Predifferentiated amniotic fluid mesenchymal stem cells enhance lung alveolar epithelium regeneration and reverse elastase-induced pulmonary emphysema

Abstract

Introduction: Pulmonary emphysema is a major component of chronic obstructive pulmonary disease (COPD). Emphysema progression attributed not only to alveolar structure loss and pulmonary regeneration impairment, but also to excessive inflammatory response, proteolytic and anti-proteolytic activity imbalance, lung epithelial cells apoptosis, and abnormal lung remodeling. To ameliorate lung damage with higher efficiency in lung tissue engineering and cell therapy, pre-differentiating graft cells into more restricted cell types before transplantation could enhance their ability to anatomically and functionally integrate into damaged lung. In this study, we aimed to evaluate the regenerative and repair ability of lung alveolar epithelium in emphysema model by using lung epithelial progenitors which pre-differentiated from amniotic fluid mesenchymal stem cells (AFMSCs).

Methods: Pre-differentiation of eGFP-expressing AFMSCs to lung epithelial progenitor-like cells (LEPLCs) was established under a modified small airway growth media (mSAGM) for 7-day induction. Pre-differentiated AFMSCs were intratracheally injected into porcine pancreatic elastase (PPE)-induced emphysema mice at day 14, and then inflammatory-, fibrotic-, and emphysema-related indices and pathological changes were assessed at 6 weeks after PPE administration.

Results: An optimal LEPLCs pre-differentiation condition has been achieved, which resulted in a yield of approximately 20% lung epithelial progenitors-like cells from AFMSCs in a 7-day period. In PPE-induced emphysema mice, transplantation of LEPLCs significantly improved regeneration of lung tissues through integrating into the lung alveolar structure, relieved airway inflammation, increased expression of growth factors such as vascular endothelial growth factor (VEGF), and reduced matrix metalloproteinases and lung remodeling factors when compared with mice injected with AFMSCs. Histopathologic examination observed a significant amelioration in DNA damage in alveolar cells, detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL), the mean linear intercept, and the collagen deposition in the LEPLC-transplanted groups.

Conclusion: Transplantation of predifferentiated AFMSCs through intratracheal injection showed better alveolar regeneration and reverse elastase-induced pulmonary emphysema in PPE-induced pulmonary emphysema mice.

Keywords: Amniotic fluid mesenchymal stem cell; Elastase-induced pulmonary emphysema; Predifferentiation.

Fig. 1

Characterization of predifferentiated AFMSCs (LEPLCs) under modified small airway growth medium (mSAGM) induction. AFMSCs were differentiated for 7 days and then immunostained with lung epithelial progenitor-like markers TTF-1, SPC, AQP-5, and CCSP. a Flow cytometry analysis was performed. b Immunocytochemistry staining for endogenous eGFP (green) and lung epithelial progenitor-like markers (red) in differentiated cells at 7 days. Scale bar = 200 μm

Fig. 2

Transplantation of LEPLCs reduced emphysematous histopathologic changes and downregulated the expression of emphysema-related genes in the PPE-induced pulmonary emphysema mouse model. Effects of LEPLCs treatment on tissue regeneration and reversal of lung emphysema. Lung tissues were collected at week 6 post-PPE treatment. a H&E stained histological sections from each group. Scale bar = 100 μm. b H&E staining was calculated as histopathological scores, n ≥ 5. Quantitative real-time RT-PCR was performed to analyze the mRNA expression levels of emphysema-related genes, c elastin, d, Vegfa, e Icam-1, f Mmp-9, and g Spa. Values were normalized to the glyceraldehyde-3-phosphate dehydrogenase (Gapdh) gene and expressed in relation to the PBS group. Data are represented as the mean ± SD; n ≥ 5 per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001

Fig. 3

Transplantation of LEPLCs downregulated the expression of inflammatory mediators in the PPE-induced pulmonary emphysema mouse model. Effects of LEPLCs treatment on the inflammatory response. Lung tissues were collected at week 6 post-PPE treatment for inflammatory-related genes, and mRNA expression levels of a pro-Il-1β, b, Il-6, c Inos, and d Mcp-1 were determined by quantitative real-time RT-PCR. Values were normalized to the Gapdh gene and expressed in relation to the PBS group. Data are represented as the mean ± SD; n ≥ 5 per group. *p < 0.05; **p < 0.01

Fig. 4

Transplantation of LEPLCs reduced fibrotic histopathologic changes and downregulated the expression of fibrotic-related genes in the PPE-induced pulmonary emphysema mouse model. Effects of LEPLCs treatment on extracellular matrix production and fibrosis-related genes. Lung tissues were collected at week 6 post-PPE treatment. a Masson’s trichrome, and c Sirius red stained histological sections from each group. Scale bar = 100 μm. The boxed regions in the a upper panel are magnified in the lower panel, and the violet staining indicates collagenous material. The fibrillar collagen appears as red structures (arrow). b, d Masson trichrome and Sirius red staining were calculated as histopathological scores. In all sub figs., n ≥ 5. Quantitative real-time RT-PCR was performed to analyze the mRNA expression of fibrotic-related genes e ColI, f, ColIII, and g Tgf-β1. Values were normalized to the Gapdh gene and expressed in relation to the PBS group. Data are represented as the mean ± SD; n ≥ 5 per group. * p < 0.05; ** p < 0.01

Fig. 5

Expression and localization of eGFP-labeled transplanted cells in the lung of PPE-induced pulmonary emphysema mouse model. a Quantitative real-time RT-PCR and b Western blot analysis were performed to detect GFP expression in the lung tissues of mice after 4-week period of PBS, AFMSCs, or LEPLCs treatment following PPE administration. Values were normalized to Gapdh or β-actin levels and were expressed relative to the PBS group. *p < 0.05, **p < 0.01, and ***p < 0.001. Each dot represents an individual mouse with the mean shown for n > 5 per group. c Immunofluorescence staining for the distribution of GFP in lung tissues. Green signal observed in the lung section indicates GFP-positive cells and nuclei were stained with DAPI (blue). Magnified views with 20 μm bars (third row) expand on the boxed regions in the low-magnified images with 100 μm bars (second row)

Fig. 6

Transplantation of LEPLCs enhanced the expression of lung epithelial progenitor-like markers in the PPE-induced pulmonary emphysema mouse model. a Quantitative real-time RT-PCR and b Western blots analysis expression of SPC and AQP-5 in whole lung tissues of animals that received PBS, AFMSCs, or LEPLCs after 4-week period of PBS, AFMSCs, or LEPLCs treatment following PPE administration. Values were normalized to the Gapdh values and were expressed relative to the PBS group. The histogram on the right shows the semiquantitative densitometry of the Western blot analysis determined using ImageJ. Data are expressed as the mean ± SD; n ≥ 5 per group. *p < 0.05, **p < 0.01, and ***p < 0.001. c, d Immunofluorescence staining of (c) GFP-positive cells (green) and type II alveolar epithelial markers, pro-SPC (red); d type I alveolar epithelial markers, AQP-5 (green), and nucleus (DAPI; blue) in mouse lung tissues from each group treated as described above. The white dotted line areas in the (c) upper panel are magnified in the lower panel with 10 μm bars, and the expressions of pro-SPC and GFP were co-localized (yellow, arrowheads). Scale bar = 100 μm

Fig. 7

Transplantation of LEPLCs attenuated apoptosis in the PPE-induced pulmonary emphysema mouse model. C57BL/6 mice received PBS, AFMSCs, or LEPLCs after 4-week period of PBS, AFMSCs, or LEPLCs treatment following PPE administration. Lung tissues were subjected to immunofluorescence staining to determine the colocalization of apoptotic cells (TUNEL stain; green) and nucleus (DAPI; blue). Scale bar = 100 μm