In vitro human cell-based models: What can they do and what are their limitations?

It is said that all models are wrong, but some are useful. In vitro human cell-based models are a prime example of this maxim. We asked researchers: when is your model system useful? How can it be made more useful? What are its limitations?

Probing the killing potency of tumor-infiltrating lymphocytes on microarrayed colorectal cancer tumoroids.

Immunotherapy has emerged as a new standard of care for certain cancer patients with specific cellular and molecular makeups. However, there is still an unmet need for ex vivo models able to readily assess the effectiveness of immunotherapeutic treatments in a high-throughput and patient-specific manner. To address this issue, we have developed a microarrayed system of patient-derived tumoroids with recreated immune microenvironments that are optimized for the high-content evaluation of tumor-infiltrating lymphocyte functionality. Here we show that this system offers unprecedented opportunities to evaluate tumor immunogenicity, characterize the response to immunomodulators, and explore novel approaches for personalized immuno-oncology.

Thymic epithelial organoids mediate T cell development.

Although the advent of organoids opened unprecedented perspectives for basic and translational research, immune system-related organoids remain largely underdeveloped. Here we established organoids from the thymus, the lymphoid organ responsible for T cell development. We identified conditions enabling thymic epithelial progenitor cell proliferation and development into organoids with diverse cell populations and transcriptional profiles resembling in vivo thymic epithelial cells (TECs) more closely than traditional TEC cultures. Contrary to these two-dimensional cultures, thymic epithelial organoids maintained thymus functionality in vitro and mediated physiological T cell development upon reaggregation with T cell progenitors. The reaggregates showed in vivo-like epithelial diversity and ability to attract T cell progenitors. Thymic epithelial organoids are the first organoids originating from the stromal compartment of a lymphoid organ. They provide new opportunities to study TEC biology and T cell development in vitro, paving the way for future thymic regeneration strategies in ageing or acute injuries.

Patient-derived mini-colons enable long-term modeling of tumor-microenvironment complexity.

Existing organoid models fall short of fully capturing the complexity of cancer because they lack sufficient multicellular diversity, tissue-level organization, biological durability and experimental flexibility. Thus, many multifactorial cancer processes, especially those involving the tumor microenvironment, are difficult to study ex vivo. To overcome these limitations, we herein implemented tissue-engineering and microfabrication technologies to develop topobiologically complex, patient-specific cancer avatars. Focusing on colorectal cancer, we generated miniature tissues consisting of long-lived gut-shaped human colon epithelia ('mini-colons') that stably integrate cancer cells and their native tumor microenvironment in a format optimized for real-time, high-resolution evaluation of cellular dynamics. We demonstrate the potential of this system through several applications: a comprehensive evaluation of drug effectivity, toxicity and resistance in anticancer therapies; the discovery of a mechanism triggered by cancer-associated fibroblasts that drives cancer invasion; and the identification of immunomodulatory interactions among different components of the tumor microenvironment. Similar approaches should be feasible for diverse tumor types.

Bioengineered human colon organoids with in vivo-like cellular complexity and function.

Organoids and organs-on-a-chip have emerged as powerful tools for modeling human gut physiology and disease in vitro. Although physiologically relevant, these systems often lack the environmental milieu, spatial organization, cell type diversity, and maturity necessary for mimicking human intestinal mucosa. To instead generate models closely resembling in vivo tissue, we herein integrated organoid and organ-on-a-chip technology to develop an advanced human organoid model, called "mini-colons." By employing an asymmetric stimulation with growth factors, we greatly enhanced tissue longevity and replicated in vivo-like diversity and patterning of proliferative and differentiated cell types. Mini-colons contain abundant mucus-producing goblet cells and, signifying mini-colon maturation, single-cell RNA sequencing reveals emerging mature and functional colonocytes. This methodology is expanded to generate microtissues from the small intestine and incorporate additional microenvironmental components. Finally, our bioengineered organoids provide a precise platform to systematically study human gut physiology and pathology, and a reliable preclinical model for drug safety assessment.

Nanoplasmonic Single-Tumoroid Microarray for Real-Time Secretion Analysis.

Organoid tumor models have emerged as a powerful tool in the fields of biology and medicine as such 3D structures grown from tumor cells recapitulate better tumor characteristics, making these tumoroids unique for personalized cancer research. Assessment of their functional behavior, particularly protein secretion, is of significant importance to provide comprehensive insights. Here, a label-free spectroscopic imaging platform is presented with advanced integrated optofluidic nanoplasmonic biosensor that enables real-time secretion analysis from single tumoroids. A novel two-layer microwell design isolates tumoroids, preventing signal interference, and the microarray configuration allows concurrent analysis of multiple tumoroids. The dual imaging capability combining time-lapse plasmonic spectroscopy and bright-field microscopy facilitates simultaneous observation of secretion dynamics, motility, and morphology. The integrated biosensor is demonstrated with colorectal tumoroids derived from both cell lines and patient samples to investigate their vascular endothelial growth factor A (VEGF-A) secretion, growth, and movement under various conditions, including normoxia, hypoxia, and drug treatment. This platform, by offering a label-free approach with nanophotonics to monitor tumoroids, can pave the way for new applications in fundamental biological studies, drug screening, and the development of therapies.

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.

Open-source microscope add-on for structured illumination microscopy.

Super-resolution techniques expand the abilities of researchers who have the knowledge and resources to either build or purchase a system. This excludes the part of the research community without these capabilities. Here we introduce the openSIM add-on to upgrade existing optical microscopes to Structured Illumination super-resolution Microscopes (SIM). The openSIM is an open-hardware system, designed and documented to be easily duplicated by other laboratories, making super-resolution modality accessible to facilitate innovative research. The add-on approach gives a performance improvement for pre-existing lab equipment without the need to build a completely new system.

Bioengineered Tubular Biliary Organoids.

Liver organoids have emerged as promising in vitro models for toxicology, drug discovery, and disease modeling. However, conventional 3D epithelial organoid culture systems suffer from significant drawbacks, including limited culture duration, a nonphysiological 3D cystic anatomy with an inaccessible apical surface, and lack of in vivo-like cellular organization. To address these limitations, herein a hydrogel-based organoid-on-a-chip model for the development functional tubular biliary organoids is reported. The resulting constructs demonstrate long-term stability for a minimum duration of 45 d, while retaining their biliary organoid identity and exhibiting key cholangiocyte characteristics including transport activities, formation of primary cilia, and protective glycocalyx. Additionally, tubular organoids are susceptible to physical and chemical injury, which cannot be applied in such resolution to classical organoids. To enhance tissue-level complexity, in vitro formation of a perfusable branching network is induced using a predetermined geometry that faithfully mimics the intricate structure of the intrahepatic biliary tree. Finally, cellular complexity is augmented through co-culturing with vascular endothelial cells and fibroblasts. The models described in this study offer valuable opportunities for investigating biliary morphogenesis and elucidating associated pathophysiological mechanisms.

ISSCR standards for the use of human stem cells in basic research.

The laboratory culture of human stem cells seeks to capture a cellular state as an in vitro surrogate of a biological system. For the results and outputs from this research to be accurate, meaningful, and durable, standards that ensure reproducibility and reliability of the data should be applied. Although such standards have been previously proposed for repositories and distribution centers, no widely accepted best practices exist for laboratory research with human pluripotent and tissue stem cells. To fill that void, the International Society for Stem Cell Research has developed a set of recommendations, including reporting criteria, for scientists in basic research laboratories. These criteria are designed to be technically and financially feasible and, when implemented, enhance the reproducibility and rigor of stem cell research.

Embryo-uterine interaction coordinates mouse embryogenesis during implantation.

Embryo implantation into the uterus marks a key transition in mammalian development. In mice, implantation is mediated by the trophoblast and is accompanied by a morphological transition from the blastocyst to the egg cylinder. However, the roles of trophoblast-uterine interactions in embryo morphogenesis during implantation are poorly understood due to inaccessibility in utero and the remaining challenges to recapitulate it ex vivo from the blastocyst. Here, we engineer a uterus-like microenvironment to recapitulate peri-implantation development of the whole mouse embryo ex vivo and reveal essential roles of the physical embryo-uterine interaction. We demonstrate that adhesion between the trophoblast and the uterine matrix is required for in utero-like transition of the blastocyst to the egg cylinder. Modeling the implanting embryo as a wetting droplet links embryo shape dynamics to the underlying changes in trophoblast adhesion and suggests that the adhesion-mediated tension release facilitates egg cylinder formation. Light-sheet live imaging and the experimental control of the engineered uterine geometry and trophoblast velocity uncovers the coordination between trophoblast motility and embryo growth, where the trophoblast delineates space for embryo morphogenesis.

Multimodal characterization of murine gastruloid development.

Gastruloids are 3D structures generated from pluripotent stem cells recapitulating fundamental principles of embryonic pattern formation. Using single-cell genomic analysis, we provide a resource mapping cell states and types during gastruloid development and compare them with the in vivo embryo. We developed a high-throughput handling and imaging pipeline to spatially monitor symmetry breaking during gastruloid development and report an early spatial variability in pluripotency determining a binary response to Wnt activation. Although cells in the gastruloid-core revert to pluripotency, peripheral cells become primitive streak-like. These two populations subsequently break radial symmetry and initiate axial elongation. By performing a compound screen, perturbing thousands of gastruloids, we derive a phenotypic landscape and infer networks of genetic interactions. Finally, using a dual Wnt modulation, we improve the formation of anterior structures in the existing gastruloid model. This work provides a resource to understand how gastruloids develop and generate complex patterns in vitro.

Primate-specific transposable elements shape transcriptional networks during human development.

The human genome contains more than 4.5 million inserts derived from transposable elements (TEs), the result of recurrent waves of invasion and internal propagation throughout evolution. For new TE copies to be inherited, they must become integrated in the genome of the germline or pre-implantation embryo, which requires that their source TE be expressed at these stages. Accordingly, many TEs harbor DNA binding sites for the pluripotency factors OCT4, NANOG, SOX2, and KLFs and are transiently expressed during embryonic genome activation. Here, we describe how many primate-restricted TEs have additional binding sites for lineage-specific transcription factors driving their expression during human gastrulation and later steps of fetal development. These TE integrants serve as lineage-specific enhancers fostering the transcription, amongst other targets, of KRAB-zinc finger proteins (KZFPs) of comparable evolutionary age, which in turn corral the activity of TE-embedded regulatory sequences in a similarly lineage-restricted fashion. Thus, TEs and their KZFP controllers play broad roles in shaping transcriptional networks during early human development.

Erratum: An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates.

[This corrects the article DOI: 10.1016/j.crmeth.2022.100244.].

Self-organizing in vitro mouse neural tube organoids mimic embryonic development.

The embryonic neural tube is the origin of the entire adult nervous system, and disturbances in its development cause life-threatening birth defects. However, the study of mammalian neural tube development is limited by the lack of physiologically realistic three-dimensional (3D) in vitro models. Here, we report a self-organizing 3D neural tube organoid model derived from single mouse embryonic stem cells that exhibits an in vivo-like tissue architecture, cell type composition and anterior-posterior (AP) patterning. Moreover, maturation of the neural tube organoids showed the emergence of multipotent neural crest cells and mature neurons. Single-cell transcriptome analyses revealed the sequence of transcriptional events in the emergence of neural crest cells and neural differentiation. Thanks to the accessibility of this model, phagocytosis of migrating neural crest cells could be observed in real time for the first time in a mammalian model. We thus introduce a tractable in vitro model to study some of the key morphogenetic and cell type derivation events during early neural development.

c-MAF coordinates enterocyte zonation and nutrient uptake transcriptional programs.

Small intestinal villi are structural and functional units present in higher vertebrates and uniquely adapted to nutrient absorption. Villus enterocytes are organized in transcriptional "zones" dedicated to specialized tasks such as absorption of specific nutrients. We report that the transcription factor c-MAF is expressed in differentiated lower and mid-villus enterocytes and is a target of BMP signaling. Maf inactivation perturbed the villus zonation program by increasing carbohydrate-related transcripts while suppressing transcripts linked to amino-acid and lipid absorption. The formation of cytoplasmic lipid droplets, shuttling dietary fat to chylomicrons, was impaired upon Maf loss indicating its role in dietary lipid handling. Maf inactivation under homeostatic conditions expanded tuft cells and led to compensatory gut lengthening, preventing weight loss. However, delayed Maf-/- enterocyte maturation impaired weight recovery after acute intestinal injury, resulting in reduced survival. Our results identify c-MAF as a regulator of the intestinal villus zonation program, while highlighting the importance of coordination between stem/progenitor and differentiation programs for intestinal regeneration.

Organoids as tools for fundamental discovery and translation-a Keystone Symposia report.

Complex three-dimensional in vitro organ-like models, or organoids, offer a unique biological tool with distinct advantages over two-dimensional cell culture systems, which can be too simplistic, and animal models, which can be too complex and may fail to recapitulate human physiology and pathology. Significant progress has been made in driving stem cells to differentiate into different organoid types, though several challenges remain. For example, many organoid models suffer from high heterogeneity, and it can be difficult to fully incorporate the complexity of in vivo tissue and organ development to faithfully reproduce human biology. Successfully addressing such limitations would increase the viability of organoids as models for drug development and preclinical testing. On April 3-6, 2022, experts in organoid development and biology convened at the Keystone Symposium "Organoids as Tools for Fundamental Discovery and Translation" to discuss recent advances and insights from this relatively new model system into human development and disease.

Gastruloids as in vitro models of embryonic blood development with spatial and temporal resolution.

Gastruloids are three-dimensional embryonic organoids that reproduce key features of early mammalian development in vitro with unique scalability, accessibility, and spatiotemporal similarity to real embryos. Recently, we adapted the gastruloid culture conditions to promote cardiovascular development. In this work, we extended these conditions to capture features of embryonic blood development through a combination of immunophenotyping, detailed transcriptomics analysis, and identification of blood stem/progenitor cell potency. We uncovered the emergence of blood progenitor and erythroid-like cell populations in late gastruloids and showed the multipotent clonogenic capacity of these cells, both in vitro and after transplantation into irradiated mice. We also identified the spatial localization near a vessel-like plexus in the anterior portion of gastruloids with similarities to the emergence of blood stem cells in the mouse embryo. These results highlight the potential and applicability of gastruloids to the in vitro study of complex processes in embryonic blood development with spatiotemporal fidelity.

An automated do-it-yourself system for dynamic stem cell and organoid culture in standard multi-well plates.

We present a low-cost, do-it-yourself system for complex mammalian cell culture under dynamically changing medium formulations by integrating conventional multi-well tissue culture plates with simple microfluidic control and system automation. We demonstrate the generation of complex concentration profiles, enabling the investigation of sophisticated input-response relations. We further apply our automated cell-culturing platform to the dynamic stimulation of two widely employed stem-cell-based in vitro models for early mammalian development: the conversion of naive mouse embryonic stem cells into epiblast-like cells and mouse 3D gastruloids. Performing automated medium-switch experiments, we systematically investigate cell fate commitment along the developmental trajectory toward mouse epiblast fate and examine symmetry-breaking, germ layer formation, and cardiac differentiation in mouse 3D gastruloids as a function of time-varying Wnt pathway activation. With these proof-of-principle examples, we demonstrate a highly versatile and scalable tool that can be adapted to specific research questions, experimental demands, and model systems.