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Generation of splanchnic mesoderm-like progenitors from human PSCs a Schematic of the protocol to differentiate hPSCs into SM subtypes. Factors in red indicate signals predicted from the mouse single-cell signaling roadmap. b RT-PCR of markers with enriched expression in specific SM subtypes based on the mouse single-cell data: cardiac (NKX2-5), early SM (FOXF1, HOXA1); liver stm/mesothelium (WT1, UKP1B), liver-fibroblast (MSX1), respiratory SM (NKX6-1+, MSC−), esophageal/gastric (MSC, BARX1). The histogram shows the means ± S.D. Statistical significance was calculated using a two-sided Tukey’s multiple comparisons test. *p < 0.05, **p < 0.005, ***p < 0.0005. Exact p-values were provided in Source data file. n = 3 independent biological samples. Similar results were obtained from five independent experiments. c Representative images of Day 7 cell cultures immunostaining. Similar results were obtained from three independent experiments. Scale bar; 50 μm (upper panels), 10 μm (lower panels). d Quantification of % cells positive for the indicated immunostaining or RNA-scope in situ hybridization. Histograms show the means ± S.D. n = 3 independent fields. Immunostaining quantification results were similar for two separate experiments and RNA-scope validation was performed once. Statistical significance was calculated using two-sided Dunnett’s multiple comparisons test. *p < 0.05, **p < 0.005, ***p < 0.0005. Exact p-values were provided in Source data file. ns; not significant, nt; not tested. Source data are provided as a Source data file.
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Visceral organs, such as the lungs, stomach and liver, are derived from the fetal foregut through a series of inductive interactions between the definitive endoderm (DE) and the surrounding splanchnic mesoderm (SM). While DE patterning is fairly well studied, the paracrine signaling controlling SM regionalization and how this is coordinated with ep...
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Micropatterned human pluripotent stem cells (hPSCs) treated with BMP4 (2D gastruloids) are among the most widely used stem cell models for human gastrulation. Due to its simplicity and reproducibility, this system is ideal for high throughput quantitative studies of tissue patterning and has led to many insights into the mechanisms of mammalian gas...
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... Moreover, pharmacological interruption and genetic manipulation were further applied to validate the critical roles of these signals during lung fate specification [42]. Actually, the critical inductive role for the mesenchyme in gut tube organogenesis was firstly established in the 1960s, where it showed that SM transplanted from different anterior-posterior regions of the embryo could direct the adjacent epithelium to adopt the organ identity consistent with the original SM position [43,44]. Amanda et al. were able to identify that it is cardiac mesoderm derived fibroblast growth factor (FGF) signals are required for the patterning of ventral foregut cells into lung cells by using embryo tissue explants [45]. ...
... Beyond the mesoderm-derived signals can regulate the cell differentiation of gut tube, signals derived from endoderm cells can also pattern the surrounding splanchnic mesoderm. Han et al., dissected the foregut cells that are spatially located between the posterior pharynx and the midgut from three embryonic stages spanning the period of early patterning and lineage induction: E8.5 (5-10 somite), E9.0 (12-15 somite), E9.5 (25-30 somite), and then applied single cell transcriptomic profiling of these foregut single cells [44]. Based on the established transcriptomic atlas, they have identified the diversity of transcriptome features of distinct organ-specific epithelium related splanchnic mesoderm lineages, and inferred a spatiotemporal signaling network of endoderm-mesoderm interactions that orchestrate foregut organogenesis. ...
... Meanwhile, Han et al., micro-dissected the early foregut tissues, which are located between the posterior pharynx and the midgut, at three time-points that span the period of early patterning and lineage induction: E8.5 (5-10 somites; s), E9.0 (12-15 s), and E9.5 (25-30 s). They found that an extensive diversification of the early splanchnic mesoderm into distinct organ-specific mesenchyme subtypes, the diversities of splanchnic mesoderm cell types are closely registered with the organ-specific epithelium, and underscore the importance of endodermderived signals in mesoderm patterning [44]. It's noteworthy that the lung development process is not completely evolutionary conserved between the mouse model and humans [88,160,161]. ...
Mammalian lung development starts from a specific cluster of endodermal cells situated within the ventral foregut region. With the orchestrating of delicate choreography of transcription factors, signaling pathways, and cell–cell communications, the endodermal diverticulum extends into the surrounding mesenchyme, and builds the cellular and structural basis of the complex respiratory system. This review provides a comprehensive overview of the current molecular insights of mammalian lung development, with a particular focus on the early stage of lung cell fate differentiation and spatial patterning. Furthermore, we explore the implications of several congenital respiratory diseases and the relevance to early organogenesis. Finally, we summarize the unprecedented knowledge concerning lung cell compositions, regulatory networks as well as the promising prospect for gaining an unbiased understanding of lung development and lung malformations through state-of-the-art single-cell omics.
... www.nature.com/scientificreports/ of disease relevant cell types. The formation of the foregut and the subsequent organ morphogenesis during embryonic development relies on the interplay between two major populations of the definitive endoderm and splanchnic mesoderm cells 15 . We know little about whether genes associated with esophageal malformations equally affect both cell types or if one has a more prominent role in their development. ...
... This heterogeneity is characterized by some cells exhibiting a preferential expression of disease-associated genes, while others do not (Fig. 1A, Table S2). To see the generalizability of our results, we calculated the single-cell disease scores of EM-associated genes among the cell types of the second atlas that contained 31,000 cells of developing mouse foregut, within 26 subclusters of definitive endoderm (E) and 36 sub-clusters of splanchnic mesoderm (M) at the three time points of E8.5, E9.0 and E9.5 (Table S3, Fig. S1) 15 . We identified a marked enrichment of our disease-relevant genes in nine mesodermal and three endodermal cell clusters (Fig. 1B, Table S3). ...
... We further looked at the disease association of mesodermal cell types throughout the development as these cell types had the highest preferential expression among all cell types of the foregut. We used both the calculated cell lineages (Fig. 3A), and the cell fate tree (Fig. 3B) of mesodermal cell types that were previously constructed by a single-cell voting approach 15 . We found that the cell types in the lateral plate mesoderm, which eventually develop into the lateral-ventral cells of the anterior foregut, show the most significant disease association. ...
Understanding the molecular mechanisms of congenital diseases is challenging due to their occurrence within specific developmental stages. Esophageal malformations are examples of such conditions, characterized by abnormalities in the development of esophagus during embryogenesis. These developmental malformations encompass a range of anomalies, including esophageal atresia, and tracheoesophageal fistula. Here, we investigated the preferential expression of 29 genes that are implicated in such malformations and their immediate interactome (a total of 67 genes). We conducted our analyses across several single-cell atlases of embryonic development, encompassing approximately 150,000 cells from the mouse foregut, 180,000 cells from human embryos, and 500,000 cells from 24 human organs. Our study, spanning diverse mesodermal and endodermal cell populations and early developmental stages, shows that the genes associated with esophageal malformations show their highest cell-type specific expression in lateral plate mesoderm cells and at the developmental stage of E8.75–E9.0 days. In human embryos, these genes show a significant cell-type specific expression among subpopulations of epithelial cells, fibroblasts and progenitor cells including basal cells. Notably, members of the forkhead-box family of transcription factors, namely FOXF1, FOXC1, and FOXD1, as well as the SRY-box transcription factor, SOX2, demonstrate the most significant preferential expression in both mouse and human embryos. Overall, our findings provide insights into the temporal and cellular contexts contributing to esophageal malformations.
... Positive selection is mediated by cTEC at the double positive (DP) stage and leads to the selection of thymocytes with a functional T cell receptor (TCR) (Kadouri et al., 2019). Negative selection filters out potential autoreactive thymocytes at the single-positive (SP) stage in the medulla (Klein et al., 2014(Klein et al., , 2000 and is mediated by HLA-DR hi mTECs expressing a great diversity of peripheral tissues antigens (PTAs) progress at the single-cell level (Han et al., 2020;Magaletta et al., 2022;Nowotschin et al., 2019). ...
... To overcome this weakness and robustly assess factor effects, we performed bulk RNA-seq on the DOE samples and compared their transcriptome to two scRNA-seq atlases of pharyngeal development. The one by Han et al. (Han et al., 2020) corresponds to early stages of development and covers the AFE and 3PPE transitions. The second by Magaletta et al. (Magaletta et al., 2022) focuses on later stages of development including the 3PPE and TEC transitions. ...
The thymus is a primary lymphoid organ playing a crucial role in immune tolerance, by educating thymocytes through a selection mediated by thymic epithelial cells (TEC). Recent advances in gene editing and immunotherapies have made in vitro generation of iPSC-derived T cells a crucial issue with promising therapeutic applications. Current state-of-the-art approaches often fail to accurately replicate the thymic niche, resulting in impaired T cell generation, particularly for CD4+ T cells. Here we address the production of functional mature iPSc-derived TECs that are able to support in vitro T cell generation. We designed a protocol allowing iPSc differentiation into thymic epithelial progenitors (TEP) through an unbiased multifactorial method based on optimal experimental design. Modulation of signaling pathways known to regulate embryonic thymus organogenesis resulted in the obtention of TEPs expressing typical thymic markers. We achieved TEP maturation into medullary and cortical TECs by setting up a hydrogel-based 3D culture supplemented in RANK ligand, resulting in the formation of thymic organoids. To assess the functionality of the generated TECs, primary hematopoietic progenitors were co-cultured in the organoids and showed significantly improved maturation in single-positive CD4 or CD8 T cells. Remarkably, our thymic organoid model shows multilineage differentiation potential, with generation of distinct dendritic cells populations in addition to the T lineages. Thus, generation of functional TECs and thymic organoids offers a practical platform for the study of thymic cellular interactions and paves the road to future cellular therapies by producing mature thymic immune cells of clinical interest.
... Given the close association of Nkx2-1 KO epithelial cells to the Krt8 + state in Nkx2-1 WT AEP-O, we used Seurat module scoring to compare Nkx2-1 KO and WT cells. Cells in Nkx2-1 KO organoids lost AEP-associated gene expression (Fig. 6O) we did not detect the protein expression of non-lung foregut endoderm markers by IHC, Nkx2-1 KO epithelial cells did express low levels of non-lung endodermal genes at the RNA level 76 , consistent with loss of the instructive activity of Nkx2-1 in constraining lung fate. Finally, we evaluated the overall chromatin organization of Nkx2-1 KO epithelial cells using scATACseq ( Supplementary Fig. S11A Fig. S14). ...
Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8⁺ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8⁺ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs.
... Subsequently, the medial and lateral structural domains fuse during foregut closure, leading to endodermal cells differentiating into liver under the influence of induced signals and genetic regulatory factors. 27 Fibroblast growth factor and BMP4 signals from the mesoderm trigger the formation of the liver from the foregut endoderm at somatic cell stages 5-6. 28 Hepatoblasts form liver buds by changing their morphology to columnar and invading the mesenchyme of the transverse septum. ...
Retinoic acid (RA) serves as a metabolic intermediate of vitamin A. It plays a crucial physiological role in regulating cell proliferation, differentiation, apoptosis, embryonic development, and immunomodulation. Once vitamin A enters the body in the form of retinol, it undergoes conversion into RA through the intestinal epithelium and liver. Subsequently, it interacts with retinoic acid receptors and retinoid X receptors within the cell nucleus, thereby regulating gene expression. Throughout liver development, RA exerts precise temporal control, stimulating liver growth, inducing RALDH2 expression in liver somatic epithelial cells, and influencing hepatocyte differentiation. Recent studies have consistently demonstrated the indispensable connection between RA deficiency and the development of liver diseases, including nonalcoholic fatty liver disease, chronic hepatitis, liver fibrosis, and liver tumors. Studying the mechanisms underlying the relationship between RA and disease can enhance our understanding and improve disease treatment. This paper provides a comprehensive review of the role of RA signaling in liver development and liver diseases.
... Importantly, the different cell numbers observed at different Z-depths indicate a pattern of differential potential distribution within the multilayer. This finding is consistent with research highlighting the importance of spatial organization and cell-cell interactions in mixed cell cultures [48]. Future research should focus on characterizing the spatial arrangement and intercellular communication between different cell types within the composite layer. ...
The aim of this study was to investigate the survival, distribution and reaction of different cell types on a monolayer disk, as well as their behavior under bioreactor treatment. Specifically, porcine EEC and porcine fibroblasts (PCF) were labeled with GFT and Texas Red, respectively, to track their viability and distribution. The experiments involved monitoring the cells using various microscopy techniques and comparing the results with controls. These findings have important implications for understanding cell behavior and potential applications for Discrete Subaortic Stenosis. This paper aims to discuss the implications of the findings in the context of existing literature and future research directions.
... Indeed, recent publications highlight the importance of organ-specific mesenchyme in organoid development. [105][106][107][108][109] Also, OMCs not only influence the differentiation of developing hair cells, but potentially could be a source of regeneration, as tympanic border cells are self-renewing in a WNT-dependent manner, and able to trans-differentiate into supporting cell-like and hair cell-like cells. 110 This further indicates that characterization of the type I basilar membrane OMC gene regulatory network may lead to the discovery of novel genes necessary for a hair cell regeneration pathway. ...
The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. Otic mesenchyme cells (OMCs), often considered a homogeneous cell type, are essential for normal cochlear development and hearing. Despite being the most numerous cell type in the developing cochlea, OMCs are poorly understood. OMCs are known to differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Here, we show that OMCs are transcriptionally and functionally heterogeneous and can be divided into four distinct populations that spatially correspond to OMC-derived cochlear structures. We also show that this heterogeneity and complexity of OMCs commences during early phases of cochlear development. Finally, we describe the cell-cell communication network of the developing cochlea, inferring a major role for OMC in outgoing signaling.
... The foregut is an important starting organ for the formation of the digestive and respiratory tracts. Visceral organs, such as lung, stomach, and liver, are formed through differentiation of the embryonic foregut [40]. Hence, PSCs usually are firstly induced into foregut progenitors for PSC-induced liver organoids formation [41,42]. ...
Liver fibrosis was initially considered to be an irreversible process which will eventually lead to the occurrence of liver cancer. So far there has been no effective therapeutic approach to treat liver fibrosis although scientists have put tremendous efforts into the underlying mechanisms of this disease. Therefore, in-depth research on novel and safe treatments of liver fibrosis is of great significance to human health. Pluripotent stem cells (PSCs) play important roles in the study of liver fibrosis due to their unique features in self-renewal ability, pluripotency, and paracrine function. This article mainly reviews the applications of PSCs in the study of liver fibrosis in recent years. We discuss the role of PSC-derived liver organoids in the study of liver fibrosis, and the latest research advances on the differentiation of PSCs into hepatocytes or macrophages. We also highlight the importance of exosomes of PSCs for the treatment of liver fibrosis.
Graphical Abstract
... The lung mesenchyme plays important roles in lung development and disease, yet knowledge is limited about the biology of lung mesenchymal progenitors, or how they initiate disease. In the mouse, lineage specification of the respiratory mesenchyme from splanchnic mesodermal precursors has been reported to occur around embryonic day (E) 9-9.5, a process coordinated through bi-directional endodermalmesodermal signaling interactions [1][2][3][4] . Mouse models have helped to identify several key regulators potentially involved in lung mesenchyme specification and differentiation, such as Retinoic Acid (RA), Wnt/ β-catenin, Sonic hedgehog (SHH), and BMP4 1,3,5-14 . ...
... Our lab and others have established protocols for the directed differentiation of both mouse and human iPSCs/ESCs into Nkx2-1+ lung epithelial progenitors, as well as toward more mature distal and proximal epithelial respiratory lineages [17][18][19][20][21][22][23][24][25][26][27][28] . In contrast, only two research groups have attempted to generate lung-specific mesenchyme by directed differentiation through a mesodermal progenitor 2,9,29 . While these studies have found an upregulation of lung mesenchymal markers by RT-qPCR in bulk cell extracts, they lacked specific reporters for cell tracking or purification, leaving unanswered questions about the efficiency of the differentiation protocol and the transcriptomic similarity of in vitro differentiated cells to primary lung mesenchyme. ...
... In contrast most of these markers were expressed at lower levels in iPSC-derived cluster 2 ( Supplementary Fig. 2d). Analysis of the combined expression of a list of 13 lung mesenchymal markers (Supplementary Data 2) known from the literature including Foxf1, Tbx4/5, Wnt2/2b and Hh targets 2,5,7,12,14,39,40 (further referred to as LgM gene set), as well as a published set consisting of 61 genes by Han et al. (Supplementary Data 2) found to be enriched in E9.5 lung mesenchyme 2 further showed that cluster 1 iPSC-derived cells are more transcriptionally similar to primary E12.5 lung mesenchyme than iPSC-derived cluster 2 cells (Fig. 2c). In addition, we also analyzed the expression of both gene sets in co-developed lung mesenchyme (cLM) and found significantly lower expression levels of these gene signatures in cLM compared to iPSCderived Tbx4-LER GFP + cells generated through directed mesenchymal differentiation ( Supplementary Fig. 2a, e, f). ...
While the generation of many lineages from pluripotent stem cells has resulted in basic discoveries and clinical trials, the derivation of tissue-specific mesenchyme via directed differentiation has markedly lagged. The derivation of lung-specific mesenchyme is particularly important since this tissue plays crucial roles in lung development and disease. Here we generate a mouse induced pluripotent stem cell (iPSC) line carrying a lung-specific mesenchymal reporter/lineage tracer. We identify the pathways (RA and Shh) necessary to specify lung mesenchyme and find that mouse iPSC-derived lung mesenchyme (iLM) expresses key molecular and functional features of primary developing lung mesenchyme. iLM recombined with engineered lung epithelial progenitors self-organizes into 3D organoids with juxtaposed layers of epithelium and mesenchyme. Co-culture increases yield of lung epithelial progenitors and impacts epithelial and mesenchymal differentiation programs, suggesting functional crosstalk. Our iPSC-derived population thus provides an inexhaustible source of cells for studying lung development, modeling diseases, and developing therapeutics.
... (18-22 somites) in mice and 4 weeks of gestation in humans [48,102]. During subsequent stages (embryonic, pseudoglandular, canalicular, saccular, and alveolar), developmental processes, such as tracheoesophageal septation, formation and elongation of the Irx2 and Foxp2 [36,48], are also expressed in the lung primordium. Future work will certainly decipher TF interactions that uniquely define the lung primordial identity and are involved in early epithelial cell fate decisions in lung development. ...
... Zorn et al. has recently used scRNA-seq to describe signaling networks governing interactions between mesenchymal and epithelial domains in early foregut development (E8.5-E9.5) [36]. This study revealed several mesenchymal cell clusters corresponding to domain-specific mesenchymal populations that engage in signaling crosstalk with the corresponding epithelial populations. ...
... In the developing lung mesenchyme, there was expression of previously described lung mesenchymal markers, such as Wnt2 [32], Osr1 [86], Foxf1 [19,84], and Tbx5 [13]. Nkx6-1, a known pancreatic epithelial TF, was also expressed in the respiratory mesenchymal domain (both by transcript and protein), and it was one of the key TFs on the cellstate trajectory from foregut lateral mesoderm to lung mesenchyme [36]. ...
Transient, tissue-specific, embryonic progenitors are important cell populations in vertebrate development. In the course of respiratory system development, multipotent mesenchymal and epithelial progenitors drive the diversification of fates that results to the plethora of cell types that compose the airways and alveolar space of the adult lungs. Use of mouse genetic models, including lineage tracing and loss-of-function studies, has elucidated signaling pathways that guide proliferation and differentiation of embryonic lung progenitors as well as transcription factors that underlie lung progenitor identity. Furthermore, pluripotent stem cell-derived and ex vivo expanded respiratory progenitors offer novel, tractable, high-fidelity systems that allow for mechanistic studies of cell fate decisions and developmental processes. As our understanding of embryonic progenitor biology deepens, we move closer to the goal of in vitro lung organogenesis and resulting applications in developmental biology and medicine.
