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SUMMARYNephron progenitor cells (NPCs) self-renew and differentiate into nephrons, the functional units of the kidney. Here we report manipulation of p38 and YAP activity creates a synthetic niche that allows the long-term clonal expansion of primary mouse and human NPCs, and induced NPCs (iNPCs) from human pluripotent stem cells. Cultured iNPCs resemble closely primary human NPCs, generating nephron organoids with abundant distal convoluted tubule cells, which are not observed in published kidney organoids. The synthetic niche reprograms differentiated nephron cells into NPC state, recapitulating the plasticity of developing nephronin vivo. Scalability and ease of genome-editing in the cultured NPCs allow for genome-wide CRISPR screening, identi-fying novel genes associated with kidney development and disease. A rapid, efficient, and scala-ble organoid model for polycystic kidney disease was derived directly from genome-edited NPCs, and validated in drug screen. These technological platforms have broad applications to kidney development, disease, plasticity, and regeneration.

Half-Integer High Spin (HIHS) systems with zero-field splitting (ZFS) parameters below 1 GHz are generally dominated by the spin |─1/2>→|+1/2> central transition (CT). Accordingly, most pulsed Electron Paramagnetic Resonance (EPR) experiments are performed at this position for maximum sensitivity. However, in certain cases it can be desirable to detect higher spin transitions away from the CT in such systems. Here, we describe the use of frequency swept Wideband, Uniform Rate, Smooth Truncation (WURST) pulses for transferring spin population from the CT, and other transitions, of Gd(III) to the neighbouring higher spin transition |─3/2>→|─1/2> at Q- and W-band frequencies. Specifically, we demonstrate this approach to enhance the sensitivity of 1H Mims Electron-Nuclear Double Resonance (ENDOR) measurements on two model Gd(III) aryl substituted 1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) complexes, focusing on transitions other than the CT. We show that an enhancement factor greater than 2 is obtained for both complexes at Q- and W-band frequencies by the application of two polarising pulses prior to the ENDOR sequence. This is in agreement with simulations of the spin dynamics of the system during WURST pulse excitation. The technique demonstrated here should allow more sensitive experiments to be measured away from the CT at higher operating temperatures, and be combined with any relevant pulse sequence.

SummaryCardiac lymphatic vessels play important roles in fluid homeostasis, inflammation, disease, and regeneration of the heart. The developing cardiac lymphatics in human fetal hearts are closely associated with coronary arteries, similar to those in zebrafish hearts. We identified a population of cardiac lymphatic endothelial cells that reside in the epicardium. Single nuclei multiomic analysis of the human fetal heart revealed the plasticity and heterogeneity of the cardiac endothelium. Furthermore, we found that VEGFC is highly expressed in arterial endothelial cells, providing a molecular basis for the arterial association of cardiac lymphatic development. Using a cell-type-specific integrative analysis, we identified a novel population of cardiac lymphatic endothelial cells marked by the PROX1, the lymphangiocrine RELN, and enriched in binding motifs of ETV transcriptional factors. We report the first in vivo molecular characterization of human cardiac lymphatics and provide a valuable resource to understand fetal heart development.

As a highly regenerative organ, the intestine is a promising source for cellular reprogramming for replacing lost pancreatic β cells in diabetes. Gut enterochromaffin cells can be converted to insulin-producing cells by forkhead box O1 (FoxO1) ablation, but their numbers are limited. In this study, we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine. Accordingly, lineage-tracing experiments show that, upon genetic or pharmacologic FoxO1 ablation, the Paneth/goblet lineage can also undergo conversion to the insulin lineage. We designed a screening platform in gut organoids to accurately quantitate β-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process in mice and human adult intestinal organoids. We identified a triple blockade of FOXO1, Notch, and TGF-β that, when tested in insulin-deficient streptozotocin (STZ) or NOD diabetic animals, resulted in near normalization of glucose levels, associated with the generation of intestinal insulin-producing cells. The findings illustrate a therapeutic approach for replacing insulin treatment in diabetes.

Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack RB1 mutations but have MYCN amplification ( MYCN A ), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCN A retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3 – ) and maturing ARR3 + cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc–overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell–specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16 INK4A expression. Orthotopic xenografts of N-Myc–overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN -transduced retinae and MYCN A retinoblastomas in patients. These findings demonstrate the MYCN A retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage–specific cell signaling circuitries to drive retinoblastoma tumorigenesis.

The TGF-β signaling pathway plays a pivotal role in controlling organogenesis during fetal development. Although the role of TGF-β signaling in promoting lung alveolar epithelial growth has been determined, mesenchymal TGF-β signaling in regulating lung development has not been studied in vivo due to a lack of genetic tools for specifically manipulating gene expression in lung mesenchymal cells. Therefore, the integral roles of TGF-β signaling in regulating lung development and congenital lung diseases are not completely understood. Using a Tbx4 lung enhancer-driven Tet-On inducible Cre transgenic mouse system, we have developed a mouse model in which lung mesenchyme-specific deletion of TGF-β receptor 2 gene ( Tgfbr2) is achieved. Reduced airway branching accompanied by defective airway smooth muscle growth and later peripheral cystic lesions occurred when lung mesenchymal Tgfbr2 was deleted from embryonic day 13.5 to 15.5, resulting in postnatal death due to respiratory insufficiency. Although cell proliferation in both lung epithelium and mesenchyme was reduced, epithelial differentiation was not significantly affected. Tgfbr2 downstream Smad-independent ERK1/2 may mediate these mesenchymal effects of TGF-β signaling through the GSK3β-β-catenin-Wnt canonical pathway in fetal mouse lung. Our study suggests that Tgfbr2-mediated TGF-β signaling in prenatal lung mesenchyme is essential for lung development and maturation, and defective TGF-β signaling in lung mesenchyme may be related to abnormal airway branching morphogenesis and congenital airway cystic lesions.

Lung maturation is not limited to proper structural development but also includes differentiation and functionality of various highly specialized alveolar cell types. Alveolar type 1 (AT1s) cells occupy nearly 95% of the alveolar surface and are critical for establishing efficient gas exchange in the mature lung. AT1 cells arise from progenitors specified during the embryonic stage as well as alveolar epithelial progenitors expressing surfactant protein C (Sftpcpos cells) during postnatal and adult stages. Previously, we found that Wnt5a, a non-canonical Wnt ligand, is required for differentiation of AT1 cells during the saccular phase of lung development. To further investigate the role of Wnt5a in AT1 cell differentiation, we generated and characterized a conditional Wnt5a gain-of-function mouse model. Neonatal Wnt5a gain-of-function disrupted alveologenesis through inhibition of cell proliferation. In this setting Wnt5a downregulated β-catenin-dependent canonical Wnt signaling, repressed AT2 (anti-AT2) and promoted AT1 (pro-AT1) lineage-specific gene expression. In addition, we identified 2 subpopulations of Sftpchigh and Sftpclow alveolar epithelial cells. In Sftpclow cells, Wnt5a exhibits pro-AT1 and anti-AT2 effects, concurrent with inhibition of canonical Wnt signaling. Interestingly, in the Sftpchigh subpopulation, although increasing AT1 lineage-specific gene expression, Wnt5a gain-of-function did not change AT2 gene expression, nor inhibit canonical Wnt signaling. Using primary epithelial cells isolated from human fetal lungs, we demonstrate that this property of Wnt5a is evolutionarily conserved. Wnt5a therefore serves as a selective regulator that ensures proper AT1/AT2 balance in the developing lung.

Insulin is the essential treatment of Type 1 (T1D) and is often used in Type 2 Diabetes. For nearly five decades, efforts have been focused on replenishing β-cells in T1D patients as a more durable treatment. Gut endocrine cells can be converted into insulin-producing cells, but their numbers are limited. In this study we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine, in addition to enteroendocrine cells. Accordingly, lineage tracing experiments show that, besides enterochromaffin cells, the Paneth/goblet lineage can undergo conversion to the insulin lineage upon genetic or pharmacologic Foxo1 ablation in mice. We leveraged these data to design a screening platform in organoids to accurately quantitate β-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process by expanding the intestinal secretory lineage. We identified a triple blockade of FoxO1, Notch, and Tgfβ that, when tested in insulin-deficient diabetic animals resulted in a near-normalization of glucose levels, associated with the appearance of gut insulin-producing cells. The findings illustrate a therapeutic approach to replace insulin treatment in diabetes.

The objectives of this study were to demonstrate the neurotoxicity of α-amylase and to compare the enzyme concentration and activity in the amniotic fluid of rats with retinoic acid induced MMC to a healthy control population.Timed pregnant Sprague Dawley rats were gavage fed all-trans retinoic acid (60 mg/kg) in olive oil on gestational day E10 to induce a MMC defect. Control rats received olive oil. Amniotic fluid was collected on embryonic days E15, E17, E19, and E21. The amniotic fluid amylase concentration and relative activity were measured at each gestational age, and levels were compared between the MMC and control groups using Wilcoxon Rank Sum and Kruskal-Wallis tests. In a subset of dams sacrificed on E10.5, neuroepithelial cells were isolated from control embryos and exposed to α-amylase in increasing concentrations. Percentage of cell survival was assessed with CellProfiler software.Amniotic fluid amylase activity for embryonic days E15, E17, E19, and E21 was determined for MMC and control pups. Amylase activity increased significantly from E15 to E21 in both control (Amylase concentration and activity level were higher in the amniotic fluid of rats with retinoic acid induced MMC compared to controls with advancing gestational age. As amylase is toxic to neural epithelial cells, the higher activity of this digestive enzyme in fetuses with MMC may be a contributor to neural tube damage

Significant progress has been made in recent years in characterizing human multipotent progenitor cells (hMPCs) of the early pancreas; however, the identity and persistence of these cells during the second trimester, after the initiation of branching morphogenesis, remain elusive. Additionally, studies on hMPCs have been hindered by few isolation methods that allow for the recovery of live cells. Here, we investigated the tip progenitor domain in the branched epithelium of human fetal pancreas between 13.5 and 17.5 gestational weeks by immunohistological staining. We also used a novel RNA‐based technology to isolate live cells followed by gene expression analyses. We identified cells co‐expressing SOX9 and PTF1A, two transcription factors known to be important for pancreatic MPCs, within the tips of the epithelium and observed a decrease in their proportions over time. Pancreatic SOX9+/PTF1A+ cells were enriched for MPC markers, including MYC and GATA6. These cells were proliferative and appeared active in branching morphogenesis and matrix remodeling, as evidenced by gene set enrichment analysis. We identified a hub of genes pertaining to the expanding tip progenitor niche, such as FOXF1, GLI3, TBX3, FGFR1, TGFBR2, ITGAV, ITGA2, and ITGB3. YAP1 of the Hippo pathway emerged as a highly enriched component within the SOX9+/PTF1A+ cells. Single‐cell RNA‐sequencing further corroborated the findings by identifying a cluster of SOX9+/PTF1A+ cells with multipotent characteristics. Based on these results, we propose that the SOX9+/PTF1A+ cells in the human pancreas are uncommitted MPC‐like cells that reside at the tips of the expanding pancreatic epithelium, directing self‐renewal and inducing pancreatic organogenesis. stem cells translational medicine 2019;8:1249&1264, Schematic representation of SOX9+/PTF1A+ cell isolation and characterization.

Summary Analysis of kidney disease-causing genes and pathology resulting from systemic diseases highlight the importance of the kidney's filtering system, the renal corpuscles. To elucidate the developmental processes that establish the renal corpuscle, we performed single-nucleus droplet-based sequencing of the human fetal kidney. This enabled the identification of nephron, interstitial, and vascular cell types that together generate the renal corpuscles. Trajectory analysis identified transient developmental gene expression, predicting precursors or mature podocytes express FBLN2, BMP4, or NTN4, in conjunction with recruitment, differentiation, and modeling of vascular and mesangial cell types into a functional filter. In vitro studies provide evidence that these factors exhibit angiogenic or mesangial recruiting and inductive properties consistent with a key organizing role for podocyte precursors in kidney development. Together these studies define a spatiotemporal developmental program for the primary filtration unit of the human kidney and provide novel insights into cell interactions regulating co-assembly of constituent cell types., Graphical Abstract, Highlights • Single-nuclear RNA-seq analysis of human fetal kidney development • Co-ordinated programs of podocyte-driven glomerular development • Secreted podocyte factors act on endothelial and interstitial cells, Biological Sciences; Cell Biology; Developmental Biology; Omics

The development of the first synapse of the visual system between photoreceptors and bipolar cells in the outer plexiform layer (OPL) of the human retina is crucial for visual processing but poorly understood. By studying the maturation state and spatial organization of photoreceptors, depolarizing bipolar cells and horizontal cells in the human fetal retina, we establish a pseudo-temporal staging system for OPL development that we term OPL-Stages 0 to 4. This was validated through quantification of increasingly precise subcellular localization of bassoon to the OPL with each stage (P

Current kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney’s collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generated de novo from human pluripotent stem cells. In chemically-defined culture conditions, UB organoids generate CD organoids, with differentiated principal and intercalated cells adopting spatial assemblies reflective of the adult kidney’s collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoids in vitro results in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Applying an efficient gene editing strategy to remove RET activity, we demonstrate genetically modified UB organoids can model congenital anomalies of kidney and urinary tract. Taken together, these platforms will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting duct system., Here, the authors model the collecting duct system in kidneys by taking ureteric bud (UB) progenitor cells from both mouse and human primary tissues, as well as from hESC and hiPSC to generate organoids, which can model congenital anomalies of the kidney and urinary tract.

Postnatal organ-specific stem and progenitor cells are an attractive potential donor cell for tissue-engineering because they can be harvested autologous from the recipient and have sufficient potential to regenerate the tissue of interest with less risk for ectopic growth or tumor formation compared to donor cells from embryonic or fetal sources. We describe the generation of tissue-engineered larynx and trachea (TELT) from human and mouse postnatal organoid units (OU) as well as from human fetal OU. Mouse TELT contained differentiated respiratory epithelium lining large lumens, cartilage and smooth muscle. In contrast, human postnatal TE trachea, formed small epithelial lumens with rare differentiation, in addition to smooth muscle and cartilage. Human fetal TELT contained the largest epithelial lumens with all differentiated cell types as well as smooth muscle and cartilage. Increased epithelial cytokeratin 14 was identified in both human fetal and postnatal TELT compared to native trachea, consistent with regenerative basal cells. Cilia in TELT epithelium also demonstrated function with beating movements. While both human postnatal and fetal progenitors have the potential to generate TELT, there is more epithelial growth and differentiation from fetal progenitors, highlighting fundamental differences in these cell populations.

Homology-directed repair (HDR) of a DNA break allows copying of genetic material from an exogenous DNA template and is frequently exploited in CRISPR-Cas9 genome editing. However, HDR is in competition with other DNA repair pathways, including non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), and the efficiency of HDR outcomes is not predictable. Consequently, to optimize HDR editing, panels of CRISPR-Cas9 guide RNAs (gRNAs) and matched homology templates must be evaluated. We report here that CRISPR-Cas9 indel signatures can instead be used to identify gRNAs that maximize HDR outcomes. Specifically, we show that the frequency of deletions resulting from MMEJ repair, characterized as deletions greater than or equal to 3 bp, better predicts HDR frequency than consideration of total indel frequency. We further demonstrate that tools that predict gRNA indel signatures can be repurposed to identify gRNAs to promote HDR. Finally, by comparing indels generated by S. aureus and S. pyogenes Cas9 targeted to the same site, we add to the growing body of data that the targeted DNA sequence is a major factor governing genome editing outcomes.

Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. How GEC affect the pathology of Alport syndrome (AS) however, is unclear. We characterized GEC from wild type (WT) and col4α5 knockout AS mice, a hereditary disorder characterized by progressive renal failure. We used endothelial-specific Tek-tdTomato reporter mice to isolate GEC by FACS and performed transcriptome analysis on them from WT and AS mice, followed by in vitro functional assays and confocal and intravital imaging studies. Biopsies from patients with chronic kidney disease, including AS were compared with our findings in mice. We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. In AS mice, the brighttdT cell number increased and presented differential expression of endothelial markers compared to WT. RNA-seq analysis revealed differences in the immune and metabolic signaling pathways. In AS mice, dimtdT and brighttdT cells had different expression profiles of matrix-associated genes (Svep1, Itgβ6), metabolic activity (Apom, Pgc1α) and immune modulation (Apelin, Icam1) compared to WT mice. We confirmed a new pro-inflammatory role of Apelin in AS mice and in cultured human GEC. Gene modulations were identified comparable to the biopsies from patients with AS and focal segmental glomerulosclerosis, possibly indicating that the same mechanisms apply to humans. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans. This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets.

SummaryCongenital abnormalities of the kidney and urinary tract are amongst the most common birth defects affecting 3% of newborns. The human kidney develops over a 30-week period in which a nephron progenitor pool gives rise to around a million nephrons. To establish a framework for human nephrogenesis, we spatially resolved a stereotypical process by which equipotent nephron progenitors generate a nephron anlagen, then applied data-driven approaches to construct three-dimensional protein maps on anatomical models of the nephrogenic program. Single cell RNA sequencing identified novel progenitor states which were spatially mapped to the nephron anatomy enabling the generation of functional gene-networks predicting interactions within and between nephron cell-types. Network mining identified known developmental disease genes and predicts new targets of interest. The spatially resolved nephrogenic program made available through the Human Nephrogenesis Atlas (https://sckidney.flatironinstitute.org/) will facilitate an understanding of kidney development and disease, and enhance efforts to generate new kidney structures.

Kidney organoids model development and diseases of the nephron but not the contiguous epithelial network of the kidney’s collecting duct (CD) system. Here, we report the generation of an expandable, 3D branching ureteric bud (UB) organoid culture model that can be derived from primary UB progenitors from mouse and human fetal kidneys, or generatedde novofrom pluripotent human stem cells. UB organoids differentiate into CD organoidsin vitro, with differentiated cell types adopting spatial assemblies reflective of the adult kidney collecting system. Aggregating 3D-cultured nephron progenitor cells with UB organoidsin vitroresults in a reiterative process of branching morphogenesis and nephron induction, similar to kidney development. Combining efficient gene editing with the UB organoid model will facilitate an enhanced understanding of development, regeneration and diseases of the mammalian collecting system.One sentence summaryCollecting duct organoids derived from primary mouse and human ureteric bud progenitor cells and human pluripotent stem cells provide anin vitroplatform for genetic dissection of development, regeneration and diseases of the mammalian collecting system.

Most single cell RNA sequencing protocols start with single cells dispersed from intact tissue. High-throughput processing of the separated cells is enabled using microfluidics platforms. However, dissociation of tissue results in loss of information about cell location and morphology and potentially alters the transcriptome. An alternative approach for collecting RNA from single cells is to re-purpose the electrophysiological technique of patch clamp recording. A hollow patch pipette is attached to individual cells, enabling the recording of electrical activity, after which the cytoplasm may be extracted for single cell RNA-Seq (“Patch-Seq”). Since the tissue is not disaggregated, the location of cells is readily determined, and the morphology of the cells is maintained, making possible the correlation of single cell transcriptomes with cell location, morphology and electrophysiology. Recent Patch-Seq studies utilizes PCR amplification to increase amount of nucleic acid material to the level required for current sequencing technologies. PCR is prone to create biased libraries – especially with the extremely high degrees of exponential amplification required for single cell amounts of RNA. We compared a PCR-based approach with linear amplifications and demonstrate that aRNA amplification (in vitro transcription, IVT) is more sensitive and robust for single cell RNA collected by a patch clamp pipette.

Human kidney function is underpinned by approximately 1,000,000 nephrons, although the number varies substantially, and low nephron number is linked to disease. Human kidney development initiates around 4 weeks of gestation and ends around 34-37 weeks of gestation. Over this period, a reiterative inductive process establishes the nephron complement. Studies have provided insightful anatomic descriptions of human kidney development, but the limited histologic views are not readily accessible to a broad audience. In this first paper in a series providing comprehensive insight into human kidney formation, we examined human kidney development in 135 anonymously donated human kidney specimens. We documented kidney development at a macroscopic and cellular level through histologic analysis, RNA in situ hybridization, immunofluorescence studies, and transcriptional profiling, contrasting human development (4-23 weeks) with mouse development at selected stages (embryonic day 15.5 and postnatal day 2). The high-resolution histologic interactive atlas of human kidney organogenesis generated can be viewed at the GUDMAP database (www.gudmap.org) together with three-dimensional reconstructions of key components of the data herein. At the anatomic level, human and mouse kidney development differ in timing, scale, and global features such as lobe formation and progenitor niche organization. The data also highlight differences in molecular and cellular features, including the expression and cellular distribution of anchor gene markers used to identify key cell types in mouse kidney studies. These data will facilitate and inform in vitro efforts to generate human kidney structures and comparative functional analyses across mammalian species.

The nephron is the functional unit of the kidney, but the mechanism of nephron formation during human development is unclear. We conducted a detailed analysis of nephron development in humans and mice by immunolabeling, and we compared human and mouse nephron patterning to describe conserved and divergent features. We created protein localization maps that highlight the emerging patterns along the proximal–distal axis of the developing nephron and benchmark expectations for localization of functionally important transcription factors, which revealed unanticipated cellular diversity. Moreover, we identified a novel nephron subdomain marked by Wnt4 expression that we fate-mapped to the proximal mature nephron. Significant conservation was observed between human and mouse patterning. We also determined the time at which markers for mature nephron cell types first emerge—critical data for the renal organoid field. These findings have conceptual implications for the evolutionary processes driving the diversity of mammalian organ systems. Furthermore, these findings provide practical insights beyond those gained with mouse and rat models that will guide in vitro efforts to harness the developmental programs necessary to build human kidney structures.

Lung morphogenesis relies on a number of important processes, including proximal-distal patterning, cell proliferation, migration and differentiation, as well as epithelial-mesenchymal interactions. In mouse lung development, SOX2+ cells are localized in the proximal epithelium, whereas SOX9+ cells are present in the distal epithelium. We show that, in human lung, expression of these transcription factors differs, in that during the pseudoglandular stage distal epithelial progenitors at the tips coexpress SOX2 and SOX9. This double-positive population was no longer present by the canalicular stages of development. As in mouse, the human proximal epithelial progenitors express solely SOX2 and are surrounded by smooth muscle cells (SMCs) both in the proximal airways and at the epithelial clefts. Upon Ras-related C3 botulinum toxin substrate 1 inhibition, we noted decreased branching, as well as increased SMC differentiation, attenuated peristalsis, and a reduction in the distal double-positive SOX2/SOX9 progenitor cell population. Thus, the presence of SOX2/SOX9 double-positive progenitor cells in the distal epithelium during the pseudoglandular stage of human lung development appears to be critical to proximal-distal patterning and lung branching. Moreover, SMCs promote a SOX2 proximal phenotype and seem to suppress the SOX9+ population.

Zika virus (ZIKV) infection is associated with microcephaly in fetuses, but the pathogenesis of ZIKV-related microcephaly is not well understood. Here we show that ZIKV infects the subventricular zone in human fetal brain tissues and that the tissue tropism broadens with the progression of gestation. Our research demonstrates also that intermediate progenitor cells (IPCs) are the main target cells for ZIKV. Post-mitotic committed neurons become susceptible to ZIKV infection as well at later stages of gestation. Furthermore, activation of microglial cells, DNA fragmentation, and apoptosis of infected or uninfected cells could be found in ZIKV-infected brain tissues. Our studies identify IPCs as the main target cells for ZIKV. They also suggest that immune activation after ZIKV infection may play an important role in the pathogenesis of ZIKV-related microcephaly.

Background & Aims For patients with short-bowel syndrome, intestinal adaptation is required to achieve enteral independence. Although adaptation has been studied extensively in animal models, little is known about this process in human intestine. We hypothesized that analysis of matched specimens with and without luminal flow could identify new potential therapeutic pathways. Methods Fifteen paired human ileum samples were collected from children aged 2–20 months during ileostomy-reversal surgery after short-segment intestinal resection and diversion. The segment exposed to enteral feeding was denoted as fed, and the diverted segment was labeled as unfed. Morphometrics and cell differentiation were compared histologically. RNA Sequencing and Gene Ontology Enrichment Analysis identified over-represented and under-represented pathways. Immunofluorescence staining and Western blot evaluated proteins of interest. Paired data were compared with 1-tailed Wilcoxon rank-sum tests with a P value less than .05 considered significant. Results Unfed ileum contained shorter villi, shallower crypts, and fewer Paneth cells. Genes up-regulated by the absence of mechanoluminal stimulation were involved in digestion, metabolism, and transport. Messenger RNA expression of LGR5 was significantly higher in unfed intestine, accompanied by increased levels of phosphorylated signal transducer and activator of transcription 3 protein, and CCND1 and C-MYC messenger RNA. However, decreased proliferation and fewer LGR5+, OLFM4+, and SOX9+ intestinal stem cells (ISCs) were observed in unfed ileum. Conclusions Even with sufficient systemic caloric intake, human ileum responds to the chronic absence of mechanoluminal stimulation by up-regulating brush-border enzymes, transporters, structural genes, and ISC genes LGR5 and ASCL2. These data suggest that unfed intestine is primed to replenish the ISC population upon re-introduction of enteral feeding. Therefore, the elucidation of pathways involved in these processes may provide therapeutic targets for patients with intestinal failure. RNA sequencing data are available at Gene Expression Omnibus series GSE82147., Graphical abstract

The signaling cross talk between the tracheal mesenchyme and epithelium has not been researched extensively, leaving a substantial gap of knowledge in the mechanisms dictating embryonic development of the proximal airways by the adjacent mesenchyme. Recently, we reported that embryos lacking mesenchymal expression of Sox9 did not develop tracheal cartilage rings and showed aberrant differentiation of the tracheal epithelium. Here, we propose that tracheal cartilage provides local inductive signals responsible for the proper differentiation, metabolism, and inflammatory status regulation of the tracheal epithelium. The tracheal epithelium of mesenchyme-specific Sox9Δ/Δmutant embryos showed altered mRNA expression of various epithelial markers such as Pb1fa1, surfactant protein B ( Sftpb), secretoglobulin, family 1A, member 1 ( Scgb1a1), and trefoil factor 1 ( Tff1). In vitro tracheal epithelial cell cultures confirmed that tracheal chondrocytes secrete factors that inhibit club cell differentiation. Whole gene expression profiling and ingenuity pathway analysis showed that the tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), and transforming growth factor-β (TGF-β) signaling pathways were significantly altered in the Sox9 mutant trachea. TNF-α and IFN-γ interfered with the differentiation of tracheal epithelial progenitor cells into mature epithelial cell types in vitro. Mesenchymal knockout of Tgf-β1 in vivo resulted in altered differentiation of the tracheal epithelium. Finally, mitochondrial enzymes involved in fat and glycogen metabolism, cytochrome c oxidase subunit VIIIb ( Cox8b) and cytochrome c oxidase subunit VIIa polypeptide 1 ( Cox7a1), were strongly upregulated in the Sox9 mutant trachea, resulting in increases in the number and size of glycogen storage vacuoles. Our results support a role for tracheal cartilage in modulation of the differentiation and metabolism and the expression of inflammatory-related genes in the tracheal epithelium by feeding into the TNF-α, IFN-γ, and TGF-β signaling pathways.

Summary The renal corpuscle of the kidney comprises a glomerular vasculature embraced by podocytes and supported by mesangial myofibroblasts, which ensure plasma filtration at the podocyte-generated slit diaphragm. With a spectrum of podocyte-expressed gene mutations causing chronic disease, an enhanced understanding of podocyte development and function to create relevant in vitro podocyte models is a clinical imperative. To characterize podocyte development, scRNA-seq was performed on human fetal kidneys, identifying distinct transcriptional signatures accompanying the differentiation of functional podocytes from progenitors. Interestingly, organoid-generated podocytes exhibited highly similar, progressive transcriptional profiles despite an absence of the vasculature, although abnormal gene expression was pinpointed in late podocytes. On transplantation into mice, organoid-derived podocytes recruited the host vasculature and partially corrected transcriptional profiles. Thus, human podocyte development is mostly intrinsically regulated and vascular interactions refine maturation. These studies support the application of organoid-derived podocytes to model disease and to restore or replace normal kidney functions.

There is growing evidence of ongoing, in utero neurological damage in fetuses with myelomeningocele (MMC). Phospholipase A2 (PLA2) has known neurotoxic properties and is predominantly present in its secretory isoform (sPLA2) in meconium, the passage of which is increased in MMC fetuses. The objective of this study was to determine if amniotic fluid (AF) levels of PLA2 are elevated in a rat model of MMC.Timed pregnant Sprague-Dawley rats were gavage fed 60 mg/kg/bodyweight retinoic acid (RA) in olive oil at embryonic day 10 (E10). Amniocentesis was performed at multiple gestational time points on MMC fetuses, RA-exposed fetuses without MMC and control fetuses. AF PLA2 levels were analyzed by a fluorescent enzyme activity assay. PLA2 isoforms were determined by measuring activity in the presence of specific inhibitors.There was no difference in AF PLA2 activity between groups on E15. PLA2 activity was significantly increased in MMC fetuses on E17, E19 and E21 (p 0.001). Secretory PLA2 primarily accounted for the overall greater activity.PLA2 levels are elevated in the AF of fetal rats with MMC and may contribute to ongoing neural injury. This pathway may be a useful drug target to limit ongoing damage and better preserve neurologic function.

The mesenchyme gives rise to multiple distinct cell lineages in the mature respiratory system, including smooth muscle cells of the airway and vasculature, vascular endothelial cells, and parenchymal fibroblasts. However, a thorough understanding of the specification and the inter-relationships among the diverse mesenchymally derived cells in the human lung is lacking. We used single cell RNAseq of human fetal lung cells, to characterize cellular phenotypes in the pseudoglandular (11.5 weeks) and early canalicular (18.5 weeks) stage human lung. Confirmed live cells obtained from protease-dispersed tissue were captured and sequenced using the Chromium 10X system. Expression analysis was performed using Seurat 2.0 and functional analysis was performed using ToppFun. At these early stages, we were able to identify molecularly distinct cell populations representing fetal human lung endothelial cells, pericytes and smooth muscle cells. Early endothelial lineages expressed “classic” endothelial cell markers (PECAM, CDH5, VWF) as well as CD34, KIT and CLDN5. Cells with pericyte characteristics were evident early in development, and defined by expression of PDGFRB, THY1, notch signaling, and broad expression of basement membrane molecules (COL4, laminin, proteoglycans). Smooth muscle cells, defined by expression of the canonical lineage marker ACTA2, demonstrated evidence for Hedgehog signaling, with high expression of HHIP and PTCH1. At both 11 and 18 weeks, we identified a large population of undefined mesenchymal cells characterized by expression of COL1, PDGFRA and elastin fiber genes (ELN, MFAP4, FBLN1, LOXL1). Our data suggest that early specification of distinct mesenchymal lineages occurs in the human lung.

Most retinoblastomas initiate in response to the inactivation of the RB1 gene and loss of functional RB protein. The tumors may form with few additional genomic changes and develop after a premalignant retinoma phase. Despite this seemingly straightforward etiology, mouse models have not recapitulated the genetic, cellular, and stage-specific features of human retinoblastoma genesis. For example, whereas human retinoblastomas appear to derive from cone photoreceptor precursors, current mouse models develop tumors that derive from other retinal cell types. To investigate the basis of the human cone-specific oncogenesis, we compared developmental stage-specific cone precursor responses to RB loss in human and murine retina cultures and in cone-specific Rb1-knockout mice. We report that RB-depleted maturing (ARR3(+)) but not immature (ARR3(−)) human cone precursors enter the cell cycle, proliferate, and form retinoblastoma-like lesions with Flexner–Wintersteiner rosettes, then form low or nonproliferative premalignant retinoma-like lesions with fleurettes and p16(INK4A) and p130 expression, and finally form highly proliferative retinoblastoma-like masses. In contrast, in murine retina, only RB-depleted immature (Arr3(−)) cone precursors entered the cell cycle, and they failed to progress from S to M phase. Moreover, whereas intrinsically highly expressed MDM2 and MYCN contribute to RB-depleted maturing (ARR3(+)) human cone precursor proliferation, ectopic MDM2 and Mycn promoted only immature (Arr3(−)) murine cone precursor cell-cycle entry. These findings demonstrate that developmental stage-specific as well as species- and cell type-specific features sensitize to RB1 inactivation and reveal the human cone precursors’ capacity to model retinoblastoma initiation, proliferation, premalignant arrest, and tumor growth.

Most retinoblastomas initiate in response to the inactivation of the RB1 gene and loss of functional RB protein. The tumors may form without additional genomic changes and develop after a pre-malignant retinoma phase. Despite this seemingly straightforward etiology, mouse models have not recapitulated the genetic, cellular, and stage-specific features of human retinoblastoma genesis. For example, whereas human retinoblastomas appear to derive from cone photoreceptor precursors, current mouse models develop tumors that derive from other retinal cell types. To investigate the basis of the human cone-specific oncogenesis, we compared developmental-stage-specific cone precursor responses to RB loss in human and murine retina cultures and in cone-specific Rb1 knockout mice. We report that RB-depleted maturing (ARR3+) but not immature (ARR3-) human cone precursors enter the cell cycle, proliferate, and form retinoblastoma-like lesions characterized by Flexner-Wintersteiner rosettes, then form low or non-proliferative pre-malignant retinoma-like lesions with fleurettes and high p16INK4A and p130 expression, and finally form highly proliferative retinoblastoma-like masses. In contrast, in murine retina, only RB-depleted immature (Arr3-) cone precursors entered the cell cycle and they failed to progress from S to M phase. Moreover, whereas the intrinsically highly expressed MDM2 and MYCN contribute to RB-depleted maturing (ARR3+) human cone precursor proliferation, ectopic MDM2 and Mycn promoted only immature (Arr3-) murine cone precursor cell cycle entry. These findings demonstrate that developmental-stage-specific as well as speciesand cell-type-specific features sensitize to RB1 inactivation and reveal the human cone precursors’ capacity to model retinoblastoma initiation, proliferation, pre-malignant arrest, and tumor growth.Significance StatementRetinoblastoma is a childhood tumor that forms in response to mutations in the RB1 gene and loss of functional RB protein. Prior studies suggested that retinoblastomas arise from cone photoreceptor precursors, whereas mouse models yield tumors deriving from other retinal cell types and lacking human retinoblastoma features. Here, we show that in cultured human retinae, retinoblastomas initiate from RB-depleted cone precursors that are in a specific maturation state and form pre-malignant “retinomas” prior to retinoblastoma lesions, as is believed to occur in retinoblastoma patients. In contrast, Rb-deficient mouse cone precursors of similar maturation state and supplemented with human-cone-precursor-specific oncoproteins fail to proliferate. Thus, human species-specific developmental features underlie retinoblastomagenesis and may challenge the production of accurate mouse retinoblastoma models.

Fibroblast growth factor (FGF) signaling plays an important role in lung organogenesis. Over recent decades, FGF signaling in lung development has been extensively studied in animal models. However, little is known about the expression, localization, and functional roles of FGF ligands during human fetal lung development. Therefore, we aimed to determine the expression and function of several FGF ligands and receptors in human lung development. Using in situ hybridization (ISH) and RNA sequencing, we assessed their expression and distribution in native human fetal lung. Human fetal lung explants were treated with recombinant FGF7, FGF9, or FGF10 in air-liquid interface culture. Explants were analyzed grossly to observe differences in branching pattern as well as at the cellular and molecular level. ISH demonstrated that FGF7 is expressed in both the epithelium and mesenchyme; FGF9 is mainly localized in the distal epithelium, whereas FGF10 demonstrated diffuse expression throughout the parenchyma, with some expression in the smooth muscle cells (SMCs). FGFR2 expression was high in both proximal and distal epithelial cells as well as the SMCs. FGFR3 was expressed mostly in the epithelial cells, with lower expression in the mesenchyme, while FGFR4 was highly expressed throughout the mesenchyme and in the distal epithelium. Using recombinant FGFs, we demonstrated that FGF7 and FGF9 had similar effects on human fetal lung as on mouse fetal lung; however, FGF10 caused the human explants to expand and form cysts as opposed to inducing epithelial branching as seen in the mouse. In conjunction with decreased branching, treatment with recombinant FGF7, FGF9, and FGF10 also resulted in decreased double-positive SOX2/SOX9 progenitor cells, which are exclusively present in the distal epithelial tips in early human fetal lung. Although FGF ligand localization may be somewhat comparable between developing mouse and human lungs, their functional roles may differ substantially. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Cellular interactions among nephron, interstitial, and collecting duct progenitors drive mammalian kidney development. In mice, Six2(+) nephron progenitor cells (NPCs) and Foxd1(+) interstitial progenitor cells (IPCs) form largely distinct lineage compartments at the onset of metanephric kidney development. Here, we used the method for analyzing RNA following intracellular sorting (MARIS) approach, single-cell transcriptional profiling, in situ hybridization, and immunolabeling to characterize the presumptive NPC and IPC compartments of the developing human kidney. As in mice, each progenitor population adopts a stereotypical arrangement in the human nephron-forming niche: NPCs capped outgrowing ureteric branch tips, whereas IPCs were sandwiched between the NPCs and the renal capsule. Unlike mouse NPCs, human NPCs displayed a transcriptional profile that overlapped substantially with the IPC transcriptional profile, and key IPC determinants, including FOXD1, were readily detected within SIX2(+) NPCs. Comparative gene expression profiling in human and mouse Six2/SIX2(+) NPCs showed broad agreement between the species but also identified species-biased expression of some genes. Notably, some human NPC-enriched genes, including DAPL1 and COL9A2, are linked to human renal disease. We further explored the cellular diversity of mesenchymal cell types in the human nephrogenic niche through single-cell transcriptional profiling. Data analysis stratified NPCs into two main subpopulations and identified a third group of differentiating cells. These findings were confirmed by section in situ hybridization with novel human NPC markers predicted through the single-cell studies. This study provides a benchmark for the mesenchymal progenitors in the human nephrogenic niche and highlights species-variability in kidney developmental programs.

Summary Acquired or congenital disruption in enteric nervous system (ENS) development or function can lead to significant mechanical dysmotility. ENS restoration through cellular transplantation may provide a cure for enteric neuropathies. We have previously generated human pluripotent stem cell (hPSC)-derived tissue-engineered small intestine (TESI) from human intestinal organoids (HIOs). However, HIO-TESI fails to develop an ENS. The purpose of our study is to restore ENS components derived exclusively from hPSCs in HIO-TESI. hPSC-derived enteric neural crest cell (ENCC) supplementation of HIO-TESI establishes submucosal and myenteric ganglia, repopulates various subclasses of neurons, and restores neuroepithelial connections and neuron-dependent contractility and relaxation in ENCC-HIO-TESI. RNA sequencing identified differentially expressed genes involved in neurogenesis, gliogenesis, gastrointestinal tract development, and differentiated epithelial cell types when ENS elements are restored during in vivo development of HIO-TESI. Our findings validate an effective approach to restoring hPSC-derived ENS components in HIO-TESI and may implicate their potential for the treatment of enteric neuropathies., Graphical Abstract, Highlights • ENCC implantation restores enteric glial and neural subpopulations in HIO-TESI • ENCC differentiate into diverse neuronal subtypes and synapse with luminal ECC • ENCC-HIO-TESI demonstrates neuron-dependent contractility and relaxation • Early in vivo ENCC implantation alters the developing HIO-TESI transcriptome, Human intestinal organoid and enteric neural crest cell co-culture restores enteric nervous system (ENS) function. Schlieve and colleagues developed an in vivo approach to establish ENS elements in tissue-engineered small intestine that demonstrates neuron-dependent functional integration. This method could be applied to other organ systems and represent a future cellular therapy for human enteric neuropathies.

Summary Pluripotent stem cells display significant heterogeneity in gene expression, but whether this diversity is an inherent feature of the pluripotent state remains unknown. Single-cell gene expression analysis in cell subsets defined by surface antigen expression revealed that human embryonic stem cell cultures exist as a continuum of cell states, even under defined conditions that drive self-renewal. The majority of the population expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity, consistently high transcripts for all pluripotency-related genes studied, and no lineage priming. This subpopulation was characterized by its expression of a particular set of intercellular signaling molecules whose genes shared common regulatory features. Our data support a model of an inherently metastable self-renewing population that gives rise to a continuum of intermediate pluripotent states, which ultimately become primed for lineage specification., Highlights • Single-cell transcript profiles characteristic of distinct substates of pluripotency • Prospective isolation of substate with high self-renewal, no lineage priming • Self-renewing subpopulation marked by expression of specific ligands and receptors, Pera and colleagues used cell surface antigen expression and single-cell gene expression profiles to define cell substates of human pluripotent stem cells. Most cells expressed canonical pluripotency transcription factors and could differentiate into derivatives of all three germ layers. A minority subpopulation of cells displayed high self-renewal capacity, consistently high levels of transcripts for all pluripotency-related genes studied, and no lineage priming.

Lung branching morphogenesis relies on a number of factors, including proper epithelial cell proliferation and differentiation, cell polarity, and migration. Rac1, a small Rho GTPase, orchestrates a number of these cellular processes, including cell proliferation and differentiation, cellular alignment, and polarization. Furthermore, Rac1 modulates both noncanonical and canonical Wnt signaling, important pathways in lung branching morphogenesis. Culture of embryonic mouse lung explants in the presence of the Rac1 inhibitor (NSC23766) resulted in a dose-dependent decrease in branching. Increased cell death and BrdU uptake were notably seen in the mesenchyme, while no direct effect on the epithelium was observed. Moreover, vasculogenesis was impaired following Rac1 inhibition as shown by decreased Vegfa expression and impaired LacZ staining in Flk1-Lacz reporter mice. Rac1 inhibition decreased Fgf10 expression in conjunction with many of its associated factors. Moreover, using the reporter lines TOPGAL and Axin2-LacZ, there was an evident decrease in canonical Wnt signaling in the explants treated with the Rac1 inhibitor. Activation of canonical Wnt pathway using WNT3a or WNT7b only partially rescued the branching inhibition. Moreover, these results were validated on human explants, where Rac1 inhibition resulted in impaired branching and decreased AXIN2 and FGFR2b expression. We therefore conclude that Rac1 regulates lung branching morphogenesis, in part through canonical Wnt signaling. However, the exact mechanisms by which Rac1 interacts with canonical Wnt in human and mouse lung requires further investigation.

Mature nephrons originate from a small population of uninduced nephrogenic progenitor cells (NPs) within the cap mesenchyme. These cells are characterized by the coexpression of SIX2 and CITED1. Many studies on mouse models as well as on human pluripotent stem cells have advanced our knowledge of NPs, but very little is known about this population in humans, since it is exhausted before birth and strategies for its direct isolation are still limited. Here we report an efficient protocol for direct isolation of human NPs without genetic manipulation or stepwise induction procedures. With the use of RNA-labeling probes, we isolated SIX2+CITED1+ cells from human fetal kidney for the first time. We confirmed their nephrogenic state by gene profiling and evaluated their nephrogenic capabilities in giving rise to mature renal cells. We also evaluated the ability to culture these cells without complete loss of SIX2 and CITED1 expression over time. In addition to defining the gene profile of human NPs, this in vitro system facilitates studies of human renal development and provides a novel tool for renal regeneration and bioengineering purposes.

Lipid-containing alveolar interstitial fibroblasts, or simply lipofibroblasts, are increasingly recognized as an important component of the epithelial stem-cell niche in the rodent lung. Although lipofibroblasts were initially believed to merely assist type 2 alveolar epithelial cells in surfactant production during neonatal life, recent evidence suggests that these cells are indispensable for the survival and growth of epithelial stem cells during adult life. Despite the increasing interest in lipofibroblast biology, little is known about their cellular origin or the molecular pathways controlling their formation during embryonic development. Here, we show that a population of lipid-droplet-containing stromal cells emerges in the developing mouse lung between E15.5 and E16.5. This event is accompanied by significant upregulation, in the lung mesenchyme, of peroxisome proliferator-activated receptor gamma (the master switch of lipogenesis), adipose differentiation-related protein (marker of mature lipofibroblasts) and fibroblast growth factor 10 (previously shown by our group to identify a subpopulation of lipofibroblast progenitors). We also demonstrate that although only a subpopulation of total embryonic lipofibroblasts derives from Fgf10-positive progenitor cells, in vivo knockdown of Fgfr2b ligand activity as well as reduction in Fgf10 expression lead to global reduction in the expression levels of lipofibroblast markers at E18.5. Constitutive Fgfr1b knockouts and mutants with conditional partial inactivation of Fgfr2b in the lung mesenchyme reveal the involvement of both receptors in lipofibroblast formation and suggest a possible compensation between the two receptors. We also provide data from human fetal lungs to demonstrate the relevance of our discoveries to humans. Our results reveal an essential role for Fgf10 signaling in the formation of lipofibroblasts during late lung development.