✍🏼 Claire Gizowski, Galina Popova, Heather Shin, Marius M Mader, Wendy Craft, Wenjun Kong, Yohei Shibuya, Bernd J Wranik, Yuheng C Fu, Constanze Depp, Tzuhua D Lin, Baby Martin-McNulty, Yongjin Yoo, Po-Han Tai, Maximilian Hingerl, Kayla Leung, Micaiah Atkins, Nicole Fong, Devyani Jogran, Agnieszka Wendorff, David Hendrickson, Astrid Gillich, Andy Chang, Beth Stevens, Marius Wernig, Oliver Hahn

🏠 Calico Life Sciences, South San Francisco, CA, USA

📑 bioRxiv (2025)

Abstract
Aging, the key risk factor for cognitive decline, impacts the brain in a region-specific manner, with microglia among the most affected cell types. However, it remains unclear whether this is intrinsically mediated or driven by age-related changes in neighboring cells. Here, we describe a scalable, genetically modifiable system for in vivo heterochronic myeloid cell replacement. We find reconstituted myeloid cells adopt region-specific transcriptional, morphological and tiling profiles characteristic of resident microglia. Young donor cells in aged brains rapidly acquired aging phenotypes, particularly in the cerebellum, while old cells in young brains adopted youthful profiles. We identified STAT1-mediated signaling as one axis controlling microglia aging, as STAT1-loss prevented aging trajectories in reconstituted cells. Spatial transcriptomics combined with cell ablation models identified rare natural killer cells as necessary drivers of interferon signaling in aged microglia. These findings establish the local environment, rather than cell-autonomous programming, as a primary driver of microglia aging phenotypes.

How the WOLF was used in this study
In this article, the WOLF G2 was used to assess blood chimerism in reconstituted mice following engineered hematopoietic stem cell (eHSC) transplantation. Peripheral blood was collected by tail nick, red blood cells were lysed, and leukocytes were filtered to generate a single-cell suspension for analysis. Cells were then analyzed on the WOLF G2 and gated based on size and GFP fluorescence to identify donor-derived, genetically modified cells expressing an EGFP reporter. This flow cytometric analysis enabled quantitative evaluation of engraftment efficiency and chimerism levels over time, providing a non-terminal means to confirm successful hematopoietic reconstitution prior to experimental takedown. The authors note that relatively low EGFP expression from the reporter construct may have led to conservative estimates of true chimerism, underscoring the sensitivity requirements of this analysis step.