In vitro 3D organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition ...of organoids and demonstrated organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor organoids, as well as on mouse mammary gland organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From organoid harvest to image analysis, the protocol takes 3 d.
Extending the success of cellular immunotherapies against blood cancers to the realm of solid tumors will require improved in vitro models that reveal therapeutic modes of action at the molecular ...level. Here we describe a system, called BEHAV3D, developed to study the dynamic interactions of immune cells and patient cancer organoids by means of imaging and transcriptomics. We apply BEHAV3D to live-track >150,000 engineered T cells cultured with patient-derived, solid-tumor organoids, identifying a 'super engager' behavioral cluster comprising T cells with potent serial killing capacity. Among other T cell concepts we also study cancer metabolome-sensing engineered T cells (TEGs) and detect behavior-specific gene signatures that include a group of 27 genes with no previously described T cell function that are expressed by super engager killer TEGs. We further show that type I interferon can prime resistant organoids for TEG-mediated killing. BEHAV3D is a promising tool for the characterization of behavioral-phenotypic heterogeneity of cellular immunotherapies and may support the optimization of personalized solid-tumor-targeting cell therapies.
Background
Nephron progenitor cells (NPCs) undergo a stepwise process to generate all mature nephron structures. Mesenchymal to epithelial transition (MET) is considered a multistep process of NPC ...differentiation to ensure progressive establishment of new nephrons. However, despite this important role, to date, no marker for NPCs undergoing MET in the nephron exists.
Results
Here, we identify LGR6 as a NPC marker, expressed in very early cap mesenchyme, pre‐tubular aggregates, renal vesicles, and in segments of S‐shaped bodies, following the trajectory of MET. By using a lineage tracing approach in embryonic explants in combination with confocal imaging and single‐cell RNA sequencing, we provide evidence for the multiple fates of LGR6+ cells during embryonic nephrogenesis. Moreover, by using long‐term in vivo lineage tracing, we show that postnatal LGR6+ cells are capable of generating the multiple lineages of the nephrons.
Conclusions
Given the profound early mesenchymal expression and MET signature of LGR6+ cells, together with the lineage tracing of mesenchymal LGR6+ cells, we conclude that LGR6+ cells contribute to all nephrogenic segments by undergoing MET. LGR6+ cells can therefore be considered an early committed NPC population during embryonic and postnatal nephrogenesis with potential regenerative capability.
Key Findings
Lgr6 is expressed in the earliest cap mesenchyme pool, a niche where nephrogenic progenitor cells (NPCs) are found.
Lgr6 marks NPCs undergoing mesenchymal to epithelial transition, following the main process of nephron development.
Using ex vivo and vivo lineage tracing, we show that mesenchymal Lgr6 expressing cells give rise to multiple types of mesenchymal derived nephron segments, including specialized glomerular epithelium, such as podocytes.
Abstract
Diffuse Midline Glioma (DMG) is a rare and aggressive pediatric cancer with no chance of survival, demonstrating a critical need for therapy development. Predictive preclinical models that ...take into account the unique developmental background and anatomical environment of DMG, are so far lacking. We developed a new regionally patterned brain organoid protocol based on a timely regulated sequence of morphogens. The patterned organoids give rise to pontine and medulla oblongata identity, as confirmed by transcriptomic profiling and immunofluorescence imaging. As the tissue matures, the complexity inherently takes shape and gives rise to pons-specific neurons and glial cells, offering the first human in vitro model for this region of the brain. Introducing H3K27M, including its partner mutations P53 and PDGFRA-D842V via electroporation forms de novo H3K27M diffuse midline glioma with a similar genetic make-up as found in patients. Indeed, contrary to common cell lines or PDX models, single cell sequencing and 3D imaging reveal that Organoid-DMG resembles WHO-defined primary patient cancer, including its diverse cell population heterogeneity and invasive nature. Utilizing barcode-based lineage tracing allows us to unravel and track the cancerous developmental evolution. Thus, we can now model early emerging populations and identify their transcriptomic signatures, which is conceptually impossible to conduct with patient-derived tumor tissue. In summary, we generated a bona fide DMG in vitro model which will give insights into tumorigenesis of DMG and ultimately contribute to pre-clinical drug- and immunotherapy development.
Tracking and Tracing: Lgr6 marks nephron progenitor cells during kidney development. From:
LGR6 marks nephron progenitor cells
; Ravian L. van Ineveld, Thanasis Margaritis, Berend A.P. Kooiman, et ...al.;
https://doi.org/10.1002/dvdy.346
; DevDyn 250:11; pp. 1568–1583.
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