Monoallelic KRAS (G13C) mutation triggers dysregulated expansion in induced pluripotent stem cell-derived hematopoietic progenitor cells.

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.

Integration of Kupffer cells into human iPSC-derived liver organoids for modeling liver dysfunction in sepsis.

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.

Incorporation of human iPSC-derived stromal cells creates a pancreatic cancer organoid with heterogeneous cancer-associated fibroblasts.

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.

EFFICACY AND SAFETY OF A DEXAMETHASONE-BASED MOUTHWASH TO PREVENT CHEMOTHERAPY-INDUCED STOMATITIS IN WOMEN WITH BREAST CANCER: A MULTICENTRE, OPEN-LABEL, RANDOMISED PHASE 2 STUDY.

PURPOSE: No standard approach other than oral care is available for preventing chemotherapy-induced stomatitis in patients with breast cancer. In this randomized, controlled phase 2 trial, we aimed to assess the efficacy and safety of a dexamethasone-based mouthwash in preventing chemotherapy-induced stomatitis in patients with early breast cancer. BASIC PROCEDURES: Patients with breast cancer scheduled for epirubicin and cyclophosphamide (EC) or docetaxel and cyclophosphamide (TC) therapy were selected and allocated in a 1:1 ratio to the intervention and control groups. The intervention group received chemotherapy, oral care, and a dexamethasone-based mouthwash, whereas the control group received chemotherapy and oral care. The primary endpoint was the incidence of stomatitis. This was a phase 2 study, and the significance level for the analysis of the primary endpoint was set a priori at 0.2. MAIN FINDINGS: Data pertaining to 58 patients in the control group and 59 patients in the intervention group were analyzed. Stomatitis incidence was 55% and 38% in the control and intervention groups, respectively (risk ratio, 0.68; 80% confidence interval, 0.52-0.88; P = .052). Stomatitis severity was lower in the intervention group than in the control group (P = .03). The proportion of patients who adhered to the mouthwash regimen was 87% (interquartile range, 67.8%-95.3%). No severe oral infections were observed. PRINCIPAL CONCLUSIONS: The dexamethasone-based mouthwash safely reduced stomatitis incidence and severity in patients receiving chemotherapy for early breast cancer. Phase 3 clinical trials are warranted for validating our results.

Preparation of mechanically patterned hydrogels for controlling the self-condensation of cells.

Synthetic protocols providing mechanical patterns to culture substrate are essential to control the self-condensation of cells for organoid engineering. Here, we present a protocol for preparing hydrogels with mechanical patterns. We describe steps for hydrogel synthesis, mechanical evaluation of the substrate, and time-lapse imaging of cell self-organization. This protocol will facilitate the rational design of culture substrates with mechanical patterns for the engineering of various functional organoids. For complete details on the use and execution of this protocol, please refer to Takebe et al. (2015) and Matsuzaki et al. (2014, 2022).1,2,3.

Functional evaluation of rare OASL variants by analysis of SLE patient-derived iPSCs.

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 patients 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 between 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 information to unveil the heritability of the pathogenesis of autoimmune diseases.

PDGF Receptors and Signaling Are Required for 3D-Structure Formation and Differentiation of Human iPSC-Derived Hepatic Spheroids.

Human iPSC-derived liver organoids (LO) or hepatic spheroids (HS) have attracted widespread interest, and the numerous studies on them have recently provided various production protocols. However, the mechanism by which the 3D structures of LO and HS are formed from the 2D-cultured cells and the mechanism of the LO and HS maturation remain largely unknown. In this study, we demonstrate that PDGFRA is specifically induced in the cells that are suitable for HS formation and that PDGF receptors and signaling are required for HS formation and maturation. Additionally, in vivo, we show that the localization of PDGFRα is in complete agreement with mouse E9.5 hepatoblasts, which begin to form the 3D-structural liver bud from the single layer. Our results present that PDGFRA play important roles for 3D structure formation and maturation of hepatocytes in vitro and in vivo and provide a clue to elucidate the hepatocyte differentiation mechanism.

Stabilized generation of human iPSC-derived liver organoids using a modified coating approach.

Human-induced pluripotent stem cell (hiPSC)-derived hepatic cells are useful tools for regenerative medicine, and various culture substrates are currently used for their differentiation. We differentiated hiPSC-derived hepatic endoderm (HE), endothelial cells (ECs), and mesenchymal cells (MCs) using Laminin-511 (LN) coating to generate liver organoids, hiPSC-liver buds (hiPSC-LBs), which exhibited therapeutic effects when transplanted into disease model animals. Stably producing significant amounts of hiPSC-LBs is necessary for sufficient therapeutic effects. However, general precoating (standard coating) requires quick manipulation, often causing failure for inexperienced cell cultures, we thus tested direct LN addition to the culture medium (Direct coating). Using quantitative gene expression, flow cytometry, albumin secretion, and ammonia metabolism, we demonstrated that Standard and Direct coating similarly induce hiPSC-derived hepatocyte, mesodermal cell, EC, and MC differentiation. Standard and Direct coating-differentiated cells generated iPSC-LBs with equivalent hepatic functions. Furthermore, Direct coating enabled stable induction of differentiation independent of individual culture skills and reduced total amount of LN use as the same differentiated cell quality can be obtained upon LN supplementation at lower concentrations. In summary, the results of this study suggest that Direct coating could enable stable hiPSC-LB production at a low cost, thereby yielding mass cell production using hiPSCs.

Establishment of experimental salivary gland cancer models using organoid culture and patient-derived xenografting.

PURPOSE: Depending on its histological subtype, salivary gland carcinoma (SGC) may have a poor prognosis. Due to the scarcity of preclinical experimental models, its molecular biology has so far remained largely unknown, hampering the development of new treatment modalities for patients with these malignancies. The aim of this study was to generate experimental human SGC models of multiple histological subtypes using patient-derived xenograft (PDX) and organoid culture techniques. METHODS: Tumor specimens from surgically resected SGCs were processed for the preparation of PDXs and patient-derived organoids (PDOs). Specimens from SGC PDXs were also processed for PDX-derived organoid (PDXO) generation. In vivo tumorigenicity was assessed using orthotopic transplantation of SGC organoids. The pathological characteristics of each model were compared to those of the original tumors using immunohistochemistry. RNA-seq was used to analyze the genetic traits of our models. RESULTS: Three series of PDOs, PDXs and PDXOs of salivary duct carcinomas, one series of PDOs, PDXs and PDXOs of mucoepidermoid carcinomas and PDXs of myoepithelial carcinomas were successfully generated. We found that PDXs and orthotopic transplants from PDOs/PDXOs showed similar histological features as the original tumors. Our models also retained their genetic traits, i.e., transcription profiles, genomic variants and fusion genes of the corresponding histological subtypes. CONCLUSION: We report the generation of SGC PDOs, PDXs and PDXOs of multiple histological subtypes, recapitulating the histological and genetical characteristics of the original tumors. These experimental SGC models may serve as a useful resource for the development of novel therapeutic strategies and for investigating the molecular mechanisms underlying the development of these malignancies.

In vitro elastic cartilage reconstruction using human auricular perichondrial chondroprogenitor cell-derived micro 3D spheroids.

Morphologically stable scaffold-free elastic cartilage tissue is crucial for treating external ear abnormalities. However, establishing adequate mechanical strength is challenging, owing to the difficulty of achieving chondrogenic differentiation in vitro; thus, cartilage reconstruction is a complex task. Auricular perichondrial chondroprogenitor cells exhibit high proliferation potential and can be obtained with minimal invasion. Therefore, these cells are an ideal resource for elastic cartilage reconstruction. In this study, we aimed to develop a novel in vitro scaffold-free method for elastic cartilage reconstruction, using human auricular perichondrial chondroprogenitor cells. Inducing chondrogenesis by using microscopic spheroids similar to auricular hillocks significantly increased the chondrogenic potential. The size and elasticity of the tissue were maintained after craniofacial transplantation in immunodeficient mice, suggesting that the reconstructed tissue was morphologically stable. Our novel tissue reconstruction method may facilitate the development of future treatments for external ear abnormalities.

Mechanical guidance of self-condensation patterns of differentiating progeny.

Spatially controlled self-organization represents a major challenge for organoid engineering. We have developed a mechanically patterned hydrogel for controlling self-condensation process to generate multi-cellular organoids. We first found that local stiffening with intrinsic mechanical gradient (IG > 0.008) induced single condensates of mesenchymal myoblasts, whereas the local softening led to stochastic aggregation. Besides, we revealed the cellular mechanism of two-step self-condensation: (1) cellular adhesion and migration at the mechanical boundary and (2) cell-cell contraction driven by intercellular actin-myosin networks. Finally, human pluripotent stem cell-derived hepatic progenitors with mesenchymal/endothelial cells (i.e., liver bud organoids) experienced collective migration toward locally stiffened regions generating condensates of the concave to spherical shapes. The underlying mechanism can be explained by force competition of cell-cell and cell-hydrogel biomechanical interactions between stiff and soft regions. These insights will facilitate the rational design of culture substrates inducing symmetry breaking in self-condensation of differentiating progeny toward future organoid engineering.

Evaluation of Urea Cycle Activity by Metabolic Flux Analysis Using Mass Spectrometry.

Hepatocytes play an important role in maintaining homeostasis in living organisms by carrying out various metabolic functions. The urea cycle, one of the metabolic pathways taking place in hepatocytes, is an important metabolic pathway that converts toxic ammonia to nontoxic urea. Performing quantitative assessments of individual metabolite levels using a mass spectrometer is useful for assessing the metabolic state of the urea cycle in hepatocytes. In addition, metabolic flux analysis using stable isotopes and a mass spectrometer is a new technique for measuring the metabolic state. It enables conducting specific, objective, and quantitative measurement of the activated state of the target metabolic pathway regardless of external disturbing factors. This section describes the technical background and methodology of performing metabolic flux analysis of the urea cycle by mass spectrometry.

Efficacy of 3% hydrogen peroxide solution in cleaning tongue coating before and after surgery: a randomized phase II study.

BACKGROUND: Increased bacterial presence in the tongue coating and thereby, the saliva, may be a risk factor for postoperative complications such as surgical site infection or postoperative pneumonia after cancer surgery. However, no method for cleaning tongue coating has been established experimentally. The purpose of this study was to verify the effect of brushing with 3% hydrogen peroxide on suppression of the number of bacteria in tongue coating. METHODS: Sixteen patients with gastric cancer or colorectal cancer undergoing surgery were randomly allocated to control and intervention groups. In the control group, the tongue was brushed for 30 s with a water-moistened toothbrush, while in the intervention group, the tongue was brushed for 30 s with a toothbrush moistened with 3% hydrogen peroxide. Bacterial counts on tongue coating were measured before and 30 s after cleaning the tongue coating using the Rapid Oral Bacteria Quantification System. RESULTS: In the control group, the number of bacteria on the tongue did not decrease significantly after tongue cleaning on the day before surgery, but did on the day after surgery. In contrast, in the intervention group, the number of bacteria on the tongue decreased significantly after tongue cleaning both on the day before and the day after surgery. Furthermore, when comparing the control and intervention groups, the intervention group had a greater reduction effect. CONCLUSIONS: Tongue brushing with 3% hydrogen peroxide is a useful method to reduce the number of bacteria on the tongue in patients with gastrointestinal cancer undergoing surgery. Trial registration jRCTs071200020 (July 3, 2020).

Highly Sensitive Detection of Human Pluripotent Stem Cells by Loop-Mediated Isothermal Amplification.

For safe regenerative medicines, contaminated or remaining tumorigenic undifferentiated cells in cell-derived products must be rigorously assessed through sensitive assays. Although in vitro nucleic acid tests offer particularly sensitive tumorigenicity-associated assays, the human pluripotent stem cell (hPSC) detectability is partly constrained by the small input amount of RNA per test. To overcome this limitation, we developed reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays that are highly gene specific and robust against interfering materials. LAMP could readily assay microgram order of input sample per test and detected an equivalent model of 0.00002% hiPSC contamination in a simple one-pot reaction. For the evaluation of cell-derived total RNA, RT-LAMP detected spiked-in hPSCs among hPSC-derived trilineage cells utilizing multiple pluripotency RNAs. We also developed multiplex RT-LAMP assays and further applied for in situ cell imaging, achieving specific co-staining of pluripotency proteins and RNAs. Our attempts uncovered the utility of RT-LAMP approaches for tumorigenicity-associated assays, supporting practical applications of regenerative medicine.

Usefulness of criteria for intraoperative Management of Postoperative Nausea and Vomiting.

BACKGROUND: Postoperative nausea and vomiting (PONV) delays postoperative recovery, prolongs hospital stays, and hinders patients' return to society, thus making it a major cause of increased healthcare costs. It is also the most troubling postoperative complication in female patients undergoing surgery. However, in Japan, guidelines for the management of PONV have not been established, and the management protocol for PONV is left to each institution and anesthesiologist. Therefore, we developed criteria for intraoperative management of PONV. METHODS: In female surgical patients, the usefulness of the criteria was evaluated by comparing the implementation rate of intraoperative management and PONV incidence before and after the establishment of the criteria. An Apfel simplified score (Apfel score) ≥2 was set as an indication for intraoperative management of PONV. RESULTS: The implementation rate of intraoperative management increased from 91.2 to 96.0% after the introduction of the criteria. In patients with an Apfel score of 2, the intraoperative management implementation rate significantly increased from 81.1 to 94.7% (p = 0.016), while PONV incidence significantly decreased from 44.6 to 34.1% after the introduction of the criteria (p = 0.040). CONCLUSIONS: The criteria for intraoperative management of PONV increased the implementation rate of intraoperative management and decreased PONV incidence, indicating the usefulness of the criteria.

Development of the nervous system in mouse liver.

BACKGROUND: The role of the hepatic nervous system in liver development remains unclear. We previously created functional human micro-hepatic tissue in mice by co-culturing human hepatic endodermal cells with endothelial and mesenchymal cells. However, they lacked Glisson's sheath [the portal tract (PT)]. The PT consists of branches of the hepatic artery (HA), portal vein, and intrahepatic bile duct (IHBD), collectively called the portal triad, together with autonomic nerves. AIM: To evaluate the development of the mouse hepatic nervous network in the PT using immunohistochemistry. METHODS: Liver samples from C57BL/6J mice were harvested at different developmental time periods, from embryonic day (E) 10.5 to postnatal day (P) 56. Thin sections of the surface cut through the hepatic hilus were examined using protein gene product 9.5 (PGP9.5) and cytokeratin 19 (CK19) antibodies, markers of nerve fibers (NFs), and biliary epithelial cells (BECs), respectively. The numbers of NFs and IHBDs were separately counted in a PT around the hepatic hilus (center) and the peripheral area (periphery) of the liver, comparing the average values between the center and the periphery at each developmental stage. NF-IHBD and NF-HA contacts in a PT were counted, and their relationship was quantified. SRY-related high mobility group-box gene 9 (SOX9), another BEC marker; hepatocyte nuclear factor 4α (HNF4α), a marker of hepatocytes; and Jagged-1, a Notch ligand, were also immunostained to observe the PT development. RESULTS: HNF4α was expressed in the nucleus, and Jagged-1 was diffusely positive in the primitive liver at E10.5; however, the PGP9.5 and CK19 were negative in the fetal liver. SOX9-positive cells were scattered in the periportal area in the liver at E12.5. The Jagged-1 was mainly expressed in the periportal tissue, and the number of SOX9-positive cells increased at E16.5. SOX9-positive cells constructed the ductal plate and primitive IHBDs mainly at the center, and SOX-9-positive IHBDs partly acquired CK19 positivity at the same period. PGP9.5-positive bodies were first found at E16.5 and HAs were first found at P0 in the periportal tissue of the center. Therefore, primitive PT structures were first constructed at P0 in the center. Along with remodeling of the periportal tissue, the number of CK19-positive IHBDs and PGP9.5-positive NFs gradually increased, and PTs were also formed in the periphery until P5. The numbers of NFs and IHBDs were significantly higher in the center than in the periphery from E16.5 to P5. The numbers of NFs and IHBDs reached the adult level at P28, with decreased differences between the center and periphery. NFs associated more frequently with HAs than IHBDs in PTs at the early phase after birth, after which the number of NF-IHBD contacts gradually increased. CONCLUSION: Mouse hepatic NFs first emerge at the center just before birth and extend toward the periphery. The interaction between NFs and IHBDs or HAs plays important roles in the morphogenesis of PT structure.

High temporal resolution proteome and phosphoproteome profiling of stem cell-derived hepatocyte development.

Primary human hepatocytes are widely used to evaluate liver toxicity of drugs, but they are scarce and demanding to culture. Stem cell-derived hepatocytes are increasingly discussed as alternatives. To obtain a better appreciation of the molecular processes during the differentiation of induced pluripotent stem cells into hepatocytes, we employ a quantitative proteomic approach to follow the expression of 9,000 proteins, 12,000 phosphorylation sites, and 800 acetylation sites over time. The analysis reveals stage-specific markers, a major molecular switch between hepatic endoderm versus immature hepatocyte-like cells impacting, e.g., metabolism, the cell cycle, kinase activity, and the expression of drug transporters. Comparing the proteomes of two- (2D) and three-dimensional (3D)-derived hepatocytes with fetal and adult liver indicates a fetal-like status of the in vitro models and lower expression of important ADME/Tox proteins. The collective data enable constructing a molecular roadmap of hepatocyte development that serves as a valuable resource for future research.

Improving the repopulation capacity of elderly human hepatocytes by decoding aging-associated hepatocyte plasticity.

BACKGROUND AND AIMS: The loss of liver regenerative capacity is the most dramatic age-associated alteration. Because of an incomplete mechanistic understanding of the liver aging process, a successful therapeutic strategy to improve liver regeneration in the elderly has not been developed so far. Hepatocyte plasticity is a principal mechanism for producing new hepatocytes and cholangiocytes during regeneration. This study aims to promote the repopulation capacity of elderly hepatocytes by decoding the underlying mechanism about the regulation of aging on human hepatocyte plasticity. APPROACH AND RESULTS: To understand the age-related mechanisms, we established a hepatocyte aging model from human-induced pluripotent stem cells and developed a method for ex vivo characterization of hepatocyte plasticity. We found that hepatocyte plasticity was gradually diminished with aging, and the impaired plasticity was caused by age-induced histone hypoacetylation. Notably, selective inhibition of histone deacetylases could markedly restore aging-impaired plasticity. Based on these findings, we successfully improved the plasticity of elderly primary human hepatocytes that enhanced their repopulation capacity in the liver injury model. CONCLUSIONS: This study suggests that age-induced histone hypoacetylation impairs hepatocyte plasticity, and hepatocyte plasticity might be a therapeutic target for promoting the regenerative capacity of the elderly liver.

Evaluation of immunosuppression protocols for MHC-matched allogeneic iPS cell-based transplantation using a mouse skin transplantation model.

BACKGROUND: Off-the-shelf major histocompatibility complex (MHC)-matched iPS cells (iPSC) can potentially initiate host immune responses because of the existence of numerous minor antigens. To suppress allo-immune responses, combination of immunosuppressants is usually used, but its efficacy to the allogeneic iPSC-based transplantation has not been precisely evaluated. METHODS: Three transplantation models were used in this study; MHC-matched, minor antigen-mismatched mouse skin or iPSC-graft transplantation, and fully allogeneic human iPSC-derived liver organoid transplantation in immune-humanized mice. The recipients were treated with triple drugs combination (TDC; tacrolimus, methylprednisolone, and mycophenolate mofetil) or co-stimulatory molecule blockade (CB) therapy with some modifications. Graft survival as well as anti-donor T and B cell responses was analyzed. RESULTS: In the mouse skin transplantation model, immunological rejection caused by the minor antigen-mismatch ranged from mild to severe according to the donor-recipient combination. The TDC treatment could apparently control the mild skin graft rejection when combined with a transient T cell depletion, but unexpected anti-donor T or B cell response was observed. On the other hand, CB therapy, particularly when combined with rapamycin treatment, was capable of attenuating both mild and severe skin graft rejection and allowing them to survive long-term without any unfavorable anti-donor immune responses. The efficacy of the CB therapy was confirmed in both mouse and human iPSC-derived graft transplantation. CONCLUSIONS: The findings suggest that the CB-based treatment seems suitable to well manage the MHC-matched allogeneic iPSC-based transplantation. The TDC-based treatment may be also used to suppress the rejection, but screening of its severity prior to the transplantation seems to be needed.

Establishment of a mouse model of pancreatic cancer using human pancreatic cancer cell line S2-013-derived organoid.

A well-established preclinical model of pancreatic cancer needs to be established to facilitate research on new therapeutic targets. Recently established animal models of pancreatic cancer, including patient-derived tumor models and organoid models, are used for pre-clinical drug testing and biomarker discovery. These models have useful characteristics over conventional xenograft mouse models based on cell lines in preclinical studies, but still cannot accurately predict the clinical outcomes of new treatments and have not yet been broadly implemented in research. We employed pancreatic cancer organoid culture methods using the pancreatic cancer cell line S2-013, and performed pathological and immunohistochemical analyses to characterize tumor xenografts obtained from a mouse model implanted with S2-013 cell line-derived organoids. Serum levels of the pancreatic cancer tumor marker CA19-9 were measured by ELISA. We generated human pancreatic cancer organoids using a co-culture of S2-013 cells, human endothelial cells derived from human umbilical vein endothelial cells, and human mesenchymal stem cells, and established a mouse model with subcutaneously transplanted human pancreatic cancer organoids (S2-013-organoid model). Although blood clotting crater-like formation developed in the middle of subcutaneous xenografts in the S2-013-conventional model, created by subcutaneously injecting S2-013 cells into the right flank of nude mice, the size of xenografts in the S2-013-organoid model gradually increased without crater-like formation. Importantly, tumor xenografts obtained from the S2-013-organoid model exhibited a clinical human pancreatic cancer tissue-like cellular morphology, tissue architecture, and polarity, and actively formed cancer stroma containing mature blood vessels with the high expression of the vascular tight junction marker CD31. In subcutaneous xenografts of S2-013-conventional mice, no blood vessel density or widely expanding areas of necrotic regions were present. Consequently, serum levels of CA19-9 in the S2-013-organoid model correlated with tumor volumes. In addition, epithelial-mesenchymal transition, the conversion of epithelial cells to the mesenchymal phenotype, was observed in tumor xenografts of the S2-013-organoid model. The S2-013-organoid model provides tumor xenografts consisting of clinical human pancreatic cancer-like tissue formation with the effective development of vascularized stroma, and may be valuable for facilitating studies on pre-clinical drug testing and biomarker discovery.