Organoid Cultures Research Articles

Advances in the study of gastric organoids as disease models

Gastric organoids are models created in the laboratory using stem cells and sophisticated three-dimensional cell culture techniques. These models have shown great promise in providing valuable insights into gastric physiology and advanced disease research. This review comprehensively summarizes and analyzes the research advances in culture methods and techniques for adult stem cells and induced pluripotent stem cell-derived organoids, and patient-derived organoids. The potential value of gastric organoids in studying the pathogenesis of stomach-related diseases and facilitating drug screening is initially discussed. The construction of gastric organoids involves several key steps, including cell extraction and culture, three-dimensional structure formation, and functional expression. Simulating the structure and function of the human stomach by disease modeling with gastric organoids provides a platform to study the mechanism of gastric cancer induction by Helicobacter pylori . In addition, in drug screening and development, gastric organoids can be used as a key tool to evaluate drug efficacy and toxicity in preclinical trials. They can also be used for precision medicine according to the specific conditions of patients with gastric cancer, to assess drug resistance, and to predict the possibility of adverse reactions. However, despite the impressive progress in the field of gastric organoids, there are still many unknowns that need to be addressed, especially in the field of regenerative medicine. Meanwhile, the reproducibility and consistency of organoid cultures are major challenges that must be overcome. These challenges have had a significant impact on the development of gastric organoids. Nonetheless, as technology continues to advance, we can foresee more comprehensive research in the construction of gastric organoids. Such research will provide better solutions for the treatment of stomach-related diseases and personalized medicine.

Biomimetic hydrogels with mesoscale collagen architecture for patient-derived tumor organoids culture

Patient-derived tumor organoids (PDTOs) shows great potential as a preclinical model. However, the current methods for establishing PDTOs primarily focus on modulating local properties, such as sub-micrometer topographies. Nevertheless, they neglect to capture the global millimeter or intermediate mesoscale architecture that have been demonstrated to influence tumor response to therapeutic treatment and tumor progression. In this study, we present a rapid technique for generating collagen bundles with an average length of 90 ± 27 μm and a mean diameter of 5 ± 1.5 μm from tumor tissue debris that underwent mechanical agitation following enzymatic digestion. The collagen bundles were subsequently utilized for the fabrication of biomimetic hydrogels, incorporating microbial transglutaminase (mTG) crosslinked gelatin. These biomimetic hydrogels, referred to as MC-gel, were specifically designed for patient-derived tumor organoids. The lung cancer organoids cultured in MC-gel exhibited larger diameters and higher cell viability compared to those cultured in gels lacking the mesoscale collagen bundle; moreover, their irregular morphology more closely resembled that observed in vivo. The MC-gel-based lung cancer organoids effectively replicated the histology and mutational landscapes observed in the original donor patient’s tumor tissue. Additionally, these lung cancer organoids showed a remarkable similarity in their gene expression and drug response across different matrices. This recently developed model holds great potential for investigating the occurrence, progression, metastasis, and management of tumors, thereby offering opportunities for personalized medicine and customized treatment options.

The efficiency of stem cell differentiation into functional beta cells for treating insulin-requiring diabetes: Recent advances and current challenges.

In recent years, the potential of stem cells (SCs) to differentiate into various types of cells, including β-cells, has led to a significant boost in development. The efficiency of this differentiation process and the functionality of the cells post-transplantation are crucial factors for the success of stem cell therapy in diabetes. Herein, this article reviews the current advances and challenges faced by stem cell differentiation into functional β-cells for diabetes treatment. In vitro, researchers have sought to enhance the differentiation efficiency of functional β-cells by mimicking the normal pancreatic development process, using gene manipulation, pharmacological and culture conditions stimulation, three-dimensional (3D) and organoid culture, or sorting for functional β-cells based on mature islet cell markers. Furthermore, in vivo studies have also looked at suitable transplantation sites, the enhancement of the transplantation microenvironment, immune modulation, and vascular function reconstruction to improve the survival rate of functional β-cells, thereby enhancing the treatment of diabetes. Despite these advancements, developing stem cells to produce functional β-cells for efficacious diabetes treatment is a continuous research endeavor requiring significant multidisciplinary collaboration, for the stem-cell-derived beta cells to evolve into an effective cellular therapy.

Modulators of the Nrf2 Signaling Pathway Enhance the Cytotoxic Effect of Standard Chemotherapeutic Drugs on Organoids of Metastatic Colorectal Cancer.

The activity of known modulators of the Nrf2 signaling pathway (bardoxolone and brusatol) was studied on cultures of tumor organoids of metastatic colorectal cancer previously obtained from three patients. The effect of modulators was studied both as monotherapy and in combination with standard chemotherapy drugs used to treat colorectal cancer. The Nrf2 inhibitor brusatol and the Nrf2 activator bardoxolone have antitumor activity. Moreover, bardoxolone and brusatol also significantly enhance the effect of the chemotherapy drugs 5-fluorouracil, oxaliplatin, and irinotecan metabolite SN-38. Thus, bardoxolone and brusatol can be considered promising candidates for further preclinical and clinical studies in the treatment of colorectal cancer.

Gastric Organoid, a Promising Modeling for Gastric Stem Cell Homeostasis and Therapeutic Application.

The elucidation of the pathophysiology underlying various diseases necessitates the development of research platforms that faithfully mimic in vivo conditions. Traditional model systems such as two-dimensional cell cultures and animal models have proven inadequate in capturing the complexities of human disease modeling. However, recent strides in organoid culture systems have opened up new avenues for comprehending gastric stem cell homeostasis and associated diseases, notably gastric cancer. Given the significance of gastric cancer, a thorough understanding of its pathophysiology and molecular underpinnings is imperative. To this end, the utilization of patient-derived organoid libraries emerges as a remarkable platform, as it faithfully mirrors patient-specific characteristics, including mutation profiles and drug sensitivities. Furthermore, genetic manipulation of gastric organoids facilitates the exploration of molecular mechanisms underlying gastric cancer development. This review provides a comprehensive overview of recent advancements in various adult stem cell-derived gastric organoid models and their diverse applications.

Abstract Wrk2-01: Organoid Models of Cancer and the Tumor Microenvironment

Abstract Both cell-intrinsic and microenvironmental influences vitally govern tumor progression. Mutational, chromosomal and epigenetic alterations represent the former, while the latter is influenced by diverse stromal populations including fibroblasts, vasculature and immune cells. In vitro organoid culture provides an experimentally tractable platform for cancer research that is applicable to cell-autonomous and tumor microenvironmental studies. The first part of this lecture will describe our work using organoid culture to functionally validate tumor cell-intrinsic genomic alterations. Notably, cancer research has traditionally lacked in vitro methods where tumors can be holistically cultured in a non-reconstitutive manner that co-preserves neoplastic cells together with endogenous stromal components. Thus, the second part of this lecture will describe advances in 3D tumor organoid culture that allow growth of cancer cells in a manner that preserves their native interaction with diverse tumor-infiltrating immune cells and allows analysis of immune checkpoint inhibition. Citation Format: C. Kuo. Organoid Models of Cancer and the Tumor Microenvironment [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr Wrk2-01.

Abstract PO5-14-06: Transcriptomics-based drug screening in 3D ex vivo patient-drived breast cancer model and patient biopsy for personalized therapy

Abstract Breast cancer is a leading cause of cancer-related mortality for women worldwide. Hormone receptor-positive (HR+) breast tumors, which represent 70% of all breast cancer cases, are treated with endocrine therapy. However, not all patients benefit from this treatment regimen because of patient-to-patient variation. Therefore, high throughput drug screening system is warranted to enable personalized medicine. Patient-derived organoids, which serve as a screening platform, lose their hormone receptors and response upon ex vivo culturing, and may not be adequate models for HR+ breast tumors. Moreover, unidentified biological components in the widely-used basement membrane matrix, Matrigel, result in high batch-to-batch variations and poor reproducibility in organoid cultures. Here, we propose a hydrogel-based 3D ex vivo model with defined structural and chemical properties to test hormone and drug sensitivity of HR+ breast tumors from patient-derived xenografts (PDXs) and patient tumor biopsies using microfluidics. Our data demonstrate the feasibility of this model to preserve cell proliferation and hormone receptor expression over 7 days. We also demonstrate that responses to hormones and FDA-approved drugs are faithfully maintained in this model. Finally, to establish a high throughput hormone and drug testing workflow with transcriptomic readout, we multiplexed barcoded- and drug-treated tumor samples in a single experiment with bulk RNA-sequencing. Our preliminary data demonstrate patient-specific responses to hormones and drugs that correspond to patient genetic mutation profiles, treatment history, disease stages and subtypes. This platform also enables testing of drugs in clinical trials that shows promising therapeutic outcomes for breast cancer, such as CDK4/6i, AKTi, PARPi, mTORi and their combination with endocrine therapy, thanks to the high throughput of the screening system and the low consumption of patient-derived or patient tissue. Given the capability of combining this physiologically-relevant 3D ex vivo model with RNA-seq for HR+ breast tumors, this platform holds potential for high throughput compound testing and transcriptomic profiling of patient biopsies for personalized medicine. Citation Format: Yueyun Zhang, Carlos Henrique Venturi Ronchi, Giovanna Ambrosini, Yuanlong Liu, Patrick Aouad, Daria Matvienko, Christoph Merten, Cathrin Brisken. Transcriptomics-based drug screening in 3D ex vivo patient-drived breast cancer model and patient biopsy for personalized therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-14-06.

Abstract PO4-28-01: Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC

Abstract Background: Chemotherapeutic resistance observed in triple-negative breast cancer (TNBC) poses a substantial clinical hurdle, emphasizing the imperative to enhance our comprehension of the underlying mechanisms with the aim of potentially mitigating or reversing this phenomenon. Methods: Doxorubicin-resistant (DoxR) TNBC models (MDA-MB-231 and BT-549) were established through continuous exposure to increasing concentrations of doxorubicin. RNA-Seq was conducted using the Illumina platform, while bioinformatics analyses were performed using CLC genomics workbench 20.2 and iDEP.91. Colony forming unit (CFU), flow cytometry and fluorescent microscopy were used to assess cell proliferation, cell cycle distribution, and cell death, respectively. Western blotting was used to confirm the expression and phosphorylation of protein targets. Ingenuity pathway analysis (IPA) and STRING database v 11.5 were used for network and pathway analyses, while Kaplan-Meier Plotter was used for survival analyses. Results: Herein, we provide a comprehensive functional and transcriptomic characterization of DoxR TNBC models, revealing multiple affected signaling networks and functional categories. The defense and immune response, response to stress, response to cytokine and external stimulus and cytokine-mediated signaling pathways were among the top activated, while suppression of cholesterol, sterol and lipid biosynthesis and metabolism were the hallmarks of DoxR cells. Upstream regulator analysis revealed IL-1B cytokine network activation in DoxR cells. Concordantly, disease and function analysis on the downregulated genes in DoxR TNBC cells revealed the most pronounced enrichment in pathways associated with cell invasion, colony formation, as well as lipid and carbohydrate metabolism. A number of upregulated genes were validated by qRT-PCR, which correlated with unfavorable overall survival (OS). Functionally, DoxR cells exhibited remarkable suppression of cell proliferation, as evidenced by diminished CFU potential, sphere formation and growth under 3D organoid culture, and cell migration. Although DoxR TNBC cells exhibited a slow cell cycling under normal culture conditions, wild-type TNBC cells exhibited remarkable arrest in the G2M phase when exposed to doxorubicin, which was less evident in DoxR TNBC models, suggesting DoxR cells undergo a quiescent state in response to chemotherapeutic challenge. In agreement with those data, increased phosphorylation of CHK2 and p53 proteins was remarkable in DoxR cells, thus promoting cellular dormancy. Conclusion: Our data revealed cellular dormancy and suppression of lipid metabolism as the hallmark associated with doxorubicin-resistant TNBC cells. On the other hand, cytokine signaling and stress response were the most enriched functional categories in DoxR TNBC cells. Therapeutic targeting to reverse the quiescent state or suppression of cytokine signaling networks could enhance the efficacy of doxorubicin-based therapeutics. Citation Format: Vishnubalaji Radhakrishnan, Nehad Alajez. Molecular profiling revealed activation of cytokine signaling and suppression of lipid metabolism as the hallmarks of doxorubicin-resistant TNBC [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-01.

Crosstalk between endothelial cells and dermal papilla entails hair regeneration and angiogenesis during aging

IntroductionTissues maintain their function through interaction with microenvironment. During aging, both hair follicles and blood vessels (BV) in skin undergo degenerative changes. However, it is elusive whether the changes are due to intrinsic aging changes in hair follicles or blood vessels respectively, or their interactions. ObjectiveTo explore how hair follicles and blood vessels interact to regulate angiogenesis and hair regeneration during aging. MethodsSingle-cell RNA-sequencing (scRNA-seq) analyses were used to identify the declined ability of dermal papilla (DP) and endothelial cells (ECs) during aging. CellChat and CellCall were performed to investigate interaction between DP and ECs. Single-cell metabolism (scMetabolism) analysis and iPATH were applied to analyze downstream metabolites in DP and ECs. Hair-plucking model and mouse cell organoid model were used for functional studies. ResultsDuring aging, distance and interaction between DP and ECs are decreased. DP interacts with ECs, with decreased EDN1-EDNRA signaling from ECs to DP and CTF1-IL6ST signaling from DP to ECs during aging. ECs-secreted EDN1 binds to DP-expressed EDNRA which enhances Taurine (TA) metabolism to promote hair regeneration. DP-emitted CTF1 binds to ECs-expressed IL6ST which activates alpha-linolenic acid (ALA) metabolism to promote angiogenesis. Activated EDN1-EDNRA-TA signaling promotes hair regeneration in aged mouse skin and in organoid cultures, and increased CTF1-IL6ST-ALA signaling also promotes angiogenesis in aged mouse skin and organoid cultures. ConclusionsOur finding reveals reciprocal interactions between ECs and DP. ECs releases EDN1 sensed by DP to activate TA metabolism which induces hair regeneration, while DP emits CTF1 signal received by ECs to enhance ALA metabolism which promotes angiogenesis. Our study provides new insights into mutualistic cellular crosstalk between hair follicles and blood vessels, and identifies novel signaling contributing to the interactions of hair follicles and blood vessels in normal and aged skin.

Hyaluronan improves photoreceptor differentiation and maturation in human retinal organoids

Human stem cell-derived organoids enable both disease modeling and serve as a source of cells for transplantation. Human retinal organoids are particularly important as a source of human photoreceptors; however, the long differentiation period required and lack of vascularization in the organoid often results in a necrotic core and death of inner retinal cells before photoreceptors are fully mature. Manipulating the in vitro environment of differentiating retinal organoids through the incorporation of extracellular matrix components could influence retinal development. We investigated the addition of hyaluronan (HA), a component of the interphotoreceptor matrix, as an additive to promote long-term organoid survival and enhance retinal maturation. HA treatment had a significant reduction in the proportion of proliferating (Ki67+) cells and increase in the proportion of photoreceptors (CRX+), suggesting that HA accelerated photoreceptor commitment in vitro. HA significantly upregulated genes specific to photoreceptor maturation and outer segment development. Interestingly, prolonged HA-treatment significantly decreased the length of the brush border layer compared to those in control retinal organoids, where the photoreceptor outer segments reside; however, HA-treated organoids also had more mature outer segments with organized discs structures, as revealed by transmission electron microscopy. The brush border layer length was inversely proportional to the molar mass and viscosity of the hyaluronan added. This is the first study to investigate the role of exogenous HA, viscosity, and polymer molar mass on photoreceptor maturation, emphasizing the importance of material properties on organoid culture. Statement of SignificanceRetinal organoids are a powerful tool to study retinal development in vitro, though like many other organoid systems, can be highly variable. In this work, Shoichet and colleagues investigated the use of hyaluronan (HA), a native component of the interphotoreceptor matrix, to improve photoreceptor maturation in developing human retinal organoids. HA promoted human photoreceptor differentiation leading to mature outer segments with disc formation and more uniform and healthy retinal organoids. These findings highlight the importance of adding components native to the developing retina to generate more physiologically relevant photoreceptors for cell therapy and in vitro models to drive drug discovery and uncover novel disease mechanisms.

Vitamin D opposes multilineage cell differentiation induced by Notch inhibition and BMP4 pathway activation in human colon organoids.

Understanding the mechanisms involved in colonic epithelial differentiation is key to unraveling the alterations causing inflammatory conditions and cancer. Organoid cultures provide an unique tool to address these questions but studies are scarce. We report a differentiation system toward enterocytes and goblet cells, the two major colonic epithelial cell lineages, using colon organoids generated from healthy tissue of colorectal cancer patients. Culture of these organoids in medium lacking stemness agents resulted in a modest ultrastructural differentiation phenotype with low-level expression of enterocyte (KLF4, KRT20, CA1, FABP2) and goblet cell (TFF2, TFF3, AGR2) lineage markers. BMP pathway activation through depletion of Noggin and addition of BMP4 resulted in enterocyte-biased differentiation. Contrarily, blockade of the Notch pathway using the γ-secretase inhibitor dibenzazepine (DBZ) favored goblet cell differentiation. Combination treatment with BMP4 and DBZ caused a balanced strong induction of both lineages. In contrast, colon tumor organoids responded poorly to BMP4 showing only weak signals of cell differentiation, and were unresponsive to DBZ. We also investigated the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on differentiation. Calcitriol attenuated the effects of BMP4 and DBZ on colon normal organoids, with reduced expression of differentiation genes and phenotype. Consistently, in normal organoids, calcitriol inhibited early signaling by BMP4 as assessed by reduction of the level of phospho-SMAD1/5/8. Our results show that BMP and Notch signaling play key roles in human colon stem cell differentiation to the enterocytic and goblet cell lineages and that calcitriol modulates these processes favoring stemness features.

Cytokine CCL9 Mediates Oncogenic KRAS-Induced Pancreatic Acinar-to-Ductal Metaplasia by Promoting Reactive Oxygen Species and Metalloproteinases.

Pancreatic ductal adenocarcinoma (PDAC) can originate from acinar-to-ductal metaplasia (ADM). Pancreatic acini harboring oncogenic Kras mutations are transdifferentiated to a duct-like phenotype that further progresses to become pancreatic intraepithelial neoplasia (PanIN) lesions, giving rise to PDAC. Although ADM formation is frequently observed in KrasG12D transgenic mouse models of PDAC, the exact mechanisms of how oncogenic KrasG12D regulates this process remain an enigma. Herein, we revealed a new downstream target of oncogenic Kras, cytokine CCL9, during ADM formation. Higher levels of CCL9 and its receptors, CCR1 and CCR3, were detected in ADM regions of the pancreas in p48cre:KrasG12D mice and human PDAC patients. Knockdown of CCL9 in KrasG12D-expressed pancreatic acini reduced KrasG12D-induced ADM in a 3D organoid culture system. Moreover, exogenously added recombinant CCL9 and overexpression of CCL9 in primary pancreatic acini induced pancreatic ADM. We also showed that, functioning as a downstream target of KrasG12D, CCL9 promoted pancreatic ADM through upregulation of the intracellular levels of reactive oxygen species (ROS) and metalloproteinases (MMPs), including MMP14, MMP3 and MMP2. Blockade of MMPs via its generic inhibitor GM6001 or knockdown of specific MMP such as MMP14 and MMP3 decreased CCL9-induced pancreatic ADM. In p48cre:KrasG12D transgenic mice, blockade of CCL9 through its specific neutralizing antibody attenuated pancreatic ADM structures and PanIN lesion formation. Furthermore, it also diminished infiltrating macrophages and expression of MMP14, MMP3 and MMP2 in the ADM areas. Altogether, our results provide novel mechanistic insight into how oncogenic Kras enhances pancreatic ADM through its new downstream target molecule, CCL9, to initiate PDAC.

Extracellular vesicles secreted by 3D tumor organoids are enriched for immune regulatory signaling biomolecules compared to conventional 2D glioblastoma cell systems.

Newer 3D culturing approaches are a promising way to better mimic the in vivo tumor microenvironment and to study the interactions between the heterogeneous cell populations of glioblastoma multiforme. Like many other tumors, glioblastoma uses extracellular vesicles as an intercellular communication system to prepare surrounding tissue for invasive tumor growth. However, little is known about the effects of 3D culture on extracellular vesicles. The aim of this study was to comprehensively characterize extracellular vesicles in 3D organoid models and compare them to conventional 2D cell culture systems. Primary glioblastoma cells were cultured as 2D and 3D organoid models. Extracellular vesicles were obtained by precipitation and immunoaffinity, with the latter allowing targeted isolation of the CD9/CD63/CD81 vesicle subpopulation. Comprehensive vesicle characterization was performed and miRNA expression profiles were generated by smallRNA-sequencing. In silico analysis of differentially regulated miRNAs was performed to identify mRNA targets and corresponding signaling pathways. The tumor cell media and extracellular vesicle proteome were analyzed by high-resolution mass spectrometry. We observed an increased concentration of extracellular vesicles in 3D organoid cultures. Differential gene expression analysis further revealed the regulation of twelve miRNAs in 3D tumor organoid cultures (with nine miRNAs down and three miRNAs upregulated). MiR-23a-3p, known to be involved in glioblastoma invasion, was significantly increased in 3D. MiR-7-5p, which counteracts glioblastoma malignancy, was significantly decreased. Moreover, we identified four miRNAs (miR-323a-3p, miR-382-5p, miR-370-3p, miR-134-5p) located within the DLK1-DIO3 domain, a cancer-associated genomic region, suggesting a possible importance of this region in glioblastoma progression. Overrepresentation analysis identified alterations of extracellular vesicle cargo in 3D organoids, including representation of several miRNA targets and proteins primarily implicated in the immune response. Our results show that 3D glioblastoma organoid models secrete extracellular vesicles with an altered cargo compared to corresponding conventional 2D cultures. Extracellular vesicles from 3D cultures were found to contain signaling molecules associated with the immune regulatory signaling pathways and as such could potentially change the surrounding microenvironment towards tumor progression and immunosuppressive conditions. These findings suggest the use of 3D glioblastoma models for further clinical biomarker studies as well as investigation of new therapeutic options.

Spatiotemporally resolved colorectal oncogenesis in mini-colons ex vivo

Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1–3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms.

Lipid Deficiency Contributes to Impaired Alveolar Progenitor Cell Function in Aging and Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an aging-associated interstitial lung disease resulting from repeated epithelial injury and inadequate epithelial repair. Alveolar type II cells (AEC2) are progenitor cells that maintain epithelial homeostasis and repair the lung after injury. In the current study, we assessed lipid metabolism in AEC2s from human lungs of IPF patients and healthy donors, as well as AEC2s from bleomycin-injured young and old mice. Through single cell RNA sequencing (scRNA-seq), we observed that lipid metabolism-related genes were downregulated in IPF AEC2s and bleomycin-injured mouse AEC2s. Aging aggravated this decrease and hindered recovery of lipid metabolism gene expression in AEC2s after bleomycin injury. Pathway analyses revealed down-regulation of genes related to lipid biosynthesis and fatty acid -oxidation in AEC2s from IPF lungs and bleomycin-injured, aged mouse lungs compared to the respective controls. We confirmed decreased cellular lipid content in AEC2s from IPF lungs and bleomycin-injured, aged mouse lungs using immunofluorescence staining and flow cytometry. We further show that lipid metabolism was associated with AEC2 progenitor function. Lipid supplementation and peroxisome proliferator activated receptor gamma (PPARγ) activation promoted progenitor renewal capacity of both human and mouse AEC2s in 3D organoid cultures. Lipid supplementation also increased AEC2 proliferation and expression of SFTPC in AEC2s. In summary, we identified a lipid metabolism deficiency in AEC2s from lungs of patients with IPF and bleomycin-injured aged mice. Restoration of lipid metabolism homeostasis in AEC2s might promote AEC2 progenitor function and offer new opportunities for therapeutic approaches to IPF.

[Research on pediatric hereditary kidney disease: from now to the future].

Hereditary kidney disease is an important cause of chronic kidney disease in children. With the progress of genome sequencing, single-cell technology, and organoid cultures, the research on hereditary kidney disease has entered a new era. How to integrate big data resources, discover new disease-causing genes, and develop effective treatment methods will be the focus of future research. This article discusses the classification, research progress, challenges and prospects of pediatric hereditary kidney disease, so as to provide valuable insights into the research of hereditary kidney disease in children.

Nfe2l2/NRF2 deletion attenuates tumorigenesis and increases bacterial diversity in a mouse model of Lynch syndrome.

Lynch syndrome (LS) is the most prevalent heritable form of colorectal cancer (CRC). Its early onset and high lifetime risk for CRC emphasize the necessity for effective chemoprevention. NFE2L2 (NRF2) is often considered a potential druggable target, and many chemopreventive compounds do induce NRF2. However, while NRF2 counteracts oxidative stress, it is also overexpressed in CRC and may promote tumorigenesis. Herein, we evaluated the role of NRF2 in prevention of LS-associated neoplasia. We found an increased levels of NRF2 in intestinal epithelia of mice with intestinal epithelial-specific Msh2 deletion (MSH2ΔIEC) as compared to C57BL/6 (wild type) mice, as well as an increase in downstream NRF2 targets Nqo1 and Gclc. Likewise, NRF2 levels were increased in human MSH2-deficient LS tumors compared to healthy controls. In silico analysis of a publicly accessible RNA-sequencing LS dataset also found an increase in downstream NRF2 targets. Upon crossing MSH2ΔIEC with Nrf2null mice (MSH2ΔIECNrf2null), we unexpectedly found reduced tumorigenesis in MSH2ΔIECNrf2null compared to MSH2ΔIEC after 40 weeks. This occurred despite an increase in oxidative damage in MSH2ΔIECNrf2null mice. Loss of NRF2 impaired proliferation as seen by Ki67 intestinal staining and in organoid cultures. This was accompanied by diminished WNT/β-catenin signaling. Apoptosis was unaffected. Microbial alpha-diversity increased over time with loss of NRF2 based upon 16S rRNA gene amplicon sequencing of murine fecal samples. Altogether, we show that NRF2 protein levels are increased in MSH2-deficiency and associated neoplasia, but loss of NRF2 attenuates tumorigenesis. Activation of NRF2 may not be a feasible strategy for chemoprevention in LS.

[Dental pulp stem cells in tooth regeneration: advancement and emerging directions].

Regenerating tissues similar to dental structure with normal function are putatively to be the aim in tooth regeneration filed. Currently, researchers preliminarily achieved tooth regeneration by applying dental pulp stem cell (DPSC) and stem cell from human exfoliated deciduous teeth (SHED). However, the regeneration efficiency remains unstable and needs further investigation. The development of single-cell RNA sequencing and organoid culture system provide potential of precise, targeted and controllable functional regeneration. This article reviews the current state of DPSC/SHED on tooth regeneration, and analyzes characteristics and hotspots of them, aiming to shed light on clinical translational application of stable and efficient tooth regeneration.

Evaluation of EGFR and COX pathway inhibition in human colon organoids of serrated polyposis and other hereditary cancer syndromes.

Serrated polyposis syndrome (SPS) presents with multiple sessile serrated lesions (SSL) in the large intestine and confers increased colorectal cancer (CRC) risk. However, the etiology of SPS is not known. SSL-derived organoids have not been previously studied but may help provide insights into SPS pathogenesis and identify novel biomarkers and chemopreventive strategies. This study examined effects of EGFR and COX pathway inhibition in organoid cultures derived from uninvolved colon and polyps of SPS patients. We also compared with organoids representing the hereditary gastrointestinal syndromes, Familial Adenomatous Polyposis (FAP) and Lynch syndrome (LS). Eighteen total organoid colon cultures were generated from uninvolved colon and polyps in SPS, FAP, LS, and non-syndromic screening colonoscopy patients. BRAF and KRAS mutation status was determined for each culture. Erlotinib (EGFR inhibitor) and sulindac (COX inhibitor) were applied individually and in combination. A 44-target gene custom mRNA panel (including WNT and COX pathway genes) and a 798-gene microRNA gene panel were used to quantitate organoid RNA expression by NanoString analysis. Erlotinib treatment significantly decreased levels of mRNAs associated with WNT and MAPK kinase signaling in organoids from uninvolved colon from all four patient categories and from all SSL and adenomatous polyps. Sulindac did not change the mRNA profile in any culture. Our findings suggest that EGFR inhibitors may contribute to the chemopreventive treatment of SSLs. These findings may also facilitate clinical trial design using these agents in SPS patients. Differentially expressed genes identified in our study (MYC, FOSL1, EGR1, IL33, LGR5 and FOXQ1) may be used to identify other new molecular targets for chemoprevention of SSLs.

Modeling HIV-1 infection and NeuroHIV in hiPSCs-derived cerebral organoid cultures.

The human immunodeficiency virus (HIV) epidemic is an ongoing global health problem affecting 38million people worldwide with nearly 1.6million new infections every year. Despite the advent of combined antiretroviral therapy (cART), a large percentage of people with HIV (PWH) still develop neurological deficits, grouped into the term of HIV-associated neurocognitive disorders (HAND). Investigating the neuropathology of HIV is important for understanding mechanisms associated with cognitive impairment seen in PWH. The major obstacle for studying neuroHIV is the lack of suitable in vitro human culture models that could shed light into the HIV-CNS interactions. Recent advances in induced pluripotent stem cell (iPSC) culture and 3D brain organoid systems have allowed the generation of 2D and 3D culture methods that possess a potential to serve as a model of neurotropic viral diseases, including HIV. In this study, we first generated and characterized several hiPSC lines from healthy human donor skin fibroblast cells. hiPSCs were then used for the generation of microglia-containing human cerebral organoids (hCOs). Once fully characterized, hCOs were infected with HIV-1 in the presence and absence of cART regimens and viral infection was studied by cellular, molecular/biochemical, and virological assays. Our results revealed that hCOs were productively infected with HIV-1 as evident by viral p24-ELISA in culture media, RT-qPCR and RNAscope analysis of viral RNA, as well as ddPCR analysis of proviral HIV-1 in genomic DNA samples. More interestingly, replication and gene expression of HIV-1 were also greatly suppressed by cART in hCOs as early as 7 days post-infections. Our results suggest that hCOs derived from hiPSCs support HIV-1 replication and gene expression and may serve as a unique platform to better understand neuropathology of HIV infection in the brain.