tau pathology, comprising homogenous populations of hiPSC-derived neurons and primary human astrocytes in microwell format. Using confocal, electron and live microscopy, we validate the procedures by showing that neurons in the 3D co-culture form pre- and postsynapses and display spontaneous calcium transients within 4 weeks. Astrocytes in the 3D co-culture display bipolar and stellate morphologies with extensive processes that ensheath neuronal somas, spatially align with axons and dendrites and can be found perisynaptically. The complex morphology of astrocytes and the interaction with neurons in the 3D co-culture mirrors that in the human brain, indicating the model’s potential to study physiological and pathological neuron-astrocyte interaction in vitro. Finally, we successfully implemented a methodology to introduce seed-independent intraneuronal tau aggregation in the 3D co-culture, enabling study of neuron-astrocyte interaction in early tau pathogenesis. Conclusions Altogether, these data provide proof-of-concept for the utility of this rapid, miniaturized, and standardized 3D model for cell type-specific manipulations, such as the intraneuronal pathology that is associated with neurodegenerative disorders.

A Human Neuron/Astrocyte Co‐culture to Model Seeded and Spontaneous Intraneuronal Tau Aggregation

“…These data, as well the data presented in our primary research paper (Batenburg et al., 2022), demonstrate the feasibility of FTDtau 1+2 to model tau aggregation without the use of seeds that have thus far been a requirement for the induction of tau aggregation in human iPSC‐derived neurons (Verheyen et al., 2015). In addition, we have showed that expression of FTDtau 1+2 induces tau aggregation in a three‐dimensional neuron/astrocyte co‐culture (Batenburg et al., 2023), highlighting the versatility of this approach to induce spontaneous tau aggregation in more advanced cell culture models. Although seeds may be heterogeneous and directly affect neurons and astrocytes independent of intraneuronal tau pathology, their use represents an established paradigm in cellular models and may be of interest for specific research questions.…”

“…Human iPSCs should harbor a stably integrated doxycycline-inducible rtTA/Ngn2 transgene cassette. We used a previously generated clonal human iPSC line stably expressing rtTA/Ngn2 (Frega et al, 2017) that was maintained as previously described (Batenburg et al, 2022(Batenburg et al, , 2023. For culture procedures for human iPSCs, see Support Protocol 1.…”

“…previously described (Batenburg et al, 2022(Batenburg et al, , 2023. For the culture procedures of human primary astrocytes, see Support Protocol 2.…”

“…Finally, biochemical extraction and subsequent western blot analysis can be performed to confirm the formation of sarkosyl-insoluble tau aggregates. Examples of such analyses can be found in the primary research papers (Batenburg et al, 2022(Batenburg et al, , 2023.…”

“…We have also validated that tau pathology can be addressed using the fluorescence intensity standard deviation of fluorescently labelled FTDtau (Batenburg et al., 2022). Thiophene dyes such as HS169 (Shirani et al., 2015) can also be used to label stacked beta sheets of pathological FTDtau (Batenburg et al., 2022, 2023). Finally, biochemical extraction and subsequent western blot analysis can be performed to confirm the formation of sarkosyl‐insoluble tau aggregates.…”