Plasma membrane vesicles of human umbilical cord mesenchymal stem cells ameliorate acetaminophen-induced damage in HepG2 cells: a novel stem cell therapy

Abstract

Background: Acetaminophen (APAP) overdose is the common cause of acute liver failure (ALF) due to the oxidative damage of multiple cellular components. This study aimed to investigate whether plasma membrane vesicles (PMVs) from human umbilical cord mesenchymal stem cells (hUCMSCs) could be exploited as a novel stem cell therapy for APAP-induced liver injury.

Methods: PMVs from hUCMSCs were prepared with an improved procedure including a chemical enucleation step followed by a mechanical extrusion. PMVs of hUCMSCs were characterized and supplemented to hepatocyte cultures. Rescue of APAP-induced hepatocyte damage was evaluated.

Results: The hUCMSCs displayed typical fibroblastic morphology and multipotency when cultivated under adipogenic, osteogenic, or chondrogenic conditions. PMVs of hUCMSCs maintained the stem cell phenotype, including the presence of CD13, CD29, CD44, CD73, and HLA-ABC, but the absence of CD45, CD117, CD31, CD34, and HLA-DR on the plasma membrane surface. RT-PCR and transcriptomic analyses showed that PMVs were similar to hUCMSCs in terms of mRNA profile, including the expression of stemness genes GATA4/5/6, Nanog, and Oct1/2/4. GO term analysis showed that the most prominent reduced transcripts in PMVs belong to integral membrane components, extracellular vesicular exosome, and extracellular matrix. Immunofluorescence labeling/staining and confocal microscopy assays showed that PMVs enclosed cellular organelles, including mitochondria, lysosomes, proteasomes, and endoplasmic reticula. Incorporation of the fusogenic VSV-G viral membrane glycoprotein stimulated the endosomal release of PMV contents into the cytoplasm. Further, the addition of PMVs and a mitochondrial-targeted antioxidant Mito-Tempo into cultures of APAP-treated HepG2 cells resulted in reduced cell death, enhanced viability, and increased mitochondrial membrane potential. Lastly, this study demonstrated that the redox state and activities of aminotransferases were restored in APAP-treated HepG2 cells.

Conclusions: The results suggest that PMVs from hUCMSCs could be used as a novel stem cell therapy for the treatment of APAP-induced liver injury.

Keywords: Acetaminophen (APAP); Acute liver failure (ALF); Human umbilical cord mesenchymal stem cells (hUCMSCs); Plasma membrane vesicles (PMVs); Stem cell therapy.

Fig. 1

Characterization of hUCMSCs and PMVs.a Microphotographs show cells of passages 1, 2, and 5 (upper panel). Passage 4 hUCMSCs were induced under adipogenic, osteogenic, and chondrogenic conditions for 2–3 weeks, and undifferentiated and differentiated of hUCMSCs were examined by Oil Red O, Alizarin Red, and Alcian Blue staining, respectively (lower panel), scale bar = 50 μm. b hUCMSCs were incubated with indicated PE-conjugated antibodies and then analyzed by flow cytometry; n = 3. An isotype-match antibody was used as a negative control. c PMVs were generated from hUCMSCs after staining with respective fluorescence-conjugated antibodies and then examined by confocal microscopy, scale bar = 10 μm. d Total RNA was harvested from hUCMSCs and PMVs. Transcripts of genes characteristic of hUCMSCs were amplified by RT-PCR and then analyzed by electrophoresis. The experiment had been repeated at least three times

Fig. 2

Bioinformatic analysis of the transcript profile of hUCMSCs and PMVs. Total RNAs were harvested from two preparations of hUCMSCs and PMVs and sent to Hengchuangjiyin Technology (China) for bioinformatic analysis (a, b) Diagrams (left) show the number of differentially expressed genes (DEGs) from preparations 1 and 2 of hUCMSCs vs. PMVs. The overlap of 2 circles represents common genes in both PMVs and hUCMSCs. Downregulated and upregulated DEGs were indicated with an arrow. Bar graphs (right) show genes with at least a 4-fold difference in the transcript levels for hUCMSCs vs. PMVs. The heat map of Euclidean distance (c) and hierarchical clustering profile (d) show the similarity between the transcripts of hUCMSCs and PMVs. The vertical distance reflects the similarity between samples (c). e, f GO term analysis of DEGs with at least 4-fold difference in the transcript levels between hUCMSCs and PMVs of preparations 1 and 2

Fig. 3

Detection of cellular organelles in hUCMSCs and PMVs by confocal microscopy. a–c Microphotographs show MitoTracker-Green (a, mitochondria), LysoTracker-Red (b, lysosome), and JC-1 (c, mitochondrial membrane potential) staining of hUCMSCs (upper panel, scale bar = 20 μm) and PMVs (lower panel, scale bar = 5 μm). d, e Microphotographs show the proteasome (d) and the endoplasmic reticulum (e) in hUCMSCs (upper panel, scale bar = 20 μm) and PMVs (lower panel, scale bar = 5 μm) detected by immunofluorescence staining for proteasome 20Sα5 (green) and calnexin (green), respectively. The images were taken by confocal microscopy after DAPI staining (blue)

Fig. 4

Phagocytosis and endosomal escape of PMVs. a PMVs were generated from hUCMSCs after incubation with Calcein-AM (green) for 30 min. Fusogenic VSV-G viral membrane glycoprotein was prepared from Ad293 cells after transfection with pLP-VSVG for 48 h. PMVs were incubated with or without VSV-G for 20 min and then added into HepG2 cell culture in 96-wells. Cells were stained with Hoechst (blue) at 24 h, harvested, and examined by confocal microscopy (upper panel, scale bar = 20 μm). An enlarged view of typical endosomal release is shown in the lower panel (scale bar = 10 μm). b Percentage of cells with fluorescence green signal in the cytoplasm (top, indicating endosomal escape of PMVs) and green dot per cell (bottom, number of PMVs being phagocytosed) are shown as mean ± SEM (n = 3 experiments). Student’s t test is performed between cultures with or without the addition of VSV-G. ***P < 0.001; **P < 0.01; *P < 0.05. c PMVs were generated from hUCMSCs after staining with MitoTracker-Green for 30 min. PMVs were incubated with or without VSV-G for 20 min and then added to HepG2 cell culture in 96-wells. Cells were stained with Hoechst (blue) at 24 h, harvested, and examined by confocal microscopy, scale bar = 10 μm

Fig. 5

PMVs increase the viability of HepG2 cells treated with APAP. HepG2 were treated with 90 mM APAP for 3 h. After washing, cells were harvested and re-seeded in 96-wells precoated with ECM (extracellular matrix) prepared from HTB9 cells. Mito-Tempo (MT) or PMVs from hUCMSCs (with VSV-G) were added at 4 h. PBS was used as the negative control. a Cells were stained with Calcein-AM (green) and Hoechst (blue) at 48 h. Cells in the attachment (scale bar = 100 μm) and in suspension (scale bar = 20 μm) were analyzed by confocal microscopy with a × 10 or × 63 objective. Total fluorescence intensity and fluorescence intensity of individual cells are shown as mean ± SEM (n = 3 experiments). Bars sharing a letter in common are not significantly different (P > 0.05) (one-way ANOVA Student’s t test). b Cells were stained with TMRE (red) and Hoechst (blue) at 48 h. Cells in the attachment (scale bar = 100 μm) and in suspension (scale bar = 20 μm) were analyzed by confocal microscopy with a × 10 or × 63 objective. Total fluorescence intensity and fluorescence intensity of individual cells are shown as mean ± SEM (n = 3 experiments). Bars sharing a letter in common are not significantly different (P < 0.05) (one-way ANOVA Student’s t test)

Fig. 6

PMVs rescue HepG2 cells from APAP-induced necrotic cell death. HepG2 were treated with 90 mM APAP for 3 h. After washing, cells were harvested and re-seeded in 96-wells precoated with ECM (extracellular matrix) prepared from HTB9 cells. Mito-Tempo (MT) or PMVs from hUCMSCs (with VSV-G) were added at 4 h. PBS was used as the negative control. a Cells were stained with Annexin V-FITC (green), propidium iodide (red), and Hoechst (blue) at 48 h, and subsequently, cells in an attachment were analyzed by confocal microscopy, scale bar = 50 μm. b, c Percentage of necrotic cells and viable cell number are shown as mean ± SEM (n = 3 experiments). Bars sharing a letter in common are not significantly different (P > 0.05) (one-way ANOVA Student’s t test)

Fig. 7

PMVs restored cellular physiology in APAP-treated HepG2 cells. HepG2 were treated with 90 mM APAP for 3 h. After washing, cells were harvested and re-seeded in 96-wells precoated with ECM (extracellular matrix) prepared from HTB9 cells. Mito-Tempo (MT) or PMVs from hUCMSCs (with VSV-G) were added at 4 h. PBS was used as the negative control. Intracellular levels of AST (a), ALT (b), total (T-) GSH (c), and GSSG (d) were measured using respective colorimetric approaches. GSH (e) and the ratio of GSH to GSSG (f) were calculated as described in the “Materials and methods” section. The values are shown as mean ± SEM (n = 3 experiments). Bars sharing a letter in common are not significantly different (P > 0.05) (one-way ANOVA Student’s t test)