Generation of Stem Cell-Derived Kupffer Cells for Application in Human In Vitro Inflammatory Liver Model

There is an evident, unmet need to develop a commercially available in vitro system that can model inflammatory states of the liver and predict immune-mediated hepatotoxicity of drugs and xenobiotics taken under inflamed conditions. Hepatocyte-Kupffer cell co-cultures can model inflammation-mediated hepatotoxicity; however, Kupffer cell (KC) source remains an important bottleneck for the development of such models. Primary human Kupffer cells (PHKCs) are costly, limited in availability and exhibit donor variability. An alternative cell source for KCs has not been reported. Important paradigm shift from the classical dogma of adult blood-circulating monocyte-derived macrophages to intrahepatic precursor/fetal monocyte-derived macrophages has shed new light into the origin of KCs in vivo. Based on these recent findings, we report here, a novel method to generate human KCs in vitro from stem cells (hPSC-KCs) via fetal monocytes. hPSC-KCs expressed macrophage markers, CD11, CD14, CD68, CD163 and CD32 at gene and protein level and exhibited functional properties such as phagocytosis and Interleukin-6 and Tumor Necrosis Factor-4alpha production upon activation. Importantly, molecular signature, liver-macrophage specific CLEC-4F expression and cytokines production levels of hPSC-KCs were similar to PHKCs but different from non-liver macrophages. We used an inflammatory liver co-culture model to demonstrate that activated hPSC-KCs, but not non-liver macrophages, were able to recapitulate effects of PHKCs when stimulated with paradigm hepatotoxicants. hPSC-KCs developed in this study offer a renewable human cell source for liver-specific macrophages which can be used to develop in vitro systems for modelling the inflammatory state of the liver. Gene expression profiles of 9 samples were determined using Human Gene 2.0 ST Array. These 9 samples included three replicates each of PHKCs (primary human Kupffer cells), human pluripotent stem cell-dervied Kupffer cells (hPSC-KCs) and non-liver macrophages (NL-Mφ). Human pluripotent stem cells were differentiated into Kupffer cells (hPSC-KCs). To analyze the molecular signature of hPSC-KCs, microarray was performed on three independent replicates (hPSC-KCs_1, hPSC-KCs_2 and hPSC-KCs_3). Microarray was also used to obtain gene expression profiles of primary human Kupffer cells (PHKCs) and non-liver macrophages (NL-Mφ) for comparison with hPSC-KCs. The data was normalized together with and compared to previously published data on hPSC-IMR90, bone marrow-derived monocytes, blood monocyte derived macrophages, alveolar macrophages and microglia from Zimmerlin L, et al. (PMID: 27660325), Abbas AR, et al. (PMID: 15789058), Beyer M, et al. (PMID: 23029029), Worgall S, et al. (PMID: 16041053) and Rock R, et al. (PMID: 16163375).