Hubrecht Institute

Researchers have found that specific gut cells, BEST4/CA7+ cells, regulate electrolyte and water balance in response to bacterial toxins that cause diarrhea. Their findings show that these cells greatly increase in number when exposed to the cytokine interferon- (IFN ), presenting a promising target for therapeutic strategies. Researchers from the Organoid group at the Hubrecht Institute have found that specific gut cells, BEST4/CA7+ cells, regulate electrolyte and water balance in response to bacterial toxins that cause diarrhea. Their findings, published in Cell Stem Cell, show that these cells greatly increase in number when exposed to the cytokine interferon-γ (IFNγ), presenting a promising target for therapeutic strategies. In the gut, a variety of cell types collaborate to keep a balance of electrolyte and water. Bacterial infections can disrupt this balance, leading to diarrhea. Yet, it was unclear which cells were mainly affected by these toxins. "In this study, we looked at the recently identified BEST4/CA7+ cells, a specific type of cell in the intestinal lining that highly expresses CFTR, an important ion channel for electrolyte balance," says Daisong Wang, lead author of the study. Understanding how BEST4/CA7+ cells develop While researchers previously identified the presence of BEST4/CA7+ cells, their development and function are not well understood due to the lack of study models. Wang and his colleagues overcame this by creating human intestinal organoids, mini organ-like structures cultured in a dish. They used these organoids to experimentally assess the response of BEST4/CA7+ cells to various specific signals. "Using cell-type specific reporter organoids and CRISPR-mediated genetic modifications, we found that the Notch signaling pathway and a master regulator named SPIB are crucial for these cells' development," Wang notes. BEST4/CA7+ cells: key targets in diarrhea The team previously observed swelling of the organoid when the ion channel CFTR was activated. "However, we saw that organoids lacking BEST4/CA7+ cells did not show this swelling, confirming the vital role of these cells in controlling fluid balance," Wang explains. This shows that BEST4/CA7+ cells are key targets in diarrhea. Unexpected growth in response to IFNγ The researchers then added the type I immune factor called IFNγ which is present during bacterial infections. What they observed was remarkable: "Though these cells only represent a small portion of the intestinal lining, we observed their numbers increase over eightfold in organoids exposed to IFNγ," Wang explains. "This is the first time that a type I immune-responsive cell type has been identified in the human gut and the significant growth of BEST4/CA7+ cells underscore their roles in immunity. On the other hand, when treated with a drug called rapamycin, we saw that the number of these cells goes down." he adds. This could suggest a promising pharmacological strategy for naturally regulating the number of BEST4/CA7+ cell. Future implications The insights from this study not only advance our understanding of gut biology but also open new doors for therapeutic strategies. "Identifying BEST4/CA7+ cells as the primary targets of bacterial toxins might allow us to control fluid release more effectively, either by changing the number of BEST4/CA7+ cells or by targeting their intracellular functional mechanisms." Wang notes. While initial laboratory results are promising, further research is necessary to determine the efficacy of these strategies in real-world applications.

OTC2024类器官前沿应用与3D细胞培养论坛圆满落幕，点击图片可查看会后报告，咨询OTC2025类器官论坛请联系：王晨 180 1628 8769。 文章来源：华夏源类器官 “ 人类肠道内分泌细胞（EEC）是一种罕见的胃肠道上皮细胞，由于低表达，多亚型，种间差异大，缺乏有效的研究模型。近日，Hans Clevers团队在顶级期刊《Science》（IF：44.7）发表研究论文[1]，全面描述并验证了肠道内分泌细胞传感器（受体）功能，揭示其在调节激素分泌中的关键作用。研究中开发的胃类器官模型，补充了现有肠道类器官培养协议，有望为糖尿病、肥胖症等代谢疾病治疗提供新策略。 肠道是重要的消化器官，也是人体最大的免疫器官，具有营养吸收、能量代谢、保护屏障等重要生理作用。这道身体“长城”既可以保护身体免受有害细菌和高度动态PH值的侵害，又可以通过消化、吸收食物中的营养成分，使其进入血液，为身体提供能量。更重要的是，肠道还是分泌多种激素以调节身体机能的内分泌细胞的“家园”。 人类肠道内分泌细胞（EEC）是肠道上皮细胞的一个特殊亚群，相对稀有，所占数量不足整个胃肠道（GI）上皮细胞的1%。EEC作为专门的激素分泌细胞，可细分为五个主要亚型，且每种亚型产生不同的肽类激素和/或神经递质（如GLP-1和GIP等），激素的释放通过代谢物传感蛋白调控。 EEC通过分泌激素来响应肠道内容物，从而调节包括食欲、胰岛素释放和肠道运动等重要生理活动。尽管如此，由于EEC的稀有性，低表达，种间差异大（小鼠 EEC 响应的信号可能与人类 EECs响应的信号不同），亚型多样，以及对EEC的药理学研究多集中在治疗糖尿病和肥胖症等方面，目前鲜有实验模型能全面反映EEC在代谢调节中的重要作用，为深入研究带来挑战。 令人惊喜的是，近日由荷兰Hubrecht研究所和罗氏人类生物学研究所（Institute of Human Biology，IHB）领导的研究团队开发了全新的胃肠类器官模型，全面描述并验证了人类肠内分泌细胞（EEC）的传感器（受体）功能，揭示了其在调节激素分泌中的作用。该研究区分了人类胃EEC，并整合了胃和肠道类器官的研究协议，超越了以往主要基于小鼠模型或仅专注于肠道类器官的研究。 该实验阐明了个别人类EEC传感器在激素分泌中的重要作用，这些传感器有望成为影响食欲、肠道运动、胰岛素敏感性和黏膜免疫的潜在药物靶点，有望为治疗代谢疾病（如糖尿病和肥胖症等）提供全新的干预途径。 01 胃类器官EEC功能验证 开辟代谢疾病治疗新路径 肠道内分泌细胞（Enteroendocrine cells, EEC）是分布在胃、小肠和结肠上皮中的激素分泌细胞。EEC根据产生的肽类激素（或神经递质）不同被分为5个亚型。例如，L细胞产生胰高血糖素样肽1（Glucagon-like peptide 1, GLP-1），而K细胞产生胃抑制蛋白（Gastric inhibitory protein, GIP）。这些激素通过G蛋白偶联受体（G protein-coupled receptors, GPCR）以及营养状态来控制细胞内钙水平，进而调节激素的分泌。 由“类器官鼻祖”Hans Clevers教授带领的Hubrecht研究团队在此前已经开发出在类器官中获取大量EEC的方法。考虑到EEC在肠道的不同区域具有不同的传感蛋白和激素谱，研究这些稀有细胞需要制作所有这些不同区域的富集EEC的类器官。 在最新研究中，研究团队设法在消化系统的其他部分（包括胃）构建类器官模型。Hans Clevers团队利用类器官技术和CRISPR基因筛选，揭示了调控EEC分化的关键因子。首先，他们通过诱导神经元生成因子-3——NEUROG-3（一种对EEC分化至关重要的转录因子）的过量表达，实现胃EEC的生产，并成功分化出人类胃类器官中的关键EEC亚型。 这些肠道内分泌细胞在受到刺激时能够分泌大约20种活性激素，包括5-羟色胺（5-HT）、生长抑素（somatostatin, SST）、神经肽Y（neuropeptide Y, NPY）等，它们通过内分泌和旁分泌途径作用于远处或邻近的靶器官。 △  人类胃器官，可以看到一种罕见的紫色生长激素释放肽产生细胞 与真实的胃部一样，这些胃类器官也会对已知的激素释放诱导剂做出反应，并分泌大量的胃饥饿素（Ghrelin），这种激素向大脑发出饥饿信号方面起着关键作用。 这证实了胃类器官可用于研究EEC的激素分泌，以此作为现有肠道类器官培养技术的补充。 △ 胃EEC生成的示意图 由于人类肠道内分泌细胞较为罕见，研究团队一直在努力绘制许多EEC的轮廓图。 在最新实验中，研究人员在EEC表面上发现了一种名为CD200的标志物，利用这一表面标志物从类器官中分离出大量人类EEC，并研究它们的传感器（受体）蛋白。 CD200表面标志物用于不同区域（胃、小肠和结肠组织）人类胃肠道（GI）EEC的深度转录组分析，克服了之前研究此类罕见细胞的困难。研究人员结合大规模RNA测序和蛋白质组分析，绘制了人类EEC的单细胞转录组图谱，并验证了类器官和组织中EEC的受体表达概况。 △ 胃、小肠和结肠中人类EEC亚型的免疫荧光染色 此外，研究人员利用CRISPR/Cas9基因编辑技术构建了一个包含22个受体缺陷类器官生物样本库。引入一种主要针对G蛋白偶联受体（GPCR）功能的新型筛选方式，研究这些受体缺失的类器官在不同配体刺激下的激素分泌情况（如GLP-1、5-羟色胺和生长激素释放肽GHRL）。例如，识别了多种控制GLP-1分泌的GPCR和通道蛋白，包括磺脲类药物受体ABCC8和色氨酸受体CasR。 △ 生成用于功能验证的人类功能丧失GPCR生物样本库的实验概述图 GPCR的功能验证提供了对这些受体如何影响肠道激素分泌的机制性理解，为开发能够激活这些传感器的新药物提供了机会，特别是通过靶向局部激素分泌（如GLP-1）作为当前系统性激素模拟物疗法的替代方案，可能引发小分子口服药物的开发，或将开辟代谢疾病（如糖尿病和肥胖症等）的治疗新思路。 综上，这些研究不仅加深了我们对EEC在代谢和免疫调节中作用的理解，还为开发新的治疗代谢性疾病的策略提供了重要基础。类器官体外平台已然成为连接基础科研与临床应用之间的桥梁。 02 医学科研革新工具 类器官技术大放异彩 肠道内分泌细胞（EEC）在调节消化、吸收、食欲和代谢等方面的关键作用，通过人类胃类器官体外模型的构建，补充了现有肠道类器官培养技术，为研究EEC的分化和功能提供了新工具。通过全面描述和验证人类EEC传感器（受体）功能，揭示其在调节激素分泌中的作用，为调控食欲、肠道运动、胰岛素敏感性和粘膜免疫提供了潜在药物靶点。 作为体内疾病的绝佳体外研究场所，类器官模型的构建已成为更多疾病治疗研究策略的新途径。 胃食管癌独特类器官模型 胃食管交界，是指食管远端和胃近端贲门交界处一段非常短的解剖学区域。胃食管交界处（GEJ）癌就是发生在这一区域的恶性肿瘤。临床上，GEJ癌既不属于胃癌也不属于食管癌范畴，治疗方案也因此差异很大。 2022年11月，来自南加利福尼亚大学等机构的科学家们通过研究利用人类组织开发了一种在实验室生长的三维类器官模型和CRISPR编辑转化的GEJ类器官模型。[2]该研究提供了对早期 GEJ 肿瘤中与TP53/CDKN2A失活相关的原发性机制的见解，确定了GEJ模型肿瘤发生过程中发生的关键代谢和表观基因组变化，并揭示了潜在的癌症治疗策略。 △ 人类胃食管交界处来源的类器官图像，通过使用CRISPR/Cas9 基因编辑技术双敲除关键抑癌基因 (TP53/CDKN2A) 进行修饰，导致细胞变得更加癌变 腹膜假性粘液瘤类器官样本库 腹膜假黏液瘤（PMP）是一种罕见的恶性疾病，通常始于阑尾，由于黏液性肿瘤细胞在腹腔内广泛扩散所致。该疾病由于极其罕见，容易误诊，且治疗手段相对复杂，缺乏有效的体外研究模型，因而迫切需要新的研究模型和治疗策略。 2024年8月，西班牙Vall d 'Hebron肿瘤研究所等医疗机构的研究人员发表最新研究成果[3]，他们通过120个PMP样本生成了50个患者衍生的类器官（PDO）和异种移植（PDX）模型，深入研究了该疾病并揭示了KRAS和BRFA药物靶点，为PMP的高通量药物筛选和个性化治疗奠定实验基础。通过实验数据和临床前模型的收集，为临床评估患者的系统性靶向治疗铺平道路。 △ 生成PMP-PDO、PDX和PDXO的模型集合，这些模型具有可识别的KRAS和BRAF药物靶点，以指导分子靶向治疗的选择 类器官与人体器官拥有高度相似的组织学特征，并能在体外重现其生理功能。不仅可以模拟疾病发育，也为更多疾病研究构建了全新体外模型，在个性化治疗，药物筛选，器官移植等方面表现出良好的应用潜力，成为再生医学领冉冉升起的新星。 Write in the last 写在最后 类器官技术将在医学基础研究和临床前转化方面扮演愈来愈重要的角色。随着类器官技术的持续精进和应用范围的拓展，预计未来将有更多类器官、患者来源类器官生物样本库的出现，有望推动医疗技术的革新，助力疾病治疗、药物筛选、新药研发、精准治疗等领域的进步，为疾病治疗提供靶向性、个性化的新选择。 参考资料（可上下滑动查看）： [1] Joep Beumer et al, Description and functional validation of human enteroendocrine cell sensors, Science (2024). DOI: 10.1126/science.adl1460. www.science.org/doi/10.1126/science.adl1460 [2] HUA ZHAO,YULAN CHENG,ANDREW KALRA, et al. Generation and multiomic profiling of a TP53/CDKN2A double-knockout gastroesophageal junction organoid model, Science Translational Medicine (2022). DOI:10.1126/scitranslmed.abq6146 [3] Jordi Martínez-Quintanilla et al, Precision oncology and systemic targeted therapy in Pseudomyxoma Peritonei, Clinical Cancer Research (2024). DOI: 10.1158/1078-0432.CCR-23-4072 END 免责声明：本文仅作知识交流与分享及科普目的，不涉及商业宣传，不作为相关医疗指导或用药建议。文章如有侵权请联系删除。 OTC2024类器官前沿应用与3D细胞培养论坛圆满落幕，点击图片可查看会后报告，咨询OTC2025类器官论坛请联系：王晨 180 1628 8769

Researchers have made a surprising new discovery in the structure of the centromere, a structure that is involved in ensuring that chromosomes are segregated properly when a cell divides. Mistakes in chromosome segregation can lead to cell death and cancer development. The researchers discovered that the centromere consists of two subdomains. This fundamental finding has important implications for the process of chromosome segregation and provides new mechanisms underlying erroneous divisions in cancer cells. The research was published in Cell on May 13th 2024. Researchers from the Kops group in collaboration with researchers from the University of Edinburgh, made a surprising new discovery in the structure of the centromere, a structure that is involved in ensuring that chromosomes are segregated properly when a cell divides. Mistakes in chromosome segregation can lead to cell death and cancer development. The researchers discovered that the centromere consists of two subdomains. This fundamental finding has important implications for the process of chromosome segregation and provides new mechanisms underlying erroneous divisions in cancer cells. The research was published in Cell on May 13th 2024. Our bodies consist of trillions of cells, most of which have a limited life span and therefore need to reproduce to replace the old ones. This reproduction process is referred to as cell division or mitosis. During mitosis, the parent cell will duplicate its chromosomes in order to pass down the genetic material to the daughter cells. The resulting identical pairs of chromosomes, the sister chromatids, are held together by a structure called the centromere. The sister chromatids then need to be evenly split over the two daughter cells to ensure that each daughter cell is an exact copy of the parent cell. If errors happen during the segregation, one daughter cell will have too many chromosomes, while the other has too few. This can lead to cell death or cancer development. The role of the centromere The centromere is a part of the chromosome that plays a vital role in chromosome segregation during mitosis. The process of dividing the sister chromatids over the cells is guided by the interaction between the centromeres and structures known as spindle microtubules. These spindle microtubules are responsible for pulling the chromatids apart and thus separating the two sister chromatids. Carlos Sacristan Lopez, the first author of this study, explains: 'If the attachment of the centromere to the spindle microtubules does not occur properly it leads to chromosome segregation mistakes which are frequently observed in cancer.' Understanding the structure of the centromere can contribute to more insights into the function of the centromere and its role in erroneous chromosomal segregation. A surprising discovery To investigate the centromere structure, the researchers used a combination of imaging and sequencing techniques. The super-resolution microscopy imaging took place at the Hubrecht Institute, while the group of Bill Earnshaw performed the sequencing. This collaboration led to a surprising new discovery in the centromere structure. Previously believed to consist of a compact structure attaching to multiple spindle microtubules, it was instead revealed that the centromere consists of two subdomains. Carlos explains: 'This discovery was very surprising, as subdomains bind microtubules independently of each other. Yet, to form correct attachments, they must remain closely connected. In cancer cells, however, we often observe that subdomains uncouple, resulting in erroneous attachments and chromosome segregation errors.' This very exciting and fundamental discovery contributes to our understanding of the origin of chromosome segregation errors which are frequently seen in cancer.