J. Gray Camp

Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells

Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.

Human cerebral organoids recapitulate gene expression programs of fetal neocortex development

Significance We have used single-cell RNA sequencing to compare human cerebral organoids and fetal neocortex. We find that, with relatively few exceptions, cells in organoid cortex-like regions use genetic programs very similar to fetal tissue to generate a structured cerebral cortex. Our study is of interest, as it shows which genetic features underlying human cortical development can be accurately studied in organoid culture systems. This is important because although cerebral organoids have great promise for modeling human neurodevelopment, the extent to which organoids recapitulate neural progenitor proliferation and differentiation networks in vivo remained unclear. Cerebral organoids—3D 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 previously unidentified 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 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.

Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states1–3. However, the intermediates through which individual cells progress are largely undefined. Here we used single-cell RNA-seq4–7 at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts (MEFs) to induced neuronal (iN) cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts suggesting the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

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.

Differences and similarities between human and chimpanzee neural progenitors during cerebral cortex development

Human neocortex expansion likely contributed to the remarkable cognitive abilities of humans. This expansion is thought to primarily reflect differences in proliferation versus differentiation of neural progenitors during cortical development. Here, we have searched for such differences by analysing cerebral organoids from human and chimpanzees using immunohistofluorescence, live imaging, and single-cell transcriptomics. We find that the cytoarchitecture, cell type composition, and neurogenic gene expression programs of humans and chimpanzees are remarkably similar. Notably, however, live imaging of apical progenitor mitosis uncovered a lengthening of prometaphase-metaphase in humans compared to chimpanzees that is specific to proliferating progenitors and not observed in non-neural cells. Consistent with this, the small set of genes more highly expressed in human apical progenitors points to increased proliferative capacity, and the proportion of neurogenic basal progenitors is lower in humans. These subtle differences in cortical progenitors between humans and chimpanzees may have consequences for human neocortex evolution. DOI: http://dx.doi.org/10.7554/eLife.18683.001

Human development: Advances in mini-brain technology

Two studies integrate cutting-edge techniques to grow and analyse 3D cultured tissues that resemble human brain structures, enabling examination of how brain regions interact and neurons mature. See Articles p.48 & p.54 Three-dimensional cellular models of the human brain, or organoids, enable the in vitro study of cerebral development and disease, but exactly which cells are generated and how much of the brains complexity they recreate is undefined. To investigate in depth the nature of cells in human cerebral organoids differentiated from pluripotent stem cells, Paola Arlotta and colleagues carried out droplet-based single-cell expression analysis on cells isolated from over 30 organoids at developmental stages ranging from 3 to 9 months and beyond. They identify a wide diversity of neurons and progenitors and show that the more mature organoids formed dendritic spines as well as electrically active networks, which responded to light stimulation. The authors suggest that organoids may facilitate the study of circuit function using physiological sensory mechanisms. Elsewhere in this issue, Sergiu Pasca and colleagues show that re-assembling ventral and dorsal forebrain spheroids obtained separately in vitro allows the migration of human interneurons and the formation of functional synapses. GABAergic neurons play important roles in brain function and are implicated in numerous psychiatric disorders. They migrate long distances from the ventral to the dorsal forebrain before integrating to cortical circuits. In vitro modelling of GABAergic neuronal differentiation during this interaction would allow us to investigate the cause of human brain disorders associated with defects in neuronal migration, but this has so far been difficult. Sergiu Pasca and colleagues have developed an approach for generating neural three-dimensional spheroids resembling either the ventral or dorsal forebrain. They show that assembling the two types of spheroids separately in vitro allows the saltatory migration of human interneurons into the cortex, as seen in human development, and the formation of functional synapses with the dorsally derived cortical glutamatergic neurons. In this context, they find that interneurons from Timothy syndrome patients exhibit perturbation in migration patterns. Elsewhere in this issue, Paola Arlotta and colleagues carried out single cell expression analysis on cells from human brain organoids to investigate the nature of cells generated by these three-dimensional models.

Human organomics: a fresh approach to understanding human development using single-cell transcriptomics

Innovative methods designed to recapitulate human organogenesis from pluripotent stem cells provide a means to explore human developmental biology. New technologies to sequence and analyze single-cell transcriptomes can deconstruct these ‘organoids’ into constituent parts, and reconstruct lineage trajectories during cell differentiation. In this Spotlight article we summarize the different approaches to performing single-cell transcriptomics on organoids, and discuss the opportunities and challenges of applying these techniques to generate organ-level, mechanistic models of human development and disease. Together, these technologies will move past characterization to the prediction of human developmental and disease-related phenomena. Summary: This Spotlight article summarizes the different approaches to performing single-cell transcriptomics on organoids, highlighting how they can generate mechanistic models of human development and disease.

Human stem cell resources are an inroad to Neandertal DNA functions

Pluripotent stem cells from diverse humans offer the potential to study human functional variation in controlled culture environments. A portion of this variation originates from ancient admixture between modern humans and Neandertals, which introduced alleles that left a phenotypic legacy on individual humans today. Here we show that a large repository of human induced pluripotent stem cells (iPSCs) harbors extensive Neandertal DNA, including most known functionally relevant Neandertal alleles present in modern humans. This resource contains Neandertal DNA that contributes to human phenotypes and diseases, encodes hundreds of amino acid changes, and alters gene expression in specific tissues. Human iPSCs thus provide an opportunity to experimentally explore the Neandertal contribution to present-day phenotypes, and potentially study Neandertal traits.

Optimal Hypoxia Regulates Human iPSC-Derived Liver Bud Differentiation through Intercellular TGFB Signaling

Summary Timely controlled oxygen (O2) delivery is crucial for the developing liver. However, the influence of O2 on intercellular communication during hepatogenesis is unclear. Using a human induced pluripotent stem cell-derived liver bud (hiPSC-LB) model, we found hypoxia induced with an O2-permeable plate promoted hepatic differentiation accompanied by TGFB1 and TGFB3 suppression. Conversely, extensive hypoxia generated with an O2-non-permeable plate elevated TGFBs and cholangiocyte marker expression. Single-cell RNA sequencing revealed that TGFB1 and TGFB3 are primarily expressed in the human liver mesenchyme and endothelium similar to in the hiPSC-LBs. Stromal cell-specific RNA interferences indicated the importance of TGFB signaling for hepatocytic differentiation in hiPSC-LB. Consistently, during mouse liver development, the Hif1a-mediated developmental hypoxic response is positively correlated with TGFB1 expression. These data provide insights into the mechanism that hypoxia-stimulated signals in mesenchyme and endothelium, likely through TGFB1, promote hepatoblast differentiation prior to fetal circulation establishment.

Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration

How the axolotl makes a new limb Unlike most vertebrate limbs, the axolotl limb regenerates the skeleton after amputation. Dermal and interstitial fibroblasts have been thought to provide sources for skeletal regeneration, but it has been unclear whether preexisting stem cells or dedifferentiation of fibroblasts formed the blastema. Gerber et al. developed transgenic reporter animals to compare periskeletal cell and fibroblast contributions to regeneration. Callus-forming periskeletal cells extended existing bone, but fibroblasts built new limb segments. Single-cell transcriptomics and Brainbow-based lineage tracing revealed the lack of a preexisting stem cell. Instead, the heterogeneous population of fibroblasts lost their adult features to form a multipotent skeletal progenitor expressing the embryonic limb program. Science, this issue p. eaaq0681 Deconstructing cell composition, reconstructing lineage relationships, and tracing tissue reprogramming in limb regeneration are explored. INTRODUCTION Axolotls (Ambystoma mexicanum) and other salamanders are the only tetrapods that can regenerate whole limbs. During this complex process, changes in gene expression regulate the outgrowth of a new appendage, but how injury induces limb cells to form regenerative progenitors that differentiate into diverse cell fates is poorly understood. Tracking and molecular profiling of individual cells during limb regeneration would resolve distinct differentiation pathways and provide clues for how cells convert from a mature resting state into regenerative cell lineages. RATIONALE Axolotl limbs are composed of many different cell types originating from neural, myogenic, epidermal, and connective tissue (CT) lineages. Upon limb amputation, cells from nearby the amputation plane accumulate in a distinctive tissue called the blastema, which serves as a progenitor cell source to build the new limb. Transgenic axolotl strains in which descendants of distinct adult cell types can be labeled, tracked, and isolated during the regenerative process provide an opportunity to understand how particular cell lineages progress during blastema formation and subsequent limb regrowth. Combining transgenic axolotl strains with single-cell RNA sequencing (scRNA-seq) enables the tracking of individual cell types, as well as the reconstruction of the molecular steps underlying the regeneration process for these particular cell lineages. CT cells, descendants of lateral plate mesoderm, are the most abundant lineage contributing to the blastema and encompass bone and cartilage, tendons, periskeleton, and dermal and interstitial fibroblasts. These cells detect the position of the amputation site, leading to the regeneration of appropriate limb parts and making the CT a key cell lineage for deciphering and understanding molecular programs of regeneration. RESULTS We used an inducible Cre-loxP fluorescence system to establish genetically marked transgenic axolotl strains for isolating CT cells from adult limb tissue as well as CT descendants in the blastema. We used scRNA-seq to molecularly profile CT cells along a dense time course of blastema formation and the outgrowth of the regenerated arm, as well as stages of embryonic limb development. This profiling indicated that CT cells express adult phenotypes that are lost upon the induction of regeneration. The heterogeneous population of CT-derived cells converges into a homogeneous and transient blastema progenitor state that at later stages recapitulates an embryonic limb bud–like program. Notably, we did not find evidence of CT stem cells or blastema-like precursors in the mature arm. We found that CT subtypes have spatially restricted contributions to proximal and distal compartments in the regenerated limb. Specifically, a particular CT subtype—periskeletal cells—extended the severed skeleton at the amputation site whereas fibroblastic CT cells de novo regenerated distal skeletal segments. By using high-throughput single-cell transcriptomics and Brainbow axolotl-based clonal lineage tracing, we could follow the redifferentiation trajectories of CT lineages during the final stages of regeneration. These findings established the formation of a multipotent skeletal progenitor cell that contributed to tendons, ligaments, skeleton, periskeleton, and fibroblasts. CONCLUSION CT cells are a key cell type for understanding regeneration because they form the patterned limb skeleton that guides the regeneration of the other limb tissues, such as muscle. Because of the cells’ heterogeneity and intermingling with other cell types, it had been difficult to study how CT forms regenerative blastema cells. The use of these newly generated transgenic reporter strains combined with single-cell transcriptomic analysis and clonal tracing have allowed us to determine that CT cells with diverse molecular features traverse through a distinctive molecular state as they dedifferentiate into a common, multipotent progenitor resembling an embryonic limb bud cell. In the future, it will be important to test which components of the transition state are required for the dedifferentiation process. Furthermore, this work opens the possibility to examine how regeneration-associated genes and their associated chromatin structure are regulated during this transition. Lastly, the work raises the possibility that the limited regeneration seen among mammals is due to an inability to reprogram CT to such embryonic states. Regeneration of the axolotl upper arm CT. Transgenic axolotl strains were used to isolate CT cells at different stages during limb regeneration. Single-cell transcriptomes were generated, and transcriptional signatures were used to track the fate of cells during regeneration. We found that heterogeneous CT cell types in the adult upper arm funnel into a homogeneous multipotent skeletal progenitor state that recapitulates axolotl limb development, before their redifferentiation into the heterogeneous CT cell subtypes. Amputation of the axolotl forelimb results in the formation of a blastema, a transient tissue where progenitor cells accumulate prior to limb regeneration. However, the molecular understanding of blastema formation had previously been hampered by the inability to identify and isolate blastema precursor cells in the adult tissue. We have used a combination of Cre-loxP reporter lineage tracking and single-cell messenger RNA sequencing (scRNA-seq) to molecularly track mature connective tissue (CT) cell heterogeneity and its transition to a limb blastema state. We have uncovered a multiphasic molecular program where CT cell types found in the uninjured adult limb revert to a relatively homogenous progenitor state that recapitulates an embryonic limb bud–like phenotype including multipotency within the CT lineage. Together, our data illuminate molecular and cellular reprogramming during complex organ regeneration in a vertebrate.