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PANCREAS Publication-derived

Type 1 Diabetes Pancreatic Organoids

Full SOP with Academic or a plan

Source Yoshihara et al., 2020 · Salk Institute for Biological Studies · 10.1038/s41586-020-2631-z

Authors: Eiji Yoshihara, Carolyn O'Connor, Emanuel Gasser, Zong Wei, Tae Gyu Oh, Tiffany W. Tseng, Dan Wang, Fritz Cayabyab, Yang Dai, Ruth T. Yu, Christopher Liddle, Annette R. Atkins, Michael Downes, Ronald M. Evans Duration: 90 days 8 phases Cell source: Human iPSC

Abstract

This protocol generates glucose-responsive human islet-like organoids (HILOs) from induced pluripotent stem cells (iPSCs) that can be engineered to express PD-L1 for immune evasion. The organoids contain functional endocrine-like cells that restore glucose homeostasis when transplanted into diabetic mice and represent a potential therapeutic alternative to cadaveric islet transplantation.

Cell source
Human iPSC
Application
Disease modeling; Diabetes therapy

Protocol overview

30 steps across 8 phases

Phase 1 · iPSC Maintenance and Reporter Line Generation Days 0-14
  1. a Maintain hiPSCs on matrigel-coated plates
  2. b Infect hiPSCs with insulin GFP reporter lentivirus
  3. c Generate PD-L1-expressing hiPSC clones
Phase 2 · Definitive Endoderm Induction (Stage 1) Days 0-4
  1. a Prepare single-cell suspension and initiate 3D culture
  2. b Begin Stage 1 differentiation (Definitive Endoderm)
  3. c Continue Stage 1 with Activin A only
Phase 3 · Pancreatic Specification (Stages 2-3) Days 4-19
  1. a Stage 2: Pancreatic lineage specification
  2. b Stage 3a: Pancreatic endocrine progenitor induction (early phase)
  3. c Stage 3b: Pancreatic endocrine progenitor induction (late phase)
  4. d Stage 4: Pancreatic endocrine differentiation
Phase 4 · Endocrine Progenitor Expansion (Stages 5) Days 14-30
  1. a Stage 5a: Early endocrine progenitor expansion
  2. b Stage 5b: Transition to maturation phase
Phase 5 · WNT4-Driven Metabolic Maturation (Days 26-36) Days 26-36
  1. a Add WNT4 for metabolic maturation
  2. b Optional: Prepare WNT5A-conditioned media
Phase 6 · Immune Evasion Engineering (Days 30-42) Days 30-42
  1. a Prepare wHILOs for immune evasion (IFN-γ pulse stimulation protocol)
  2. b Apply multiple pulse stimulation (MPS) for sustained immune evasion
  3. c Alternative: Exogenous PD-L1 overexpression
Phase 7 · Quality Control and Functional Assays (Days 30-50) Days 30-50
  1. a Glucose-stimulated insulin secretion (GSIS) assay
  2. b Oxygen consumption rate (OCR) measurement
  3. c Immunohistochemistry for cell markers
  4. d Flow cytometry for endocrine cell composition
  5. e Quantitative RT-PCR for gene expression verification
  6. f Single-cell RNA sequencing (optional, for detailed characterization)
Phase 8 · Transplantation and In Vivo Validation (Days 50+) Days 50-90+
  1. a Preparation of mice and induction of diabetes
  2. b Prepare HILOs for transplantation
  3. c Surgical transplantation under kidney capsule
  4. d Monitor blood glucose post-transplantation
  5. e Measure serum human c-peptide levels
  6. f Graft removal (nephrectomy) to confirm HILO function
  7. g Immune profiling of recovered grafts

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Attribution

This SOP was written by Organthis based on the published method in Yoshihara et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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