Kirill Chesnov

All Publications

• Defined human tri-lineage brain microtissues. bioRxiv : the preprint server for biology Uenaka, T., Jung, S., Kumar, I., Vodehnal, K., Rastogi, M., Yoo, Y., Koontz, M., Thome, C., Li, W., Chan, T., Green, E. M., Chesnov, K., Sun, Z., Zhang, S., Wang, J., Venida, A., Mellier, A. M., Atkins, M., Jackrel, M., Skotheim, J. M., Wyss-Coray, T., Abu-Remaileh, M., Lashuel, H. A., Bassik, M. C., Südhof, T. C., Del Sol, A., Ullian, E., Wernig, M. 2025

  Abstract

  Microglia are the immune cells of the central nervous system and are thought to be key players in both physiological and disease conditions. Several microglial features are poorly conserved between mice and human, such as the function of the neurodegeneration-associated immune receptor Trem2. Induced pluripotent stem cell (iPSC)-derived microglia offer a powerful opportunity to generate and study human microglia. However, human iPSC-derived microglia often exhibit activated phenotypes in vitro, and assessing their impact on other brain cell types remains challenging due to limitations in current co-culture systems. Here, we developed fully defined brain microtissues, composed of human iPSC-derived neurons, astrocytes, and microglia, co-cultured in 2D or 3D formats. Our microtissues are stable and self-sufficient over time, requiring no exogenous cytokines or growth factors. All three cell types exhibit morphologies characteristic of their in vivo environment and show functional properties. Co-cultured microglia develop more homeostatic phenotypes compared to microglia exposed to exogenous cytokines. Hence, these tri-cultures provide a unique approach to investigate cell-cell interactions between brain cell types. We found that astrocytes and not neurons are sufficient for microglial survival and maturation, and that astrocyte-derived M-CSF is essential for microglial survival. Single-cell and single-nucleus RNA sequencing analyses nominated a network of reciprocal communication between cell types. Brain microtissues faithfully recapitulated pathogenic α-synuclein seeding and aggregation, suggesting their usefulness as human cell models to study not only normal but also pathological cell biological processes.

  View details for DOI 10.1101/2025.08.05.668605

  View details for PubMedID 40799568

  View details for PubMedCentralID PMC12340862

• Generation of human excitatory forebrain neurons by cooperative binding of proneural NGN2 and homeobox factor EMX1. Proceedings of the National Academy of Sciences of the United States of America Ang, C. E., Olmos, V. H., Vodehnal, K., Zhou, B., Lee, Q. Y., Sinha, R., Narayanaswamy, A., Mall, M., Chesnov, K., Dominicus, C. S., Sudhof, T., Wernig, M. 2024; 121 (11): e2308401121

  Abstract

  Generation of defined neuronal subtypes from human pluripotent stem cells remains a challenge. The proneural factor NGN2 has been shown to overcome experimental variability observed by morphogen-guided differentiation and directly converts pluripotent stem cells into neurons, but their cellular heterogeneity has not been investigated yet. Here, we found that NGN2 reproducibly produces three different kinds of excitatory neurons characterized by partial coactivation of other neurotransmitter programs. We explored two principle approaches to achieve more precise specification: prepatterning the chromatin landscape that NGN2 is exposed to and combining NGN2 with region-specific transcription factors. Unexpectedly, the chromatin context of regionalized neural progenitors only mildly altered genomic NGN2 binding and its transcriptional response and did not affect neurotransmitter specification. In contrast, coexpression of region-specific homeobox factors such as EMX1 resulted in drastic redistribution of NGN2 including recruitment to homeobox targets and resulted in glutamatergic neurons with silenced nonglutamatergic programs. These results provide the molecular basis for a blueprint for improved strategies for generating a plethora of defined neuronal subpopulations from pluripotent stem cells for therapeutic or disease-modeling purposes.

  View details for DOI 10.1073/pnas.2308401121

  View details for PubMedID 38446849