BACKGROUND:Organ transplantation remains the only curative treatment for end-stage organ failure. However, a critical shortage of donor organs presents a major challenge. Human induced pluripotent stem cell (hiPSC)-derived organoids represent a promising technology for generating transplantable human tissues. This study aimed to create functional and scalable tissue from hiPSC-derived liver organoids (hiPSC-LOs) by establishing an oxygenation system using a decellularized liver (DL), which retains the parenchymal and vascular extracellular matrix to support cell adhesion and medium perfusion. METHODS:First, the culture condition of hiPSC-LOs was refined to enhance their engraftment in rat DLs. Next, an oxygenation system for hiPSC-LO-engrafted DLs (hiPSC-liver tissue [hiPSC-LT]) was established using perfusion of oxygen-enriched medium containing artificial red blood cells. Finally, the oxygenated hiPSC-LTs were transplanted onto the liver surface of immunodeficient mice. RESULTS:Functional hiPSC-LTs were successfully generated by infusing day 8 precultured hiPSC-LOs into DLs. Perfusion with oxygenated medium containing artificial red blood cells suppressed cell death and promoted hepatic function of hiPSC-LTs by mimicking the physiological oxygen concentration found in the fetal liver. Notably, oxygenated hiPSC-LTs demonstrated improved engraftment in vivo, as evidenced by increased human albumin levels in recipient mouse serum compared with nonoxygenated controls. CONCLUSIONS:The oxygenation system using DLs and artificial red blood cells effectively supported the generation of transplantable, functional hiPSC-LT. This culture strategy enhances both the enlargement and functionality of hiPSC-organoids and represents a promising step toward developing alternatives to conventional organ transplantation.

Bioengineered livers using decellularized extracellular matrix (ECM) scaffolds hold promise for transplantation therapies. However, conventional recellularization methods involving cell injection via blood vessels encounter challenges in recapitulating the complexity of hepatic tissues, including high cellularity, differentiation hierarchy, and cellular heterogeneity. Here, we developed a methodology termed “combined single-cell and organoid (CSO) injection.” We injected human liver organoids (HLOs) and dispersed organoid cells via direct puncture and vascular injection into a decellularized porcine ECM scaffold, respectively. This process facilitated the construction of densely populated and self-organized liver-like tissues (CSO Livers) that formed cell-cell/cell-ECM interactions. The tissues displayed hepatic characteristics, including ALBUMIN secretion, robust expression of liver-associated genes, and cellular heterogeneity comprising hepatocytes, cholangiocytes, as well as stellate-like and endothelial cells. Furthermore, we demonstrated that CSO-Livers functioned as grafts in immunodeficient mice and in a microminipig model of liver fibrosis through transplantation via vascular anastomosis. These findings indicate the applicability of the combined single-cell and organoid (CSO) injection method for generating bioengineered liver grafts using HLOs.

Summary The specific contribution of tissue-resident macrophages (TRMs) to pancreatic ductal adenocarcinoma (PDAC) progression remains unclear. Here, we found that a high abundance of TRM-derived tumor-associated macrophages (TRM-TAMs) is an independent indicator of poor prognosis in patients with PDAC. To elucidate the underlying mechanism, we established an advanced organoid platform (iMac-FPCO), which incorporates macrophages derived from human induced pluripotent stem cells to reflect the differentiation process of TRMs. Single-cell transcriptomic analysis revealed this model recapitulates the transcriptional identity of TRM-TAMs in patient tissue. We demonstrated that TRM-TAMs drive cancer cell proliferation, while maintaining chemoresistance, and identified TRM-derived insulin-like growth factor 1 (IGF1) as the critical mediator. This result provides a rationale for why previous trials targeting IGF1 receptor (IGF1R) failed to improve survival in unselected patient populations. We hypothesize that stratifying patients by TRM-TAM abundance could help identify a responsive subgroup, thereby reviving IGF1R-targeted therapy as a viable treatment for PDAC.

Pancreatic ductal adenocarcinoma (PDAC) organoids that simulate the tumor microenvironment (TME) are an effective tool to identify how TME affects PDAC malignancy. We present a protocol for generating a fused pancreatic cancer organoid (FPCO) that partly reproduces the TME, including heterogeneous cancer-associated fibroblasts (CAFs), using patient-derived PDAC cells and human-induced pluripotent cell-derived endothelial and mesenchymal cells. We also describe the procedure for analyzing FPCO characteristics. FPCO can provide a platform for establishing a reliable drug screening system.For complete details on the use and execution of this protocol, please refer to Takeuchi et al.1

Pancreatic ductal adenocarcinoma (PDAC) is a progressive cancer with a poor prognosis. It contains a complex tumor microenvironment (TME) that includes various stromal cell types. Comprehending cellular communications within the TME is difficult due to a lack of research models that can recapitulate human PDAC-TME. Previously, we recapitulated, in part, the PDAC-TME containing a diversity of cancer-associated fibroblasts (CAFs) in vitro. This was done by establishing a PDAC organoid by co-culturing patient-derived cancer cells with human induced pluripotent stem cell (hiPSC)-derived mesenchymal and endothelial cells, which was designated the fused pancreatic cancer organoid (FPCO). We further incorporated macrophages derived from the THP-1 cell line, which are the source of tumor-associated macrophages (TAMs), a major TME component, into FPCO, which was designated M0-FPCO. Bulk RNA sequencing (RNAseq) analysis revealed that macrophages in M0-FPCO (FPCO-Mac) lost their pro-inflammatory features but acquired pro-angiogenic features. Consistently, the formation of an endothelial cell network was enhanced in M0-FPCO. Single-cell RNA-seq (scRNA-seq) analysis revealed that M0-FPCO contained five TAM subpopulations similar to the corresponding TAM in human PDAC tissue in the integrated analysis, including SPP1+-TAM, which has been correlated with tumor angiogenesis and cell proliferation. Focusing on PDAC cells, we found that they could survive longer within the organoid in the presence of TAM. Consistent with the prolonged proliferation and survival of PDAC cells, PDAC subclusters were characterized by proliferative features, such as increased M0-FPCO. Therefore, by establishing a PDAC organoid with macrophages, we recapitulated the diversity of TAMs and identified the role of TAM in endothelial network formation as well as in the modulation of PDAC cell properties. Significance PDAC organoids, including macrophages using hiPSC, showed that PDAC-TAM has angiogenic features and contributes to PDAC cell survival.

Integration of resident immune cells into in vitro organoid models is important for accurately recapitulating native tissue physiology. Here, we present a protocol for integrating liver-resident macrophages, Kupffer cells, into liver organoid models derived from human induced pluripotent stem cells (iPSCs). We describe procedures for generating Kupffer cell progenitors and hepatic endoderm from iPSCs, followed by detailed steps for establishing liver organoids containing Kupffer cells (KuLOs). This protocol provides a platform for investigating the roles of Kupffer cells in liver development and diseases. For complete details on the use and execution of this protocol, please refer to Li et al.1

Lysosomal stress due to the accumulation of nucleic acids (NAs) activates endosomal TLRs in macrophages. Here, we show that lysosomal RNA stress, caused by the lack of RNase T2, induces macrophage accumulation in multiple organs such as the spleen and liver through TLR13 activation by microbiota-derived ribosomal RNAs. TLR13 triggered emergency myelopoiesis, increasing the number of myeloid progenitors in the bone marrow and spleen. Splenic macrophages continued to proliferate and mature into macrophages expressing the anti-inflammatory cytokine IL-10. In the liver, TLR13 activated monocytes/macrophages to proliferate and mature into monocyte-derived KCs (moKCs), in which, the liver X receptor (LXR) was activated. In accumulated moKCs, tissue clearance genes such as MerTK, AXL, and apoptosis inhibitor of macrophage (AIM) were highly expressed, while TLR-dependent production of proinflammatory cytokines was impaired. Consequently, Rnaset2-/- mice were resistant to acute liver injuries elicited by acetaminophen (APAP) and LPS with D-galactosamine. These findings suggest that TLR13 activated by lysosomal RNA stress promotes the replenishment of tissue-protective Kupffer cells.

Organoids derived from human induced pluripotent stem cells (hiPSC) are potentially applicable for regenerative medicine. However, the applications have been hampered by limited organoid size and function as a consequence of a lack of progenitor expansion. Here, we report the recapitulation of progenitor expansion in hiPSC-liver organoids based on the analysis of mouse development. Visualization of blood perfusion and oxygen levels in mouse embryos reveals a transient hypoxic environment during hepatoblast expansion, despite active blood flow. During this specific stage, the placenta expresses various growth factors. Human and mouse placenta-liver interaction analysis identifies various placenta-derived factors. Among them, IL1α efficiently induces the growth in hiPSC-liver organoids as well as mouse fetal livers following progenitor expansion under hypoxia. Furthermore, subsequent oxygenation demonstrates that progenitors expanded by IL1α contribute to hiPSC-liver organoid size and function. Taken together, we demonstrate that treatment with the placenta-derived factor under hypoxia is a crucial human organoid culture technique that efficiently induces progenitor expansion. Applications of hPSC-derived organoids have been hampered by limited organoid size and function. Here, authors report that treatment with IL1α, a placenta-derived factor, under hypoxia induces progenitor expansion in the hPSC-derived liver organoids.

Integration of resident immune cells into in vitro organoid models is important for accurately recapitulating native tissue physiology. Here, we present a protocol for integrating liver-resident macrophages, Kupffer cells, into liver organoid models derived from human induced pluripotent stem cells (iPSCs). We describe procedures for generating Kupffer cell progenitors and hepatic endoderm from iPSCs, followed by detailed steps for establishing liver organoids containing Kupffer cells (KuLOs). This protocol provides a platform for investigating the roles of Kupffer cells in liver development and diseases. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Human-induced pluripotent stem cell (hiPSC)-derived liver organoids (LOs) are valuable for studying human liver organogenesis but face challenges in faithfully recapitulating certain processes, like vasculogenesis, due to the lack of specific cell components. Hepatic stellate cells (HSCs), which are liver-specific pericytes and might be crucial for liver vasculogenesis, remain underutilized in developmental studies because of their disease-related status and inefficient generation process. Here, we present an efficient method for generating hiPSC-derived HSCs (hiPSC-HSCs) resembling the transcriptomic profiles of fetal human HSCs. These hiPSC-HSCs exhibit exceptional expandability (>105-fold) while maintaining essential cellular features. Additionally, in entirely hiPSC-derived LOs consisting of HSCs, hepatic endoderm, and endothelial cells, hiPSC-HSCs play a vital role in LO maturation and vascularization, both in vitro and in vivo. This work represents a significant advancement in understanding HSC roles in human liver development, and LOs containing hiPSC-HSCs hold potential in modeling congenital human liver diseases.

Cell migration assays, also known as chemotaxis assays, are widely used to measure the migratory capacities of cancer cells, leukocytes, macrophages, and other motile cell types. In these assays, fluorescently labeled cells are seeded onto cell culture inserts with microporous membranes that block light transmission from 490 to 700 nm. The migrated cells are then observed and quantified from the bottom of the microporous membrane using a fluorescence microscope. In this study, we conducted cell migration assays using macrophages as the motile cells. We discovered that the commonly employed fluorescent labeling method using calcein acetoxymethyl ester (calcein AM) can lead to the time-dependent attenuation of fluorescent signals in certain cell types during migration assays, potentially compromising assay stability. This study overcame this limitation by utilizing PKH26, which fluorescently labels cell surfaces through a mechanism distinct from that of calcein AM. With this modification, we observed a consistent increase in the number of migrating macrophages over time. We also demonstrated that the gradient of chemoattractants is key to the success of cell migration assays. Our improved protocol provided reliable and stable results for cell migration assays.

Background Although oncogenic RAS mutants are thought to exert mutagenic effects upon blood cells, it remains uncertain how a single oncogenic RAS impacts non-transformed multipotent hematopoietic stem or progenitor cells (HPCs). Such potential pre-malignant status may characterize HPCs in patients with RAS-associated autoimmune lymphoproliferative syndrome-like disease (RALD). This study sought to elucidate the biological and molecular alterations in human HPCs carrying monoallelic mutant KRAS (G13C) with no other oncogene mutations. Methods We utilized induced pluripotent stem cells (iPSCs) derived from two unrelated RALD patients. Isogenic HPC pairs harboring either wild-type KRAS or monoallelic KRAS (G13C) alone obtained following differentiation enabled reliable comparative analyses. The compound screening was conducted with an established platform using KRAS (G13C) iPSCs and differentiated HPCs. Results Cell culture assays revealed that monoallelic KRAS (G13C) impacted both myeloid differentiation and expansion characteristics of iPSC-derived HPCs. Comprehensive RNA-sequencing analysis depicted close clustering of HPC samples within the isogenic group, warranting that comparative studies should be performed within the same genetic background. When compared with no stimulation, iPSC-derived KRAS (G13C)-HPCs showed marked similarity with the wild-type isogenic control in transcriptomic profiles. After stimulation with cytokines, however, KRAS (G13C)-HPCs exhibited obvious aberrant cell-cycle and apoptosis responses, compatible with "dysregulated expansion," demonstrated by molecular and biological assessment. Increased BCL-xL expression was identified amongst other molecular changes unique to mutant HPCs. With screening platforms established for therapeutic intervention, we observed selective activity against KRAS (G13C)-HPC expansion in several candidate compounds, most notably in a MEK- and a BCL-2/BCL-xL-inhibitor. These two compounds demonstrated selective inhibitory effects on KRAS (G13C)-HPCs even with primary patient samples when combined. Conclusions Our findings indicate that a monoallelic oncogenic KRAS can confer dysregulated expansion characteristics to non-transformed HPCs, which may constitute a pathological condition in RALD hematopoiesis. The use of iPSC-based screening platforms will lead to discovering treatments that enable selective inhibition of RAS-mutated HPC clones.

Maximizing the potential of human liver organoids (LOs) for modeling human septic liver requires the integration of innate immune cells, particularly resident macrophage Kupffer cells. In this study, we present a strategy to generate LOs containing Kupffer cells (KuLOs) by recapitulating fetal liver hematopoiesis using human induced pluripotent stem cell (hiPSC)-derived erythro-myeloid progenitors (EMPs), the origin of tissue-resident macrophages, and hiPSC-derived LOs. Remarkably, LOs actively promote EMP hematopoiesis toward myeloid and erythroid lineages. Moreover, supplementing with macrophage colony-stimulating factor (M-CSF) proves crucial in sustaining the hematopoietic population during the establishment of KuLOs. Exposing KuLOs to sepsis-like endotoxins leads to significant organoid dysfunction that closely resembles the pathological characteristics of the human septic liver. Furthermore, we observe a notable functional recovery in KuLOs upon endotoxin elimination, which is accelerated by using Toll-like receptor-4-directed endotoxin antagonist. Our study represents a comprehensive framework for integrating hematopoietic cells into organoids, facilitating in-depth investigations into inflammation-mediated liver pathologies.

Donor organ shortages for transplantation remain a serious global concern, and alternative treatment is in high demand. Fetal cells and tissues have considerable therapeutic potential as, for example, organoid technology that uses human induced pluripotent stem cells (hiPSCs) to generate unlimited human fetal-like cells and tissues. We previously reported the in vivo vascularization of early fetal liver–like hiPSC-derived liver buds (LBs) and subsquent improved survival of recipient mice with subacute liver failure. Here, we show hiPSC–liver organoids (LOs) that recapitulate midgestational fetal liver promote de novo liver generation when grafted onto the surface of host livers in chemical fibrosis models, thereby recovering liver function. We found that fetal liver, a hematopoietic tissue, highly expressed macrophage-recruiting factors and antifibrotic M2 macrophage polarization factors compared with the adult liver, resulting in fibrosis reduction because of CD163 + M2-macrophage polarization. Next, we created midgestational fetal liver–like hiPSC-LOs by fusion of hiPSC-LBs to induce static cell-cell interactions and found that these contained complex structures such as hepatocytes, vasculature, and bile ducts after transplantation. This fusion allowed the generation of a large human tissue suitable for transplantation into immunodeficient rodent models of liver fibrosis. hiPSC-LOs showed superior liver function compared with hiPSC-LBs and improved survival and liver function upon transplantation. In addition, hiPSC-LO transplantation ameliorated chemically induced liver fibrosis, a symptom of liver cirrhosis that leads to organ dysfunction, through immunomodulatory effects, particularly on CD163 + phagocytic M2-macrophage polarization. Together, our results suggest hiPSC-LO transplantation as a promising therapeutic option for liver fibrosis.

In fetal development, tissue interaction such as the interplay between blood vessel (BV) and epithelial tissue is crucial for organogenesis. Here we recapitulate the spatial arrangement between liver epithelial tissue and the portal vein to observe the formation of intrahepatic bile ducts (BDs) from human induced pluripotent stem cells (hiPSC). We co-culture hiPSC-liver progenitors on the artificial BV consisting of immature smooth muscle cells and endothelial cells, both derived from hiPSCs. After 3 weeks, liver progenitors within hiPSC-BV-incorporated liver organoids (BVLO) differentiate to cholangiocytes and acquire epithelial characteristics, including intercellular junctions, microvilli on the apical membrane, and secretory functions. Furthermore, liver surface transplanted-BVLO temporarily attenuates cholestatic injury symptoms. Single cell RNA sequence analysis suggests that BD interact with the BV in BVLO through TGFβ and Notch pathways. Knocking out JAG1 in hiPSC-BV significantly attenuates bile duct formation, highlighting BVLO potential as a model for Alagille syndrome, a congenital biliary disease. Overall, we develop a novel 3D co-culture method that successfully establishes functional human BDs by emulating liver epithelial-BV interaction.

Background: Systemic lupus erythematosus (SLE) is a chronic systemic autoimmune disease characterized by genetic heterogeneity and an interferon (IFN) signature. The overall landscapes of the heritability of SLE remains unclear.Objectives: To identify and elucidate the biological functions of rare variants underlying SLE, we conducted analyses of patient-derived induced pluripotent stem cells (iPSCs) in combination with genetic analysis.Methods: Two familial SLE patient-and two healthy donor (HD)-derived iPSCs were established. Type 1 IFN-secreting dendritic cells (DCs) were differentiated from iPSCs. Genetic analyses of SLE-iPSCs, and 117 SLE pa-tients and 107 HDs in the ImmuNexUT database were performed independently. Genome editing of the variants on iPSCs was performed with the CRISPR/Cas9 system.Results: Type 1 IFN secretion was significantly increased in DCs differentiated from SLE-iPSCs compared to HD-iPSCs. Genetic analyses revealed a rare variant in the 2'-5'-Oligoadenylate Synthetase Like (OASL) shared be-tween SLE-iPSCs and another independent SLE patient, and significant accumulation of OASL variants among SLE patients (HD 0.93%, SLE 6.84%, OR 8.387) in the database. Genome editing of mutated OASL 202Q to wild -type 202 R or wild-type OASL 202 R to mutated 202Q resulted in reduced or enhanced Type 1 IFN secretion of DCs. Three other OASL variants (R60W, T261S and A447V) accumulated in SLE patients had also capacities to enhance Type 1 IFN secretion in response to dsRNA.Conclusions: We established a patient-derived iPSC-based strategy to investigate the linkage of genotype and phenotype in autoimmune diseases. Detailed case-based investigations using patient-derived iPSCs provide in-formation to unveil the heritability of the pathogenesis of autoimmune diseases.

The aggressiveness of pancreatic ductal adenocarcinoma (PDAC) is affected by the tumor microenvironment (TME). In this study, to recapitulate the PDAC TME ex vivo, we cocultured patient-derived PDAC cells with mesenchymal and vascular endothelial cells derived from human induced pluripotent stem cells (hiPSCs) to create a fused pancreatic cancer organoid (FPCO) in an air-liquid interface. FPCOs were further induced to resemble two distinct aspects of PDAC tissue. Quiescent FPCOs were drug resistant, likely because the TME consisted of abundant extracellular matrix proteins that were secreted from the various types of cancer-associated fibroblasts (CAFs) derived from hiPSCs. Proliferative FPCOs could re-proliferate after anticancer drug treatment, suggesting that this type of FPCO would be useful for studying PDAC recurrence. Thus, we generated PDAC organoids that recapitulate the heterogeneity of PDAC tissue and are a potential platform for screening anticancer drugs.