TP63 basal cells are indispensable during endoderm differentiation into proximal airway cells on acellular lung scaffolds.

The use of decellularized whole-organ scaffolds for bioengineering of organs is a promising avenue to circumvent the shortage of donor organs for transplantation. However, recellularization of acellular scaffolds from multicellular organs like the lung with a variety of different cell types remains a challenge. Multipotent cells could be an ideal cell source for recellularization. Here we investigated the hierarchical differentiation process of multipotent ES-derived endoderm cells into proximal airway epithelial cells on acellular lung scaffolds. The first cells to emerge on the scaffolds were TP63+ cells, followed by TP63+/KRT5+ basal cells, and finally multi-ciliated and secretory airway epithelial cells. TP63+/KRT5+ basal cells on the scaffolds simultaneously expressed KRT14, like basal cells involved in airway repair after injury. Removal of TP63 by CRISPR/Cas9 in the ES cells halted basal and airway cell differentiation on the scaffolds. These findings suggest that differentiation of ES-derived endoderm cells into airway cells on decellularized lung scaffolds proceeds via TP63+ basal cell progenitors and tracks a regenerative repair pathway. Understanding the process of differentiation is key for choosing the cell source for repopulation of a decellularized organ scaffold. Our data support the use of airway basal cells for repopulating the airway side of an acellular lung scaffold.

Reversal of Surfactant Protein B Deficiency in Patient Specific Human Induced Pluripotent Stem Cell Derived Lung Organoids by Gene Therapy.

Surfactant protein B (SFTPB) deficiency is a fatal disease affecting newborn infants. Surfactant is produced by alveolar type II cells which can be differentiated in vitro from patient specific induced pluripotent stem cell (iPSC)-derived lung organoids. Here we show the differentiation of patient specific iPSCs derived from a patient with SFTPB deficiency into lung organoids with mesenchymal and epithelial cell populations from both the proximal and distal portions of the human lung. We alter the deficiency by infecting the SFTPB deficient iPSCs with a lentivirus carrying the wild type SFTPB gene. After differentiating the mutant and corrected cells into lung organoids, we show expression of SFTPB mRNA during endodermal and organoid differentiation but the protein product only after organoid differentiation. We also show the presence of normal lamellar bodies and the secretion of surfactant into the cell culture medium in the organoids of lentiviral infected cells. These findings suggest that a lethal lung disease can be targeted and corrected in a human lung organoid model in vitro.

Hypoxia-inducible factor-1 stimulates postnatal lung development but does not prevent O2-induced alveolar injury.

This study investigated whether hypoxia-inducible factor (HIF)-1 influences postnatal vascularization and alveologenesis in mice and whether stable (constitutive-active) HIF could prevent hyperoxia-induced lung injury. We assessed postnatal vessel and alveolar formation in transgenic mice, expressing a stable, constitutive-active, HIF1α-subunit (HIF-1αΔODD) in the distal lung epithelium. In addition, we compared lung function, histology, and morphometry of neonatal transgenic and wild-type mice subjected to hyperoxia. We found that postnatal lungs of HIF-1αΔODD mice had a greater peripheral vessel density and displayed advanced alveolarization compared with control lungs. Stable HIF-1α expression was associated with increased postnatal expression of angiogenic factors, including vascular endothelial growth factor, angiopoietins 1 and 2, Tie2, and Ephrin B2 and B4. Hyperoxia-exposed neonatal HIF-1αΔODD mice exhibited worse lung function but had similar histological and surfactant abnormalities compared with hyperoxia-exposed wild-type controls. In conclusion, expression of constitutive-active HIF-1α in the lung epithelium was associated with increased postnatal vessel growth via up-regulation of angiogenic factors. The increase in postnatal vasculature was accompanied by enhanced alveolar formation. However, stable HIF-1α expression in the distal lung did not prevent hyperoxia-induced lung injury in neonates but instead worsened lung function.

Hypoxia-inducible regulation of placental BOK expression.

BOK (BCL-2-related ovarian killer) is a member of the pro-apoptotic BCL-2 family that is highly expressed in the human placenta. BOK excess causes increased trophoblast autophagy and apoptosis in pre-eclampsia, a pathological condition of hypoxia and oxidative stress. In the present study, we identified an HRE (hypoxia-response element) at the junction of exon-1 and intron-1 (+229 to +279) in the human BOK gene, as well as an antisense transcript driven by a promoter located in intron-2. The isolated BOK-HRE bound hypoxia-inducible HIF (hypoxia-inducible factor) proteins in vitro as well as in trophoblastic JEG3 cells and was functional in its natural position as well as in front of a heterologous promoter. Being a reverted repeat, the BOK-HRE functioned in both orientations. This directionless feature of the BOK-HRE facilitates hypoxia regulation via HIF of both BOK and its antisense transcript as demonstrated by RNAi knockdown of the HIF system. Although the antisense transcript was expressed in several human carcinoma cell lines, including choriocarcinoma-derived JEG3 cells, no antisense-regulated mechanism for BOK expression was noted. Taken together, these findings indicate that hypoxia-induced expression of BOK in placental cells is regulated via HIF and is not affected by its antisense transcript.

Mouse snail is a target gene for HIF.

The transcriptional inhibitor Snail is a critical regulator for epithelial-mesenchymal transition (EMT). Although low oxygen induces Snail transcription, thereby stimulating EMT, a direct role of hypoxia-inducible factor (HIF) in this process remains to be demonstrated. Here we show that hypoxia induces the expression of Snail via HIF. In silico analysis identified a potential hypoxia-response element (HRE) close to the minimal promoter of the human and mouse genome of the snail gene. Gel shift assays demonstrated that a specific hypoxia-inducible complex is formed with the putative HRE and that the complex contains HIF proteins. ChIP assays confirmed the interaction of HIF proteins with the putative HRE in vivo. Reporter gene analyses showed that the putative HRE responds to hypoxia in its natural position as well as in front of a heterologous promoter and that the HRE is directly activated by HIF-1α or HIF-2α. HIF knockdown with siRNA at 2% oxygen and overexpression of an oxygen-insensitive HIF (HIF-ΔODD) mutant at 21% oxygen showed that HIF regulates Snail activation and subsequent cell migration. Our findings identify snail as a HIF target gene and provide novel insights into the regulation of snail and hypoxia-induced EMT.

Maternal exposure to endotoxin delays alveolarization during postnatal rat lung development.

Maternal bacterial infections adversely affect lung development by crossing the placental barrier and infecting the developing fetus. The underlying mechanism negatively affecting pulmonary development remains unknown. Herein, we investigated whether a systemic maternal infection affects postnatal inflammation and alveolar development. Pregnant rats were injected with 2.5 mg/kg LPS on day 20 and 21 (term = 22 days). Postnatal (PN0-21) mRNA and protein expression of cytokines (IL-1beta, IL-6, IL-10, CXCL1/2, TNFalpha) and genes implicated in alveologenesis [tropoelastin, lysyl oxidase (LOX), lysyl oxidase-like (LOXL)1, tenascin-C (TNC), fibulin 5, vascular endothelial growth factor (VEGF-A), VEGF receptor (VEGFR)2, VEGFR1, platelet-derived growth factor (PDGF)A, PDGFB, and PDGFRalpha] were quantified by real-time PCR and beadlyte technology. Lung transcript and protein levels of IL-1beta, IL-6, and CXCL1/2 were significantly greater in LPS-exposed pups than those of control pups at PN0, 2, 6, 10, and 14. Bronchoalveolar lavage fluid (BALF) of LPS-exposed animals contained significantly more macrophages at PN2 and 14 than BALF of control pups. Morphometric analysis revealed that LPS-exposed animals had fewer and larger alveoli, fewer secondary septa, and decreased peripheral vessel density when compared with control pups. This morphological delay in alveolar development disappeared after PN14. Tropoelastin, LOXL1, VEGF, VEGFR2, and PDGFRalpha mRNA expression of LPS-exposed animals was significantly greater than those of control animals in PN2-14 lungs. TNC, LOX, fibulin 5, VEGFR1, PDGFA, and PDGFB expression was not affected by maternal LPS exposure. Together, the data demonstrate that maternal exposure to endotoxin results in a prolonged pulmonary inflammation postnatally, altered gene expression of molecules implicated in alveologenesis, and delayed morphological maturation of the lung.

Effect of calcium ionophore A23187 on prostaglandin synthase type 2 and 15-hydroxy-prostaglandin dehydrogenase expression in human chorion trophoblast cells.

OBJECTIVE: Prostaglandins induce parturition in humans. Prostaglandin output is regulated by the synthetic and metabolic enzymes, prostaglandin synthase type 2 (PTGS2) and 15-hydroxyprostaglandin dehydrogenase (PGDH). The role of calcium in regulating PTGS2 and PGDH expression was investigated in chorion trophoblasts. STUDY DESIGN: Cells were treated with calcium ionophore A23187 in the presence or absence of calcium chelators; changes in messenger ribonucleic acid expression were measured with real-time polymerase chain reaction and analyzed with analysis of variance. Protein expression was evaluated with Western blot and dual immunofluorescence. RESULTS: A23187 stimulated PTGS2 and suppressed PGDH expression. Effects of A23187 were reversed by calcium chelators. PTGS2 had perinuclear and cytosolic distribution, whereas PGDH was cytosolic. Some cells expressed both enzymes, some neither enzyme, and some either PTGS2 or PGDH. CONCLUSION: Chorion cells showed heterogeneity in the expression of PTGS2 and PGDH. Calcium influx regulates PTGS2 and PGDH expression, thereby promoting coordinated increased prostaglandin output in circumstances such as term and preterm labor.

Fibroblast growth factor-2 and receptor-1alpha(IIIc) regulate postnatal rat lung cell apoptosis.

RATIONALE: Fibroblast growth factor receptor-1alpha(IIIc) [FGF-R1alpha(IIIc)] regulates recovery of neonatal rat lung growth, after 95% oxygen-mediated growth arrest. Its role in normal postnatal alveologenesis is unknown. OBJECTIVE: To determine if FGF-R1alpha(IIIc) regulates normal postnatal alveologenesis. METHODS: Truncated soluble FGF-R1alpha(IIIc) or neutralizing antibodies to FGF-1 or FGF-2 were injected intraperitoneally into 3-d-old rats. The pups were killed at Day 7 for studies of alveolar development. MEASUREMENTS AND MAIN RESULTS: Injected, truncated soluble FGF-R1alpha(IIIc) inhibited phosphorylation of the endogenous FGF-R1, and downstream pathway, and paradoxically increased lung DNA content and tissue fraction while inhibiting lung cell DNA synthesis. The increase in tissue thickness was due to reduced apoptosis, as indicated by reductions in cleaved effector caspases 3 and 7. Inhibition of the intrinsic apoptosis pathway was suggested by decreases in the proapoptotic protein Bax and mitochondrial cytochrome c release, and an increase in the antiapoptotic protein Bcl-x(L). Injected antibodies to FGF-1 and FGF-2 had no effect on DNA synthesis, but both increased Bcl-x(L) content and decreased cytochrome c release and cleaved caspase-7 protein expression. However, only injection of the antibody to FGF-2 replicated the increased tissue fraction and inhibited apoptosis observed with the injection of truncated soluble FGF-R1alpha(IIIc). CONCLUSIONS: Inhibition of ligand binding, most likely of FGF-2, to the FGF-R1alpha(IIIc) inhibits normal postnatal lung cell apoptosis.

Alternative splicing and promoter usage generates an intracellular stromelysin 3 isoform directly translated as an active matrix metalloproteinase.

Human stromelysin 3 (ST3) is a matrix metalloproteinase (MMP) that has been implicated in cancer progression and in various tissue remodeling processes. Unlike most MMPs, ST3 is characterized by a distinct substrate specificity and a specific regulation and is not directly involved in extracellular matrix degradation. In the present study, we have identified an additional ST3 gene promoter that is accessible to nuclear factors such as C/EBP and retinoic acid receptors. This human specific promoter is inducible and controls the expression of a novel ST3 transcript called the beta-ST3 that is expressed in cultured cells and in placenta. This transcript encodes a 40-kDa ST3 isoform that lacks both the signal peptide common to all secreted MMPs and the prodomain that normally maintains enzyme latency. Consistent with the lack of a signal peptide, the beta-ST3 was found to be intracellular. The relative amount of the extracellular alpha-ST3 isoform was about 20-fold higher than that of the intracellular ST3 isoforms, as estimated by Western blot analysis. Furthermore, recombinant beta-ST3 produced in Escherichia coli exhibits a proteolytic activity against alpha1-proteinase inhibitor, a substrate previously shown to be inactivated by the alpha-ST3. Therefore, although it was thought that all MMPs were synthesized as inactive zymogens and functioned extracellularly, this is the first MMP isoform reported that is generated by alternative promoter usage and directly translated as an active enzyme. Although the intracellular function of the beta-ST3 remains to be investigated, these data support the idea that the functions of MMPs are not restricted to the extracellular space.