In vitro models of fetal lung development to enhance research into congenital lung diseases

Abstract Purpose This paper aims to build upon previous work to definitively establish in vitro models of murine pseudoglandular stage lung development. These can be easily translated to human fetal lung samples to allow the investigation of lung development in physiologic and pathologic conditions. Methods Lungs were harvested from mouse embryos at E12.5 and cultured in three different settings, i.e., whole lung culture, mesenchyme-free epithelium culture, and organoid culture. For the whole lung culture, extracted lungs were embedded in Matrigel and incubated on permeable filters. Separately, distal epithelial tips were isolated by firstly removing mesothelial and mesenchymal cells, and then severing the tips from the airway tubes. These were then cultured either in branch-promoting or self-renewing conditions. Results Cultured whole lungs underwent branching morphogenesis similarly to native lungs. Real-time qPCR analysis demonstrated expression of key genes essential for lung bud formation. The culture condition for epithelial tips was optimized by testing different concentrations of FGF10 and CHIR99021 and evaluating branching formation. The epithelial rudiments in self-renewing conditions formed spherical 3D structures with homogeneous Sox9 expression. Conclusion We report efficient protocols for ex vivo culture systems of pseudoglandular stage mouse embryonic lungs. These models can be applied to human samples and could be useful to paediatric surgeons to investigate normal lung development, understand the pathogenesis of congenital lung diseases, and explore novel therapeutic strategies.

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Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00226.2003 ◽

2003 ◽

Vol 285 (6) ◽

pp. L1323-L1336 ◽

Cited By ~ 30

Author(s):

John M. Shannon ◽

Kathleen McCormick-Shannon ◽

Michael S. Burhans ◽

Xiaofei Shangguan ◽

Kalpana Srivastava ◽

...

Keyword(s):

Chondroitin Sulfate ◽

Lung Development ◽

Sodium Chlorate ◽

Surfactant Protein ◽

Branching Morphogenesis ◽

Clara Cell ◽

Normal Lung ◽

Lung Growth ◽

Isolated Lung

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene ( SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein ( CC10), sonic hedgehog ( Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.

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Unique Aspects of the Developing Lung Circulation: Structural Development and Regulation of Vasomotor Tone

Pulmonary Circulation ◽

10.1086/688890 ◽

2016 ◽

Vol 6 (4) ◽

pp. 407-425 ◽

Cited By ~ 20

Author(s):

Yuangsheng Gao ◽

David N. Cornfield ◽

Kurt R. Stenmark ◽

Bernard Thébaud ◽

Steven H. Abman ◽

...

Keyword(s):

Lung Development ◽

Lung Diseases ◽

Vascular Tone ◽

Current Knowledge ◽

Normal Lung ◽

Pulmonary Vasculature ◽

Structural Development ◽

Vasomotor Tone ◽

Disease States ◽

Vasculogenesis And Angiogenesis

This review summarizes our current knowledge on lung vasculogenesis and angiogenesis during normal lung development and the regulation of fetal and postnatal pulmonary vascular tone. In comparison to that of the adult, the pulmonary circulation of the fetus and newborn displays many unique characteristics. Moreover, altered development of pulmonary vasculature plays a more prominent role in compromised pulmonary vasoreactivity than in the adult. Clinically, a better understanding of the developmental changes in pulmonary vasculature and vasomotor tone and the mechanisms that are disrupted in disease states can lead to the development of new therapies for lung diseases characterized by impaired alveolar structure and pulmonary hypertension.

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State of the art on lung organoids in mammals

Veterinary Research ◽

10.1186/s13567-021-00946-6 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Fabienne Archer ◽

Alexandra Bobet-Erny ◽

Maryline Gomes

Keyword(s):

Lung Development ◽

One Health ◽

State Of The Art ◽

Animal Health ◽

In Vitro Models ◽

Cancer Genetic ◽

3 Dimensional ◽

The One ◽

Species Specific

AbstractThe number and severity of diseases affecting lung development and adult respiratory function have stimulated great interest in developing new in vitro models to study lung in different species. Recent breakthroughs in 3-dimensional (3D) organoid cultures have led to new physiological in vitro models that better mimic the lung than conventional 2D cultures. Lung organoids simulate multiple aspects of the real organ, making them promising and useful models for studying organ development, function and disease (infection, cancer, genetic disease). Due to their dynamics in culture, they can serve as a sustainable source of functional cells (biobanking) and be manipulated genetically. Given the differences between species regarding developmental kinetics, the maturation of the lung at birth, the distribution of the different cell populations along the respiratory tract and species barriers for infectious diseases, there is a need for species-specific lung models capable of mimicking mammal lungs as they are of great interest for animal health and production, following the One Health approach. This paper reviews the latest developments in the growing field of lung organoids.

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Matrix GLA protein modulates branching morphogenesis in fetal rat lung

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00188.2003 ◽

2004 ◽

Vol 286 (6) ◽

pp. L1179-L1187 ◽

Cited By ~ 18

Author(s):

Kirk A. Gilbert ◽

Stephen R. Rannels

Keyword(s):

Mrna Expression ◽

Time Course ◽

Immunohistochemical Analysis ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Matrix Gla Protein ◽

Antibody Treatment ◽

Developmentally Regulated

The regulation of matrix γ-carboxyglutamic acid protein (MGP) expression during the process of lung branching morphogenesis and development was investigated. MGP mRNA expression was determined over an embryonic and postnatal time course and shown to be developmentally regulated. Immunohistochemical analysis revealed increased staining for MGP in peripheral mesenchyme surrounding distal epithelial tubules. Fetal lung explants were used as an in vitro growth model to examine expression and regulation of MGP during branching morphogenesis. MGP mRNA expression over the culture interval mimicked the in vivo time course. Explants cultured in the presence of antibodies against MGP showed gross dilation and reduced terminal lung bud counts, accompanied by changes in MGP, sonic hedgehog, and patched mRNA expression. Similarly, antifibronectin antibody treatment resulted in explant dilation and reduced MGP expression, providing evidence for an interaction with MGP and fibronectin. Conversely, intraluminal microinjection of anti-MGP antibodies had no effect either on explant growth or MGP expression, supporting the hypothesis that MGP exerts its effects through the mesenchyme. Taken together, the results suggest that MGP plays a role in lung growth and development, likely via temporally and spatially specific interactions with other branching morphogenesis-related proteins to influence growth processes.

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Autophagy is impaired in fetal hypoplastic lungs and rescued by administration of amniotic fluid stem cell extracellular vesicles

10.1101/2022.01.12.476078 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Lina Antounians ◽

Rebeca Lopes Figueira ◽

Martin Post ◽

Augusto Zani

Keyword(s):

Stem Cell ◽

Amniotic Fluid ◽

Extracellular Vesicles ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Lung Epithelial Cells ◽

Normal Lung ◽

Amniotic Fluid Stem Cell ◽

Human Fetal Lung

Rationale: Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by reduced branching morphogenesis, which is responsible for poor clinical outcomes. Administration of amniotic fluid stem cell extracellular vesicles (AFSC-EVs) rescues branching morphogenesis in rodent fetal models of pulmonary hypoplasia. Herein, we hypothesized that AFSC-EVs exert their regenerative potential by affecting autophagy, a process required for normal lung development. Objectives: To evaluate autophagy in hypoplastic lungs throughout gestation and establish whether AFSC-EV administration improves branching morphogenesis through autophagy-mediated mechanisms. Methods: EVs were isolated from c-kit+ AFSC conditioned medium by ultracentrifugation and characterized for size, morphology, and EV markers. Branching morphogenesis was inhibited in rat fetuses by nitrofen administration to dams and in human fetal lung explants by blocking RAC1 activity with NSC23766. Expression of autophagy activators (BECN1 and ATG5) and adaptor (SQSTM1/p62) was analyzed in vitro (rat and human fetal lung explants) and in vivo (rat fetal lungs). Mechanistic studies on rat fetal primary lung epithelial cells were conducted using inhibitors for microRNA-17 and -20a contained in the AFSC-EV cargo and known to regulate autophagy. Measurements and Main Results: Rat and human models of fetal pulmonary hypoplasia showed reduced autophagy mainly at pseudoglandular and canalicular stages. AFSC-EV administration restored autophagy in both pulmonary hypoplasia models by transferring miR-17~92 cluster members contained in the EV cargo. Conclusions: AFSC-EV treatment rescues branching morphogenesis partly by restoring autophagy through miRNA cargo transfer. This study enhances our understanding of pulmonary hypoplasia pathogenesis and creates new opportunities for fetal therapeutic intervention in CDH babies.

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Amniotic fluid stem cell extracellular vesicles promote fetal lung branching and cell differentiation in experimental congenital diaphragmatic hernia

10.1101/2022.01.10.475632 ◽

2022 ◽

Author(s):

Kasra Khalaj ◽

Rebeca Lopes Figueira ◽

Lina Antounians ◽

Sree Gandhi ◽

Matthew Wales ◽

...

Keyword(s):

Amniotic Fluid ◽

Congenital Diaphragmatic Hernia ◽

Extracellular Vesicles ◽

Diaphragmatic Hernia ◽

Lung Development ◽

Pulmonary Hypoplasia ◽

Fetal Lung ◽

Branching Morphogenesis ◽

Neuroendocrine Cells ◽

Beta Catenin

Pulmonary hypoplasia secondary to congenital diaphragmatic hernia (CDH) is characterized by impaired branching morphogenesis and differentiation. We have previously demonstrated that administration of extracellular vesicles derived from rat amniotic fluid stem cells (AFSC-EVs) rescues development of hypoplastic lungs at the pseudoglandular and alveolar stages in rodent models of CDH. Herein, we tested whether AFSC-EVs exert their regenerative effects at the canalicular and saccular stages, as these are translationally relevant for clinical intervention. To induce fetal pulmonary hypoplasia, we gavaged rat dams with nitrofen at embryonic day 9.5 and demonstrated that nitrofen-exposed lungs had impaired branching morphogenesis, dysregulated signaling pathways relevant to lung development (FGF10/FGFR2, ROBO/SLIT, Ephrin, Neuropilin 1, beta-catenin) and impaired epithelial and mesenchymal cell marker expression at both stages. AFSC-EVs administered to nitrofen-exposed lung explants rescued airspace density and increased the expression levels of key factors responsible for branching morphogenesis. Moreover, AFSC-EVs rescued the expression of alveolar type 1 and 2 cell markers at both canalicular and saccular stages, and restored markers of club, ciliated epithelial, and pulmonary neuroendocrine cells at the saccular stage. AFSC-EV treated lungs also had restored markers of lipofibroblasts and PDGFRA+ cells to control levels at both stages. EV tracking showed uptake of AFSC-EV RNA cargo throughout the fetal lung and an mRNA-miRNA network analysis identified that several miRNAs responsible for regulating lung development processes were contained in the AFSC-EV cargo. These findings suggest that AFSC-EV based therapies hold potential for restoring fetal lung growth and maturation in babies with pulmonary hypoplasia secondary to CDH.

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Regulation of retinoic acid signaling during lung morphogenesis

Development ◽

10.1242/dev.127.14.3057 ◽

2000 ◽

Vol 127 (14) ◽

pp. 3057-3067 ◽

Cited By ~ 1

Author(s):

S. Malpel ◽

C. Mendelsohn ◽

W.V. Cardoso

Keyword(s):

Retinoic Acid ◽

Lung Development ◽

Branching Morphogenesis ◽

Initial Stage ◽

Signaling Center ◽

Distal Lung ◽

Immature Lung ◽

Airway Branching ◽

Correct Expression

Little is known about how retinoic acid (RA) synthesis, utilization and metabolism are regulated in the embryonic lung and how these activities relate to lung pattern formation. Here we report that early lung bud formation and subsequent branching morphogenesis are characterized by distinct stages of RA signaling. At the onset of lung development RA signaling is ubiquitously activated in primary buds, as shown by expression of the major RA-synthesizing enzyme, RALDH-2 and activation of a RARE-lacZ transgene. Nevertheless, further airway branching appears to require downregulation of RA pathways by decreased synthesis, increased RA degradation in the epithelium via P450RAI-mediated metabolism, and inhibition of RA signaling in the mesenchyme by COUPTF-II expression. These mechanisms controlling local RA signaling may be critical for normal branching, since we show that manipulating RA levels in vitro to maintain RA signaling activated as in the initial stage, leads to an immature lung phenotype characterized by failure to form typical distal buds. We show that this phenotype likely results from RA interfering with the establishment of a distal signaling center, altering levels and distribution of Fgf10 and Bmp4, genes that are essential for distal lung formation. Furthermore, RA upregulates P450RAI expression, suggesting the presence of feedback mechanisms controlling RA availability. Our study illustrates the importance of regional mechanisms that control RA availability and utilization for correct expression of pattern regulators and normal morphogenesis during lung development.

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Fibroblast growth factor 10 (FGF10) and branching morphogenesis in the embryonic mouse lung

Development ◽

10.1242/dev.124.23.4867 ◽

1997 ◽

Vol 124 (23) ◽

pp. 4867-4878 ◽

Cited By ~ 74

Author(s):

S. Bellusci ◽

J. Grindley ◽

H. Emoto ◽

N. Itoh ◽

B.L. Hogan

Keyword(s):

Growth Factor ◽

Fibroblast Growth Factor ◽

Lung Development ◽

Expression Patterns ◽

Branching Morphogenesis ◽

Mouse Lung ◽

Fibroblast Growth ◽

Embryonic Mouse ◽

Fibroblast Growth Factor 10

During mouse lung morphogenesis, the distal mesenchyme regulates the growth and branching of adjacent endoderm. We report here that fibroblast growth factor 10 (Fgf10) is expressed dynamically in the mesenchyme adjacent to the distal buds from the earliest stages of lung development. The temporal and spatial pattern of gene expression suggests that Fgf10 plays a role in directional outgrowth and possibly induction of epithelial buds, and that positive and negative regulators of Fgf10 are produced by the endoderm. In transgenic lungs overexpressing Shh in the endoderm, Fgf10 transcription is reduced, suggesting that high levels of SHH downregulate Fgf10. Addition of FGF10 to embryonic day 11.5 lung tissue (endoderm plus mesenchyme) in Matrigel or collagen gel culture elicits a cyst-like expansion of the endoderm after 24 hours. In Matrigel, but not collagen, this is followed by extensive budding after 48–60 hours. This response involves an increase in the rate of endodermal cell proliferation. The activity of FGF1, FGF7 and FGF10 was also tested directly on isolated endoderm in Matrigel culture. Under these conditions, FGF1 elicits immediate endodermal budding, while FGF7 and FGF10 initially induce expansion of the endoderm. However, within 24 hours, samples treated with FGF10 give rise to multiple buds, while FGF7-treated endoderm never progresses to bud formation, at all concentrations of factor tested. Although exogenous FGF1, FGF7 and FGF10 have overlapping activities in vitro, their in vivo expression patterns are quite distinct in relation to early branching events. We conclude that, during early lung development, localized sources of FGF10 in the mesoderm regulate endoderm proliferation and bud outgrowth.

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Iroquoisgenes influence proximo-distal morphogenesis during rat lung development

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00293.2005 ◽

2006 ◽

Vol 290 (4) ◽

pp. L777-L789 ◽

Cited By ~ 29

Author(s):

Minke van Tuyl ◽

Jason Liu ◽

Freek Groenman ◽

Ross Ridsdale ◽

Robin N. N. Han ◽

...

Keyword(s):

Lung Development ◽

Cell Formation ◽

Surfactant Protein ◽

Branching Morphogenesis ◽

Secretory Protein ◽

Clara Cell ◽

Fgf Receptor ◽

Clara Cell Secretory Protein ◽

Morphogenetic Protein

Lung development is a highly regulated process directed by mesenchymal-epithelial interactions, which coordinate the temporal and spatial expression of multiple regulatory factors required for proper lung formation. The Iroquois homeobox ( Irx) genes have been implicated in the patterning and specification of several Drosophila and vertebrate organs, including the heart. Herein, we investigated whether the Irx genes play a role in lung morphogenesis. We found that Irx1– 3 and Irx5 expression was confined to the branching lung epithelium, whereas Irx4 was not expressed in the developing lung. Antisense knockdown of all pulmonary Irx genes together dramatically decreased distal branching morphogenesis and increased distention of the proximal tubules in vitro, which was accompanied by a reduction in surfactant protein C-positive epithelial cells and an increase in β-tubulin IV and Clara cell secretory protein positive epithelial structures. Transmission electron microscopy confirmed the proximal phenotype of the epithelial structures. Furthermore, antisense Irx knockdown resulted in loss of lung mesenchyme and abnormal smooth muscle cell formation. Expression of fibroblast growth factors (FGF) 1, 7, and 10, FGF receptor 2, bone morphogenetic protein 4, and Sonic hedgehog (Shh) were not altered in lung explants treated with antisense Irx oligonucleotides. All four Irx genes were expressed in Shh- and Gli2-deficient murine lungs. Collectively, these results suggest that Irx genes are involved in the regulation of proximo-distal morphogenesis of the developing lung but are likely not linked to the FGF, BMP, or Shh signaling pathways.

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