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CARDIAC Publication-derived

Preliminary Exposure to Histone Deacetylase Inhibitors Changes the Direction of Human iPSCs' Differentiation with the Formation of Cardiospheres Instead of Skin Organoids

Source Abdyev et al., 2023 · Koltsov Institute of Developmental Biology of Russian Academy of Sciences, Moscow · 10.1134/s1062360423060024

👤 V. K. Abdyev, A. A. Riabinin, E. D. Erofeeva, M. D. Pankratova, E. A. Vorotelak, A. V. Vasiliev ⏱ 110 days 📋 9 phases 🧫 Human iPSC

Abstract

This protocol describes the differentiation of human induced pluripotent stem cells (iPSCs) into embryoid bodies and organoids using a previously established skin organoid differentiation method. When hiPSCs are pre-exposed to histone deacetylase (HDAC) inhibitors (sodium butyrate and valproic acid), the differentiation trajectory changes, resulting in the formation of contracting cardiospheres with mesodermal origin rather than skin organoids with ectodermal origin. The contracting organoids display cardiac functional properties and express early mesodermal differentiation markers.

Cell source
Human iPSC
Application
Disease modeling; Differentiation protocol development

Protocol overview

37 steps across 9 phases

hiPSC Cultivation under Control Conditions Pre-differentiation
  1. 1 Culture hiPSCs in Essential 8 medium under normoxia
  2. 2 Passage hiPSCs at 60–80% confluence
hiPSC Cultivation under HDAC Inhibitor Conditions (Experimental) Pre-differentiation
  1. 1 Prepare modified differentiation medium with HDAC inhibitors
  2. 2 Culture hiPSCs in HDAC inhibitor-supplemented medium
  3. 3 Passage HDAC inhibitor-treated hiPSCs at 60–80% confluence
Formation of Embryoid Bodies Day 0–7
  1. 1 Form embryoid bodies from control and treated hiPSCs
Embryoid Body Differentiation and Organoid Maturation (Days 1–110) Day 7–110
  1. 1 Initiate embryoid body differentiation
  2. 2 Transfer organoids to adhesive plates during maturation phase
  3. 3 Monitor organoid development and contraction
Immunocytochemical Labeling of hiPSCs Day 5 of hiPSC culture (characterization phase)
  1. 1 Wash hiPSCs with PBS
  2. 2 Fix cells with paraformaldehyde
  3. 3 Wash fixed cells and permeabilize
  4. 4 Block nonspecific antibody binding
  5. 5 Incubate with primary antibodies
  6. 6 Wash and incubate with secondary antibodies
  7. 7 Stain nuclei and mount for microscopy
Immunohistochemical Labeling of Organoid Cryosections Day 30 and Day 110 of organoid differentiation
  1. 1 Prepare organoids for cryosectioning
  2. 2 Cryoprotect organoids with sucrose gradient
  3. 3 Cut cryosections
  4. 4 Block nonspecific antibody binding in sections
  5. 5 Incubate with primary antibodies
  6. 6 Wash and apply F-actin and secondary antibodies
  7. 7 Stain nuclei and mount
Total RNA Isolation Day 5 of hiPSC culture (characterization)
  1. 1 Extract RNA using ExtractRNA reagent
  2. 2 Separate aqueous and organic phases with chloroform
  3. 3 Precipitate RNA with isopropanol
  4. 4 Wash RNA with ethanol
  5. 5 Dissolve RNA and quantify
cDNA Synthesis via Reverse Transcription Following RNA isolation
  1. 1 Prepare reverse transcription reaction mixture 1
  2. 2 Prepare reverse transcription reaction mixture 2
  3. 3 Combine mixtures and perform reverse transcription
  4. 4 Inactivate enzyme and store cDNA
  5. 5 Dilute cDNA for RT-PCR
PCR and Real-Time qPCR Analysis Following cDNA synthesis
  1. 1 Perform qualitative PCR
  2. 2 Perform real-time quantitative PCR (qPCR)
  3. 3 Calculate relative gene expression
  4. 4 Perform statistical analysis

Full SOP

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Attribution

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

This wording is awaiting legal review.

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