scholarly journals Basal stem cell fate specification is mediated by SMAD signaling in the developing human lung

2018 ◽  
Author(s):  
Alyssa J. Miller ◽  
Qianhui Yu ◽  
Michael Czerwinski ◽  
Yu-Hwai Tsai ◽  
Renee F. Conway ◽  
...  
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Basal Cell ◽  
Human Lung ◽  
Branching Morphogenesis ◽  
Basal Cells ◽  
Smad Signaling ◽  
Cell Fate Decisions ◽  
Cell Transition

AbstractBasal stem cells (basal cells), located in the bronchi and trachea of the human lung epithelium, play a critical role in normal airway homeostasis and repair, and have been implicated in the development of diseases such as cancer1-4. Additionally, basal-like cells contribute to alveolar regeneration and fibrosis following severe injury5-8. However, the developmental origin of basal cells in humans is unclear. Previous work has shown that specialized progenitor cells exist at the tips of epithelial tubes during lung branching morphogenesis, and in mice, give rise to all alveolar and airway lineages9,10. These ‘bud tip progenitor cells’ have also been described in the developing human lung11-13, but the mechanisms controlling bud tip differentiation into specific cell lineages, including basal cells, are unknown. Here, we interrogated the bud tip-to-basal cell transition using human tissue specimens, bud tip progenitor organoid cultures11, and single-cell transcriptomics. We used single-cell mRNA sequencing (scRNAseq) of developing human lung specimens from 15-21 weeks gestation to identify molecular signatures and cell states in the developing human airway epithelium. We then inferred differentiation trajectories during bud tip-to-airway differentiation, which revealed a previously undescribed transitional cell state (‘hub progenitors’) and implicated SMAD signaling as a regulator of the bud tip-to-basal cell transition. We used bud tip progenitor organoids to show that TGFT1 and BMP4 mediated SMAD signaling robustly induced the transition into functional basal-like cells, and these in vitro-derived basal cells exhibited clonal expansion, self-renewal and multilineage differentiation. This work provides a framework for deducing and validating key regulators of cell fate decisions using single cell transcriptomics and human organoid models. Further, the identification of SMAD signaling as a critical regulator of newly born basal cells in the lung may have implications for regenerative medicine, basal cell development in other organs, and understanding basal cell misregulation in disease.

scholarly journals Progenitor identification and SARS-CoV-2 infection in long-term human distal lung organoid cultures

2020 ◽  
Author(s):  
Ameen A. Salahudeen ◽  
Shannon S. Choi ◽  
Arjun Rustagi ◽  
Junjie Zhu ◽  
Sean M. de la O ◽  
...  
Keyword(s):  
Lung Disease ◽  
Single Cell ◽  
Basal Cell ◽  
Human Lung ◽  
Basal Cells ◽  
Ciliated Cells ◽  
Club Cells ◽  
Distal Lung

ABSTRACTThe distal lung contains terminal bronchioles and alveoli that facilitate gas exchange and is affected by disorders including interstitial lung disease, cancer, and SARS-CoV-2-associated COVID-19 pneumonia. Investigations of these localized pathologies have been hindered by a lack of 3D in vitro human distal lung culture systems. Further, human distal lung stem cell identification has been impaired by quiescence, anatomic divergence from mouse and lack of lineage tracing and clonogenic culture. Here, we developed robust feeder-free, chemically-defined culture of distal human lung progenitors as organoids derived clonally from single adult human alveolar epithelial type II (AT2) or KRT5+ basal cells. AT2 organoids exhibited AT1 transdifferentiation potential, while basal cell organoids progressively developed lumens lined by differentiated club and ciliated cells. Organoids consisting solely of club cells were not observed. Upon single cell RNA-sequencing (scRNA-seq), alveolar organoids were composed of proliferative AT2 cells; however, basal organoid KRT5+ cells contained a distinct ITGA6+ITGB4+ mitotic population whose proliferation segregated to a TNFRSF12Ahi subfraction. Clonogenic organoid growth was markedly enriched within the TNFRSF12Ahi subset of FACS-purified ITGA6+ITGB4+ basal cells from human lung or derivative organoids. In vivo, TNFRSF12A+ cells comprised ~10% of KRT5+ basal cells and resided in clusters within terminal bronchioles. To model COVID-19 distal lung disease, we everted the polarity of basal and alveolar organoids to rapidly relocate differentiated club and ciliated cells from the organoid lumen to the exterior surface, thus displaying the SARS-CoV-2 receptor ACE2 on the outwardly-facing apical aspect. Accordingly, basal and AT2 “apical-out” organoids were infected by SARS-CoV-2, identifying club cells as a novel target population. This long-term, feeder-free organoid culture of human distal lung alveolar and basal stem cells, coupled with single cell analysis, identifies unsuspected basal cell functional heterogeneity and exemplifies progenitor identification within a slowly proliferating human tissue. Further, our studies establish a facile in vitro organoid model for human distal lung infectious diseases including COVID-19-associated pneumonia.

scholarly journals Deciphering cell lineage specification during male sex determination with single-cell RNA sequencing

2017 ◽  
Author(s):  
Isabelle Stévant ◽  
Yasmine Neirjinck ◽  
Christelle Borel ◽  
Jessica Escoffier ◽  
Lee B. Smith ◽  
...  
Keyword(s):  
Sex Determination ◽  
Single Cell ◽  
Progenitor Cells ◽  
Rna Sequencing ◽  
Cell Fate ◽  
Time Course ◽  
Cell Lineage ◽  
Lineage Specification ◽  
Cell Fate Decisions ◽  
Single Cell Rna Sequencing

SummaryThe gonad is a unique biological system for studying cell fate decisions. However, major questions remain regarding the identity of somatic progenitor cells and the transcriptional events driving cell differentiation. Using time course single cell RNA sequencing on XY mouse gonads during sex determination, we identified a single population of somatic progenitor cells prior sex determination. A subset of these progenitors differentiate into Sertoli cells, a process characterized by a highly dynamic genetic program consisting of sequential waves of gene expression. Another subset of multipotent cells maintains their progenitor state but undergo significant transcriptional changes that restrict their competence towards a steroidogenic fate required for the differentiation of fetal Leydig cells. These results question the dogma of the existence of two distinct somatic cell lineages at the onset of sex determination and propose a new model of lineage specification from a unique progenitor cell population.

scholarly journals GATA-targeted compounds modulate cardiac subtype cell differentiation in dual reporter stem cell line

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mika J. Välimäki ◽  
Robert S. Leigh ◽  
Sini M. Kinnunen ◽  
Alexander R. March ◽  
Ana Hernández de Sande ◽  
...  
Keyword(s):  
Gene Expression ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Reporter Gene ◽  
Gene Regulatory Networks ◽  
Regulatory Networks ◽  
Cell Fate Decisions ◽  
Dual Reporter ◽  
Gene Regulatory ◽  
Reporter Gene Assays

AbstractBackgroundPharmacological modulation of cell fate decisions and developmental gene regulatory networks holds promise for the treatment of heart failure. Compounds that target tissue-specific transcription factors could overcome non-specific effects of small molecules and lead to the regeneration of heart muscle following myocardial infarction. Due to cellular heterogeneity in the heart, the activation of gene programs representing specific atrial and ventricular cardiomyocyte subtypes would be highly desirable. Chemical compounds that modulate atrial and ventricular cell fate could be used to improve subtype-specific differentiation of endogenous or exogenously delivered progenitor cells in order to promote cardiac regeneration.MethodsTranscription factor GATA4-targeted compounds that have previously shown in vivo efficacy in cardiac injury models were tested for stage-specific activation of atrial and ventricular reporter genes in differentiating pluripotent stem cells using a dual reporter assay. Chemically induced gene expression changes were characterized by qRT-PCR, global run-on sequencing (GRO-seq) and immunoblotting, and the network of cooperative proteins of GATA4 and NKX2-5 were further explored by the examination of the GATA4 and NKX2-5 interactome by BioID. Reporter gene assays were conducted to examine combinatorial effects of GATA-targeted compounds and bromodomain and extraterminal domain (BET) inhibition on chamber-specific gene expression.ResultsGATA4-targeted compounds 3i-1000 and 3i-1103 were identified as differential modulators of atrial and ventricular gene expression. More detailed structure-function analysis revealed a distinct subclass of GATA4/NKX2-5 inhibitory compounds with an acetyl lysine-like domain that contributed to ventricular cells (%Myl2-eGFP+). Additionally, BioID analysis indicated broad interaction between GATA4 and BET family of proteins, such as BRD4. This indicated the involvement of epigenetic modulators in the regulation of GATA-dependent transcription. In this line, reporter gene assays with combinatorial treatment of 3i-1000 and the BET bromodomain inhibitor (+)-JQ1 demonstrated the cooperative role of GATA4 and BRD4 in the modulation of chamber-specific cardiac gene expression.ConclusionsCollectively, these results indicate the potential for therapeutic alteration of cell fate decisions and pathological gene regulatory networks by GATA4-targeted compounds modulating chamber-specific transcriptional programs in multipotent cardiac progenitor cells and cardiomyocytes. The compound scaffolds described within this study could be used to develop regenerative strategies for myocardial regeneration.

scholarly journals Resolution of Cell Fate Decisions Revealed by Single-Cell Gene Expression Analysis from Zygote to Blastocyst

2010 ◽  
Vol 18 (4) ◽  
pp. 675-685 ◽  
Author(s):  
Guoji Guo ◽  
Mikael Huss ◽  
Guo Qing Tong ◽  
Chaoyang Wang ◽  
Li Li Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Fate ◽  
Expression Analysis ◽  
Gene Expression Analysis ◽  
Cell Fate Decisions ◽  
Cell Gene Expression ◽  
Cell Gene

scholarly journals Single-cell transcriptional analysis to uncover regulatory circuits driving cell fate decisions in early mouse development

2014 ◽  
Vol 31 (7) ◽  
pp. 1060-1066 ◽  
Author(s):  
Haifen Chen ◽  
Jing Guo ◽  
Shital K. Mishra ◽  
Paul Robson ◽  
Mahesan Niranjan ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Transcriptional Analysis ◽  
Mouse Development ◽  
Cell Fate Decisions ◽  
Regulatory Circuits ◽  
Early Mouse

scholarly journals Depletion of L3MBTL1 promotes the erythroid differentiation of human hematopoietic progenitor cells: possible role in 20q− polycythemia vera

Blood ◽  
2010 ◽  
Vol 116 (15) ◽  
pp. 2812-2821 ◽  
Author(s):  
Fabiana Perna ◽  
Nadia Gurvich ◽  
Ruben Hoya-Arias ◽  
Omar Abdel-Wahab ◽  
Ross L. Levine ◽  
...  
Keyword(s):  
Polycythemia Vera ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Myeloproliferative Neoplasms ◽  
Erythroid Differentiation ◽  
Polycomb Group ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cell Fate Decisions ◽  
Cd34 Cells

Abstract L3MBTL1, the human homolog of the Drosophila L(3)MBT polycomb group tumor suppressor gene, is located on chromosome 20q12, within the common deleted region identified in patients with 20q deletion-associated polycythemia vera, myelodysplastic syndrome, and acute myeloid leukemia. L3MBTL1 is expressed within hematopoietic CD34+ cells; thus, it may contribute to the pathogenesis of these disorders. To define its role in hematopoiesis, we knocked down L3MBTL1 expression in primary hematopoietic stem/progenitor (ie, CD34+) cells isolated from human cord blood (using short hairpin RNAs) and observed an enhanced commitment to and acceleration of erythroid differentiation. Consistent with this effect, overexpression of L3MBTL1 in primary hematopoietic CD34+ cells as well as in 20q− cell lines restricted erythroid differentiation. Furthermore, L3MBTL1 levels decrease during hemin-induced erythroid differentiation or erythropoietin exposure, suggesting a specific role for L3MBTL1 down-regulation in enforcing cell fate decisions toward the erythroid lineage. Indeed, L3MBTL1 knockdown enhanced the sensitivity of hematopoietic stem/progenitor cells to erythropoietin (Epo), with increased Epo-induced phosphorylation of STAT5, AKT, and MAPK as well as detectable phosphorylation in the absence of Epo. Our data suggest that haploinsufficiency of L3MBTL1 contributes to some (20q−) myeloproliferative neoplasms, especially polycythemia vera, by promoting erythroid differentiation.

scholarly journals Nucleocytoplasmic Transport of RNA-Binding Proteins Regulates Neural Stem Cell Fates 

2020 ◽  
Author(s):  
Melissa J. MacPherson ◽  
Sarah L Erickson ◽  
Drayden Kopp ◽  
Pengqiang Wen ◽  
Mohammadreza Aghanoori ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Neurodevelopmental Disorder ◽  
Neural Progenitor ◽  
Nucleocytoplasmic Transport ◽  
Transcriptional Level ◽  
Cell Fates ◽  
Cell Fate Decisions

Abstract The formation of the cerebral cortex requires balanced expansion and differentiation of neural progenitor cells, the fate choice of which requires regulation at many steps of gene expression. As progenitor cells often exhibit transcriptomic stochasticity, the ultimate output of cell fate-determining genes must be carefully controlled at the post-transcriptional level, but how this is orchestrated is poorly understood. Here we report that de novo missense variants in an RNA-binding protein CELF2 cause human cortical malformations and perturb neural progenitor cell fate decisions in mice by disrupting the nucleocytoplasmic transport of CELF2. In self-renewing neural progenitors, CELF2 is localized in the cytoplasm where it binds and coordinates mRNAs that encode cell fate regulators and neurodevelopmental disorder-related factors. The translocation of CELF2 into the nucleus releases mRNAs for translation and thereby triggers neural progenitor differentiation. Our results reveal a mechanism by which transport of CELF2 between discrete subcellular compartments orchestrates an RNA regulon to instruct cell fates in cerebral cortical development.

Extrinsic and intrinsic factors control the genesis of amacrine and cone cells in the rat retina

Development ◽  
1999 ◽  
Vol 126 (3) ◽  
pp. 555-566 ◽  
Author(s):  
M.J. Belliveau ◽  
C.L. Cepko
Keyword(s):  
Progenitor Cells ◽  
Cell Fate ◽  
Progenitor Cell ◽  
Amacrine Cell ◽  
Amacrine Cells ◽  
Environment Change ◽  
Rat Retina ◽  
Cell Fate Decisions ◽  
Cone Cells ◽  
Postnatal Environment

The seven major classes of cells of the vertebrate neural retina are generated from a pool of multipotent progenitor cells. Recent studies suggest a model of retinal development in which both the progenitor cells and the environment change over time (Cepko, C. L., Austin, C. P., Yang, X., Alexiades, M. and Ezzeddine, D. (1996). Proc. Natl. Acad. Sci. USA 93, 589–595). We have utilized a reaggregate culture system to test this model. A labeled population of progenitors from the embryonic rat retina were cultured with an excess of postnatal retinal cells and then assayed for their cell fate choices. We found that the postnatal environment had at least two signals that affected the embryonic cells' choice of fate; one signal inhibited the production of amacrine cells and a second affected the production of cone cells. No increase in cell types generated postnatally was observed. The source of the inhibitor of the amacrine cell fate appeared to be previously generated amacrine cells, suggesting that amacrine cell number is controlled by feedback inhibition. The progenitor cell lost its ability to be inhibited for production of an amacrine cell as it entered M phase of the cell cycle. We suggest that postmitotic cells influence progenitor cell fate decisions, but that they do so in a manner restricted by the intrinsic biases of progenitor cells.

Abstract 39: Fate Of Renin Cell Progenitors In The Developing Kidney Vasculature

Hypertension ◽  
2021 ◽  
Vol 78 (Suppl_1) ◽  
Author(s):  
Alexandre Martini ◽  
Ariel R Gomez ◽  
Maria Luisa Sequeira Lopez
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Fluorescent Protein ◽  
Enrichment Analysis ◽  
Cell Fate Decisions ◽  
Mural Cells ◽  
Developing Kidney ◽  
Green Fluorescent ◽  
Old Mice ◽  
Accessible Chromatin

The unique spatial arregement of the kidney arterioles is an essential event for its development. However, the mechanisms that govern this process are still poorly understood. During nephrogenesis, a group of stromal cells expressing the Forkhead Box D1 ( FoxD1 ) transcription factor (TF) will give rise to the metanephric progenitors for the mural cells of the kidneys arteries and arterioles. We aim to identify the core TFs involved in the cell fate along the differentiaton pathways of the developing kidney vasculature. Therefore, we generated Foxd1-cre; mTmG mice, whose Foxd1 derivative cells are labeled with green fluorescent protein (GFP). GFP+ cells were isolated from 5 (P5) or 30 (P30) days old mice kidneys, and processed either for single-cell RNA-Seq (scRNA-Seq) or for single-cell Assay for Transposase-Accessible Chromatin (scATAC-Seq ). The top5 highly expressed TFs on scRNA-Seq at P5 are: Tcf21, Zeb2, Meis2, Cebpd and Nme3 (p_adjusted_value(padj)= 0, 3.8E-187, 3.9E-180, 4E-172, 4.1E-172 and 3.2E-154, respectively). They are involved in developmental processes and cell proliferation. At P30, the top5 highly expressed TFs are: Atf3, klf2, Fos, Nr4a2 and Junb (padj= 4.2E-294, 2.1E-200, 3.5E-182, 1.7E-52 and 0.2E-24, respectively). They are implicated with calcium-signaling pathway and inflammation. Additionally, scATAC-Seq identifies regions of accessible chromatin for pontential TFs binding, leading to changes in gene expression content and cell identity. At P30, scATAC-Seq showed differential accessible regions with subsequent putative motif enrichment analysis for the TF N4a2 (padj: 4E-297). This is in accordance with our scRNA-Seq results and might play a role in the Foxd1 progenitors cell fate decisions. Our results tracks the fate of the Foxd1+ cells during the kidney vasculature assembly and suggest a new transcription factors network that might play a role to orchestrate cell fate decisions during kidney vascular development.

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