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LIVER Publication-derived

Placenta Mesenchymal Stem Cell-Derived Extracellular Vesicles Alleviate Liver Fibrosis by Inactivating Hepatic Stellate Cells through a miR-378c/SKP2 Axis

Source Zheng et al., 2023 · Nantong University Affiliated Hospital and Wake Forest Institute for Regenerative Medicine · 10.1186/s41232-023-00297-z

👤 Wenjie Zheng, Saiyan Bian, Shi Qiu, Colin E. Bishop, Meimei Wan, Nuo Xu, Xieyin Sun, Russel Clive Sequeira, Anthony Atala, Zhifeng Gu, Weixin Zhao ⏱ 16 days 📋 10 phases 🧫 Human placenta-derived MSCs; primary human hepatocytes, Kupffer cells, hepatic stellate cells, and liver sinusoidal endothelial cells

Abstract

This protocol describes the construction of multicellular liver organoids and hepatic stellate cell spheroids to model TGF-β1-induced liver fibrosis and evaluate the therapeutic effects of placenta-derived mesenchymal stem cell extracellular vesicles (Pd-MSCs-EVs). The protocol demonstrates that Pd-MSCs-EVs alleviate fibrotic phenotypes and inactivate hepatic stellate cells through a miR-378c/SKP2-dependent mechanism.

Cell source
Human placenta-derived MSCs; primary human hepatocytes, Kupffer cells, hepatic stellate cells, and liver sinusoidal endothelial cells
Application
Disease modeling (liver fibrosis); extracellular vesicle therapeutic evaluation; drug screening

Protocol overview

55 steps across 10 phases

MSC Characterization and Culture Days 1–7+
  1. 1 Culture Placenta-Derived MSCs
  2. 2 Confirm MSC Surface Marker Expression by Flow Cytometry
  3. 3 Verify MSC Differentiation Capacity
Extracellular Vesicle Extraction and Characterization Days 2–4
  1. 1 Collect Conditioned Medium from Pd-MSCs
  2. 2 Differential Centrifugation
  3. 3 Filtration
  4. 4 Ultracentrifugation and Resuspension
  5. 5 Measure Exosome Concentration
  6. 6 Transmission Electron Microscopy Characterization
  7. 7 Nanoparticle Size Characterization
  8. 8 Confirm Exosome Markers by Western Blotting
Liver Organoid Construction Days 1–7+
  1. 1 Prepare and Thaw Primary Liver Cells
  2. 2 Seed Liver Organoid Precursors
  3. 3 Culture Organoids with Medium Replacement
  4. 4 Assess Organoid Viability and Function
Hepatic Stellate Cell Spheroid Construction Days 1–7+
  1. 1 Culture Human Hepatic Stellate Cells
  2. 2 Seed HSC-Derived Spheroids
  3. 3 Maintain HSC-Derived Spheroids
TGF-β1-Induced Fibrosis Modeling Days 10–16
  1. 1 Optimize TGF-β1 Concentration
  2. 2 Measure Albumin and Inflammatory Markers
  3. 3 Assess Pro-Collagen Expression
  4. 4 Confirm Fibrosis at mRNA Level
  5. 5 Confirm Fibrosis by Immunofluorescence
Pd-MSCs-EV Treatment and Efficacy Assessment Days 15–16 (organoids); variable (2D HSCs)
  1. 1 Determine Optimal EV Uptake Conditions
  2. 2 Treat Fibrotic Organoids with Pd-MSCs-EVs
  3. 3 Assess Growth Status and Viability
  4. 4 Measure Recovery of Albumin and Inflammatory Markers
  5. 5 Confirm Reversal of Fibrotic Markers at mRNA Level
  6. 6 Confirm Reduction of Fibrotic Protein Expression
  7. 7 Perform Immunohistochemical Staining
miR-378c Functional Studies Variable (typically Days 1–7 for transfection experiments)
  1. 1 Identify Differential miRNAs by Small RNA Sequencing
  2. 2 Validate Differential miRNA Expression by qRT-PCR
  3. 3 Generate EVs with miR-378c Inhibitor
  4. 4 Evaluate Impact of miR-378c Depletion on Fibrosis Reversal
  5. 5 Assess EMT Markers in Response to miR-378c
HSC 2D Culture Studies and miR-378c Target Validation Variable (Days 1–5 for HSC experiments)
  1. 1 Activate HSCs with TGF-β1
  2. 2 Transfect miR-378c Mimics into HSCs
  3. 3 Measure HSC Proliferation by CCK-8 Assay
  4. 4 Assess HSC Migration by Transwell Assay
  5. 5 Perform Wound Healing Assay
  6. 6 Measure Fibrotic and EMT Marker Expression in HSCs
  7. 7 Assess Cell Cycle Distribution by Flow Cytometry
  8. 8 Assess Apoptosis by Flow Cytometry
SKP2 Target Validation and Mechanism Elucidation Variable (Days 1–7 for mechanistic studies)
  1. 1 Predict miR-378c Target Genes
  2. 2 Validate miR-378c Regulation of SKP2 in 293T Cells
  3. 3 Validate miR-378c Regulation of SKP2 in HSCs
  4. 4 Assess SKP2-Mediated E-cadherin Ubiquitination
  5. 5 Perform Cycloheximide-Based Protein Stability Assay
  6. 6 Evaluate SKP2 Rescue Experiments
  7. 7 Assess Clinical Correlation in Human Fibrotic Tissues
Supporting Assays and Quality Control Throughout study
  1. 1 Perform EdU Proliferative Assay
  2. 2 Perform Live/Dead Viability Assay on Organoids and Spheroids
  3. 3 Perform H&E Staining of Organoid Sections
  4. 4 Verify EV Labeling for Uptake Studies
  5. 5 Measure Albumin Secretion by Organoids

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Zheng et al., 2023. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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