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PANCREAS Publication-derived

A Human Pluripotent Stem Cell-based Platform to Study SARS-CoV-2 Tropism and Model Virus Infection in Human Cells and Organoids

Source Yang et al., 2020 · Weill Cornell Medicine · 10.1016/j.stem.2020.06.015

👤 Liuliu Yang, Yuling Han, Benjamin E. Nilsson-Payant, Vikas Gupta, Pengfei Wang, Xiaohua Duan, Xuming Tang, Jiajun Zhu, Zeping Zhao, Fabrice Jaffré, Tuo Zhang, Tae Wan Kim, Oliver Harschnitz, David Redmond, Sean Houghton, Chengyang Liu, Ali Naji, Gabriele Ciceri, Sudha Guttikonda, Yaron Bram, Duc-Huy T. Nguyen, Michele Cioffi, Vasuretha Chandar, Daisy A. Hoagland, Yaoxing Huang, Jenny Xiang, Hui Wang, David Lyden, Alain Borczuk, Huanhuan Joyce Chen, Lorenz Studer, Fong Cheng Pan, David D. Ho, Benjamin R. tenOever, Todd Evans, Robert E. Schwartz, Shuibing Chen ⏱ 48 days 📋 8 phases 🧫 Human iPSC and Human ESC

Abstract

This protocol describes the generation of multiple hPSC-derived cell types and organoids representing three germ layers to study SARS-CoV-2 tropism. The platform enables investigation of viral entry, replication, and cellular immune responses across pancreatic endocrine cells, liver organoids, cardiomyocytes, and dopaminergic neurons, with validation using primary adult human islets and liver organoids.

Cell source
Human iPSC and Human ESC
Application
Disease modeling (COVID-19), viral tropism study

Protocol overview

28 steps across 8 phases

hPSC Maintenance and Pancreatic Differentiation (Definitive Endoderm) Days 0-3
  1. 1 hPSC Culture and Seeding
  2. 2 Day 0: Definitive Endoderm Induction
  3. 3 Day 1: Medium Change
  4. 4 Day 3: Primitive Gut Tube Specification
Pancreatic Lineage Specification (Posterior Foregut to Pancreatic Endoderm) Days 5-9
  1. 1 Day 5: Posterior Foregut Induction
  2. 2 Day 7: Pancreatic Endoderm Specification
  3. 3 Day 10: Pancreatic Endocrine Precursor Specification
Pancreatic Endocrine Cell Maturation and Differentiation Days 13-34
  1. 1 Day 13: Endocrine Precursor Maturation (First 7 Days)
  2. 2 Day 20: Continued Maturation (Days 20-26)
  3. 3 Day 27: Final Maturation Medium (Days 27-34)
SARS-CoV-2 Pseudo-Entry Virus Infection Variable (24-48 hours post-infection)
  1. 1 Prepare SARS-CoV-2 Pseudo-Entry Virus
  2. 2 Seed Cells and Infect with Pseudo-Entry Virus
  3. 3 Harvest Cells at 24 Hours Post-Infection
Live SARS-CoV-2 Virus Infection 24 hours post-infection
  1. 1 Prepare Live SARS-CoV-2 Inoculum
  2. 2 Infect hPSC-Derived Cells or Organoids
  3. 3 Harvest Infected Cells
Viral Replication Assessment by qRT-PCR 24 hours post-infection (analysis day)
  1. 1 Extract Total RNA
  2. 2 Perform qRT-PCR for Viral sgRNA
  3. 3 Data Analysis and Normalization
Immunofluorescence Staining and Imaging Variable (same day as fixation or later)
  1. 1 Permeabilization
  2. 2 Primary Antibody Staining
  3. 3 Secondary Antibody Staining and Nuclear Counterstain
  4. 4 Confocal Imaging
RNA-Sequencing and Transcriptomics Analysis Post-infection (variable)
  1. 1 Prepare RNA for Library Construction
  2. 2 Prepare cDNA Libraries
  3. 3 Sequence Libraries
  4. 4 Quality Control and Read Processing
  5. 5 Differential Expression Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yang et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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