Assembloid CRISPR screens reveal impact of disease genes in human neurodevelopment.

The assembly of cortical circuits involves the generation and migration of interneurons from the ventral to the dorsal forebrain1-3, which has been challenging to study at inaccessible stages of late gestation and early postnatal human development4. Autism spectrum disorder and other neurodevelopmental disorders (NDDs) have been associated with abnormal cortical interneuron development5, but which of these NDD genes affect interneuron generation and migration, and how they mediate these effects remains unknown. We previously developed a platform to study interneuron development and migration in subpallial organoids and forebrain assembloids6. Here we integrate assembloids with CRISPR screening to investigate the involvement of 425 NDD genes in human interneuron development. The first screen aimed at interneuron generation revealed 13 candidate genes, including CSDE1 and SMAD4. We subsequently conducted an interneuron migration screen in more than 1,000 forebrain assembloids that identified 33 candidate genes, including cytoskeleton-related genes and the endoplasmic reticulum-related gene LNPK. We discovered that, during interneuron migration, the endoplasmic reticulum is displaced along the leading neuronal branch before nuclear translocation. LNPK deletion interfered with this endoplasmic reticulum displacement and resulted in abnormal migration. These results highlight the power of this CRISPR-assembloid platform to systematically map NDD genes onto human development and reveal disease mechanisms.

Generation of Urine-Derived Induced Pluripotent Stem Cells and Cerebral Organoids for Modeling Down Syndrome.

Down syndrome (DS, or trisomy 21, T21), is the most common genetic cause of intellectual disability. Alterations in the complex process of cerebral cortex development contribute to the neurological deficits in DS, although the underlying molecular and cellular mechanisms are not completely understood. Human cerebral organoids (COs) derived from three-dimensional (3D) cultures of induced pluripotent stem cells (iPSCs) provide a new avenue for gaining a better understanding of DS neuropathology. In this study, we aimed to generate iPSCs from individuals with DS (T21-iPSCs) and euploid controls using urine-derived cells, which can be easily and noninvasively obtained from most individuals, and examine their ability to differentiate into neurons and astrocytes grown in monolayer cultures, as well as into 3D COs. We employed nonintegrating episomal vectors to generate urine-derived iPSC lines, and a simple-to-use system to produce COs with forebrain identity. We observed that both T21 and control urine-derived iPSC lines successfully differentiate into neurons and astrocytes in monolayer, as well as into COs that recapitulate early features of human cortical development, including organization of neural progenitor zones, programmed differentiation of excitatory and inhibitory neurons, and upper-and deep-layer cortical neurons as well as astrocytes. Our findings demonstrate for the first time the suitability of using urine-derived iPSC lines to produce COs for modeling DS.

Development and characterization of human fetal female reproductive tract organoids to understand Müllerian duct anomalies.

Müllerian ducts are paired tubular structures that give rise to most of the female reproductive organs. Any abnormalities in the development and differentiation of these ducts lead to anatomical defects in the female reproductive tract organs categorized as Müllerian duct anomalies. Due to the limited access to fetal tissues, little is understood of human reproductive tract development and the associated anomalies. Although organoids represent a powerful model to decipher human development and disease, such organoids from fetal reproductive organs are not available. Here, we developed organoids from human fetal fallopian tubes and uteri and compared them with their adult counterparts. Our results demonstrate that human fetal reproductive tract epithelia do not express some of the typical markers of adult reproductive tract epithelia. Furthermore, fetal organoids are grossly, histologically, and proteomically different from adult organoids. While external supplementation of WNT ligands or activators in culture medium is an absolute requirement for the adult reproductive tract organoids, fetal organoids are able to grow in WNT-deficient conditions. We also developed decellularized tissue scaffolds from adult human fallopian tubes and uteri. Transplantation of fetal organoids onto these scaffolds led to the regeneration of the adult fallopian tube and uterine epithelia. Importantly, suppression of Wnt signaling, which is altered in patients with Müllerian duct anomalies, inhibits the regenerative ability of human fetal organoids and causes severe anatomical defects in the mouse reproductive tract. Thus, our fetal organoids represent an important platform to study the underlying basis of human female reproductive tract development and diseases.

Growth factor dependency in mammary organoids regulates ductal morphogenesis during organ regeneration.

Signaling pathways play an important role in cell fate determination in stem cells and regulate a plethora of developmental programs, the dysregulation of which can lead to human diseases. Growth factors (GFs) regulating these signaling pathways therefore play a major role in the plasticity of adult stem cells and modulate cellular differentiation and tissue repair outcomes. We consider murine mammary organoid generation from self-organizing adult stem cells as a tool to understand the role of GFs in organ development and tissue regeneration. The astounding capacity of mammary organoids to regenerate a gland in vivo after transplantation makes it a convenient model to study organ regeneration. We show organoids grown in suspension with minimal concentration of Matrigel and in the presence of a cocktail of GFs regulating EGF and FGF signaling can recapitulate key epithelial layers of adult mammary gland. We establish a toolkit utilizing in vivo whole animal imaging and ultrasound imaging combined with ex vivo approaches including tissue clearing and confocal imaging to study organ regeneration and ductal morphogenesis. Although the organoid structures were severely impaired in vitro when cultured in the presence of individual GFs, ex vivo imaging revealed ductal branching after transplantation albeit with significantly reduced number of terminal end buds. We anticipate these imaging modalities will open novel avenues to study mammary gland morphogenesis in vivo and can be beneficial for monitoring mammary tumor progression in pre-clinical and clinical settings.

Suspension culture promotes serosal mesothelial development in human intestinal organoids.

Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium.

Biocompatible micro tweezers for 3D hydrogel organoid array mechanical characterization.

This study presents novel biocompatible Polydimethylsiloxane (PDMS)-based micromechanical tweezers (µTweezers) capable of the stiffness characterization and manipulation of hydrogel-based organoids. The system showed great potential for complementing established mechanical characterization methods such as Atomic Force Microscopy (AFM), parallel plate compression (PPC), and nanoindentation, while significantly reducing the volume of valuable hydrogels used for testing. We achieved a volume reduction of ~0.22 µl/sample using the µTweezers vs. ~157 µl/sample using the PPC, while targeting high-throughput measurement of widely adopted micro-mesoscale (a few hundred µm-1500 µm) 3D cell cultures. The µTweezers applied and measured nano-millinewton forces through cantilever' deflection with high linearity and tunability for different applications; the assembly is compatible with typical inverted optical microscopes and fit on standard tissue culture Petri dishes, allowing mechanical compression characterization of arrayed 3D hydrogel-based organoids in a high throughput manner. The average achievable output per group was 40 tests per hour, where 20 organoids and 20 reference images in one 35 mm petri dish were tested, illustrating efficient productivity to match the increasing demand on 3D organoids' applications. The changes in stiffness of collagen I hydrogel organoids in four conditions were measured, with ovarian cancer cells (SKOV3) or without (control). The Young's modulus of the control group (Control-day 0, E = 407± 146, n = 4) measured by PPC was used as a reference modulus, where the relative elastic compressive modulus of the other groups based on the stiffness measurements was also calculated (control-day 0, E = 407 Pa), (SKOV3-day 0, E = 318 Pa), (control-day 5, E = 528 Pa), and (SKOV3-day 5, E = 376 Pa). The SKOV3-embedded hydrogel-based organoids had more shrinkage and lowered moduli on day 0 and day 5 than controls, consistently, while SKOV3 embedded organoids increased in stiffness in a similar trend to the collagen I control from day 0 to day 5. The proposed method can contribute to the biomedical, biochemical, and regenerative engineering fields, where bulk mechanical characterization is of interest. The µTweezers will also provide attractive design and application concepts to soft membrane-micro 3D robotics, sensors, and actuators.

Integrated stem cells from apical papilla in a 3D culture system improve human embryonic stem cell derived retinal organoid formation.

AIM: Eye organoids are 3D models of the retina that provide new possibilities for studying retinal development, drug toxicity and the molecular mechanisms of diseases. Although there are several protocols that can be used to generate functional tissues, none have been used to assemble human retinal organoids containing mesenchymal stem cells (MSCs). MAIN METHODS: In this study we intend to assess the effective interactions of MSCs and human embryonic stem cells (hESCs) during retinal organoid formation. We evaluated the inducing activities of bone marrow MSCs (BM-MSCs), trabecular meshwork (TM), and stem cells from apical papilla (SCAP)-derived MSCs in differentiation of hESCs in a three-dimensional (3D) direct co-culture system. KEY FINDINGS: In comparison with the two other MSC sources, the induction potential of SCAP was confirmed in the co-culture system. Although the different SCAP cell ratios did not show any significant morphology changes during the first seven days, increasing the number of SCAPs improved formation of the optic vesicle (OV) structure, which was confirmed by assessment of specific markers. The OVs subsequently developed to an optic cup (OC), which was similar to the in vivo environment. These arrangements expressed MITF in the outer layer and CHX10 in the inner layer. SIGNIFICANCE: We assessed the inducing activity of SCAP during differentiation of hESCs towards a retinal fate in a 3D organoid system. However, future studies be conducted to gather additional details about the development of the eye field, retinal differentiation, and the molecular mechanisms of diseases.

Tissue geometry drives deterministic organoid patterning.

Epithelial organoids are stem cell­derived tissues that approximate aspects of real organs, and thus they have potential as powerful tools in basic and translational research. By definition, they self-organize, but the structures formed are often heterogeneous and irreproducible, which limits their use in the lab and clinic. We describe methodologies for spatially and temporally controlling organoid formation, thereby rendering a stochastic process more deterministic. Bioengineered stem cell microenvironments are used to specify the initial geometry of intestinal organoids, which in turn controls their patterning and crypt formation. We leveraged the reproducibility and predictability of the culture to identify the underlying mechanisms of epithelial patterning, which may contribute to reinforcing intestinal regionalization in vivo. By controlling organoid culture, we demonstrate how these structures can be used to answer questions not readily addressable with the standard, more variable, organoid models.

Human brain organogenesis: Toward a cellular understanding of development and disease.

The construction of the human nervous system is a distinctly complex although highly regulated process. Human tissue inaccessibility has impeded a molecular understanding of the developmental specializations from which our unique cognitive capacities arise. A confluence of recent technological advances in genomics and stem cell-based tissue modeling is laying the foundation for a new understanding of human neural development and dysfunction in neuropsychiatric disease. Here, we review recent progress on uncovering the cellular and molecular principles of human brain organogenesis in vivo as well as using organoids and assembloids in vitro to model features of human evolution and disease.

Dynamics of transcription regulatory network during mice-derived retina organoid development.

The retina is a complex system containing several neuron types arranged in distinct layers. Many aspects of the retina's development and the molecular events in the human light-sensing system have been previously unveiled. However, there is limited information about regulatory networks governing the transitional stages during retina development. To address this issue, we have studied the transcriptome dynamics of mice-derived retinal organoid development in 10 successive time-points, from stem cell to functional retina. For the first time, we have identified the main modules of genes related to different stages of development and predicted all possible transcription factors. A major shift in the transcriptome occurs during the transition of cells from D0 to D10 and again at the late stages of retina development. Transcription, nervous system development, cell cycle, neurotransmitter transport, glycosylation, and lipid metabolisms are the most important biological processes during retina development. Altogether, we have identified and reported 15 TFs, including Irx2, Irx3, Lmo2, Tead2, Tbx20, and Zeb1, which are potentially involved in the regulation of retinal organoid development. In conclusion, using several rigorous analyses, we have found main stage-specific biological processes in the retina development and predicted TFs with strong potency in controlling this structure.

SIRT1 induces the accumulation of TAMs at colorectal cancer tumor sites via the CXCR4/CXCL12 axis.

Our previous work suggested that high SIRT1 expression by cancer cells predicted a poor colorectal cancer (CRC) prognosis, but its role in the tumor microenvironment was unclear. Here, we examined tumor-infiltrating lymphocytes (TILs) in CRC expressing different levels of SIRT1. We also established a co-culture system with monocytes, CD8+ T cells and patient-derived tumor organoids (PDOs) to study the relationships between immune cells and cancer cells. The percentage of CD8+ T cells was decreased and the percentage of macrophages was increased in SIRT1-high (SIRT1-hi) CRC. Co-culture results showed that tumor-associated macrophages (TAMs) from SIRT1-hi CRC inhibited the proliferation and anti-tumor activity of CD8+ T cells. Importantly, SIRT1-hi CRC were shown to modulate the migration and the activity of TAMs. RNA sequencing revealed that CD14+ monocytes in SIRT1-hi patients expressed higher levels of CXCR4. Mechanistically, SIRT1 expression was shown to promote CXCL12 expression by inhibiting the acetylation of p53. Our findings indicate that SIRT1 in CRC induces TAM migration through the CXCR4/CXCL12 pathway, and inhibits the proliferation and activity of CD8+ T cells, resulting in promotion of CRC progression.

Single-cell transcriptomics captures features of human midbrain development and dopamine neuron diversity in brain organoids.

Three-dimensional brain organoids have emerged as a valuable model system for studies of human brain development and pathology. Here we establish a midbrain organoid culture system to study the developmental trajectory from pluripotent stem cells to mature dopamine neurons. Using single cell RNA sequencing, we identify the presence of three molecularly distinct subtypes of human dopamine neurons with high similarity to those in developing and adult human midbrain. However, despite significant advancements in the field, the use of brain organoids can be limited by issues of reproducibility and incomplete maturation which was also observed in this study. We therefore designed bioengineered ventral midbrain organoids supported by recombinant spider-silk microfibers functionalized with full-length human laminin. We show that silk organoids reproduce key molecular aspects of dopamine neurogenesis and reduce inter-organoid variability in terms of cell type composition and dopamine neuron formation.

NF-κB determines Paneth versus goblet cell fate decision in the small intestine.

Although the role of the transcription factor NF-κB in intestinal inflammation and tumor formation has been investigated extensively, a physiological function of NF-κB in sustaining intestinal epithelial homeostasis beyond inflammation has not been demonstrated. Using NF-κB reporter mice, we detected strong NF-κB activity in Paneth cells, in '+4/+5' secretory progenitors and in scattered Lgr5+ crypt base columnar stem cells of small intestinal (SI) crypts. To examine NF-κB functions in SI epithelial self-renewal, mice or SI crypt organoids ('mini-guts') with ubiquitously suppressed NF-κB activity were used. We show that NF-κB activity is dispensable for maintaining SI epithelial proliferation, but is essential for ex vivo organoid growth. Furthermore, we demonstrate a dramatic reduction of Paneth cells in the absence of NF-κB activity, concomitant with a significant increase in goblet cells and immature intermediate cells. This indicates that NF-κB is required for proper Paneth versus goblet cell differentiation and for SI epithelial homeostasis, which occurs via regulation of Wnt signaling and Sox9 expression downstream of NF-κB. The current study thus presents evidence for an important role for NF-κB in intestinal epithelial self-renewal.

Characterization of primary models of human trophoblast.

Two recently developed models, trophoblast organoids and trophoblast stem cells (TSCs), are useful tools to further the understanding of human placental development. Both differentiate from villous cytotrophoblast (VCT) to either extravillous trophoblast (EVT) or syncytiotrophoblast (SCT). Here, we compare the transcriptomes and miRNA profiles of these models to identify which trophoblast they resemble in vivo. Our findings indicate that TSCs do not readily undergo SCT differentiation and closely resemble cells at the base of the cell columns from where EVT derives. In contrast, organoids are similar to VCT and undergo spontaneous SCT differentiation. A defining feature of human trophoblast is that VCT and SCT are human leukocyte antigen (HLA) null, whereas EVT expresses HLA-C, -G and -E molecules. We find that trophoblast organoids retain these in vivo characteristics. In contrast, TSCs express classical HLA-A and HLA-B molecules, and maintain their expression after EVT differentiation, with upregulation of HLA-G. Furthermore, HLA expression in TSCs differs when grown in 3D rather than in 2D, suggesting that mechanical cues are important. Our results can be used to select the most suitable model for the study of trophoblast development, function and pathology.

Bioengineering Approaches for the Advanced Organoid Research.

Recent advances in 3D cell culture technology have enabled scientists to generate stem cell derived organoids that recapitulate the structural and functional characteristics of native organs. Current organoid technologies have been striding toward identifying the essential factors for controlling the processes involved in organoid development, including physical cues and biochemical signaling. There is a growing demand for engineering dynamic niches characterized by conditions that resemble in vivo organogenesis to generate reproducible and reliable organoids for various applications. Innovative biomaterial-based and advanced engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of organoid research. The recent advances in organoid engineering, including extracellular matrices and genetic modulation, are comprehensively summarized to pinpoint the parameters critical for organ-specific patterning. Moreover, perspective trends in developing tunable organoids in response to exogenous and endogenous cues are discussed for next-generation developmental studies, disease modeling, and therapeutics.

Microfluidic device with brain extracellular matrix promotes structural and functional maturation of human brain organoids.

Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases.

Regulatory Mechanisms of Retinal Photoreceptors Development at Single Cell Resolution.

Photoreceptors are critical components of the retina and play a role in the first step of the conversion of light to electric signals. With the discovery of the intrinsically photosensitive retinal ganglion cells, which regulate non-image-forming visual processes, our knowledge of the photosensitive cell family in the retina has deepened. Photoreceptor development is regulated by specific genes and proteins and involves a series of molecular processes including DNA transcription, post-transcriptional modification, protein translation, and post-translational modification. Single-cell sequencing is a promising technology for the study of photoreceptor development. This review presents an overview of the types of human photoreceptors, summarizes recent discoveries in the regulatory mechanisms underlying their development at single-cell resolution, and outlines the prospects in this field.

Characterization of an in vitro 3D intestinal organoid model by using massive RNAseq-based transcriptome profiling.

Organoids culture provides unique opportunities to study human diseases and to complement animal models. Several organs and tissues can be in vitro cultured in 3D structures resembling in vivo tissue organization. Organoids culture contains most of the cell types of the original tissue and are maintained by growth factors mimicking the in vivo state. However, the system is yet not fully understood, and specific in vivo features especially those driven by cell-extrinsic factors may be lost in culture. Here we show a comprehensive transcriptome-wide characterization of mouse gut organoids derived from different intestinal compartments and from mice of different gender and age. RNA-seq analysis showed that the in vitro culture strongly influences the global transcriptome of the intestinal epithelial cells (~ 60% of the total variance). Several compartment-, age- and gender-related transcriptome features are lost after culturing indicating that they are driven by niche or systemic factors. However, certain intrinsic transcriptional programs, for example, some compartment-related features and a minority of gender- and aging- related features are maintained in vitro which suggested possibilities for these features to be studied in this system. Moreover, our study provides knowledge about the cell-extrinsic or cell-intrinsic origin of intestinal epithelial transcriptional programs. We anticipated that our characterization of this in vitro system is an important reference for scientists and clinicians using intestinal organoids as a research model.