Human fetal brain self-organizes into long-term expanding organoids.

Human brain development involves an orchestrated, massive neural progenitor expansion while a multi-cellular tissue architecture is established. Continuously expanding organoids can be grown directly from multiple somatic tissues, yet to date, brain organoids can solely be established from pluripotent stem cells. Here, we show that healthy human fetal brain in vitro self-organizes into organoids (FeBOs), phenocopying aspects of in vivo cellular heterogeneity and complex organization. FeBOs can be expanded over long time periods. FeBO growth requires maintenance of tissue integrity, which ensures production of a tissue-like extracellular matrix (ECM) niche, ultimately endowing FeBO expansion. FeBO lines derived from different areas of the central nervous system (CNS), including dorsal and ventral forebrain, preserve their regional identity and allow to probe aspects of positional identity. Using CRISPR-Cas9, we showcase the generation of syngeneic mutant FeBO lines for the study of brain cancer. Taken together, FeBOs constitute a complementary CNS organoid platform.

Temporal morphogen gradient-driven neural induction shapes single expanded neuroepithelium brain organoids with enhanced cortical identity.

Pluripotent stem cell (PSC)-derived human brain organoids enable the study of human brain development in vitro. Typically, the fate of PSCs is guided into subsequent specification steps through static medium switches. In vivo, morphogen gradients are critical for proper brain development and determine cell specification, and associated defects result in neurodevelopmental disorders. Here, we show that initiating neural induction in a temporal stepwise gradient guides the generation of brain organoids composed of a single, self-organized apical-out neuroepithelium, termed ENOs (expanded neuroepithelium organoids). This is at odds with standard brain organoid protocols in which multiple and independent neuroepithelium units (rosettes) are formed. We find that a prolonged, decreasing gradient of TGF-ß signaling is a determining factor in ENO formation and allows for an extended phase of neuroepithelium expansion. In-depth characterization reveals that ENOs display improved cellular morphology and tissue architectural features that resemble in vivo human brain development, including expanded germinal zones. Consequently, cortical specification is enhanced in ENOs. ENOs constitute a platform to study the early events of human cortical development and allow interrogation of the complex relationship between tissue architecture and cellular states in shaping the developing human brain.

The cellular composition and function of the bone marrow niche after allogeneic hematopoietic cell transplantation.

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative therapy for patients with a variety of malignant and non-malignant diseases. Despite its life-saving potential, HCT is associated with significant morbidity and mortality. Reciprocal interactions between hematopoietic stem cells (HSCs) and their surrounding bone marrow (BM) niche regulate HSC function during homeostatic hematopoiesis as well as regeneration. However, current pre-HCT conditioning regimens, which consist of high-dose chemotherapy and/or irradiation, cause substantial short- and long-term toxicity to the BM niche. This damage may negatively affect HSC function, impair hematopoietic regeneration after HCT and predispose to HCT-related morbidity and mortality. In this review, we summarize current knowledge on the cellular composition of the human BM niche after HCT. We describe how pre-HCT conditioning affects the cell types in the niche, including endothelial cells, mesenchymal stromal cells, osteoblasts, adipocytes, and neurons. Finally, we discuss therapeutic strategies to prevent or repair conditioning-induced niche damage, which may promote hematopoietic recovery and improve HCT outcome.

Multispectral confocal 3D imaging of intact healthy and tumor tissue using mLSR-3D.

Revealing the 3D composition of intact tissue specimens is essential for understanding cell and organ biology in health and disease. State-of-the-art 3D microscopy techniques aim to capture tissue volumes on an ever-increasing scale, while also retaining sufficient resolution for single-cell analysis. Furthermore, spatial profiling through multi-marker imaging is fast developing, providing more context and better distinction between cell types. Following these lines of technological advance, we here present a protocol based on FUnGI (fructose, urea and glycerol clearing solution for imaging) optical clearing of tissue before multispectral large-scale single-cell resolution 3D (mLSR-3D) imaging, which implements 'on-the-fly' linear unmixing of up to eight fluorophores during a single acquisition. Our protocol removes the need for repetitive illumination, thereby allowing larger volumes to be scanned with better image quality in less time, also reducing photo-bleaching and file size. To aid in the design of multiplex antibody panels, we provide a fast and manageable intensity equalization assay with automated analysis to design a combination of markers with balanced intensities suitable for mLSR-3D. We demonstrate effective mLSR-3D imaging of various tissues, including patient-derived organoids and xenografted tumors, and, furthermore, describe an optimized workflow for mLSR-3D imaging of formalin-fixed paraffin-embedded samples. Finally, we provide essential steps for 3D image data processing, including shading correction that does not require pre-acquired shading references and 3D inhomogeneity correction to correct fluorescence artefacts often afflicting 3D datasets. Together, this provides a one-week protocol for eight-fluorescent-marker 3D visualization and exploration of intact tissue of various origins at single-cell resolution.