Engineering Immunocompetent Systems for Modeling, Modulating, and Treating Inflammatory Diseases

Dysregulated inflammation is a central driver of a significant fraction of all human diseases, including chronic metabolic conditions, tumor metastasis, autoimmune disorders, and aging. Despite numerous advancements in tissue engineering and disease modeling, our ability to accurately capture cell-mediated tissue inflammation in vitro that faithfully mimics in vivo human physiology remains limited. Here, we propose to combine human pluripotent stem cell (hPSCs) and genetic engineering tools to create immunocompetent tissue models that replicate the natural behavior of tissue-resident macrophages with on-demand control over inflammatory states. Specifically, we suggest an immunoengineering strategy to generate human microtissues in a dish containing bona fide resting tissue-resident macrophages. We aim to accomplish this using a “progenitor- based assembly” tissue engineering approach, where myeloid progenitors derived from hPSCs are combined with their developmentally matched tissue and vascular counterparts. Target model systems for this research will include skeletal muscle, liver, and adipose microtissues, as these are all known to exhibit dysregulated inflammation in the context of metabolic diseases—a globally pressing clinical need. Along with strategies to generate and characterize these tissues, we propose to establish bioprocessing procedures to enhance the scale and ability to cryopreserve key progenitors that will enable the dissemination of these tools to labs focused on tissue inflammation research but lack expertise in tissue engineering or stem cell biology. With the development of these microtissues, we then aim to develop and employ genetic engineering tools to overcome known limitations in conventional controllable gene induction systems that are not functionally compatible with hPSCs and their derivatives. Using these tools and a novel lineage tracing and retrieval approach, we further propose to develop multi-lineage CRISPR/Cas9 screens that will identify cell-mediated inflammatory mechanisms that modulate neighboring metabolic tissue cells – the core essence of immunoregulation. Additionally, we will employ these approaches to drive cell-mediated inflammatory and anti-inflammatory states within tissues, overcoming challenges associated with the limited efficacy of in vitro macrophage via recombinant cytokines. Lastly, we will explore using our tool sets to evaluate adoptive hPSC-macrophage transfer strategies as a test bed for immunotherapy development. As part of this effort, we will test hypotheses regarding the “open niche” dependency for successful transfer, the long-term fate and durability of macrophage phenotypes after transfer, and develop additional, more therapeutically relevant genetic tools to modulate these processes. This research will ultimately deepen our understanding of tissue–macrophage biology, create new tools for studying immunoregulatory processes, and develop putative therapeutic strategies for metabolic and inflammatory diseases. Collectively, we aim to greatly expand our understanding of macrophage biology and establish transformative new tools for stem cell-derived tissue modeling and regenerative immunotherapy. Project Number: 1R35GM162637-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Andrew Khalil | Institution: UNIVERSITY OF MINNESOTA, MINNEAPOLIS, MN | Award Amount: $398,144 | Activity Code: R35 | Study Section: Special Emphasis Panel[ZRG1 MCST-Q (55)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11272683