The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages.

Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.

CRISPR/Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat.

RATIONALE: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice missing critical morphogenetic genes. While epithelial cell lineages were efficiently generated from ESC, other cell types were mosaic. A complete contribution of donor ESC to lung tissue has never been achieved. The mouse lung has never been generated in a rat. OBJECTIVE: To generate the mouse lung in a rat. METHODS: CRISPR/Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat 1-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESC into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESC to the lung tissue was examined by immunostaining, flow cytometry and single-cell RNA sequencing. MEASUREMENTS AND MAIN RESULTS: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR/Cas9-mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1-/- blastocysts with mouse ESC restored pulmonary and thyroid structures in mouse-rat chimeras leading to a near 99% contribution of ESC to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. CONCLUSIONS: A combination of CRISPR/Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors".

Fluorinated amphiphilic Poly(ß-Amino ester) nanoparticle for highly efficient and specific delivery of nucleic acids to the Lung capillary endothelium.

Endothelial cell dysfunction occurs in a variety of acute and chronic pulmonary diseases including pulmonary hypertension, viral and bacterial pneumonia, bronchopulmonary dysplasia, and congenital lung diseases such as alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). To correct endothelial dysfunction, there is a critical need for the development of nanoparticle systems that can deliver drugs and nucleic acids to endothelial cells with high efficiency and precision. While several nanoparticle delivery systems targeting endothelial cells have been recently developed, none of them are specific to lung endothelial cells without targeting other organs in the body. In the present study, we successfully solved this problem by developing non-toxic poly(ß-amino) ester (PBAE) nanoparticles with specific structure design and fluorinated modification for high efficiency and specific delivery of nucleic acids to the pulmonary endothelial cells. After intravenous administration, the PBAE nanoparticles were capable of delivering non-integrating DNA plasmids to lung microvascular endothelial cells but not to other lung cell types. IVIS whole body imaging and flow cytometry demonstrated that DNA plasmid were functional in the lung endothelial cells but not in endothelial cells of other organs. Fluorination of PBAE was required for lung endothelial cell-specific targeting. Hematologic analysis and liver and kidney metabolic panels demonstrated the lack of toxicity in experimental mice. Thus, fluorinated PBAE nanoparticles can be an ideal vehicle for gene therapy targeting lung microvascular endothelium in pulmonary vascular disorders.

Demonstration of Safety in Wild Type Mice of npFOXF1, a Novel Nanoparticle-Based Gene Therapy for Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins.

Introduction: Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins (ACDMPV) is a fatal congenital disease resulting from a pulmonary vascular endothelial deficiency of FOXF1, producing abnormal morphogenesis of alveolar capillaries, malpositioned pulmonary veins and disordered development of lung lobes. Affected neonates suffer from cyanosis, severe breathing insufficiency, pulmonary hypertension, and death typically within days to weeks after birth. Currently, no treatment exists for ACDMPV, although recent murine research in the Kalinichenko lab demonstrates nanoparticle delivery improves survival and reconstitutes normal alveolar-capillary architecture. The aim of the present study is to investigate the safety of intravenous administration of FOXF1-expressing PEI-PEG nanoparticles (npFOXF1), our pioneering treatment for ACDMPV. Methods: npFOXF1 was constructed, validated, and subsequently administered in a single dose to postnatal day 14 (P14) mice via retro-orbital injection. Biochemical, serologic, and histologic safety were monitored at postnatal day 16 (P16) and postnatal day 21 (P21). Results: With treatment we observed no lethality, and the general condition of mice revealed no obvious abnormalities. Serum chemistry, whole blood, and histologic toxicity was assayed on P16 and P21 and revealed no abnormality. Discussion: In conclusion, npFOXF1 has a very good safety profile and combined with preceding studies showing therapeutic efficacy, npFOXF1 can be considered as a good candidate therapy for ACDMPV in human neonates.

In vivo generation of bone marrow from embryonic stem cells in interspecies chimeras.

Generation of bone marrow (BM) from embryonic stem cells (ESCs) promises to accelerate the development of future cell therapies for life-threatening disorders. However, such approach is limited by technical challenges to produce a mixture of functional BM progenitor cells able to replace all hematopoietic cell lineages. Herein, we used blastocyst complementation to simultaneously produce BM cell lineages from mouse ESCs in a rat. Based on fluorescence-activated cell sorting analysis and single-cell RNA sequencing, mouse ESCs differentiated into multiple hematopoietic and stromal cell types that were indistinguishable from normal mouse BM cells based on gene expression signatures and cell surface markers. Receptor-ligand interactions identified Cxcl12-Cxcr4, Lama2-Itga6, App-Itga6, Comp-Cd47, Col1a1-Cd44, and App-Il18rap as major signaling pathways between hematopoietic progenitors and stromal cells. Multiple hematopoietic progenitors, including hematopoietic stem cells (HSCs) in mouse-rat chimeras derived more efficiently from mouse ESCs, whereas chondrocytes predominantly derived from rat cells. In the dorsal aorta and fetal liver of mouse-rat chimeras, mouse HSCs emerged and expanded faster compared to endogenous rat cells. Sequential BM transplantation of ESC-derived cells from mouse-rat chimeras rescued lethally irradiated syngeneic mice and demonstrated long-term reconstitution potential of donor HSCs. Altogether, a fully functional BM was generated from mouse ESCs using rat embryos as 'bioreactors'.

Transcriptomic and Metabolomic Analyses Provide Insights into the Formation of the Peach-like Aroma of Fragaria nilgerrensis Schlecht. Fruits.

Fragaria nilgerrensis Schlecht. is a wild diploid strawberry species. The intense peach-like aroma of its fruits makes F. nilgerrensis an excellent resource for strawberry breeding programs aimed at enhancing flavors. However, the formation of the peach-like aroma of strawberry fruits has not been comprehensively characterized. In this study, fruit metabolome and transcriptome datasets for F. nilgerrensis (HA; peach-like aroma) and its interspecific hybrids PA (peach-like aroma) and NA (no peach-like aroma; control) were compared. In total, 150 differentially accumulated metabolites were detected. The K-means analysis revealed that esters/lactones, including acetic acid, octyl ester, δ-octalactone, and δ-decalactone, were more abundant in HA and PA than in NA. These metabolites may be important for the formation of the peach-like aroma of F. nilgerrensis fruits. The significantly enriched gene ontology terms assigned to the differentially expressed genes (DEGs) were fatty acid metabolic process and fatty acid biosynthetic process. Twenty-seven DEGs were predicted to be associated with ester and lactone biosynthesis, including AAT, LOX, AOS, FAD, AIM1, EH, FAH, ADH, and cytochrome P450 subfamily genes. Thirty-five transcription factor genes were predicted to be associated with aroma formation, including bHLH, MYB, bZIP, NAC, AP2, GATA, and TCPfamily members. Moreover, we identified differentially expressed FAD, AOS, and cytochrome P450 family genes and NAC, MYB, and AP2 transcription factor genes that were correlated with δ-octalactone and δ-decalactone. These findings provide key insights into the formation of the peach-like aroma of F. nilgerrensis fruits, with implications for the increased use of wild strawberry resources.

The significance of serum S100 calcium-binding protein A4 in silicosis.

BACKGROUND: Silicosis is a chronic occupational pulmonary disease characterized by persistent inflammation and irreversible fibrosis. Considerable evidences now indicate that S100 calcium-binding protein A4 (S100A4) has been associated with fibrotic diseases. However, the role of S100A4 in silicosis is still unclear. METHODS: In this study, serum levels of S100A4, transforming growth factor-ß1 (TGF-ß1), connective tissue growth factor (CTGF), interleukin-6 (IL-6) and tumour necrosis factor-α (TNF-α) in patients with silicosis (n = 42) and control group (CG, n = 12) were measured by ELISA. S100A4 expression in lung tissues and primary alveolar macrophages (AMs) of mice with and without silicosis was detected by immunohistochemistry (IHC)/real-time PCR. The correlations between S100A4 and cytokines or lung function were assessed by Spearman's rank correlation analyses. RESULTS: Compared with CG, the levels of S100A4 were significantly increased in silicosis patients (70.84 (46.22, 102.46) ng/ml vs (49.84 (42.86, 60.02) ng/ml). The secretions of TGF-ß1, CTGF, IL-6 and TNF-α in silicosis group were significantly higher than that in control group (p < 0.05). Serum S100A4 levels were positively correlated with TGF-ß1 and IL-6, while were negatively correlated with lung function parameters including percentage of predicted forced vital capacity (FVC%pre), maximum vital capacity (Vcmax), deep inspiratory capacity (IC) and peak expiratory flow at 75% of vital capacity (PEF75). In receiver operating characteristic (ROC) analyses, S100A4 > 61.7 ng/ml had 63.4% sensitivity and 83.3% specificity for silicosis, and the area under the curve (AUC) was 0.707. Furthermore, immunostaining of lung tissues showed the accumulation of S100A4-positive cells in the areas of nodules of silicotic mice. The mRNA expression of S100A4 in the lung tissues and AMs of silicotic mice were significantly higher than controls. CONCLUSION: These data suggested that increased S100A4 might contribute to the pathogenesis of silicosis.

Effect of resveratrol on superovulation in mice.

Oocyte quality is one of the key factors affecting the outcome of ART. Therefore, how to improve oocyte quality has become an urgent problem in the field of ART. In this study we evaluated the effect of resveratrol (RSV), added during the process of superovulation, on embryonic development in mice. The results showed that the blastocyst rate was significantly higher in the RSV treated group than in the control group when oocytes were parthenogenetically activated in vitro (61.67 vs 41.51%, P = 0.032). In the naturally fertilized oocytes group, the rates of cleavage and blastocyst were significantly higher in the RSV treatment group than in the control group (74.47% vs 60.98%, P = 0.035; 96.19% vs 70.00%, P = 0.000, respectively). For the aged mice, the average number of oocytes, the rates of cleavage and blastocyst were also significantly higher in RSV treated groups than in the control group (19.47 ± 5.98 vs 10.30 ± 4.82, P = 0.028; 69.03 vs 50.75%, P = 0.014; 64.10% vs 44.12%, P = 0.049, respectively). Mitochondrial membrane potential and mtDNA copy number in oocytes were significantly increased after RSV treatment in both the young and aged populations. The expression of mitochondrial biogenesis related genes was significantly upregulated in cumulus cells of young and aged mice following RSV treatment. Our data suggest that supplementation of RSV during superovulation improves oocytes quality in young and aged mice, increases the number of oocytes retrieved from aged mice, and improves oocytes mitochondrial function.

Generation of Pulmonary Endothelial Progenitor Cells for Cell-based Therapy Using Interspecies Mouse-Rat Chimeras.

Rationale: Although pulmonary endothelial progenitor cells (EPCs) hold promise for cell-based therapies for neonatal pulmonary disorders, whether EPCs can be derived from pluripotent embryonic stem cells (ESCs) or induced pluripotent stem cells remains unknown.Objectives: To investigate the heterogeneity of pulmonary EPCs and derive functional EPCs from pluripotent ESCs.Methods: Single-cell RNA sequencing of neonatal human and mouse lung was used to identify the heterogeneity of pulmonary EPCs. CRISPR/Cas9 gene editing was used to genetically label and purify mouse pulmonary EPCs. Functional properties of the EPCs were assessed after cell transplantation into neonatal mice with S52F Foxf1 mutation, a mouse model of alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV). Interspecies mouse-rat chimeras were produced through blastocyst complementation to generate EPCs from pluripotent ESCs for cell therapy in ACDMPV mice.Measurements and Main Results: We identified a unique population of EPCs, FOXF1+cKIT+ EPCs, as a subset of recently described general capillary cells (gCAPs) expressing SMAD7, ZBTB20, NFIA, and DLL4 but lacking mature arterial, venous, and lymphatic markers. FOXF1+cKIT+ gCAPs are reduced in ACDMPV, and their transcriptomic signature is conserved in mouse and human lungs. After cell transplantation into the neonatal circulation of ACDMPV mice, FOXF1+cKIT+ gCAPs engraft into the pulmonary vasculature, stimulate angiogenesis, improve oxygenation, and prevent alveolar simplification. FOXF1+cKIT+ gCAPs, produced from ESCs in interspecies chimeras, are fully competent to stimulate neonatal lung angiogenesis and alveolarization in ACDMPV mice.Conclusions: Cell-based therapy using donor or ESC/induced pluripotent stem cell-derived FOXF1+cKIT+ endothelial progenitors may be considered for treatment of human ACDMPV.

Blastocyst complementation reveals that NKX2-1 establishes the proximal-peripheral boundary of the airway epithelium.

BACKGROUND: Distinct boundaries between the proximal conducting airways and more peripheral-bronchial regions of the lung are established early in foregut embryogenesis, demarcated in part by the distribution of SOX family and NKX2-1 transcription factors along the cephalo-caudal axis of the lung. We used blastocyst complementation to identify the role of NKX2-1 in the formation of the proximal-peripheral boundary of the airways in mouse chimeric embryos. RESULTS: While Nkx2-1-/- mouse embryos form primordial tracheal cysts, peripheral pulmonary structures are entirely lacking in Nkx2-1-/- mice. Complementation of Nkx2-1-/- embryos with NKX2-1-sufficient embryonic stem cells (ESCs) enabled the formation of all tissue components of the peripheral lung but did not enhance ESC colonization of the most proximal regions of the airways. In chimeric mice, a precise boundary was formed between NKX2-1-deficient basal cells co-expressing SOX2 and SOX9 in large airways and ESC-derived NKX2-1+ SOX9+ epithelial cells of smaller airways. NKX2-1-sufficient ESCs were able to selectively complement peripheral, rather than most proximal regions of the airways. ESC complementation did not prevent ectopic expression of SOX9 but restored ß-catenin signaling in Nkx2-1-/- basal cells of large airways. CONCLUSIONS: NKX2-1 and ß-catenin function in an epithelial cell-autonomous manner to establish the proximal-peripheral boundary along developing airways.

In Vivo Generation of Lung and Thyroid Tissues from Embryonic Stem Cells Using Blastocyst Complementation.

Rationale: The regeneration and replacement of lung cells or tissues from induced pluripotent stem cell- or embryonic stem cell-derived cells represent future therapies for life-threatening pulmonary disorders but are limited by technical challenges to produce highly differentiated cells able to maintain lung function. Functional lung tissue-containing airways, alveoli, vasculature, and stroma have never been produced via directed differentiation of embryonic stem cells (ESCs) or induced pluripotent stem cells. We sought to produce all tissue components of the lung from bronchi to alveoli by embryo complementation.Objectives: To determine whether ESCs are capable of generating lung tissue in Nkx2-1-/- mouse embryos with lung agenesis.Methods: Blastocyst complementation was used to produce chimeras from normal mouse ESCs and Nkx2-1-/- embryos, which lack pulmonary tissues. Nkx2-1-/- chimeras were examined using immunostaining, transmission electronic microscopy, fluorescence-activated cell sorter analysis, and single-cell RNA sequencing.Measurements and Main Results: Although peripheral pulmonary and thyroid tissues are entirely lacking in Nkx2-1 gene-deleted embryos, pulmonary and thyroid structures in Nkx2-1-/- chimeras were restored after ESC complementation. Respiratory epithelial cell lineages in restored lungs of Nkx2-1-/- chimeras were derived almost entirely from ESCs, whereas endothelial, immune, and stromal cells were mosaic. ESC-derived cells from multiple respiratory cell lineages were highly differentiated and indistinguishable from endogenous cells based on morphology, ultrastructure, gene expression signatures, and cell surface proteins used to identify cell types by fluorescence-activated cell sorter.Conclusions: Lung and thyroid tissues were generated in vivo from ESCs by blastocyst complementation. Nkx2-1-/- chimeras can be used as "bioreactors" for in vivo differentiation and functional studies of ESC-derived progenitor cells.

Decreased Soluble Receptor of Advanced Glycation End Product Levels Correlated with Inflammation in Silicosis.

Silicosis is a devastating disease caused by inhalation of silica dust that leads to inflammatory cascade and then scarring of the lung tissue. Increasing evidences indicate that soluble receptor for advanced glycation end products (sRAGE) is involved in inflammatory diseases. However, no data on the possible relationship between sRAGE and inflammation of silicosis are available. In this study, serum from subjects with silicosis (n = 59) or from healthy controls (HC, n = 14) was analyzed for the secretion of sRAGE, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), transforming growth factor-ß1 (TGF-ß1), and oxidized low-density lipoprotein (ox-LDL). The associations between sRAGE and cytokines and ox-LDL and lung function were assessed by Pearson's correlation analyses. Mean levels of serum sRAGE were lower in silicosis than those in controls (p < 0.05). The subjects who had a longer term of occupational exposure had higher levels of sRAGE (p < 0.05). The secretion of TNF-α, IL-1ß, IL-6, TGF-ß1, and ox-LDL was significantly higher in the silicosis group than that in the HC group (p < 0.05). Furthermore, the levels of sRAGE were negatively correlated with TNF-α, IL-6, IL-1ß, and ox-LDL. There is no correlation between sRAGE and TGF-ß1 and lung function. The optimal point of sRAGE for differentiating silicosis from healthy controls was 14250.02 pg/ml by ROC curve analysis. A decrease in serum sRAGE and its association with inflammatory response might suggest a role for sRAGE in the pathogenesis of silicosis.

Lineage specification revealed by single-cell gene expression analysis in porcine preimplantation embryos.

After zygotic genome activation and lineage specification, zygotes develop into late blastocysts comprising three distinct cell types. The molecular mechanisms underlying this progress are largely unknown in pigs. Here, we intended to analyze an extensive set of regulators at the single-cell level to define the events involved in the development of the porcine blastocysts. Using a quantitative microfluidics approach in single cells, we detected mRNA levels of 96 genes known to function in early embryonic development and maintenance of stem cell pluripotency simultaneously in 480 individual cells derived from porcine preimplantation embryos. The developmental transitions can be distinguished based on distinctive gene expression profiles, and we identified paired box 6 (PAX6) and aquaporin 3 (AQP3) expressed in early and late developmental stages, respectively. Two lineages can be segregated in porcine early and late blastocysts by the expression patterns of lineage-specific genes such as DAB2, clathrin adaptor protein (DAB2) for trophectoderm (TE), platelet derived growth factor receptor alpha (PDGFRA), Nanog homeobox (NANOG), fibronectin 1 (FN1), hepatocyte nuclear factor 4 alpha (HNF4A), goosecoid homeobox (GSC), nuclear receptor subfamily 5 group A member 2 (NR5A2), and lysine acetyltransferase 6A (KAT6A; previously known as MYST3) for inner cell mass (ICM). However, the epiblast and primitive endoderm cannot be identified in late blastocysts, and those TE or ICM lineage-specific genes were low expressed in blastomeres from the morula. Our results shed light on early cell fate determination in porcine preimplantation embryos and offer theoretical support for deriving porcine embryonic stem cells.

Tetraploid embryonic stem cells can contribute to the development of chimeric fetuses and chimeric extraembryonic tissues.

Our study examined the in vivo chimeric and survival capacities of chimeras created by injecting tetraploid embryonic stem cells (ESCs) expressing green fluorescent protein (GFP) into diploid embryos. At 3.5 days post-coitum (dpc) and 4.5 dpc, the tetraploid ESCs were able to contribute to the inner cell mass (ICM) just as diploid ESCs tagged with GFP. At 6.5 dpc, 8.0 dpc and 10.5 dpc, the tetraploid ESCs manifested in the same location as the diploid ESCs. The GFP cells in the extraembryonic tissues and fetuses of tetraploid ESC chimeras were tetraploid as determined by fluorescence activated cell sorting (FACS). Furthermore, tetraploid ESCs contributed to the development of the placenta, embryolemma and umbilical cord at 13.5 dpc and 16.5 dpc; however, very less GFP cells were found in the fetuses of tetraploid ESC chimeras. We further found that the proliferation of tetraploid ESCs was slower than that of diploid ESCs. In addition, the relative mRNA expression in the three germ layers and the trophoblast was abnormal in the EBs of tetraploid ESCs compared with diploid ESCs. In short, slower proliferation and abnormal differentiation potential of tetraploid ESCs might be two of the reasons for their poor survival and chimeric capacities.