Int J Stem Cells.  2020 Jul;13(2):221-236. 10.15283/ijsc19110.

Intratracheal Transplantation of Amnion-Derived Mesenchymal Stem Cells Ameliorates Hyperoxia-Induced Neonatal Hyperoxic Lung Injury via Aminoacyl-Peptide Hydrolase

• Li Z
• Gong X
• Li D
• Yang X
• Shi Q
• Ju X

• KMID: 2504335
• DOI: http://doi.org/10.15283/ijsc19110

Abstract

Background and Objectives
Bronchopulmonary dysplasia (BPD) has major effects in premature infants. Although previous literature has indicated that mesenchymal stem cells (MSCs) can alleviate lung pathology in BPD newborns and improve the survival rate, few research have been done investigating significantly differentially expressed genes in the lungs before and after MSCs therapy. The aim of this study is to identify differentially expressed genes in lung tissues before and after hAD-MSC treatment.
Methods and Results
Human amnion-derived MSCs (hAD-MSCs) were cultured and met the MSCs criteria for cell phenotype and multidirectional differentiation. Then we confirmed the size of hAD-MSCs-EXOs and their expressed markers. An intratracheal drip of living cells showed the strongest effect on NHLI compared to cellular secretions or exosomes, both in terms of ameliorating pulmonary edema and reducing inflammatory cell infiltration. Through gene chip hybridization, PCR, and western blotting, acylaminoacyl-peptide hydrolase (APEH) expression was found to be significantly decreased under hyperoxia, and significantly increased after hAD-MSC treatment.
Conclusions
The intratracheal transplantation of hAD-MSCs ameliorated NHLI in neonatal rats through APEH.

Keyword

Bronchopulmonary dysplasia; Cell therapy; Human amniotic mesenchymal stem cells; Acylaminoacyl-peptide hydrolase

Figure

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  Fig. 1 Characterization of human amniotic mesenchymal stem cells (hAD-MSCs). (A) Overall experimental design process and grouping. (B) Within days of primary culture, small debris of amniotic membrane usually become germinal centers of adherent cells. (C) Adherent cells growing from amniotic tissue mass showed typical fibrous morphology and grew tightly. Bar=100 μm. (D) Flow cytometry showed that the cells positively expressed MSC markers (CD29, CD44, CD73, CD90, and CD105), but negatively expressed CD31, CD34, and CD45.

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  Fig. 2 Differentiation capacity of hAD-MSCs and characterization of hAD-MSC-derived exosomes (EXOs). (A) After adipogenic induction, almost all cells became broad and contained fat droplets dyed by oil red O. (B) After 21 days chondrogenic induction, hAD-MSCs expressed high concentrations of type II collagen, with a brown signal. (C) Under osteogenic conditions, MSCs aggregated, and were stained with Alizarin red, and (D) most cells were found to be alkaline phosphatase-positive, with a red signal. (E) The average diameter of the exosomes was 110 nm. (F) Western blotting indicated that hAD-MSC-EXOs expressed exosomal markers such as CD63, CD9, Hsp70 and CD81. Magnification: 100×.

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  Fig. 3 hAD-MSCs effectively improved survival and body weight. (A) Survival curve. (B) Hyperoxia exposure significantly reduced the body weight of neonatal rats. Treatment with hAD-MSCs and their derivatives did not significantly increase the body weight of neonatal rats. (C) Hyperoxia exposure significantly increased the lung weight/body weight ratio. Both the direct transfusion of cells and the transfusion of cell secretions reversed this increase. At P14: *p<0.05 vs. Normoxia group, #p<0.05 vs. Hyperoxia group.

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  Fig. 4 Histological analysis of neonatal hyperoxic lung injury (NHLI) lungs. (A∼E) Compared to normoxia exposure (A), hyperoxia exposure (B) caused capillary expansion and alveolar hyperemia, as well as infiltration of neutrophils. The direct transfusion of cells (C), and the transfusion of exosomes (EXOs) (D) and conditioned media (CM) (E) reduced pulmonary edema. (F) To evaluate the effect of hAD-MSCs on NHLI, we used a 5-level evaluation system. hAD-MSCs, hAD-MSC-EXOs, and hAD-MSC-CM significantly alleviated pulmonary edema, especially in the hAD-MSC-treated group. (G) Mean linear intercept (MLI) values in the five groups. The lung tissue sections were assessed through lung morphometry. The hyperoxia group exhibited significantly higher MLI values than the normoxia and three therapy groups. hAD-MSCs treatment significantly reduced the hyperoxia-induced increase in lung injury scores and MLI values. Magnification: 100×. *p<0.05 vs. Normoxia group, **p<0.01 vs. Normoxia group, #p<0.05 vs. Hyperoxia group, ##p<0.01 vs. Hyperoxia group.

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  Fig. 5 hAD-MSCs effectively alleviated inflammatory response. Both hAD-MSCs and their derivatives reduced the levels of (A) TNF-α, (B) IL-6, (C) IL-1β, (D) MCP-1, (E) malondialdehyde (MDA), (F) overall proteins, and (G) neutrophils in bronchoalveolar lavage fluid (BALF); these were all increased after hyperoxia exposure. (H) Hyperoxia exposure significantly decreased the expression of superoxide dismutase (SOD) in BALF. Both hAD-MSCs and hAD-MSC-EXOs significantly increased SOD expression, while hAD-MSC-derived conditioned media (CM) did not. *p<0.05 vs. Normoxia group, **p<0.01 vs. Normoxia group, #p<0.05 vs. Hyperoxia group, ##p<0.01 vs. Hyperoxia group.

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  Fig. 6 Microarray gene expression signaling pathway analysis. (A) The most significantly affected signaling pathways in the hyperoxia group compared to those in the normoxia group. (B) The most significantly affected signaling pathways in the hAD-MSCs group compared to those in the hyperoxia group.

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  Fig. 7 The expression of APEH is closely related to lung injury and treatment with hAD-MSCs. (A) RT-qPCR analysis was performed to confirm the expression profiles obtained from gene chip analysis. (B) The expression of APEH was verified using western blotting. *p<0.05 vs. Normoxia group, **p<0.01 vs. Normoxia group, #p<0.05 vs. Hyperoxia group, ##p<0.01 vs. Hyperoxia group.

Reference

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