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Human liver sinusoidal endothelial cells support the development of functional human pluripotent stem cell-derived Kupffer cells

ABSTRACT: In mice, Kupffer cells (KCs) first derive from yolk sac (YS) hematopoietic progenitors prior to the emergence of the hematopoietic stem cell. To investigate human KC ontogeny, we recapitulated YS hematopoiesis from human pluripotent stem cells (hPSCs) and transplanted derivative macrophage progenitors into NSG mice previously humanized with hPSC-liver sinusoidal endothelial cells (LSECs). We demonstrate that hPSC-LSECs facilitate stable hPSC-YS-macrophage engraftment for at least 7 weeks. Single cell RNA sequencing of engrafted YS-macrophages revealed a homogenous MARCO-expressing KC gene signature and low expression of inflammatory macrophage genes. In contrast, human cord blood (CB)-derived macrophages generated grafts that contain multiple hematopoietic lineages in addition to KCs. Functional analyses showed that the engrafted KCs actively perform phagocytosis and erythrophagocytosis in vivo. Taken together, these findings demonstrate that it is possible to generate human KCs from hPSCs and show that the equivalent of the human YS hematopoietic programs represent enriched sources of progenitors for this lineage.

ORGANISM(S): Mus musculus Homo sapiens

PROVIDER: GSE250618 | GEO | 2024/08/09

REPOSITORIES: GEO

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Project description: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).

Project description:Next-generation sequencing (NGS) has significantly facilitated the analysis of the gene profile and elucidated the molecular mechanisms important for specific cell lineage differentiated from human pluripotent stem cells (hPSCs). Here we report the application of RNA-sequencing technology for transcriptome profile of primary human brain microvascular endothelial cells (BMECs) and hPSC-derived BMECs, and comparison of transcriptomes among cells, including hPSCs, mesodermal progenitors, ectodermal progenitors, endodermal progenitors, hBMECs and hPSC-derived BMECs from hPSCs. Four different BMEC samples differentiated from hPSCs by two different methods and hBMECs were performed in IIIumina HiSeq2500. The resulting sequence reads (about 20 million reads per sample) were mapped to human genome (hg19) using HISAT, and the RefSeq transcript levels (FPKMs) were quantified using the python script rpkmforgenes.py. We next analyzed the expression of a subset of genes that regulate key BBB attributes, including tight junctions and molecular transporters. The gene set comprises 20 tight junction-related genes and an unbiased list of all 25 CLDN genes, all 407 solute carrier (SLC) transporters, and all 53 ATP-binding cassette (ABC) transporters regardless of prior knowledge of BBB association. Primary human BMECs expressed 234 of these genes. BMECs differentiated from hPSCs via the defined method expressed many of these same genes (206 of 234 (88%)) as did BMECs differentiated via the UM method (208 of 234 (89%), indicating a close similarity between hPSC-derived BMECs and primary human BMECs with respect to transcripts having likely relevance to BBB function. RNA sequencing was used to compare global gene expression profiles in the hPSC-derived BMECs with BMECs generated from our previously reported co-differentiation system and primary human BMECs. As expected, hPSC-derived BMECs from three independent differentiations clustered closely and were similar to those generated from the undefined UM platform. Moreover, the hPSC-derived BMECs clustered with primary human BMECs and were distinct from undifferentiated hPSCs and hPSC-derived ectoderm, endoderm and mesodermThis study provides detailed transcriptome comparison between hBMECs and hPSC-derived BMECs and unveils important genes related BMEC phenotypes.

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Human liver sinusoidal endothelial cells support the development of functional human pluripotent stem cell-derived Kupffer cells

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