Direct contact between iPSC-derived macrophages and hepatocytes drives reciprocal acquisition of Kupffer cell identity and hepatocyte maturation

Abstract Hepatic macrophages play central roles in liver homeostasis, injury, and immune-mediated hepatotoxicity through dynamic crosstalk with hepatocytes. While monocyte-derived macrophages have been widely used in vitro, they do not fully recapitulate the biology of liver-resident Kupffer cells (KCs), which are embryonically derived and maintained locally. Recent advances suggest that induced pluripotent stem cell (iPSC)-derived macrophages (iMacs) more closely resemble embryonic macrophages and may therefore serve as a relevant platform to model KC biology. Here, we developed a human iPSC-based co-culture system by combining iMacs with iPSC-derived hepatocytes (iHeps) derived from the same donor, enabling direct cell–cell interactions. We hypothesized that such interactions would both enhance hepatocyte maturation and promote KC-like differentiation of iMacs. Indeed, co-culture induced KC-like phenotypes in iMacs and improved functional maturation of iHeps, highlighting the importance of bidirectional cellular communication. Comparative analyses with iMacs cultured in hepatocyte-conditioned media revealed that direct contact provides additional signals beyond soluble factors in driving hepatic macrophage specialization. Functionally, this co-culture system demonstrated improved physiological relevance, particularly in modeling immune-mediated drug responses, as evidenced by enhanced cytokine production profiles upon exposure to a panel of test compounds. Overall, this study establishes a novel human iPSC-derived platform that captures key aspects of hepatocyte–macrophage crosstalk, providing a more physiologically relevant model to investigate liver biology and assess immune-mediated drug toxicity.