Development Network

Development Network - Overview

We performed single-cell RNA sequencing (scRNA-seq) analysis of human post-implantation trophoblast cells. A hierarchical model was established, which was characterized by the sequential and stepwise development of two primitive cytotrophoblast cell (pCTB) subtypes after the trophectoderm (TE), two primitive syncytiotrophoblast (pSTB) subtypes, and migrative trophoblast cells (MTB). Further analysis characterized a new subtype of fusion-competent cells, which not only prepare for fusion to form primitive syncytiotrophoblast but also possess migration ability, which could be dual-function cells during implantation. Our work allows for reconstruction of sequential trophoblast cell transcriptional programs transition during implantation and provides a valuable resource to study pathologies in early pregnancy, such as recurrent implantation failure.

A Single-Cell Characterization of Human Post-implantation Embryos Cultured In Vitro Delineates Morphogenesis in Primary Syncytialization

Human limbs emerge during the fourth post-conception week as mesenchymal buds, which develop into fully formed limbs over the subsequent months. This process is orchestrated by numerous temporally and spatially restricted gene expression programmes, making congenital alterations in phenotype common. Decades of work with model organisms have defined the fundamental mechanisms underlying vertebrate limb development, but an in-depth characterization of this process in humans has yet to be performed. Here we detail human embryonic limb development across space and time using single-cell and spatial transcriptomics. We demonstrate extensive diversification of cells from a few multipotent progenitors to myriad differentiated cell states, including several novel cell populations. We uncover two waves of human muscle development, each characterized by different cell states regulated by separate gene expression programmes, and identify musculin (MSC) as a key transcriptional repressor maintaining muscle stem cell identity. Through assembly of multiple anatomically continuous spatial transcriptomic samples using VisiumStitcher, we map cells across a sagittal section of a whole fetal hindlimb. We reveal a clear anatomical segregation between genes linked to brachydactyly and polysyndactyly, and uncover transcriptionally and spatially distinct populations of the mesenchyme in the autopod. Finally, we perform single-cell RNA sequencing on mouse embryonic limbs to facilitate cross-species developmental comparison, finding substantial homology between the two species.

A human embryonic limb cell atlas resolved in space and time

Across the animal kingdom, gastrulation represents a key developmental event during which embryonic pluripotent cells diversify into lineage-specific precursors that will generate the adult organism. Here we report the transcriptional profiles of 116,312 single cells from mouse embryos collected at nine sequential time points ranging from 6.5 to 8.5 days post-fertilization. We construct a molecular map of cellular differentiation from pluripotency towards all major embryonic lineages, and explore the complex events involved in the convergence of visceral and primitive streak-derived endoderm.

A single-cell molecular map of mouse gastrulation and early organogenesis

In order to systematically define the early organ-specific progenitor cells in human development, we conducted single-cell RNA-seq on embryos between Carnegie Stage 12 and 16, when the neural tube finishes closure, the limbs emerge, and essentially all organs are laid out. Based on 103,264 single-cell transcriptomes, we identified 333 cell types in 19 tissue types. We characterized expression patterns of five major signaling pathways that are extensively involved in vertebrate embryonic patterning. We further reconstructed spatial axes in neural progenitors and demonstrated the conservation between human and mouse neural cell types. We defined precise domains for limb mesenchymal cells and provided a thorough characterization of dynamic gene expression during limb patterning. Finally, we revealed the differentiation process of retina development when combined with published data on later human development. Our study provides a compendium of early progenitor cells of human organs, which can serve as basis of lineage analysis in organogenesis as well as benchmarks for iPS and ESC-based in vitro induction systems. Overall design: 103264 single-cell transcriptomes of early human embryo between Carnegie Stage 12 and 16

A single-cell transcriptome atlas of human early embryogenesis

Single-cell RNA sequencing (scRNA-seq) is the leading technique for charting the molecular properties of individual cells. The latest methods are scalable to thousands of cells, enabling in- depth characterization of sample composition without prior knowledge. However, there are important differences between scRNA-seq techniques, and it remains unclear which are the most suitable protocols for drawing cell atlases of tissues, organs and organisms. We have generated benchmark datasets to systematically evaluate techniques in terms of their power to comprehensively describe cell types and states. We performed a multi-center study comparing 13 commonly used single-cell and single-nucleus RNA-seq protocols using a highly heterogeneous reference sample resource. Comparative and integrative analysis at cell type and state level revealed marked differences in protocol performance, highlighting a series of key features for cell atlas projects. These should be considered when defining guidelines and standards for international consortia, such as the Human Cell Atlas project. Overall design: The samples consists of two complex tissues (human PBMC and mouse colon) and three cell lines (HEK293-RFP, NIH3T3-GFP, MDCK-Turbo650). The primary PBMC and the colon sample constitute 90% and the cell lines 10%. The sample preparation aims to be standardized for all methods to allow a comparison of library preparation performance (6% HEK293T-RFP, 3% NIH3T3-GFP, 1% MDCK-Turbo650) of the sample content.

Benchmarking Single-Cell RNA Sequencing Protocols for Cell Atlas Projects

We report global transcriptional alteration and transcriptional trajectories from naive human pluripotent stem cells-derived trphectoderm to cytotrophoblast. By graph-based cell identities of total samples on the Seurat platform, we identified 9 main clusters, and further revealed the pseudotime differentiation during trophectoderm-cytotrophoblast transition. Overall design: Analyzing transcriptional profiles during naive human pluripotent stem cells-derived trophectoderm-cytotrophoblast transition using scRNA-seq. We generated chromium single cell 3' v3.1 libraries from randomly captured trophoblast-like cells and sequenced them using Illumina HiSeq 4000. Finally, we profiled 14,164 cells (nTE_D2: 2,378 cells; nTE_D3: 5,148 cells; nCT_D5:3,529 cells; nCT_D10: 3,109 cells) with a range of 56,312 – 130,971 mean reads per cell for each sample, whereby approximately 5,500–8,000 median genes per cell for each sample were detected.

Capturing human trophoblast development with naive pluripotent stem cells in vitro

Single-cell RNA sequencing was used to determine the cell type composition of all major human organs and construct a basic scheme for the human cell landscape (HCL).

Construction of A Human Cell Landscape by Single-cell mRNA-seq

Despite our growing understanding of embryonic immune development, rare early megakaryocytes (MKs) remain relatively understudied. Here we used single-cell RNA sequencing of human MKs from embryonic yolk sac (YS) and fetal liver (FL) to characterize the transcriptome, cellular heterogeneity, and developmental trajectories of early megakaryopoiesis. In the YS and FL, we found heterogeneous MK subpopulations with distinct developmental routes and patterns of gene expression that could reflect early functional specialization. Intriguingly, we identified a subpopulation of CD42b+CD14+ MKs in vivo that exhibit high expression of genes associated with immune responses and can also be derived from human embryonic stem cells (hESCs) in vitro. Furthermore, we identified THBS1 as an early marker for MK-biased embryonic endothelial cells. Overall, we provide important insights and invaluable resources for dissection of the molecular and cellular programs underlying early human megakaryopoiesis.

Decoding Human Megakaryocyte Development

Cellular heterogeneity can emerge from the expression of only one parental allele, however it has remained unknown to what degree patterns of random monoallelic expression of autosomal genes (aRME) are mitotically inherited (clonal) or stochastic (dynamic) in somatic cells. Here, we resolved this by applying allele-sensitive single-cell RNA-seq on primary mouse fibroblasts and in vivo human T-cells to simultaneously investigate clonal and dynamic aRME. Dynamic aRME affected a considerable portion of the transcriptome, with levels dependent on the cell’s transcriptional activity, but clonal aRME was surprisingly scarce (<1% of genes) and affected mainly lowly expressed genes. Consequently, the overwhelming portion of aRME occurs transiently and is scattered throughout somatic populations rather than, as previously hypothesized, confined to spatially restricted patches of clonally related cells.

Dissecting the clonal nature of allelic expression in somatic cells by single-cell RNA-seq

Cerebral organoids – three-dimensional cultures of human cerebral tissue derived from pluripotent stem cells – have emerged as models of human cortical development. However, the extent to which in vitro organoid systems recapitulate neural progenitor cell proliferation and neuronal differentiation programs observed in vivo remains unclear. Here we use single-cell RNA sequencing (scRNA-seq) to dissect and compare cell composition and progenitor-to-neuron lineage relationships in human cerebral organoids and fetal neocortex. Covariation network analysis using the fetal neocortex data reveals known and novel interactions among genes central to neural progenitor proliferation and neuronal differentiation. In the organoid, we detect diverse progenitors and differentiated cell types of neuronal and mesenchymal lineages, and identify cells that derived from regions resembling the fetal neocortex. We find that these organoid cortical cells use gene expression programs remarkably similar to those of the fetal tissue in order to organize into cerebral cortex-like regions. Our comparison of in vivo and in vitro cortical single cell transcriptomes illuminates the genetic features underlying human cortical development that can be studied in organoid cultures.

Human cerebral organoids recapitulate gene expression programs of fetal neocortex development.

Human brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution, from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood, and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development. Overall design: Single cell transcriptome and chromatin accessibility analysis of normal human prefrontal cortex development from gestation to adulthood. Consists of 29 samples for single nucleus RNA-seq (snRNA-seq) and 17 samples for single nucleus Assay for Transposase-Accessible Chromatin using sequencing (snATAC-seq), including 16 paired samples from the same donors with broad temporal coverage from fetal to adulthood stages of the Brodmann area 8, 9, 10, and 46 prefrontal cortex regions. Also includes 3 cerebral organoid snRNA-seq datasets

Human prefrontal cortex gene regulatory dynamics from gestation to adulthood at single-cell resolution

Here we describe sci-CAR, a combinatorial indexing strategy to jointly profile chromatin accessibility and mRNA in each of thousands of single cells. As a proof-of-concept, we apply sci-CAR to 4,825 cells comprising a time-series of dexamethasone treatment, as well as to 11,233 cells from the mouse kidney.

Joint profiling of chromatin accessibility and gene expression in thousands of single cells

Kidney organoids have potential uses in disease modelling, drug screening and regenerative medicine. However, novel cost-effective techniques are needed to enable scaled-up production of kidney cell types in vitro. The authors describe here a modified suspension culture method for the generation of kidney micro-organoids from human pluripotent stem cells. Optimisation of differentiation conditions allowed the formation of micro-organoids, each containing six to ten nephrons that were surrounded by endothelial and stromal populations. Single cell transcriptional profiling confirmed the presence and transcriptional equivalence of all anticipated renal cell types consistent with a previous organoid culture method.

Kidney micro-organoids in suspension culture as a scalable source of human pluripotent stem cell-derived kidney cells.

Human iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 450,118 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells. Overall design: We have 49 organoids in total. We include 4 donors (AS, ThF, N1, N2; 2 males, 2 females) at 4 timepoints: 1 replicate each at the iPSC (Day 0), 1 replicate for 2 donors (N1, N2), 2 for the other two (AS, ThF) at Day 7, 3 replicates each at Days 15 and 29. For one donor (AS), we include 2 experiments at D15 and D29. For one donor (ThF), we have extended culture experiments at D32 (3 replicates) and D51(1 replicate). For ThF, we generated D29 organoids (3 replicates) from a second protocol. We have 2 transplanted organoids at D29. Organoids were pooled on lanes using a randomized design to ensure that organoids replicates from an individual batch (donor, replicate, condition) were distributed across lanes.

Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

Hematopoietic stem cells (HSCs) give rise to diverse cell types in the blood system, yet our molecular understanding of the early trajectories that generate this enormous diversity in humans remains incomplete. Here, we leverage Drop-seq, a massively parallel single-cell RNA sequencing (scRNA-seq) approach, to individually profile 20,000 progenitor cells from human cord blood, without prior enrichment or depletion for individual lineages based on surface markers. Our data reveal a transcriptional compendium of progenitor states in human cord blood, representing four committed lineages downstream from HSC, alongside the transcriptional dynamics underlying fate commitment. We identify intermediate stages that simultaneously co-express “primed” programs for multiple downstream lineages, and also observe striking heterogeneity in the early molecular transitions between myeloid subsets. Integrating our data with a recently published scRNA-seq dataset from human bone marrow, we illustrate the molecular similarity between these two commonly used systems and further explore the chromatin dynamics of “primed” transcriptional programs based on ATAC-seq. Finally, we demonstrate that Drop-seq data can be utilized to identify new heterogeneous surface markers of cell state that correlate with functional output.

Molecular transitions in early progenitors during human cord blood hematopoiesis

The purpose of this project is to assess the relevance of pluripotent stem cell-derived cerebral and liver organoids to recapitulate the variation in cell-type specific gene expression programs between individuals. Towards this aim, we will generate reference atlases of the developing cortex and liver from multiple individuals, derive iPSC lines from these same individuals, and determine if inter-individual gene expression variation is recapitulated in cerebral and liver organoids from the same individual from which we have reference maps. In parallel we will assess the genetic contribution to variablity between organoids from different iPSCs of multiple human individuals that are available in existing iPSC resources (e.g. HipSci).

Organoid single-cell genomic atlas uncovers human-specific features of brain development

To study the developmental process of human podocytes and compare to the in vitro counterpart, we dissociated cells from the inner and outer kidney cortex as well as kidney organoids, and performed 10X Genomics single-cell RNA sequencing.

Single Cell RNA-Seq profiling of human embryonic kidney outer and inner cortical cells and kidney organoid cells

The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. To address this, we employed single cell RNA-sequencing to profile human skeletal muscle tissues from embryonic, fetal and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a “roadmap” of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols, and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. Additionally, we found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. In summary, this work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for better characterization and understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle based regenerative medicine.

Single Cell RNA-Sequencing of Human Limb Skeletal Muscle across Development and Myogenic Culture from Pluripotent Stem Cells

These files represent single cell RNA-Seq data generated on a 10x Chromium genomics platform from three biological replicates from the embryonic day (E)18.5 developing mouse kidney and three biological replicates of iPSC-derived human kidney organoids differentiated according to our published protocol (Takasato et al., Nature Protocols 2016). When aggregated, the mouse data represents >6000 cells that passed our QC, containing most major cell types known to exist in the developing mouse kidney. The aggregated human organoid data contains of >7000 cells that passed our QC and contains populations representing endothelial cells, podocytes, stroma, nephron, and off-target populations with similarity to neurons.

Single cell RNA-Seq of E18.5 developing mouse kidney and human kidney organoids

Dendritic cells (DCs) are professional antigen-presenting cells specialized in the recognition, processing and presentation of antigens to T-cells, inducing adaptive immune responses. We have identified PU.1, IRF8 and BATF3 transcription factors (TFs)  to reprogram human fibroblasts into DC type 1 (DC1). Induced DCs acquire a step-wise DC1 transcriptional signature at the single cell level as assessed with droplet based scRNA-seq. Hence, we provide evidence that antigen presentation can be dynamically programmed by a small combination of TFs in human cells. Moreover, it represents a platform for generating autologous DC1, the most attractive DC subset for cancer immunotherapy.

Single cell profiling of human induced dendritic cells generated by direct reprogramming of embryonic fibroblasts

Diverse cell types are produced from dorsal and ventral regions of the developing neural tube. In this study we describe a system for generating human inhibitory interneurons by ventralizing human embryonic stem cells in vitro and characterizing the gene expression of the cell types produced over time. We engineered a DCX-Citrine/Y hESC line to sort and characterize progenitor and neuron transcriptomics separately at both the subpopulation and single cell level. The cells generated in vitro were compared to similar populations present in human fetal brain samples by mapping gene expression data from human fetal cells onto the principal component analysis (PCA) space of in vitro-derived populations. Weighted gene co-expression network analysis (WGCNA) was used to determine the discreet cell types present at D24, D54, D100 and D125 of culture, and describe the gene expression changes that occur in progenitor and neuron populations over time. Immature lateral ganglionic eminence and medial ganglionic eminence cells are present at early timepoints, along with MGE-like and dorsal pallium-like neuronal progenitors. At later timepoints we observe the emergence of SST-expressing interneurons, as well as oligodendrocyte and astrocyte progenitors. We also identified genes that were upregulated in somatostatin-expressing interneurons as they mature. The transcriptomes of 1732 ventralized single cells were profiled by SmartSeq2 at different timepoints throughout a 125-day differentiation protocol that converted H1 human embryonic stem cells to a variety of ventrally-derived cell types.

Single-cell RNA-seq analysis  throughout a 125-day differentiation protocol that converted H1 human embryonic stem cells to a variety of ventrally-derived cell types.

We performed single cell RNA sequencing of human haemopoietic lympho-myeloid progenitors with the aim to separate functionally and transcriptionally distinct progenitors within the heterogeneous GMP, LMPP and MLP populations. We performed RNA sequencing in a total of 415 single cells. From donors 1 (CB369) and 2 (CB431), 163/166 and 157/249 cells passed quality control.We further analysed the 320 single cells that passed quality control from two  different donors (157 and 163 from each donor; 91 LMPP, 110 MLP and 119 GMP). We performed clustering using Seurat package, which identified 3 clusters of cells.  We identified genes that were differentially expressed among cells of the different clusters. The majority of the cells in cluster 1 were MLP, the majority of cluster 3 were GMP while cluster comprised of LMPP and GMP cells. Cluster 1 showed high expression of lymphoid-affiliated genes. Conversely, cluster 3 showed increased expression of myeloid genes, while cluster 2 showed a mixed transcriptional signature. PCA analysis revealed a transcriptional continuum of LMPP, MLP and GMP populations.

Single-cell analysis reveals the continuum of human lympho-myeloid progenitor cells

Aging is accompanied by a loss of muscle mass and function, termed sarcopenia, causing numerous morbidities and economic burdens in human populations. Mechanisms implicated in age-related sarcopenia include inflammation, muscle stem cell depletion, mitochondrial dysfunction and loss of motor neurons, but whether there are key drivers of sarcopenia is not yet known. To gain deeper insights into age-related sarcopenia, we performed transcriptome profiling on lower limb muscle biopsies from 72 young, old and sarcopenic subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17). Using this combined approach, we discovered novel changes in gene expression that occur with age and sarcopenia in multiple cell types comprising mature skeletal muscle. Notably, we found increased expression of the genes MYH8 and PDK4, and decreased expression of the gene IGFN1, in old muscle. We also identified a small population of nuclei that express CDKN1A, present only in aged samples, consistent with P21CIP1 senescence in this subpopulation. Our findings identify unique cellular sub-populations populations in aged and sarcopenic skeletal muscle, which will facilitate the development of new therapeutic strategies to combat age-related sarcopenia. Overall design: We performed transcriptome profiling on lower limb muscle biopsies from 72 young, old and sarcopenic subjects using bulk RNA-seq (N = 72) and single-nuclei RNA-seq (N = 17).

Single-nuclei profiling of aged skeletal muscle

Development of the human intestine is not well understood. Here we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including novel epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe new functions for these including as vascular niche cells. We pinpoint the origins of Peyer’s patches and gut-associated lymphoid tissue (GALT) and describe location specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. Overall design: Colon and terminal ileum tissue sections from 8-22 post-conceptual week fetal intestine samples were subject to single cell RNA-seq analysis.

Spatiotemporal Analysis of Human Intestinal Development at Single Cell Resolution - scRNA-Seq

To delineate the ontogeny of the mammalian endoderm, we generated 112,217 single-cell transcriptomes representing all endoderm populations within the mouse embryo until midgestation. By employing graph-based approaches, we modelled differentiating cells for spatio-temporal characterization of developmental trajectories and defined the transcriptional architecture that accompanies the emergence of the first (primitive or extra-embryonic) endodermal population and its sister pluripotent (embryonic) epiblast lineage. We uncovered a relationship between descendants of these two lineages, whereby epiblast cells differentiate into endoderm at two distinct time-points, before and during gastrulation. Trajectories of endoderm cells were mapped as they acquired embryonic versus extra-embryonic fates, and as they spatially converged within the nascent gut endoderm; revealing them to be globally similar but retaining aspects of their lineage history. We observed the regionalized identity of cells along the anterior-posterior axis of the emergent gut tube, reflecting their embryonic or extra-embryonic origin, and their coordinate patterning into organ-specific territories.

The emergent landscape of the mouse gut endoderm at single-cell resolution

Find the casual relationship between gene expression network and cellular phenotype at single cell resolution. We collected donated human pre-implatation embryos, and the embryonic stem cells derived from them, isolate individual cells, prepared single cell cDNAs, and sequenced them by HiSeq2000. Then we analyzed the expression of known RefSeq genes.

Tracing pluripotency of human early embryos and embryonic stem cells by single cell RNA-seq

Mouse studies have been instrumental in forming our current understanding of early cell-lineage decisions; however, similar insights into the early human development are severely limited. Here, we present a comprehensive transcriptional map of human embryo development, including the sequenced transcriptomes of 1,529 individual cells from 88 human preimplantation embryos. These data show that cells undergo an intermediate state of co-expression of lineage-specific genes, followed by a concurrent establishment of the trophectoderm, epiblast, and primitive endoderm lineages, which coincide with blastocyst formation. Female cells of all three lineages achieve dosage compensation of X chromosome RNA levels prior to implantation. However, in contrast to the mouse, XIST is transcribed from both alleles throughout the progression of this expression dampening, and X chromosome genes maintain biallelic expression while dosage compensation proceeds. We envision broad utility of this transcriptional atlas in future studies on human development as well as in stem cell research.

Development Network

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