BackgroundExposure to high levels of oxygen (hyperoxia) after birth leads to lung injury. Our aims were to investigate the modulation of myeloid cell sub-populations and the reduction of fibrosis in the lungs following administration of human mesenchymal stem cells (hMSC) to neonatal mice exposed to hyperoxia.MethodNewborn mice were exposed to 90% O2 (hyperoxia) or 21% O2 (normoxia) from postnatal days 0–4. A sub-group of hyperoxia mice were injected intratracheally with 2.5X105 hMSCs. Using flow cytometry we assessed pulmonary immune cells at postnatal days 0, 4, 7 and 14. The following markers were chosen to identify these cells: CD45+ (leukocytes), Ly6C+Ly6G+ (granulocytes), CD11b+CD11c+ (macrophages); macrophage polarisation was assessed by F4/80 and CD206 expression. hMSCs expressing enhanced green fluorescent protein (eGFP) and firefly luciferase (fluc) were administered via the trachea at day 4. Lung macrophages in all groups were profiled using next generation sequencing (NGS) to assess alterations in macrophage phenotype. Pulmonary collagen deposition and morphometry were assessed at days 14 and 56 respectively.ResultsAt day 4, hyperoxia increased the number of pulmonary Ly6C+Ly6G+ granulocytes and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. At days 7 and 14, hyperoxia increased numbers of CD45+ leukocytes, CD11b+CD11c+ alveolar macrophages and F4/80lowCD206low macrophages but decreased F4/80highCD206high macrophages. hMSCs administration ameliorated these effects of hyperoxia, notably reducing numbers of CD11b+CD11c+ and F4/80lowCD206low macrophages; in contrast, F4/80highCD206high macrophages were increased. Genes characteristic of anti-inflammatory ‘M2’ macrophages (Arg1, Stat6, Retnla, Mrc1, Il27ra, Chil3, and Il12b) were up-regulated, and pro-inflammatory ‘M1’ macrophages (Cd86, Stat1, Socs3, Slamf1, Tnf, Fcgr1, Il12b, Il6, Il1b, and Il27ra) were downregulated in isolated lung macrophages from hyperoxia-exposed mice administered hMSCs, compared to mice without hMSCs. Hydroxyproline assay at day 14 showed that the 2-fold increase in lung collagen following hyperoxia was reduced to control levels in mice administered hMSCs. By day 56 (early adulthood), hMSC administration had attenuated structural changes in hyperoxia-exposed lungs.ConclusionsOur findings suggest that hMSCs reduce neonatal lung injury caused by hyperoxia by modulation of macrophage phenotype. Not only did our cell-based therapy using hMSC induce structural repair, it limited the progression of pulmonary fibrosis.Electronic supplementary materialThe online version of this article (10.1186/s12931-018-0816-x) contains supplementary material, which is available to authorized users.
The in vivo engraftment of induced pluripotent stem cell (iPSC)‐derived podocytes following allogeneic transplantation into host kidneys remains a challenge. Here we investigate the survival and engraftment of human dermal fibroblasts‐derived differentiated iPSCs using a newborn mouse model, which represents a receptive immunoprivileged host environment. iPSCs were generated from skin biopsies of patients using Sendai virus reprogramming. Differentiation of nephrin (NPHS1)‐green fluorescent protein (GFP) iPSCs into kidney podocytes (iPSC‐PODs) was performed by the addition of Activin A, bone morphogenetic protein 7 (BMP7), and retinoic acid over 10 days of culture. To assess the in vivo incorporation of cells, undifferentiated iPSCs or day 10 iPSC‐PODs, were labeled with either carboxyfluorescein succinimidyl ester (CFSE) or Qdot nanocrystals (Q705). Thereafter, 1 × 105 differentiated iPSC‐PODs were injected directly into the kidneys of mouse pups at postnatal day one (P1). Using co‐expression analysis of glomerular and podocyte‐specific markers, Day 10 differentiated iPSC‐PODs that were positive for podocin, were detected following direct kidney injection into newborn mice up to 1 week after transplantation. Undifferentiated iPSC‐PODs were not detected at the same timepoint. The transplanted cells were viable and located in the outer nephrogenic zone where they were found to colocalize with, or sit adjacent to, cells positive for glomerular‐specific markers including podocin, synaptopodin, and Wilms' tumor 1 (WT1). This study provides proof‐of‐principle that transplanted iPSC–POD can survive in recipient newborn mouse kidneys due to the immature and immunoprivileged nature of the developing postnatal kidneys.
Background Lung immaturity is one of the most serious consequences of growth restriction and premature birth. Preterm babies often require mechanical ventilation to survive, but exposure to high levels of oxygen can permanently damage the lungs and interrupts normal development. As lung macrophages play an important role in hyperoxic lung injury and repair, our objective was to use next generation sequencing (NGS) to identify changes in the macrophage transcriptome following neonatal hyperoxia, with and without treatment with human mesenchymal stem cells (hMSCs). We provide the first report of RNA-sequencing of lung macrophages following neonatal hyperoxia and hMSCs therapy. Methods Neonatal mice exposed to normoxia (21%O2) or hyperoxia (90% O2) from birth to postnatal day 4 were randomized to receive either hMSCs or vehicle via intratracheal delivery on postnatal day 4. Mouse lungs from normoxia and hyperoxia groups with and without hMSCs therapy were examined at day 14. RNA-sequencing was performed on flow-cytometric CD45+CD11b+CD11c+ sorted lung macrophages. Purified total RNA was used to construct barcoded multiplex-compatible sequencing libraries using: 1) Illumina Stranded mRNA Sample Preparation chemistry (for transcriptomics) and 2) Bio Scientific NEXTFlex Small RNA chemistry (for small RNA). Results Sorted CD45+CD11b+CD11c+ lung macrophages from hyperoxia-exposed neonatal mice showed differentially expressed macrophage genes and miRNA compared to mice exposed to normoxia or hyperoxia+hMSCs. The administration of hMSCs was found to differentially upregulate 421 genes and downregulate 651 genes in CD45+CD11b+CD11c+ lung macrophages from neonatal mice exposed to hyperoxia, compared to normoxia. Integrity pathway analysis (IPA) analysis of macrophage-specific gene pathways revealed the effectiveness of hMSCs in altering macrophage function towards an anti-inflammatory ‘M2’ phenotype. Small-RNA sequencing provided further evidence on the effects of hMSCs, where 1,098 small RNAs transcriptomes were expressed as either significantly up- or down-regulated in response to hMSCs therapy following hyperoxia-induced lung damage. Conclusions Pathway analysis of the predicted mRNA targets of differentially expressed genes provides insight into miRNAs that preferentially target several important pathways. These miRNAs will be functionally relevant for lung macrophages, and will provide a greater understanding of the interaction between macrophage genotype and the associated phenotypes in the setting of inflammation or tissue repair.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.