类器官在乳腺癌相关研究中的应用进展

乳腺癌是女性最常见的癌症,目前乳腺癌的研究主要借助体内模型和传统细胞培养方法,然而研究表明,由于人类和动物之间固有的物种差异,以及器官和细胞之间组织结构的差异,使用上述两种研究方法研制出的药物,在临床试验中失败率高达90%,因此,类器官三维培养应运而生。类器官是一种具有空间结构的三维细胞复合体,它作为一种新的肿瘤研究模型,在精准医疗、器官移植、建立难治疾病模型、基因治疗和药物研发等方向具有广阔的应用前景,是未来生命科学研究的理想载体之一。乳腺癌作为一种表型复杂的异质性疾病,其患者生存率较低,而乳腺癌类器官可以重现人类乳腺癌的许多关键特征,故构建乳腺癌类器官生物库,将会为研究乳腺癌的发生、发展、转移和耐药机制提供一个新的平台。文中将系统介绍类器官的培养条件及其在乳腺癌相关研究中的应用,并对类器官的应用前景进行展望。     

The science and engineering of stem cell-derived organoids-examples from hepatic,biliary, and pancreatic tissues

The field of organoid engineering promises to revolutionize medicine with wide-ranging applications of scientific, engineering, and clinical interest, including precision and personalized medicine, gene editing, drug development, disease modelling, cellular therapy, and human development. Organoids are a three-dimensional (3D) miniature representation of a target organ, are initiated with stem/progenitor cells, and are extremely promising tools with which to model organ function. The biological basis for organoids is that they foster stem cell self-renewal, differentiation, and self-organization, recapitulating 3D tissue structure or function better than two-dimensional (2D) systems. In this review, we first discuss the importance of epithelial organs and the general properties of epithelial cells to provide a context and rationale for organoids of the liver, pancreas, and gall bladder. Next, we develop a general framework to understand self-organization, tissue hierarchy, and organoid cultivation. For each of these areas, we provide a historical context, and review a wide range of both biological and mathematical perspectives that enhance understanding of organoids. Next, we review existing techniques and progress in hepatobiliary and pancreatic organoid engineering. To do this, we review organoids from primary tissues, cell lines, and stem cells, and introduce engineering studies when applicable. We discuss non-invasive assessment of organoids, which can reveal the underlying biological mechanisms and enable improved assays for growth, metabolism, and function. Applications of organoids in cell therapy are also discussed. Taken together, we establish a broad scientific foundation for organoids and provide an in-depth review of hepatic, biliary and pancreatic organoids.     

Next generation organoids for biomedical research and applications

Organoids are in vitro cultures of miniature fetal or adult organ-like structures. Their potentials for use in tissue and organ replacement, disease modeling, toxicology studies, and drug discovery are tremendous. Currently, major challenges facing human organoid technology include (i) improving the range of cellular heterogeneity for a particular organoid system, (ii) mimicking the native micro- and matrix-environment encountered by cells within organoids, and (iii) developing robust protocols for the in vitro maturation of organoids that remain mostly fetal-like in cultures. To tackle these challenges, we advocate the principle of reverse engineering that replicates the inner workings of in vivo systems with the goal of achieving functionality and maturation of the resulting organoid structures with the input of minimal intrinsic (cellular) and environmental (matrix and niche) constituents. Here, we present an overview of organoid technology development in several systems that employ cell materials derived from fetal and adult tissues and pluripotent stem cell cultures. We focus on key studies that exploit the self-organizing property of embryonic progenitors and the role of designer matrices and cell-free scaffolds in assisting organoid formation. We further explore the relationship between adult stem cells, niche factors, and other current developments that aim to enhance robust organoid maturation. From these works, we propose a standardized pipeline for the development of future protocols that would help generate more physiologically relevant human organoids for various biomedical applications.     

类器官的应用研究进展

类器官是利用干细胞的自我更新和分化能力,在体外培养形成的一种微小组织器官类似物,在很大程度上具有体内相应器官的功能。迄今为止,在3D培养条件下,已经成功培养出多种类器官如肺、胃、肠、肝和肾等类器官。它们不仅可作为组织器官的替代品用于药物和临床研究,还可用于体内器官移植。本文综述了类器官在药物毒性检测、药效评价和新药筛选中的作用以及利用类器官建立疾病模型、研究组织器官发育和类器官在精准医疗、再生医学中的价值。     

In-Depth Comparison of Matrigel Dissolving Methods on Proteomic Profiling of Organoids

Patient-derived organoids recently emerged as promising ex vivo 3D culture models recapitulating histological and molecular characteristics of original tissues, thus proteomic profiling of organoids could be valuable for function investigation and clinical translation. However, organoids are usually cultured in murine Matrigel (served as scaffolds and matrix), which brings an issue to separate organoids from Matrigel. Because of the complex compositions of Matrigel and thousands of identical peptides shared between Matrigel and organoids, insufficiently dissolved Matrigel could influence proteomic analysis of organoids in multiple ways. Thus, how to dissolve Matrigel matrix and recovery organoid cells efficiently is vital for sample preparation. Here, we comprehensively compared three popular Matrigel dissolving methods (cell recovery solution, dispase, and PBS–EDTA buffer) and investigated the effect of undissolved Matrigel proteins on proteomic profiles of organoids. By integrative analysis of label-free proteomes of Matrigel and stable isotope labeling by amino acids in cell culture proteomes of organoids collected by three methods, respectively, we found that dispase showed an optimal efficiency, with the highest peptide yield and the highest incorporation ratio of stable isotope labeling by amino acids in cell culture labels (97.1%), as well as with the least potential Matrigel contaminants. To help analysis of proteomic profiles of organoids collected by the other two methods, we identified 312 high-confidence Matrigel contaminants, which could be filtered out to attenuate Matrigel interference with minimal loss of biological information. Together, our study identifies bioinformatics and experimental approaches to eliminate interference of Matrigel contaminants efficiently, which will be valuable for basic and translational proteomic research using organoid models.     

Bio-modulated mice epithelial endometrial organoids by low-level laser therapy serves as an invitro model for endometrial regeneration

Endometrial regeneration is a dynamic process that is not well understood. The destruction of the endometrium with the formation of intrauterine adhesions is known as Asherman’s syndrome. The lesions range from minor to severe adhesions and their impact on pregnancy is well documented. Operative hysteroscopy is the mainstay of diagnosis and treatment of intrauterine adhesions. Nevertheless, the recurrence rates remain high. It was recorded that low-level laser therapy in low doses has a stimulatory effect on different tissues while the high dose produces a suppressive effect. Organoid is a three-dimensional assembly that displays architectures and functionalities similar to in vivo organs that are being developed from human or animal stem cells or organ-specific progenitors through a self-organization process. Our prospective was to study the effect of Low-Level Laser Therapy (LLLT) on mouse epithelial endometrial organoids regarding cell proliferation and endometrial regeneration as a new modality of treatment. An in vitro clinical trial to generate mouse epithelial organoid model and testing LLLT using He:Ne 632.8 nm device on organoids proliferation, function, and their response to ovarian hormones was performed. Trying endometrial regeneration by culturing organoids with decellularized uterine matrix (DUM) and studying the LLLT effect on the regeneration process. LLLT produced a proliferative effect on the epithelial mouse organoids confirmed by Ki67 and PCNA IHC. The organoids could regenerate the epithelial layer of the endometrium in vitro on DUM and LLLT could help in this process. In conclusion, organoids whether control or bio-stimulated proved a new modality to regenerate the endometrium.     

Human iPS Cell-Derived Insulin Producing Cells Form Vascularized Organoids under the Kidney Capsules of Diabetic Mice

Type 1 diabetes (T1D) is caused by autoimmune disease that leads to the destruction of pancreatic β-cells. Transplantation of cadaveric pancreatic organs or pancreatic islets can restore normal physiology. However, there is a chronic shortage of cadaveric organs, limiting the treatment of the majority of patients on the pancreas transplantation waiting list. Here, we hypothesized that human iPS cells can be directly differentiated into insulin producing cells (IPCs) capable of secreting insulin. Using a series of pancreatic growth factors, we successfully generated iPS cells derived IPCs. Furthermore, to investigate the capability of these cells to secrete insulin in vivo, the differentiated cells were transplanted under the kidney capsules of diabetic immunodeficient mice. Serum glucose levels gradually declined to either normal or near normal levels over 150 days, suggesting that the IPCs were secreting insulin. In addition, using MRI, a 3D organoid appeared as a white patch on the transplanted kidneys but not on the control kidneys. These organoids showed neo-vascularization and stained positive for insulin and glucagon. All together, these data show that a pancreatic organ can be created in vivo providing evidence that iPS cells might be a novel option for the treatment of T1D.     

Robust circadian rhythms in organoid cultures from PERIOD2::LUCIFERASE mouse small intestine

Disruption of circadian rhythms is a risk factor for several human gastrointestinal (GI) diseases, ranging from diarrhea to ulcers to cancer. Four-dimensional tissue culture models that faithfully mimic the circadian clock of the GI epithelium would provide an invaluable tool to understand circadian regulation of GI health and disease. We hypothesized that rhythmicity of a key circadian component, PERIOD2 (PER2), would diminish along a continuum from ex vivo intestinal organoids (epithelial ‘miniguts’), nontransformed mouse small intestinal epithelial (MSIE) cells and transformed human colorectal adenocarcinoma (Caco-2) cells. Here, we show that bioluminescent jejunal explants from PERIOD2::LUCIFERASE (PER2::LUC) mice displayed robust circadian rhythms for >72 hours post-excision. Circadian rhythms in primary or passaged PER2::LUC jejunal organoids were similarly robust; they also synchronized upon serum shock and persisted beyond 2 weeks in culture. Remarkably, unshocked organoids autonomously synchronized rhythms within 12 hours of recording. The onset of this autonomous synchronization was slowed by >2 hours in the presence of the glucocorticoid receptor antagonist RU486 (20 μM). Doubling standard concentrations of the organoid growth factors EGF, Noggin and R-spondin enhanced PER2 oscillations, whereas subtraction of these factors individually at 24 hours following serum shock produced no detectable effects on PER2 oscillations. Growth factor pulses induced modest phase delays in unshocked, but not serum-shocked, organoids. Circadian oscillations of PER2::LUC bioluminescence aligned with Per2 mRNA expression upon analysis using quantitative PCR. Concordant findings of robust circadian rhythms in bioluminescent jejunal explants and organoids provide further evidence for a peripheral clock that is intrinsic to the intestinal epithelium. The rhythmic and organotypic features of organoids should offer unprecedented advantages as a resource for elucidating the role of circadian rhythms in GI stem cell dynamics, epithelial homeostasis and disease.KEY WORDS: Circadian rhythm, Intestinal organoid, PERIOD2, R-spondin, RU486     

Circulating tumor cell-derived organoids: Current challenges and promises in medical research and precision medicine

Traditional 2D cell cultures do not accurately recapitulate tumor heterogeneity, and insufficient human cell lines are available. Patient-derived xenograft (PDX) models more closely mimic clinical tumor heterogeneity, but are not useful for high-throughput drug screening. Recently, patient-derived organoid cultures have emerged as a novel technique to fill this critical need. Organoids maintain tumor tissue heterogeneity and drug-resistance responses, and thus are useful for high-throughput drug screening. Among various biological tissues used to produce organoid cultures, circulating tumor cells (CTCs) are promising, due to relative ease of ascertainment. CTC-derived organoids could help to acquire relevant genetic and epigenetic information about tumors in real time, and screen and test promising drugs. This could reduce the need for tissue biopsies, which are painful and may be difficult depending on the tumor location. In this review, we have focused on advances in CTC isolation and organoid culture methods, and their potential applications in disease modeling and precision medicine.     

Linking stem cell function and growth pattern of intestinal organoids

Intestinal stem cells (ISCs) require well-defined signals from their environment in order to carry out their specific functions. Most of these signals are provided by neighboring cells that form a stem cell niche, whose shape and cellular composition self-organize. Major features of this self-organization can be studied in ISC-derived organoid culture. In this system, manipulation of essential pathways of stem cell maintenance and differentiation results in well-described growth phenotypes.We here provide an individual cell-based model of intestinal organoids that enables a mechanistic explanation of the observed growth phenotypes. In simulation studies of the 3D structure of expanding organoids, we investigate interdependences between Wnt- and Notch-signaling which control the shape of the stem cell niche and, thus, the growth pattern of the organoids. Similar to in vitro experiments, changes of pathway activities alter the cellular composition of the organoids and, thereby, affect their shape. Exogenous Wnt enforces transitions from branched into a cyst-like growth pattern; known to occur spontaneously during long term organoid expansion. Based on our simulation results, we predict that the cyst-like pattern is associated with biomechanical changes of the cells which assign them a growth advantage. The results suggest ongoing stem cell adaptation to in vitro conditions during long term expansion by stabilizing Wnt-activity.Our study exemplifies the potential of individual cell-based modeling in unraveling links between molecular stem cell regulation and 3D growth of tissues. This kind of modeling combines experimental results in the fields of stem cell biology and cell biomechanics constituting a prerequisite for a better understanding of tissue regeneration as well as developmental processes.     

Collective behaviours in organoids

Collective behaviour emerges from interacting units within communities, such as migrating herds, swimming fish schools, and cells within tissues. At the microscopic level, collective behaviours include collective cell migration in development and cancer invasion, rhythmic gene expression in pattern formation, cell competition in homeostasis and cancer, force generation and mechano-sensing in morphogenesis. Studying the initiation and the maintenance of collective cell behaviours is key to understand the principles of development, regeneration and disease. However, the manifold influences of contributing factors in in vivo environments challenge the dissection of causalities in animal models. As an alternative model that has emerged to overcome this difficulty, in vitro three-dimensional organoid cultures provide a reductionist approach yet retain similarities with the in vivo tissue in cellular composition and tissue organisation. Here, we focus on recent progresses in studying collective behaviours in different organoid systems and discuss their advantages and the possibility of improvement for future applications.     

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.     

R‐spondin 1 and noggin facilitate expansion of resident stem cells from non‐damaged gallbladders

Pioneering studies within the last few years have allowed the in vitro expansion of tissue‐specific adult stem cells from a variety of endoderm‐derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R‐spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5‐positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non‐regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R‐spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder‐derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride‐treated mice. Human gallbladders were also a source of organoid‐forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.     

Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy

Intestinal epithelial cells (IECs) not only have a critical function in the absorption of nutrients, but also act as a physical barrier between our body and the outside world. Damage and death of the epithelial cells lead to the breakdown of this barrier function and inflammation due to access of the immune system to compounds of the intestinal flora. Intestinal epithelial damage is frequently associated with various inflammatory disorders, chemo- and radiotherapy as well as drug-mediated toxicity. Until recently, intestinal epithelial-damaging activities of drugs and treatments could be tested only in vivo in animal models because of the poor survival rate of primary IECs ex vivo. The three-dimensional culture and outgrowth of intestinal crypt stem cells into organoids have offered new possibilities to culture and study IECs ex vivo. Here we demonstrate that intestinal organoids are a useful and physiologically relevant model system to study cell death and survival in IECs. We further describe a number of microscopy-based as well as colorimetric methods to monitor and score survival and death of intestinal organoids. Finally, the comparison of organoids isolated from gene-deficient mice and wild-type mice allows investigating the role of specific genes in the regulation of IEC death. Owing to their comparable structure and behavior, intestinal organoids may serve as an interesting and physiologically relevant surrogate system for large- and mid-scale in vitro testing of intestinal epithelium-damaging drugs and toxins, and for the investigation of cell death pathways.     

皮肤类器官的构建、功能及其应用研究进展

皮肤类器官作为一种新型的类器官模型,不仅能高度模拟皮肤组织的生理结构和功能,更好地在不同体外环境下还原较真实的皮肤生态,还可以应用于皮肤发育研究、皮肤疾病病理研究及药物筛选等领域。在干细胞研究中,皮肤类器官模型可以在特殊的生境下对具有特定功能的皮肤细胞及其附属物进行重建和改造,以弥补现有体外皮肤模型在结构、功能等方面的不足。基于此,皮肤类器官将会在皮肤再生、组织修复、药物筛选及医学美容等方面扮演越来越重要的角色。本文详述了皮肤类器官构建中所参与的细胞来源及近年来的应用,并对未来皮肤类器官的发展与优化做出了展望。     

类器官的构建与应用进展

类器官是在体外经由干细胞驱动的, 形成具有来源器官显微解剖特征的多细胞三维结构且能自我更新的微组织。类器官能分化产生器官特异性的多种细胞类型,能重现对应器官的部分功能和空间架构,它的诞生为生命医学研究和临床应用注入了新动能,在癌症基础与临床研究、再生医学等领域表现出广阔的应用前景。对近些年国内外类器官研究进展进行综述,介绍其构建过程与培养体系,并详细阐述其作为体外研究模型的优缺点,为基于类器官的科学研究与应用提供了参考。     

小鼠肠道类器官三种不同条件培养基的比较研究

摘要 目的：比较三种不同条件培养基对小鼠类器官形态和增殖速度的影响。方法：取C57BL/6小鼠的小肠和结肠,EDTA法分离隐窝,以基质胶包埋,加入不同小鼠肠道类器官培养基培养7 天,使用光学显微镜记录和比较类器官形成率和出芽情况。随后进行二代类器官培养,使用TrypLE将类器官消化为单细胞,重新包埋和培养,使用光学显微镜记录和比较不同类器官培养基对二代类器官的培养效率。采用荧光定量PCR比较不同条件培养类器官中干细胞标志物Lgr5和分化标志物MUC2的表达。使用免疫荧光法检测类器官中ki-67的表达。结果：对于小肠类器官的培养,使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（18.2±4.5） %、（63.8±4.0） %和（82.1±8.4） %。其中使用IntestiCult条件培养基培养类器官的出芽率更高。对于结肠类器官的培养,使用条件培养基1、IntestiCult条件培养基和L-WRN培养基培养结肠类器官的形成率分别为（17.3±7.3） %、（58.0±6.1） %和（46.3±7.4） %。对于二代类器官的培养,IntestiCult条件培养基和L-WRN培养基都能够支持消化为单细胞后的二代类器官培养。干细胞标志物Lgr5和分化细胞（杯状细胞）标志物MUC2的表达无明显差异。使用L-WRN培养基的类器官ki-67阳性比例更高,增殖速度更快。结论：本研究比较了三种不同条件培养基对小鼠类器官形态和增殖速度的影响。经过对比,L-WRN培养基更有利于小鼠肠道类器官的形成和增殖速度。     

Organoid models of the tumor microenvironment and their applications

A small percentage of data obtained from animal/2D culture models can be translated to humans. Therefore, there is a need to using native tumour microenvironment mimicking models to improve preclinical screening and reduce this attrition rate. For this purpose, currently, the utilization of organoids is expanding. Tumour organoids can recapitulate tumour microenvironment that is including cancer cells and non-neoplastic host components. Indeed, tumour organoids, both phenotypically and genetically, resemble the tumour tissue that originated from it. The unique properties of the tumour microenvironment can significantly affect drug response and cancer progression. In this review, we will discuss about various organoid culture strategies for modelling the tumour immune microenvironment, their applications and advantages in cancer research such as testing cancer immunotherapeutics, developing novel approaches for personalized medicine, testing drug toxicity, drug screening, study cancer initiation and progression, and we will also review the limitations of organoid culture systems.     

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these ''mini-guts'', also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.