Tasnim, F. | bioRxiv Science

bioRxiv2025

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

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.

immunology↗

bioRxiv2025

Investigation of mitochondrial phenotypes in motor neurons derived by direct conversion of fibroblasts from familial ALS subjects

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease of motor neurons, leading to fatal muscle paralysis. Familial forms of ALS (fALS) account for approximately 10% of cases and are associated with mutations in numerous genes. Alterations of mitochondrial functions have been proposed to contribute to disease pathogenesis. Here, we employed a direct conversion (DC) technique to generate induced motor neurons (iMN) from skin fibroblasts to investigate mitochondrial phenotypes in a patient-derived disease relevant cell culture system. We converted 7 control fibroblast lines and 17 lines harboring the following fALS mutations, SOD1A4V, TDP-43N352S, FUSR521G, CHCHD10R15L, and C9orf72 repeat expansion. We developed new machine learning approaches to identify iMN, analyze their mitochondrial function, and follow their fate longitudinally. Mitochondrial and energetic abnormalities were observed, but not all fALS iMN lines exhibited the same alterations. SOD1A4V, C9orf72, and TDP-43N352S iMN had increased mitochondrial membrane potential, while in CHCHD10R15L cells membrane potential was decreased. TDP-43N352S iMN displayed changes in mitochondrial morphology and increased motility. SOD1A4V, TDP-43N352S, and CHCHD10R15L iMN had increased oxygen consumption rates and altered extracellular acidification rates, reflecting a hypermetabolic state similar to the one described in sporadic ALS fibroblasts. FUSR521G mutants had decreased ATP/ADP ratio, suggesting impaired energy metabolism. We then tested the viability of iMN and found decreases in survival in SOD1A4V, C9orf72, and FUSR521G, which were corrected by small molecules that target mitochondrial stress. Together, our findings reinforce the role of mitochondrial dysfunction in ALS and indicate that fibroblast-derived iMN may be useful to study fALS metabolic alterations. Strengths of the DC iMN approach include low cost, speed of transformation, and the preservation of epigenetic modifications. However, further refinement of the fibroblasts DC iMN technique is still needed to improve transformation efficiency, reproducibility, the relatively short lifespan of iMN, and the senescence of the parental fibroblasts.

neuroscience↗