
==== Front
Stem Cells Transl Med
Stem Cells Transl Med
stcltm
Stem Cells Translational Medicine
2157-6564
2157-6580
Oxford University Press US

39077914
10.1093/stcltm/szae051
szae051
Tissue Engineering and Regenerative Medicine
AcademicSubjects/MED00770
AcademicSubjects/SCI00960
Three-dimensional cultured human umbilical cord mesenchymal stem cells attenuate pulmonary fibrosis by improving the balance of mitochondrial fusion and fission
Zhai Huifang Department of Clinical Nursing, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, People’s Republic of China

Jiang Mengqi Department of Cellular and Genetic Medicine, Binzhou Medical University, Yantai 264003, People’s Republic of China

Zhao Yaqin Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, People’s Republic of China

Wang Yujie Department of Cellular and Genetic Medicine, Binzhou Medical University, Yantai 264003, People’s Republic of China

Zhang Haitong Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, People’s Republic of China

Ji Yunxia Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, People’s Republic of China

Song Xiaodong Department of Cellular and Genetic Medicine, Binzhou Medical University, Yantai 264003, People’s Republic of China

Zhang Jinjin Department of Cellular and Genetic Medicine, Binzhou Medical University, Yantai 264003, People’s Republic of China

Lv Changjun Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, People’s Republic of China

https://orcid.org/0000-0002-8995-3443
Li Minge Department of Clinical Nursing, Binzhou Medical University Hospital, Binzhou Medical University, Binzhou 256603, People’s Republic of China

Huifang Zhai, Mengqi Jiang and Yaqin Zhao contributed equally to this work.

Corresponding author: Minge Li, Department of Clinical Nursing, Binzhou Medical University Hospital, Binzhou Medical University, No. 522, Huanghe Road, Binzhou 256603, China (byfylme@163.com); or, Changjun Lv, Department of Respiratory and Critical Care Medicine, Binzhou Medical University Hospital, Binzhou Medical University, No. 522, Huanghe Road, Binzhou 256603, China (lucky_lcj@sina.com); or, Jinjin Zhang, Department of Cellular and Genetic Medicine, Binzhou Medical University, No.346, Guanhai Road, Yantai 264003, China (Jjinzhang@126.com).
9 2024
30 7 2024
30 7 2024
13 9 912926
30 11 2023
15 6 2024
© The Author(s) 2024. Published by Oxford University Press.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.

Abstract

Pulmonary fibrosis is a kind of fibrotic interstitial pneumonia with poor prognosis. Aging, environmental pollution, and coronavirus disease 2019 are considered as independent risk factors for pulmonary fibrogenesis. Consequently, the morbidity and mortality striking continues to rise in recent years. However, the clinical therapeutic efficacy is very limited and unsatisfactory. So it is necessary to develop a new effective therapeutic approach for pulmonary fibrosis. Human umbilical cord mesenchymal stem cells (hucMSCs) are considered as a promising treatment for various diseases because of their multiple differentiation and immunomodulatory function. The key bottleneck in the clinical application of hucMSCs therapy is the high-quality and large-scale production. This study used FloTrix miniSpin bioreactor, a three-dimensional (3D) cell culture system, for large-scale expansion of hucMSCs in vitro, and proved 3D cultured hucMSCs inhibited the differentiation of fibroblasts into myofibroblasts and myofibroblasts proliferation and migration, leading to slow down the development of pulmonary fibrosis. Further mechanistic studies clarified that hucMSCs reduced the amount of binding between circELP2 and miR-630, resulting in blocking YAP/TAZ translocation from cytoplasm to nucleus. This condition inhibited mitochondrial fusion and promoted mitochondrial fission, and ultimately improved fusion/fission balance and cellular homeostasis. To sum up, this work clarified the anti-fibrosis and mechanism of hucMSCs cultured from the 3D FloTrix miniSpin bioreactor. We hope to provide new ideas and new methods for the clinical transformation and industrialization of hucMSCs therapy.

Graphical Abstract

Graphical Abstract

pulmonary fibrosis
hucMSCs
circRNA
YAP/TAZ
mitochondrial fusion
mitochondrial fission
National Natural Science Foundation of China 10.13039/501100001809 82370094 82370079 42207495 82170085 81970064 81870001
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pmcSignificance statement

In recent years, the pulmonary fibrosis morbidity and mortality continue to increase, so developing a new effective therapeutic approach against pulmonary fibrosis is necessary. However, the specific mechanism of human umbilical cord mesenchymal stem cells in the treatment of pulmonary fibrosis is still unclear, and traditional two-dimensional planar culture is difficult to meet the requirements of large-scale clinical treatment. Our findings suggest that three-dimensional cultured hucMSCs attenuate pulmonary fibrosis by improving the balance of mitochondrial fusion and fission through the circELP2-miR-630-YAP/TAZ-mLST8 pathway. This study provides a new idea to fight against pulmonary fibrosis, which has potential universal applicability in the treatment of fibrosis diseases.

Introduction

Pulmonary fibrosis is a kind of end-stage change in lung diseases, characterized by epithelium injury, fibroblast-activated myofibroblast, and accumulation of extracellular matrix. These characteristics lead to the destruction of lung tissue structure, loss of elasticity, and decreased lung ventilation capacity, which all seriously affect the respiratory function of the human body, leading to dry cough, and progressive dyspnea. With the aggravation of pulmonary fibrosis, the respiratory function of patients continues to deteriorate and ultimately leads to death from respiratory failure.1-3 The prognosis of pulmonary fibrosis is very poor, and the average survival time after diagnosis is only about 2.5 years.4,5 Aging is commonly considered as an independent risk factor for pulmonary fibrosis, and the risk in people aged 70 years or over is 6.9 times more than that in people aged over 40 years.6,7 With the aggravation of environmental pollution, people pay increased attention to environmental pollution-induced pulmonary fibrosis.8-10 As one of the complications of coronavirus disease 2019, pulmonary fibrosis has received widespread attention in recent years.11,12 Consequently, the morbidity and mortality continue to increase in recent years. Antifibrotic agents pirfenidone and nintedanib are currently utilized for treating pulmonary fibrosis. However, the clinical therapeutic efficacy is very limited and unsatisfactory.13,14 So, developing a new effective therapeutic approach for pulmonary fibrosis is necessary.

Human umbilical cord mesenchymal stem cells (hucMSCs) are considered as a promising treatment for various diseases because of their multiple differentiation and immunomodulatory function.15,16 The umbilical cord belongs to newborn tissue, and the extracted stem cells are more primitive and have stronger proliferation and differentiation ability than the stem cells derived from other sources such as bone marrow, adipose, and circulating endothelial progenitor cells. Hence, research on the effect of hucMSCs on pulmonary fibrosis is increasing.17-19 However, 2 critical issues limit the clinical application of hucMSCs. First, the specific mechanism of hucMSCs in treating pulmonary fibrosis remains unclarified. Second, the traditional two-dimensional (2D) planar culture is difficult to meet the large-scale requirements of clinical hucMSCs therapy. In this study, a three-dimensional (3D) bioreactor was used to culture hucMSCs, and the anti-pulmonary fibrotic mechanism of hucMSCs was further explored.

Circular RNA (circRNA) is a special non-coding RNA with closed ring structure. It is usually derived from the precursor mRNA through back-splicing. Increasing reports found that a large number of circRNAs can control the function and regulatory mechanism of stem cells, thus highlighting the role of circRNA as a new potential therapeutic target for stem cell therapy.20,21 In the authors’ previous study, a preliminary investigation about the regulatory roles of circRNAs in idiopathic pulmonary fibrosis (IPF) was performed in plasma samples of patients with IPF. Among these significantly dysregulated circRNAs, circRNA_102348 can directly interact with miR-630 to promote pulmonary fibrosis.22 However, whether it can be a therapeutic target for stem cells against pulmonary fibrosis is not elucidated. In the present work, circRNA_102348 was renamed as circELP2, and whether the circELP2-mediated signal pathway could be the therapeutic target for hucMSCs therapy of pulmonary fibrosis was explored. This work could provide an option for high-quality and large-scale production of hucMSCs and circRNA-based therapeutic strategy.

Materials and methods

Cell culture

Human fetal lung fibroblast cell line MRC-5 was purchased from American Type Culture Collection. The cells were maintained in advanced minimum essential medium and supplemented with 10% fetal bovine serum (FBS, Hyclone), 100 U/mL penicillin, and 100 mg/mL streptomycin, and incubated at 37 °C in an incubator under a humidified atmosphere of 5% CO2 and 95% air. Passage was performed when cell density was about 90%. According to the requirements of the experiments, MRC-5 cells were divided into normal group, TGFβ1 group, TGFβ1 + hucMSCs group, TGFβ1 + hucMSCs + circELP2 blank plasmid (BP)/recombinant plasmid (RP) group, and TGFβ1 + hucMSCs + circELP2 RP + miR-630 mimic/NC group. Except the cells in the normal group, other cells were first treated with 5 ng/mL TGFβ1 and /or hucMSCs conditioned media for 72 hours. Then, the cells were transfected with overexpressed circELP2 RP plasmids (OBio, Shanghai) for 48 hours, overexpressed circELP2 BP plasmids (OBio, Shanghai) for 48 hours, miR-630 mimic (RiboBio, Guangzhou) for 48 hours, or miR-630 mimic NC (RiboBio, Guangzhou) for 48 hours.

Preparation and culture of hucMSCs

The research was approved by the Ethics Committee of Binzhou Medical University (No. 2022-423). The umbilical cords were donated by the lying-in women signed Written Informed Consent Form. hucMSCs were prepared from newborn human umbilical cords by tissue block adhesion method. Briefly, after the umbilical cord was cleaned with PBS buffer, disinfected with 75% alcohol, removed residual blood vessels and connective tissue, cut into pieces to a size of 1 mm3 and spread out on a 10 cm dish. After culture for 7 days, scattered cells appeared around the tissue block, and the cells gradually increased with time. The cells that crawled out of the tissue block were P0 generation. After the cell density was about 90%, cells were passaged with 0.25% trypsin and considered as P1 generation. When the cells spread to 3 generations, cells were collected and moved to 3D FloTrix® miniSpin bioreactor (Beijing CytoNiche Biotechnology Co. Ltd.) for 3D dynamic culture. Cells were cultured according to the manufacturer’s instructions. Briefly, 2.5 × 106 cells, 5 pieces of 3D TableTrix microslides, and 50 mL basal medium were added into the 125 mL spinning flask. The speed of the miniSpin host was set at 40 rpm/min, and the flask was placed in a 37 °C incubator with 5% CO2. After 5 days of culture, the culture flask was stood for 15 minutes to let the microcarriers with cells and culture medium stratification. The supernatant was collected for subsequent experiments. The microcarriers with cells were lysed by 3D FloTrix Digest lysate, followed by centrifugation at 1050 rpm for 5 minutes. The supernatant was discarded and cell precipitates were collected for subsequent experiments (Figure 1A).

Figure 1. hucMSCs were identified. (A) A diagram of hucMSCs culture in 3D FloTrix miniSpin bioreactor is shown. (B) hucMSCs morphology was observed under an inverted microscope. The images showed that the cells were long spindles and arranged in parallel or whirlpool. Each group requires 1 × 106 cells. (C) Representative flow cytometry analysis of cell surface markers showed that hucMSCs highly expressed CD73, CD90, and CD44; lowly expressed CD105; and did not express HLA-DR, CD34, and CD45. Each group requires 1 × 106 cells. (D) Alizarin red staining of osteogenic differentiation of hucMSCs showed the formation of bone matrix. Each group requires 3 × 105 cells. (E) Detection of fat differentiation by oil red O staining showed a large number of fat droplets in hucMSCs. Each group requires 3 × 105 cells. (F) Identification of chondrogenic induction of hucMSCs using Alisin blue staining. Each group requires 3 × 105 cells.

Adipogenic and osteogenic differentiation

Adipogenic and osteogenic differentiation were performed according to the HyCyte Human Umbilical Cord Marrow Mesenchymal Stem Cells Adipogenic Differentiation Kit and Osteogenic Differentiation Kit (Cas9X), respectively. Briefly, 4 × 104/mL hucMSCs were inoculated on 24-well plates. When the cell density was about 95%, the supernatant was discarded and the medium for adipogenic differentiation was added. The culture medium was exchanged every 3 days. After 20 days, oil red O or alizarin red dye liquor was added and observed under the microscope.

Quantitative real time polymerase chain reaction (qRT-PCR)

Total RNA was isolated from cells or tissues using TRIzol reagent. Complementary DNA synthesis was performed using an Evo M-MLV RT Premix reagent (Accurate Biology) following the manufacturer’s instructions. The PCR was performed using a 2×SYBR Green Pro Taq HS Premix (Accurate Biology) on the Rotor Gene3000 real-time PCR system (Roche, Germany). The reaction system was 20 μL and conditions were as follows: pre-denaturation at 95 °C for 10 minutes and PCR stage for 45 cycles at 95 °C for 5 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. The forward and reverse primers of circELP2 are as follows: circELP2-F 5ʹ-CTGATGAAGAGGAGCTGTTA-3ʹ and circELP2-R 5ʹ-AGAGAAAGCACTTTCTGAAAAG-3ʹ. The primers of miR-630 were synthesized by Guangzhou RiboBio Co. Ltd.

Real time cell proliferation analysis

In a 12-well plate, 1 × 105/mL MRC-5 cells were seeded. When the cell density was about 70%, the cells were treated with serum-free medium with or without TGFβ1, hucMSCs, cirELP2 RP/BP, miR-630 mimic/NC, and placed in a cell incubator. After 30 minutes, the cell samples were placed in the IncuCyte S3 live cell analysis system (Essen Bioscience) for real-time observation. The real-time proliferation of cells was automatically recorded by IncuCyte S3 instrument, and the data were analyzed according to the instrument software.

Wound healing test

5 × 104/mL MRC-5 cells were seeded in a 96-well plate. When the cells grew to more than 95% density, a scribe-line was drawn in 96 well plate with IncuCyte S3 scratcher (Sartorius, Michigan). The scratched cell residues were washed away with 1 × PBS, rinsed twice, and fresh medium was added. After incubation at 37 °C for 30 min, the cell samples were placed in an IncuCyte S3 instrument. The IncuCyte S3 instrument was pre-set to automatically repeat scanning every 6 hours. The images were exported and analyzed according to the instrument software.

Animal model

Eight weeks old C57BL/6 male mouse (average weight 20 ± 5 g) was purchased from Jinan Pengyue Experimental Animal Breeding Company. According to the experimental requirements, the mice were randomly divided into different groups (10 mice per group) by random assignment method: sham group, bleomycin (BLM) group, BLM + hucMSCs group, and BLM + hucMSCs + circELP2 BP/RP group. BLM-treated mouse was sprayed 5 mg/kg BLM with Penn-Century MicroSprayer (Penn-Century Inc.), and the sham operation mouse was sprayed with the same amount of normal saline. circELP2 BP/RP-treated mouse was sprayed 25 × 109 vg adenovirus circELP2 BP/RP. After 14 days of BLM modeling, injecting 0.1 mL were anesthetized by intraperitoneal injection of 2.5% Avertin. Lung changes of mice were assessed by using the MicroCT small animal imaging system (PerkinElmer). Lung specimens were collected for the subsequent experiments.

Bioluminescence imaging of hucMSCs

First, hucMSCs were transfected with lentiviruses carrying green fluorescent protein (GFP). The positive hucMSCs expressed GFP were screened by puromycin. Subsequently, BLM-induced pulmonary fibrosis mice were injected with positive hucMSCs via the tail vein. The images of hucMSCs distribution in mice were captured by an IVIS imaging system (Caliper PerkinElmer).

Pulmonary function analysis

After abdominal anesthesia, a small transverse incision was made in the trachea of the mice and a metal sleeve was inserted into trachea. A plastic catheter was connected to Buxco pulmonary function testing system (DSI Buxco) and mechanical ventilation was performed at a respiratory rate of 150 times/min, tidal volume of 10 mL/kg, and positive end-expiratory pressure of 3 cm H2O. Forced vital capacity (FVC) was calculated directly from the blood flow generated during lung contraction.

MicroCT imaging

MicroCT imaging system (PerkinElmer) was preheated and adjusted parameters in advance. After abdominal anesthesia, mice were placed on the MicroCT machine in the supine position and images were captured by the machine.

Flow cytometry analysis

Cell samples were collected by centrifugation. The supernatant was discarded and the precipitation was resuspended with 1 × PBS. Antibodies of CD34 (Biolegend), CD45 (Biolegend), HLA-DR (Biolegend), CD90 (Biolegend), CD105 (Biolegend), CD44 (Biolegend), and CD73 (Biolegend) were added, respectively. After 30 minutes of incubation at 4°C in the dark, each sample was added with 1mL PBS. Expression of the antibodies was detected by flow cytometry (Becton, Dickinson and Company).

Immunofluorescence observation

1 × 105/mL cells were seeded in a 24-well plate with the cell slides. The cells were washed with 1 × PBS for 3 times, immobilized with 4% histiocytic fixative for 30 minutes, and perforated with 0.3% TritonX-100. Then goat serum was incubated at room temperature for 30 minutes, primary antibody was added, and incubated at 4 °C overnight. After rewarming at 37 °C for 30 minutes, fluorescently labeled secondary antibody was added. After incubation at room temperature for 50 minutes, the cells were washed with 1 × PBS. Each well was filled with 200 μL of 0.25% DAPI staining solution, stained the nucleus for 5 minutes, and then washed with 1 × PBS. Fluorescence quenching agent resistance was dropped on the cell slides. Images were taken by Laser Scanning Confocal Microscope (Zeiss LSM880).

RNA antisense purification

RNA antisense purification (RAP) experiment was performed by using the BersinBio RAP kit (BersinBio). Briefly, the cells were collected, cross-linked with 1% formaldehyde PBS and 1.375 M glycine, lysed with lysis buffer, protease inhibitor, and RNase inhibitor. DNase salt stock, DNase, EDTA, EGTA, and DTT were added to remove DNA. The supernatant was centrifuged at 16 000g for 10 minutes at 4 °C. At the same time, the probes and magnetic beads were incubated with 10 mM Tris-HCl and pretreated with 1 × hybridization buffer. Then, the supernatant samples were incubated at 65 °C for 10 minutes until denatured. The probes were added to the denatured samples and hybridized at 37 °C for 30 minutes. The samples were eluted and purified by RNA elution buffer, 5 × protein hydrolase K buffer, protease K, phenol-chloromethane-isoamyl alcohol mixture (25:24:1), 5M NaCl, glycogen, anhydrous ethanol, and RNase-free water. Finally, reverse transcription and PCR were performed. The qRT-PCR conditions were as follows: pre-denaturation at 95 °C for 10 minutes, PCR stage for 45 cycles at 95 °C for 2 seconds, 60°C for 20 seconds, and 70°C for 10 seconds. The probes of Lac Z and circELP2 were: G C C T G A T G C G G T A T T T T C T C C T T A C G C A T C T G T G C G G T A T T T C A C A C C G C A T A T G G T G C A and A T G T C C A C A G A G A G A A A G C A C T T T C T G A A A A G T T T T T G A A C T T C A G G C C A C A A A G T A T T C.

Observation of mitochondrial morphology

On glass bottom culture dishes, 1 × 105/mL MRC-5 cells were seeded. When the cell density reached up to 70%, cells were treated with TGFβ1, TGFβ1 + hucMSCs, and TGFβ1 + hucMSCs + circELP2 BP/RP, and TGFβ1 + hucMSCs + circELP2 RP + miR-630 mimic/ NC, respectively. Then, the cells were collected and stained with Mito-Tracker Red CMXRos (Beyotime, China) at 37 °C for 15 minutes. Laser scanning confocal microscope (Zeiss lsm880, Germany) was used to observe the mitochondrial morphology of cell samples.

Measurement of mitochondrial membrane potential

Measurement of mitochondrial membrane potential (MMP) was detected by JC-1 assay kit (Beyotime). On 6 cm dishes, 3 × 105/mL MRC-5 cells were planted. When the cell density was reached up to 70%, cells were treated with TGFβ1, TGFβ1 + hucMSCs and TGFβ1 + hucMSCs + circELP2 BP/RP and TGFβ1 + hucMSCs + circELP2 RP + miR-630 mimic/ NC, respectively at the desired time. After the cells were collected, they were washed twice with PBS, stained with JC-1 staining working solution, incubated in the cell incubator at 37 °C for 20 minutes in the dark, washed twice with JC-1 staining buffer, and detected by flow cytometry (Becton, Dickinson and Company).

Statistical analysis

Data were analyzed using GraphPad Prism9.0 statistical software, expressed as mean ±  SD. Corresponding statistical analysis of the 2 groups of data was conducted by t tests, and comparison among multiple groups of data was conducted by one-way analysis of variance (one-way ANOVA) and Newman–Keuls. Statistical significance was considered at P < .05.

Results

Identification of hucMSCs

Cells were collected from 3D FloTrix dynamic culture flask to identify as hucMSCs by observation of cell morphology, osteogenic and adipogenic differentiation, and cell surface markers (Figure 1A). The cell morphology was long spindle-shaped and swirled (Figure 1B). The markers on the cell surface were identified by flow cytometry. The results showed that CD90, CD73, and CD44 were expressed positively on the cell surface, whereas CD105 was expressed lowly and HLA-DR, CD34, and CD45 were not expressed (Figure 1C). Alizarin red staining showed the cells underwent osteogenic induction and differentiation (Figure 1D). Oil red O staining showed a large number of red lipid droplets in the cells (Figure 1E). Alisin blue staining identified that the cells had the ability to differentiate into cartilage (Figure 1F). These findings demonstrated that hucMSCs were successfully cultured.

First, a TGFβ1-stimulated MRC-5 cell model was established to assess the therapeutic effect of hucMSCs in vitro. Scratch healing experiment was performed to detect the influence of hucMSCs treatment on cell migration under TGFβ1 stimulation for 0, 12, and 24 h. The results displayed that the migration speed of cells in the TGFβ1 group accelerated compared with that in the normal group. The migration rate of cells in the hucMSCs treatment group significantly decreased compared with that in the TGFβ1 group (Figure 2A). The results of cell proliferation curve showed that the proliferation ability was enhanced by TGFβ1 and decreased by hucMSCs treatment (Figure 2B). Western blot results showed that the expression levels of fibrosis-related proteins in the hucMSCs treatment group significantly decreased compared with those in the TGFβ1 group (Figure 2C). These data indicated that hucMSCs have obviously therapeutic effects on pulmonary fibrosis in vitro.

Figure 2. hucMSCs demonstrated anti-pulmonary fibrotic effect in vitro and targeted mouse lungs. (A) Scratch test results showed that the migration speed of cells in the TGFβ1 group clearly accelerated compared with that in the normal group. The migration rate of cells in the hucMSCs treatment group significantly decreased compared with that in the TGFβ1 group. Each group requires 5 × 104 cells. (B) Cell proliferation curve showed that the proliferation ability of the TGFβ1 group enhanced. The cell proliferation ability of the hucMSCs treatment group decreased compared with that of the TGFβ1 group. Each group requires 1 × 105 cells. (C) Western blot detection showed that the expression levels of fibrosis-related proteins in the hucMSCs treatment group significantly decreased compared with those in the TGFβ1 group. Each group requires 1 × 106 cells. (D) BLM or hucMSCs were administered in mice by tail vein injection and tracheal spraying. (E) Cell membrane staining confirmed residual hucMSCs in the lung. The fluorescence intensity of tail vein injection was higher than that of tracheal spraying on the 28th day. (F) In-vivo imaging of small animals showed that hucMSCs could target the lungs of mice. The whole-body images of mice were observed at 3 and 7 days after hucMSCs injection via tail vein. Each bar represents mean ± SD (n = 6), *P < .05.

Second, tracheal spraying and tail vein injection were applied to confirm the optimal treatment method of hucMSCs (Figure 2D). The hucMSCs membrane was stained with DiI dye kit. The results showed that the fluorescence intensity after tail vein injection was higher than that after tracheal spraying on the 28th day, indicating that tail vein injection is more effective (Figure 2E). In-vivo imaging of small animals showed that hucMSCs-labeled GFP targeted the mouse lungs (Figure 2F). So, tail vein injection and 28 days of hucMSCs treatment were applied to assess the anti-pulmonary fibrotic effect of hucMSCs. The images from MicroCT imaging, H&E staining, and Masson’s staining in vivo showed that the alveolar wall of hucMSCs-treated mice thinned, the collagen deposition decreased, the alveolar structure effectively improved, and alveolar inflammation was significantly alleviated compared with the BLM group (Figure 3A and B). The results of lung function test showed that the forced vital capacity (FVC) of the BLM group decreased, and hucMSCs treatment improved the FVC of mice (Figure 3C). Weight monitoring showed that compared with the normal group, the BLM group showed a significant decrease in weight, but the BLM + hucMSC group showed a significant increase in weight compared to the BLM group (Figure 3D). Western blot showed that the expression of fibrosis-related proteins, including collagen, vimentin, α-SMA, fibroblast activation protein, and S100 calcium binding protein A4 (S100A4), in the hucMSCs treatment group significantly decreased compared with those in the BLM group (Figure 3E). These data indicated that hucMSCs treatment significantly reduced the degree of pulmonary fibrosis in vivo.

Figure 3. hucMSCs had anti-pulmonary fibrosis effect in vivo. (A) MicroCT imaging for small animals depicted that the BLM group had honeycomb-like changes and uneven patchy shadows compared with the sham group. These fibrotic symptoms were remarkably alleviated in the hucMSCs treatment group. There is no significant difference between the Sham + hucMSCs group and the Sham group. (B) H&E staining and Masson’s staining detected the pathological changes in lung tissue structure after hucMSCs treatment. The results showed that the BLM group had more fibrotic lesions and damaged alveolar tissue structure than the sham group. hucMSCs alleviated these morphological abnormalities and improved the lesions and symptoms. There is no significant difference between the Sham + hucMSCs group and the Sham group. (C) FVC results showed that the lung function of the BLM group decreased, and that of the hucMSCs treatment group was higher. (D) The body weight of the BLM group significantly decreased, whereas that of the hucMSCs treatment group showed a tendency to increase. (E) Western blot results showed that the expression levels of fibrosis-related proteins collagen, vimentin, α-SMA, FAP, and S100A4 in the hucMSCs treatment group significantly decreased compared with those in the BLM group. Each bar represents mean ± SD (n = 6), *P < .05.

hucMSCs alleviated pulmonary fibrosis by targeting circELP2-miR-630

circELP2 can deteriorate pulmonary fibrosis by sponging miR-630, so whether hucMSCs block the occurrence of pulmonary fibrosis through regulating circELP2-miR-630 was further explored. qRT-PCR showed that hucMSCs treatment reduced the expression of circELP2 and increased that of miR-630 (Figure 4A, B). RNA antisense purification experiment (RAP) showed that under the action of hucMSCs, the binding of circELP2 to miR-630 decreased compared with that in TGFβ1 treatment (Figure 4C). The rescue experiment of overexpressed circELP2 illustrated that circELP2 overexpression accelerated the cell proliferation and migration and reversed the downward trend caused by hucMSCs treatment. This finding indicated that circELP2 mediated the inhibition of hucMSCs on cell proliferation and migration. The rescue experiment further illustrated that miR-630 mimic reduced the cell proliferation and migration at different timepoints and reversed the upward trend caused by circELP2 overexpression. This finding indicated that circELP2 mediated the inhibitory effect of hucMSCs on cell proliferation and migration depending on miR-630 (Figure 4D–F). Western blot detection showed that overexpressed circELP2 significantly promoted the expression levels of fibrotic proteins and reversed the effect of hucMSCs on these proteins. After miR-630 mimic was transfected into cells, the expression levels of fibrotic proteins were reduced, and the effect of overexpressed circELP2 was reversed (Figure 4G, H). All the above results indicated that circELP2 mediated the anti-pulmonary fibrotic effect of hucMSCs depending on miR-630.

Figure 4. circELP2-mediated hucMSCs therapy on pulmonary fibrosis depended on miR-630. (A) qRT-PCR results showed that circELP2 expression was significantly upregulated after MRC-5 cells were stimulated with TGFβ1 for 72 h compared with that in the normal group. hucMSCs treatment repressed circELP2 expression compared with TGFβ1 treatment. Each group requires 1 × 106 cells. (B) qRT-PCR results showed that miR-630 was highly expressed in the normal group. After MRC-5 cells were stimulated with TGFβ1 for 72 h, the expression of miR-630 was significantly downregulated. hucMSCs treatment promoted miR-630 expression compared with TGFβ1 treatment. Each group requires 1 × 106 cells. (C) RAP results showed that the binding of circELP2 to miR-630 reduced under the action of hucMSCs compared with TGFβ1 action. Each group requires 4 × 107 cells. (D) Compared with TGFβ1 + hucMSCs/circELP2 BP treatment, circELP2 overexpression promoted cell proliferation and reversed the effect of hucMSCs on cell proliferation. Each group requires 1 × 105 cells. (E) miR-630 mimic inhibited cell proliferation and reversed the effect of circELP2 overexpression on cell proliferation. Each group requires 1 × 105 cells. (F) Migration images were automatically monitored using the IncuCyte S3 Live-Cell Analysis System. Compared with hucMSCs treatment, circELP2 overexpression promoted cell migration and reversed the inhibitory effect of hucMSCs on cell migration. Meanwhile, miR-630 mimic inhibited cell migration and reversed the promoting effect of circELP2 overexpression on cell migration. Each group requires 5 × 104 cells. (G) Overexpressed circELP2 significantly increased the expression levels of fibrotic proteins and reversed the effect of hucMSCs. Each group requires 1 × 106 cells. (H) miR-630 mimic reduced the expression levels of fibrotic proteins and reversed the effect of overexpressed circELP2. RP represents overexpression of circELP2; BP represents overexpression of circELP2 empty plasmid; and mimic represents miR-630 analog. Each group requires 1 × 106 cells. Each bar represents mean ± SD (n = 6), *P < .05.

Next, rescue experiments in animals were designed to prove the therapeutic effect of hucMSCs via circELP2. The overexpressed circELP2 was packaged into the adenovirus vector to spray into the mouse lung (Figure 5A). The MicroCT images showed that circELP2 overexpression aggravated the degree of fibrosis and reversed the therapeutic effect of hucMSCs (Figure 5B). The results of H&E and Masson’s staining exhibited that compared with the hucMSCs treatment/cirELP2 BP group, the circELP2 overexpression group had thickened alveolar walls, increased collagen deposition, abnormal changes in alveolar structure, and significantly aggravated alveolar inflammation (Figure 5C). Compared with hucMSCs/cirELP2 BP treatment, circELP2 overexpression worsened lung function, increased body weight loss, and increased the expression levels of fibrosis-related proteins and trans-differentiation proteins (Figure 5D-F). These data indicated that circELP2 can reverse the inhibitory effect of hucMSCs on pulmonary fibrosis in mice, and the therapeutic effect of hucMSCs is through circELP2.

Figure 5. Rescue experiments were designed to prove the therapeutic effect of hucMSCs via circELP2 in mice. (A) Schematic diagram of drug administration in mice. (B) MicroCT imaging system for small animals showed that circELP2 overexpression reversed the therapeutic effect of hucMSCs and aggravated the degree of fibrosis. (C) H&E and Masson’s staining exhibited that the mice with circELP2 overexpression had thickened alveolar walls, increased collagen deposition, abnormal changes in alveolar structure, and significantly aggravated alveolar inflammation compared with those in the BLM + hucMSCs + cirELP2 BP group. (D) Compared with the hucMSCs and cirELP2 BP treatment groups, the circELP2 overexpression group demonstrated worsening of lung function. (E) The body weight of the BLM + hucMSCs + circELP2 RP group was significantly lower than that of the BLM + hucMSCs + circELP2 BP and BLM + hucMSCs groups. (F) Compared with the BLM + hucMSCs and BLM + hucMSCs + circELP2 BP groups, the BLM + hucMSCs + circELP2 RP group demonstrated a significant increase in the expression levels of pulmonary fibrosis-related proteins. Each bar represents mean ± SD (n = 6), *P < .05.

hucMSCs blocked nuclear translocation of YAP/TAZ by targeting circELP2-miR-630

Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) are the target genes of miR-630, so the effect of hucMSCs on YAP/TAZ was further explored. YAP/TAZ mainly exists in the nucleus to exert transcriptional function by binding the target genes’ cis-acting element, and p-YAP/TAZ mainly exists in the cytoplasm. Immunofluorescence staining was performed to observe whether hucMSCs could block nuclear translocation of YAP/TAZ from the cytoplasm to the nucleus. The immunofluorescence images showed that the YAP/TAZ and p-YAP/TAZ expression levels in the hucMSCs treatment group decreased and increased, respectively, compared with those in the compared with the control group. The expression of YAP/TAZ in the nucleus increased after TGFβ1 or overexpressed circELP2 treatment, whereas that of YAP/TAZ in the nucleus decreased after hucMSCs or miR-630 mimic treatment. Meanwhile, the expression of p-YAP/TAZ in the cytoplasm decreased after TGFβ1 or overexpressed circELP2 treatment, whereas that of p-YAP/TAZ in the cytoplasm increased after hucMSCs or miR-630 mimic treatment. The images showed that upregulated circELP2 reversed the inhibitory effect of hucMSCs treatment on YAP/TAZ nuclear translocation, and miR-630 mimic reversed the effect of hucMSCs + overexpressed circELP2 treatment (Figure 6A, B). The data suggested that hucMSCs block the nuclear translocation of YAP/TAZ by targeting circELP2-miR-630. The western blot analysis showed that hucMSCs weakened YAP/TAZ expression and enhanced p-YAP/TAZ expression in vivo and vitro (Figure 6C). The rescue experiments validated that circELP2 overexpression enhanced YAP/TAZ expression and weakened p-YAP/TAZ expression compared with TGFβ1 or BLM treatment; it also reversed the effect of hucMSCs on YAP/TAZ (Figure 6D). Meanwhile, miR-630 mimic reduced YAP/TAZ expression and enhanced p-YAP/TAZ expression compared with circELP2 overexpression + hucMSCs + TGFβ1; it also reversed the effect of circELP2 overexpression (Figure 6E). The above data indicated that the effect of hucMSCs on YAP/TAZ depended on circELP2-miR-630.

Figure 6. hucMSCs blocked nuclear translocation of YAP/TAZ by targeting circELP2-miR-630. (A) Immunofluorescence staining images showed that in the TGFβ1 group, the YAP in the nucleus increased and the p-YAP in the cytoplasm decreased. Compared with TGFβ1 treatment, hucMSCs treatment significantly decreased the expression of YAP in the nucleus and increased the cytoplasmic expression of p-YAP. Overexpressed circELP2 increased YAP in the nucleus, decreased p-YAP in the cytoplasm, and reversed the effect of hucMSCs treatment. miR-630 mimic decreased YAP in the nucleus, increased p-YAP in the cytoplasm, and reversed the effect of overexpressed circELP2 treatment. Each group requires 1 × 105 cells. (B) Immunofluorescence images showed that in the TGFβ1 group, TAZ increased in the nucleus and p-TAZ decreased in the cytoplasm compared with the normal group. hucMSCs treatment significantly decreased TAZ in the nucleus and increased the cytoplasmic expression of p-TAZ. Overexpressed circELP2 increased TAZ in the nucleus, decreased p-TAZ expression in the cytoplasm, and reversed the effect of hucMSCs treatment. miR-630 mimic decreased TAZ expression in the nucleus, increased p-TAZ expression in the cytoplasm, and reversed the effect of overexpressed circELP2 treatment. Each group requires 1 × 105 cells. (C) Western blot results showed that hucMSCs treatment significantly reduced the expression levels of YAP and TAZ and increase those of p-YAP and p-TAZ proteins increased compared TGFβ1 or BLM treatment. Each group requires 1 × 106 cells. (D) Overexpression of circELP2 increased the expression levels of YAP and TAZ proteins decreased those of p-YAP and p-TAZ proteins, and reversed the effect of hucMSCs on YAP/TAZ. Each group requires 1 × 106 cells. (E) miR-630 mimic reversed the effect of overexpressed circELP2 treatment by reducing YAP/TAZ expression and increasing p-YAP/TAZ expression. Each group requires 1 × 106 cells.

hucMSCs improved the balance of mitochondrial fusion and fission through circELP2-miR-630-YAP/TAZ pathway

YAP/TAZ can affect mitochondrial structure,23-26 so the changes of mitochondrial morphology, ROS, and mitochondrial membrane potential were detected with or without si-YAP/TAZ treatment. Our results suggest that interference with YAP/TAZ can promote cell mitochondrial division (Supplementary Figure S1), reduce cell ROS level (Supplementary Figure S2), and maintain normal mitochondrial membrane potential (Supplementary Figure S3). YAP and TAZ regulate mitochondrial function by binding the cis-acting element of mLST8, so the effect of hucMSCs on mitochondrial function was further studied. Mito-Tracker staining of the mitochondria in MRC-5 cells revealed that in the TGFβ1 group, mitochondrial fusion was more common and mitochondrial morphology was filamentous. Meanwhile, in the hucMSCs treatment group, mitochondrial fission increased and mitochondrial morphology was punctate. Overexpression of circELP2 promoted mitochondrial fusion and reversed the effect of hucMSCs. The addition of miR-630 mimic promoted mitochondrial fission and reversed the effect of circELP2 overexpression (Figure 7A). Mitochondrial membrane is an important source of intracellular reactive oxygen species (ROS). In this study, ROS was detected by fluorescent probe DCFH-DA. The ROS in the TGFβ1 group was higher than that in the normal group, whereas the ROS of the hucMSCs treatment group was reduced. Overexpression of circELP2 promoted the release of ROS and reversed the effect of hucMSCs. Adding miR-630 mimic reduced ROS and reversed the effect of circELP2 overexpression (Figure 7B). JC-1 was used as a fluorescence probe to detect the changes of mitochondrial membrane potential (MMP). When the mitochondrial membrane potential is high, JC-1 exists in the form of aggregate and can generate red fluorescence. When the mitochondrial membrane potential is low, JC-1 exists in the form of monomer and can produce green fluorescence. MMP is often measured by the relative ratio of red to green fluorescence. The MMP in the TGFβ1 group was lower than that in the normal group and increased in the hucMSCs treatment group. After circELP2 was overexpressed, MMP decreased and the effect of hucMSCs was reversed. Adding miR-630 mimic increased MMP and reversed the effect of circELP2 overexpression (Figure 7C). Western blot showed that hucMSCs reduced the expression of mLST8 compared with BLM or TGFβ1 treatment in vivo and vitro (Figure 7D). circELP2 overexpression promoted mLST8 expression and reversed the inhibition of hucMSCs on mLST8. miR-630 mimic decreased mLST8 and reversed the effect of circELP2 overexpression on mLST8 (Figure 7E).

Figure 7. hucMSCs inhibited/promoted mitochondrial fusion/fission through circELP2-mediated signal pathway. (A) Mitochondrial morphology was observed using a laser scanning confocal microscope. Compared with the normal group, the TGFβ1 and hucMSCs treatment group showed an increase in mitochondrial fusion and fission, respectively. Overexpression of circELP2 promoted mitochondrial fusion, and adding miR-630 mimic increased mitochondrial fission. Each group requires 1 × 105 cells. (B) Flow cytometry was used to detect ROS. Compared with the ROS level in the normal group, those in the TGFβ1 and hucMSCs treatment groups increased and decreased, respectively. These levels were increased by overexpression of circELP2 and decreased by the addition of miR-630 mimic. Each group requires 1 × 106 cells. (C) MMP was detected by JC-1. Compared with the normal group, the TGFβ1 and hucMSCs treatment groups had decreased and increased MMP, respectively. Overexpression of circELP2 decreased MMP and reversed the effect of hucMSCs treatment. miR-630 mimic reversed the effect of overexpressed circELP2. Each group requires 1 × 106 cells. (D) Western blot experiment showed that hucMSCs reduced mLST8 expression compared with TGFβ1 or BLM. Each group requires 1 × 106 cells. (E) Rescue experiment showed that overexpression of circELP2 increased mLST8 expression and reversed the effect of hucMSCs. miR-630 mimic decreased mLST8 expression and reversed the effect of overexpressed circELP2. Each group requires 1 × 106 cells. (F) The expression of OPA1 was higher in the TGFβ1 or BLM group than in the control group, whereas that of DRP1 was lower. DRP1 was upregulated and OPA1 was downregulated by hucMSCs treatment. Each group requires 1 × 106 cells. (G) Western blot results showed that circELP2 overexpression enhanced OPA1 expression, reduced DRP1 expression, and reserved the effect of hucMSCs treatment. miR-630 mimic reversed the effect of overexpressed circELP2. Each group requires 1 × 106 cells.

Optic atrophy 1 (OPA1) and dynamin-related protein 1 (DRP1) are essential for mitochondrial fusion/fission. The western blot assay showed that OPA1 expression was significantly increased under TGFβ1 or BLM treatment and decreased by hucMSCs treatment. However, DRP1 expression showed an opposite effect (Figure 7F). The rescue experiment showed that circELP2 overexpression reduced the expression of DRP1, increased the expression of OPA1 protein, and reversed the effect of hucMSCs. Further rescue experiments showed that miR-630 mimic significantly reduced OPA1 expression, increased DRP1 expression, and reversed the effect of hucMSCs + overexpressed circELP2 (Figure 7G). These results suggested that hucMSCs treatment can promote mitochondrial fission and inhibit mitochondrial fusion through the circELP2-miR-630-YAP/TAZ pathway.

Discussion

hucMSCs therapy is a rational therapeutic strategy with broad prospects against various diseases.27-29 However, its clinical application remains limited.30,31 The key bottleneck in the clinical application of hucMSCs therapy is the high-quality and large-scale production. Clinical cell therapy requires 1010-1012 cells per batch to meet the treatment needs, and the traditional 2D planar culture methods are difficult to support.32 3D cell culture microcarriers could be the optimal strategy for stem cell culture. So, FloTrix miniSpin bioreactor, a 3D cell culture system, was used in the present study for large-scale expansion of hucMSCs in vitro. The hucMSCs from the bioreactor expressed the immunophenotype and pluripotent markers, indicating the hucMSCs still retained their immunophenotypic and stemmed characteristics. Meanwhile, the 3D cultured hucMSCs had a homing effect and targeted lung properties. The administration of tail vein injection and tracheal spraying were evaluated in mice. Tail vein injection of hucMSCs achieved better effects than tracheal spraying. The lung tissue was assumed to have 2 sets of circulatory systems, namely, systemic circulation and pulmonary circulation, thus providing the lung with good pulmonary capillary. As a result, the hucMSCs after intravenous injection were first trapped in the lung tissue and then distributed to other organs. Nozzle diameter, pressure of tracheal sprayer, and spray speed probably induced mechanical damage to hucMSCs or the lung tissue. Therefore, tail vein injection was used in mice for the subsequent experiments. The in vivo and vitro results revealed that hucMSCs treatment effectively improved the alveolar structure, reduced collagen deposition, and inhibited the expression of fibrosis-related proteins, leading to repressed development of pulmonary fibrosis. Further mechanistic studies clarified that hucMSCs reduced the amount of binding between circELP2 and miR-630, resulting in the blocking of YAP/TAZ translocation from the cytoplasm to the nucleus. This condition inhibited mitochondrial fusion and promoted mitochondrial fission via the mLST8-mediated signal pathway, and ultimately improved the balance between fusion and fission.

YAP and TAZ, which are 2 important co-transcription factors in the Hippo signaling pathway, are involved in organ growth and plasticity during development and regeneration by regulating cell growth, death, migration, and differentiation.33-35 The activation of YAP/TAZ mechanical signals can promote fibrogenesis in multiple organs, such as lung, liver, kidney, and skin, and it is the core of mechano-sensing for fibroblast activation. Haak et al elucidated that the Gα s-coupled dopamine receptor D1 (DRD1) is preferentially expressed in lung and liver mesenchymal cells. Selective YAP/TAZ inhibition in fibroblasts via DRD1 agonism reversed tissue fibrosis in mouse models.36 A recent study investigated how epithelial YAP/TAZ accelerates lung fibrogenesis and found that increased nuclear YAP/TAZ enhanced alveolar regenerative capacity and attenuated pulmonary fibrosis. On the contrary, inactivation of YAP/TAZ in alveolar type 2 decreased alveolar epithelial regeneration and increased pulmonary fibrosis.37 These findings suggested that targeting YAP/TAZ may help promote pulmonary alveolar regeneration and treat pulmonary fibrosis. However, YAP/TAZ has many functions, so blocking YAP/TAZ activation could result in multiple adverse effects, and thus, the expected effects may not be achieved.38 While circELP2 regulation is finer than YAP/TAZ regulation, circELP2 knockdown is a good antifibrosis therapeutic strategy. This study revealed that hucMSCs blocked YAP/TAZ nuclear translocation via the circELP2-mediated signal pathway to improve the balance between mitochondrial fusion and fission.

Mitochondrial fusion and fission are interdependent processes. Fusion reduces stress response by mixing the contents of normal mitochondria and partially damaged mitochondria; fission is necessary for the formation of new mitochondria while removing damaged mitochondria. The balance between mitochondrial fission and fusion dynamically regulates cellular homeostasis. Abnormal fission can lead to mitochondrial fragmentation, and abnormal fusion can lead to prolonged mitochondrial morphology, both of which can affect mitochondrial function. The imbalance between mitochondrial fusion and fission can produce giant mitochondria, which are common in pathological cells.39-41 The imbalance is usually accompanied with increased oxidative stress, which is a hallmark of pulmonary fibrosis.42,43 In turn, oxidative stress can directly affect mitochondrial dynamics, leading to imbalanced fusion and fission processes.44 In addition, the profibrotic cytokine TGFβ can trigger different mitochondrial dysfunction, including mitochondrial fusion and fission in IPF.45 OPA1 and DRP1 are the regulatory factors involved in mitochondrial fusion and fission, respectively.46-48 OPA1 facilitates a branched network formation of elongated mitochondria through the integrity of its GTPase and C-terminal coiled-coil domain. Decreased OPA1 expression by RNA interference can produce small, fragmented, and scattered mitochondria.49 A decrease in OPA1-regulated mitochondrial dynamics can help pulmonary fibrogenesis.50 DRP1-mediated mitochondrial fission is opposed by mitochondria fusion, which is driven by OPA1. A decrease in mitochondrial fusion can shift the balance towards mitochondrial fission.51 Astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on DRP1-mediated mitochondrial fission.52 The present study discovered that hucMSCs treatment inhibited OPA1 expression and promoted DRP1 expression. Fluorescence observation confirmed that hucMSCs can promote mitochondrial fission and inhibit mitochondrial fusion, thus improving the balance between fusion and fission and cellular homeostasis.

Conclusion

FloTrix miniSpin bioreactor can produce high-quality and large-scale hucMSCs. The 3D cultured hucMSCs attenuate pulmonary fibrosis by improving the balance between mitochondrial fusion and fission through the circELP2-miR-630-YAP/TAZ-mLST8 pathway. These findings can provide an approach for hucMSCs production and a strategy for circRNA-based therapy.

Supplementary material

Supplementary material is available at Stem Cells Translational Medicine online.

szae051_suppl_Supplementary_Figures_S1-S3

Acknowledgments

All authors are acknowledged for their contribution to the study.

Author contributions

Minge Li, Changjun Lv, Jinjin Zhang: conception and design, administrative support, financial support. Huifang Zhai, Mengqi Jiang, Yaqin Zhao: carried out experiments, analyzed the data, and discussed the manuscript. Haitong Zhang, Yunxia Ji, Yujie Wang: provision of study material, collection and/or assembly of data. Huifang Zhai, Xiaodong Song: data analysis and interpretation, manuscript writing. All authors have read and approved the final manuscript and have consented to publication.

Funding

This work was supported by the National Natural Science Foundation of China (82370094, 82370079, 42207495, 82170085, 81970064, and 81870001).

Conflicts of interest

The authors declare that they have no competing interests.

Data availability

All data associated with this study are present in the paper.

Ethics approval and consent to participate

The research was approved by the Ethics Committee of Binzhou Medical University (No. 2022-423). Approved project: hucMSCs regulate the occurrence and development of pulmonary fibrosis through circELP2 adsorption of miR-630 targeting YAP/ TAZ-mitochondrial pathway, Approved Date: 11/04/2022. The lying-in women donated the umbilical cords and signed a written informed consent form. It is imperative to note that all experimental procedures employed in this study were in strict conformity with the principles stipulated in the Helsinki Declaration, ensuring the ethical integrity of research involving human subjects. Animal study was conducted in accordance with the Basel Declaration of 2010.
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