Extracellular LGALS3BP regulates neural progenitor position and relates to human cortical complexity.

Basal progenitors (BPs), including intermediate progenitors and basal radial glia, are generated from apical radial glia and are enriched in gyrencephalic species like humans, contributing to neuronal expansion. Shortly after generation, BPs delaminate towards the subventricular zone, where they further proliferate before differentiation. Gene expression alterations involved in BP delamination and function in humans are poorly understood. Here, we study the role of LGALS3BP, so far known as a cancer biomarker, which is a secreted protein enriched in human neural progenitors (NPCs). We show that individuals with LGALS3BP de novo variants exhibit altered local gyrification, sulcal depth, surface area and thickness in their cortex. Additionally, using cerebral organoids, human fetal tissues and mice, we show that LGALS3BP regulates the position of NPCs. Single-cell RNA-sequencing and proteomics reveal that LGALS3BP-mediated mechanisms involve the extracellular matrix in NPCs' anchoring and migration within the human brain. We propose that its temporal expression influences NPCs' delamination, corticogenesis and gyrification extrinsically.

Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells (HSPCs) are frequently located around the bone marrow (BM) vasculature. These so-called perivascular niches regulate HSC function both in health and disease, but they have been poorly studied in humans due to the scarcity of models integrating complete human vascular structures. Herein, we propose the stromal vascular fraction (SVF) derived from human adipose tissue as a cell source to vascularize 3D osteoblastic BM niches engineered in perfusion bioreactors. We show that SVF cells form self-assembled capillary structures, composed by endothelial and perivascular cells, that add to the osteogenic matrix secreted by BM mesenchymal stromal cells in these engineered niches. In comparison to avascular osteoblastic niches, vascularized BM niches better maintain immunophenotypically-defined cord blood (CB) HSCs without affecting cell proliferation. In contrast, HSPCs cultured in vascularized BM niches showed increased CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) numbers. The vascularization also contributed to better preserve osteogenic gene expression in the niche, demonstrating that niche vascularization has an influence on both hematopoietic and stromal compartments. In summary, we have engineered a fully humanized and vascularized 3D BM tissue to model native human endosteal perivascular niches and revealed functional implications of this vascularization in sustaining undifferentiated CB HSPCs. This system provides a unique modular platform to explore hemato-vascular interactions in human healthy/pathological hematopoiesis.

A roadmap for the Human Developmental Cell Atlas.

The Human Developmental Cell Atlas (HDCA) initiative, which is part of the Human Cell Atlas, aims to create a comprehensive reference map of cells during development. This will be critical to understanding normal organogenesis, the effect of mutations, environmental factors and infectious agents on human development, congenital and childhood disorders, and the cellular basis of ageing, cancer and regenerative medicine. Here we outline the HDCA initiative and the challenges of mapping and modelling human development using state-of-the-art technologies to create a reference atlas across gestation. Similar to the Human Genome Project, the HDCA will integrate the output from a growing community of scientists who are mapping human development into a unified atlas. We describe the early milestones that have been achieved and the use of human stem-cell-derived cultures, organoids and animal models to inform the HDCA, especially for prenatal tissues that are hard to acquire. Finally, we provide a roadmap towards a complete atlas of human development.

Charting human development using a multi-endodermal organ atlas and organoid models.

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated.

Fibroblast dedifferentiation as a determinant of successful regeneration.

Limb regeneration, while observed lifelong in salamanders, is restricted in post-metamorphic Xenopus laevis frogs. Whether this loss is due to systemic factors or an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that connective tissue (CT) cells form the post-metamorphic frog blastema, as in the case of axolotls. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells dedifferentiate and integrate to form lineages, including cartilage. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT, but not to the developing cartilage. Furthermore, using single-cell RNA-seq analysis we find that embryonic and adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration.

In Vitro and In Vivo Development of the Human Airway at Single-Cell Resolution.

Bud tip progenitor cells give rise to all murine lung epithelial lineages and have been described in the developing human lung; however, the mechanisms controlling human bud tip differentiation into specific lineages are unclear. Here, we used homogeneous human bud tip organoid cultures and identified SMAD signaling as a key regulator of the bud tip-to-airway transition. SMAD induction led to the differentiation of airway-like organoids possessing functional basal cells capable of clonal expansion and multilineage differentiation. To benchmark in vitro-derived organoids, we developed a single-cell mRNA sequencing atlas of the human lung from 11.5 to 21 weeks of development, which revealed high degrees of similarity between the in vitro-derived and in vivo airway. Together, this work sheds light on human airway differentiation in vitro and provides a single-cell atlas of the developing human lung.

Reconstructing complex lineage trees from scRNA-seq data using MERLoT.

Advances in single-cell transcriptomics techniques are revolutionizing studies of cellular differentiation and heterogeneity. It has become possible to track the trajectory of thousands of genes across the cellular lineage trees that represent the temporal emergence of cell types during dynamic processes. However, reconstruction of cellular lineage trees with more than a few cell fates has proved challenging. We present MERLoT (https://github.com/soedinglab/merlot), a flexible and user-friendly tool to reconstruct complex lineage trees from single-cell transcriptomics data. It can impute temporal gene expression profiles along the reconstructed tree. We show MERLoT's capabilities on various real cases and hundreds of simulated datasets.

Altered neuronal migratory trajectories in human cerebral organoids derived from individuals with neuronal heterotopia.

Malformations of the human cortex represent a major cause of disability1. Mouse models with mutations in known causal genes only partially recapitulate the phenotypes and are therefore not unlimitedly suited for understanding the molecular and cellular mechanisms responsible for these conditions2. Here we study periventricular heterotopia (PH) by analyzing cerebral organoids derived from induced pluripotent stem cells (iPSCs) of patients with mutations in the cadherin receptor-ligand pair DCHS1 and FAT4 or from isogenic knockout (KO) lines1,3. Our results show that human cerebral organoids reproduce the cortical heterotopia associated with PH. Mutations in DCHS1 and FAT4 or knockdown of their expression causes changes in the morphology of neural progenitor cells and result in defective neuronal migration dynamics only in a subset of neurons. Single-cell RNA-sequencing (scRNA-seq) data reveal a subpopulation of mutant neurons with dysregulated genes involved in axon guidance, neuronal migration and patterning. We suggest that defective neural progenitor cell (NPC) morphology and an altered navigation system in a subset of neurons underlie this form of PH.

Single Cell Transcriptomes Derived from Human Cervical and Uterine Tissue during Pregnancy.

This work presents the workflow for generating single cell transcriptomes derived from primary human uterine and cervical tissue obtained during planned cesarean hysterectomies. In total, a catalogue of 310 single cell transcriptomes are obtained, cell types present in these biopsies are inferred, and specific genes defining each of the cellular types present in the tissue are identified. Further validation of the inferred cell identity is also demonstrated via meta-analysis of independent repositories in literature generated by bulk sequenced data of fluorescence-activated cell sorting sorted cells.

Multilineage communication regulates human liver bud development from pluripotency.

Conventional two-dimensional differentiation from pluripotency fails to recapitulate cell interactions occurring during organogenesis. Three-dimensional organoids generate complex organ-like tissues; however, it is unclear how heterotypic interactions affect lineage identity. Here we use single-cell RNA sequencing to reconstruct hepatocyte-like lineage progression from pluripotency in two-dimensional culture. We then derive three-dimensional liver bud organoids by reconstituting hepatic, stromal, and endothelial interactions, and deconstruct heterogeneity during liver bud development. We find that liver bud hepatoblasts diverge from the two-dimensional lineage, and express epithelial migration signatures characteristic of organ budding. We benchmark three-dimensional liver buds against fetal and adult human liver single-cell RNA sequencing data, and find a striking correspondence between the three-dimensional liver bud and fetal liver cells. We use a receptor-ligand pairing analysis and a high-throughput inhibitor assay to interrogate signalling in liver buds, and show that vascular endothelial growth factor (VEGF) crosstalk potentiates endothelial network formation and hepatoblast differentiation. Our molecular dissection reveals interlineage communication regulating organoid development, and illuminates previously inaccessible aspects of human liver development.

Mapping heterogeneity in patient-derived melanoma cultures by single-cell RNA-seq.

Recent technological advances in single-cell genomics make it possible to analyze cellular heterogeneity of tumor samples. Here, we applied single-cell RNA-seq to measure the transcriptomes of 307 single cells cultured from three biopsies of three different patients with a BRAF/NRAS wild type, BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant melanoma metastasis, respectively. Analysis based on self-organizing maps identified sub-populations defined by multiple gene expression modules involved in proliferation, oxidative phosphorylation, pigmentation and cellular stroma. Gene expression modules had prognostic relevance when compared with gene expression data from published melanoma samples and patient survival data. We surveyed kinome expression patterns across sub-populations of the BRAF/NRAS wild type sample and found that CDK4 and CDK2 were consistently highly expressed in the majority of cells, suggesting that these kinases might be involved in melanoma progression. Treatment of cells with the CDK4 inhibitor palbociclib restricted cell proliferation to a similar, and in some cases greater, extent than MAPK inhibitors. Finally, we identified a low abundant sub-population in this sample that highly expressed a module containing ABC transporter ABCB5, surface markers CD271 and CD133, and multiple aldehyde dehydrogenases (ALDHs). Patient-derived cultures of the BRAF mutant/NRAS wild type and BRAF wild type/NRAS mutant metastases showed more homogeneous single-cell gene expression patterns with gene expression modules for proliferation and ABC transporters. Taken together, our results describe an intertumor and intratumor heterogeneity in melanoma short-term cultures which might be relevant for patient survival, and suggest promising targets for new treatment approaches in melanoma therapy.

Lineage-negative progenitors mobilize to regenerate lung epithelium after major injury.

Broadly, tissue regeneration is achieved in two ways: by proliferation of common differentiated cells and/or by deployment of specialized stem/progenitor cells. Which of these pathways applies is both organ- and injury-specific. Current models in the lung posit that epithelial repair can be attributed to cells expressing mature lineage markers. By contrast, here we define the regenerative role of previously uncharacterized, rare lineage-negative epithelial stem/progenitor (LNEP) cells present within normal distal lung. Quiescent LNEPs activate a ΔNp63 (a p63 splice variant) and cytokeratin 5 remodelling program after influenza or bleomycin injury in mice. Activated cells proliferate and migrate widely to occupy heavily injured areas depleted of mature lineages, at which point they differentiate towards mature epithelium. Lineage tracing revealed scant contribution of pre-existing mature epithelial cells in such repair, whereas orthotopic transplantation of LNEPs, isolated by a definitive surface profile identified through single-cell sequencing, directly demonstrated the proliferative capacity and multipotency of this population. LNEPs require Notch signalling to activate the ΔNp63 and cytokeratin 5 program, and subsequent Notch blockade promotes an alveolar cell fate. Persistent Notch signalling after injury led to parenchymal 'micro-honeycombing' (alveolar cysts), indicative of failed regeneration. Lungs from patients with fibrosis show analogous honeycomb cysts with evidence of hyperactive Notch signalling. Our findings indicate that distinct stem/progenitor cell pools repopulate injured tissue depending on the extent of the injury, and the outcomes of regeneration or fibrosis may depend in part on the dynamics of LNEP Notch signalling.

Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq.

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type 2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

In situ synthesis of lipopeptides as versatile receptors for the specific binding of nanoparticles and liposomes to solid-supported membranes.

A detailed study of the in situ coupling of small peptides such as CGGH6 (H6) and CGWK8 (K8) to maleimide functionalized phospholipid bilayers is presented. Individually addressable microstructured membranes are employed to unequivocally probe the conjugation. The in situ coupling of peptides via a terminal cysteine moiety to maleimide functionalized phospholipids is shown to be a convenient and versatile way to selectively fabricate peptide-modified phospholipid bilayers serving as specific receptor platforms for functionalized vesicles and nanoparticles. Specific binding of functional vesicles to the peptide-modified bilayers is achieved by either histidine complexation with Ni-NTA-DOGS containing vesicles or electrostatic interaction between positively charged oligolysine bearing lipopeptides and negatively charged POPC/POPG vesicles. Peptide receptors are also found to be easily accessible from the aqueous phase and not buried within the membrane interior.