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Using scRNA-seq and microscopy, we describe a cell that is enriched in the lower airways of the developing human lung and identified by the unique coexpression of SCGB3A2/SFTPB/CFTR . To functionally interrogate these cells, we apply a single-cell barcode-based lineage tracing method, called CellTagging, to track the fate of SCGB3A2/SFTPB/CFTR cells during airway organoid differentiation in vitro. Lineage tracing reveals that these cells have a distinct differentiation potential from basal cells, giving rise predominantly to pulmonary neuroendocrine cells and a subset of multiciliated cells distinguished by high C6 and low MUC16 expression. Lineage tracing results are supported by studies using organoids and isolated cells from the lower noncartilaginous airway. We conclude that SCGB3A2/SFTPB/CFTR cells are enriched in the lower airways of the developing human lung and contribute to the epithelial diversity and heterogeneity in this region.

Human intestinal organoids (HIOs) derived from pluripotent stem cells provide a valuable model for investigating human intestinal organogenesis and physiology, but they lack the immune components required to fully recapitulate the complexity of human intestinal biology and diseases. To address this issue and to begin to decipher human intestinal-immune crosstalk during development, we generated HIOs containing immune cells by transplanting HIOs under the kidney capsule of mice with a humanized immune system. We found that human immune cells temporally migrate to the mucosa and form cellular aggregates that resemble human intestinal lymphoid follicles. Moreover, after microbial exposure, epithelial microfold cells are increased in number, leading to immune cell activation determined by the secretion of IgA antibodies in the HIO lumen. This in vivo HIO system with human immune cells provides a framework for future studies on infection- or allergen-driven intestinal diseases., (© 2023. The Author(s).)

Epithelial organoids derived from intestinal tissue, called enteroids, recapitulate many aspects of the organ in vitro and can be used for biological discovery, personalized medicine, and drug development. Here, we interrogated the cell signaling environment within the developing human intestine to identify niche cues that may be important for epithelial development and homeostasis. We identified an EGF family member, EPIREGULIN (EREG), which is robustly expressed in the developing human crypt. Enteroids generated from the developing human intestine grown in standard culture conditions, which contain EGF, are dominated by stem and progenitor cells and feature little differentiation and no spatial organization. Our results demonstrate that EREG can replace EGF in vitro, and EREG leads to spatially resolved enteroids that feature budded and proliferative crypt domains and a differentiated villus-like central lumen. Multiomic (transcriptome plus epigenome) profiling of native crypts, EGF-grown enteroids, and EREG-grown enteroids showed that EGF enteroids have an altered chromatin landscape that is dependent on EGF concentration, downregulate the master intestinal transcription factor CDX2, and ectopically express stomach genes, a phenomenon that is reversible. This is in contrast to EREG-grown enteroids, which remain intestine like in culture. Thus, EREG creates a homeostatic intestinal niche in vitro, enabling interrogation of stem cell function, cellular differentiation, and disease modeling.

Many esophageal diseases can arise during development or throughout life. Therefore, well-characterized in vitro models and detailed methods are essential for studying human esophageal development, homeostasis and disease. Here, we (1) create an atlas of the cell types observed in the normal adult human esophagus; (2) establish an ancestrally diverse biobank of in vitro esophagus tissue to interrogate homeostasis and injury; and (3) benchmark in vitro models using the adult human esophagus atlas. We created a single-cell RNA sequencing reference atlas using fresh adult esophagus biopsies and a continuously expanding biobank of patient-derived in vitro cultures (n=55 lines). We identify and validate several transcriptionally distinct cell classes in the native human adult esophagus, with four populations belonging to the epithelial layer, including basal, epibasal, early differentiating and terminally differentiated luminal cells. Benchmarking in vitro esophagus cultures to the in vivo reference using single-cell RNA sequencing shows that the basal stem cells are robustly maintained in vitro, and the diversity of epithelial cell types in culture is dependent on cell density. We also demonstrate that cultures can be grown in 2D or as 3D organoids, and these methods can be employed for modeling the complete epithelial layers, thereby enabling in vitro modeling of the human adult esophagus., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Bud tip progenitors (BTPs) in the developing lung give rise to all epithelial cell types found in the airways and alveoli. This work aimed to develop an iPSC organoid model enriched with NKX2-1+ BTP-like cells. Building on previous studies, we optimized a directed differentiation paradigm to generate spheroids with more robust NKX2-1 expression. Spheroids were expanded into organoids that possessed NKX2-1+/CPM+ BTP-like cells, which increased in number over time. Single cell RNA-sequencing analysis revealed a high degree of transcriptional similarity between induced BTPs (iBTPs) and in vivo BTPs. Using FACS, iBTPs were purified and expanded as induced bud tip progenitor organoids (iBTOs), which maintained an enriched population of bud tip progenitors. When iBTOs were directed to differentiate into airway or alveolar cell types using well-established methods, they gave rise to organoids composed of organized airway or alveolar epithelium, respectively. Collectively, iBTOs are transcriptionally and functionally similar to in vivo BTPs, providing an important model for studying human lung development and differentiation., Competing Interests: Competing interests J.R.S. holds intellectual property rights pertaining to lung organoid technologies., (© 2022. Published by The Company of Biologists Ltd.)

The human respiratory epithelium is derived from a progenitor cell in the distal buds of the developing lung. These "bud tip progenitors" are regulated by reciprocal signaling with surrounding mesenchyme; however, mesenchymal heterogeneity and function in the developing human lung are poorly understood. We interrogated single-cell RNA sequencing data from multiple human lung specimens and identified a mesenchymal cell population present during development that is highly enriched for expression of the WNT agonist RSPO2, and we found that the adjacent bud tip progenitors are enriched for the RSPO2 receptor LGR5. Functional experiments using organoid models, explant cultures, and FACS-isolated RSPO2 + mesenchyme show that RSPO2 is a critical niche cue that potentiates WNT signaling in bud tip progenitors to support their maintenance and multipotency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

NOTCH signaling is a key regulator involved in maintaining intestinal stem cell (ISC) homeostasis and for balancing differentiation. Using single-cell transcriptomics, we observed that OLFM4, a NOTCH target gene present in ISCs, is first expressed at 13 weeks post-conception in the developing human intestine and increases over time. This led us to hypothesize that the requirement for NOTCH signaling is acquired across human development. To test this, we established a series of epithelium-only organoids (enteroids) from different developmental stages and used γ-secretase inhibitors (dibenzazepine [DBZ] or DAPT) to functionally block NOTCH signaling. Using quantitative enteroid-forming assays, we observed a decrease in enteroid forming efficiency in response to γ-secretase inhibition as development progress. When DBZ was added to cultures and maintained during routine passaging, enteroids isolated from tissue before 20 weeks had higher recovery rates following single-cell serial passaging. Finally, bulk RNA sequencing (RNA-seq) analysis 1 day and 3 days after DBZ treatment showed major differences in the transcriptional changes between developing or adult enteroids. Collectively, these data suggest that ISC dependence on NOTCH signaling increases as the human intestine matures., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Measuring cell identity in development, disease, and reprogramming is challenging as cell types and states are in continual transition. Here, we present Capybara, a computational tool to classify discrete cell identity and intermediate "hybrid" cell states, supporting a metric to quantify cell fate transition dynamics. We validate hybrid cells using experimental lineage tracing data to demonstrate the multi-lineage potential of these intermediate cell states. We apply Capybara to diagnose shortcomings in several cell engineering protocols, identifying hybrid states in cardiac reprogramming and off-target identities in motor neuron programming, which we alleviate by adding exogenous signaling factors. Further, we establish a putative in vivo correlate for induced endoderm progenitors. Together, these results showcase the utility of Capybara to dissect cell identity and fate transitions, prioritizing interventions to enhance the efficiency and fidelity of stem cell engineering., Competing Interests: Declaration of interests S.A.M. is on the advisory board of Cell Stem Cell., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Pluripotent-stem-cell-derived human intestinal organoids (HIOs) model some aspects of intestinal development and disease, but current culture methods do not fully recapitulate the diverse cell types and complex organization of the human intestine and are reliant on 3D extracellular matrix or hydrogel systems, which limit experimental control and translational potential for regenerative medicine. We describe suspension culture as a simple, low-maintenance method for culturing HIOs and for promoting in vitro differentiation of an organized serosal mesothelial layer that is similar to primary human intestinal serosal mesothelium based on single-cell RNA sequencing and histological analysis. Functionally, HIO serosal mesothelium has the capacity to differentiate into smooth-muscle-like cells and exhibits fibrinolytic activity. An inhibitor screen identifies Hedgehog and WNT signaling as regulators of human serosal mesothelial differentiation. Collectively, suspension HIOs represent a three-dimensional model to study the human serosal mesothelium., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Organs are composed of diverse cell types that traverse transient states during organogenesis. To interrogate this diversity during human development, we generate a single-cell transcriptome atlas from multiple developing endodermal organs of the respiratory and gastrointestinal tract. We illuminate cell states, transcription factors, and organ-specific epithelial stem cell and mesenchyme interactions across lineages. We implement the atlas as a high-dimensional search space to benchmark human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) under multiple culture conditions. We show that HIOs recapitulate reference cell states and use HIOs to reconstruct the molecular dynamics of intestinal epithelium and mesenchyme emergence. We show that the mesenchyme-derived niche cue NRG1 enhances intestinal stem cell maturation in vitro and that the homeobox transcription factor CDX2 is required for regionalization of intestinal epithelium and mesenchyme in humans. This work combines cell atlases and organoid technologies to understand how human organ development is orchestrated., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

During development, distinct progenitors contribute to the nephrons versus the ureteric epithelium of the kidney. Indeed, previous human pluripotent stem-cell-derived models of kidney tissue either contain nephrons or pattern specifically to the ureteric epithelium. By re-analyzing the transcriptional distinction between distal nephron and ureteric epithelium in human fetal kidney, we show here that, while existing nephron-containing kidney organoids contain distal nephron epithelium and no ureteric epithelium, this distal nephron segment alone displays significant in vitro plasticity and can adopt a ureteric epithelial tip identity when isolated and cultured in defined conditions. "Induced" ureteric epithelium cultures can be cryopreserved, serially passaged without loss of identity, and transitioned toward a collecting duct fate. Cultures harboring loss-of-function mutations in PKHD1 also recapitulate the cystic phenotype associated with autosomal recessive polycystic kidney disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The human intestinal stem cell niche supports self-renewal and epithelial function, but little is known about its development. We used single-cell mRNA sequencing with in situ validation approaches to interrogate human intestinal development from 7-21 weeks post conception, assigning molecular identities and spatial locations to cells and factors that comprise the niche. Smooth muscle cells of the muscularis mucosa, in close proximity to proliferative crypts, are a source of WNT and RSPONDIN ligands, whereas EGF is expressed far from crypts in the villus epithelium. Instead, an PDGFRA HI /F3 HI /DLL1 HI mesenchymal population lines the crypt-villus axis and is the source of the epidermal growth factor (EGF) family member NEUREGULIN1 (NRG1). In developing intestine enteroid cultures, NRG1, but not EGF, permitted increased cellular diversity via differentiation of secretory lineages. This work highlights the complexities of intestinal EGF/ERBB signaling and delineates key niche cells and signals of the developing intestine., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Human pluripotent stem cell (hPSC)-derived intestinal organoids (HIOs) lack some cellular populations found in the native organ, including vasculature. Using single-cell RNA sequencing (scRNA-seq), we have identified a population of endothelial cells (ECs) present early in HIO differentiation that declines over time in culture. Here, we developed a method to expand and maintain this endogenous population of ECs within HIOs (vHIOs). Given that ECs possess organ-specific gene expression, morphology, and function, we used bulk RNA-seq and scRNA-seq to interrogate the developing human intestine, lung, and kidney in order to identify organ-enriched EC gene signatures. By comparing these gene signatures and validated markers to HIO ECs, we find that HIO ECs grown in vitro share the highest similarity with native intestinal ECs relative to kidney and lung. Together, these data demonstrate that HIOs can co-differentiate a native EC population that is properly patterned with an intestine-specific EC transcriptional signature in vitro., Competing Interests: Declaration of Interests E.M.H. and J.R.S. hold a provisional patent application pertaining to the vHIOs described herein., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bud tip progenitor cells give rise to all murine lung epithelial lineages and have been described in the developing human lung; however, the mechanisms controlling human bud tip differentiation into specific lineages are unclear. Here, we used homogeneous human bud tip organoid cultures and identified SMAD signaling as a key regulator of the bud tip-to-airway transition. SMAD induction led to the differentiation of airway-like organoids possessing functional basal cells capable of clonal expansion and multilineage differentiation. To benchmark in vitro-derived organoids, we developed a single-cell mRNA sequencing atlas of the human lung from 11.5 to 21 weeks of development, which revealed high degrees of similarity between the in vitro-derived and in vivo airway. Together, this work sheds light on human airway differentiation in vitro and provides a single-cell atlas of the developing human lung., Competing Interests: Declaration of Interests J.R.S. and A.J.M. are co-inventors on patents filed by the Regents of the University of Michigan relating to the isolation and maintenance of lung bud tip progenitor cells and the differentiation of lung epithelial progenitor cells into basal cells., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Organoids are complex three-dimensional in vitro organ-like model systems. Human organoids, which are derived from human pluripotent stem cells or primary human donor tissue, have been used to address fundamental questions about human development, stem cell biology and organ regeneration. Focus has now shifted towards implementation of organoids for biological discovery and advancing existing systems to more faithfully recapitulate the native organ. This work has highlighted significant unknowns in human biology and has invigorated new exploration into the cellular makeup of human organs during development and in the adult - work that is crucial for providing appropriate benchmarks for organoid systems. In this Review, we discuss efforts to characterize human organ cellular complexity and attempts to make organoid models more realistic through co-culture, transplantation and bioengineering approaches., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

With increasing demands upon a geriatric psychiatry service, the priorities for day care and long-term hospital care have had to be re-examined. As a result, old people already in local authority residential care are receiving lower priority than those still in the community. In this study we suggest that certain behavioural characteristics and, in particular, incontinence, may present the caring staff of residential homes with special difficulties. We conclude that adequate assessment is essential to ensure that residents are suited to the level of facilities supplied.

A two-year prospective study of 11 local authority homes showed no increase in the degrees of behavioural disabilities or the prevalence of dependency or dementia among the residents. This finding, which differed from trends reported in other studies, was unlikely to be due to the design of the study and occurred in the absence of a co-ordinated service for the elderly. It was probably the effect of changes in the admission policy of the social-work services and suggests that, in some parts of Britain at least, residential homes are no longer absorbing additional numbers of dependent or demented old people in need of institutional care.

Ninety-six per cent of the residents occupying the 11 Local Authority Homes within the catchment area of one Scottish district psychiatric hospital were surveyed to estimate current levels of organic cerebral impairment and behavioural problems in the Homes. Clear evidence of dementia was shown by 27.1% of the residents but the actual prevalence of organic cerebral impairment could be as high as 66.4%. An excessively high level of socially disruptive behaviour was discovered. This study suggests that Local Authority Homes, with their present staffing levels, are having to cope with large numbers of dependent and behaviourally disturbed old people. Four possible solutions are outlined. Irrespective of what is adopted, it is essential that Health and Social Service developments are fully co-ordinated.