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Introduction <p>Neonates with congenital diaphragmatic hernia (CDH) often require surgical closure of the large diaphragmatic defect with a patch; however, artificial materials are associated with complications such as foreign body reaction, infection, and recurrence. Regenerative medicine is a promising alternative for CDH patch repair, particularly through the application of stem cells and tissue engineering techniques. Despite recent research focusing on amniotic fluid stem cells, no research has focused on the use of human induced pluripotent stem cells (iPSCs) to create a skeletal muscle sheet for diaphragmatic replacement. DAPT and SB431542 (SB) have been described to promote hypertrophy and fusion of myoblasts lines for myotube formation. In this study, we generated an iPSC-derived skeletal muscle progenitor sheet and evaluated its viability for diaphragmatic replacement in CDH.</p> Methods <p>Human iPSCs (HiPS-RIKEN-2F) were cultured in StemFit AK02N medium on iMatrix511-coated dishes and differentiated into skeletal myogenic progenitor cells using a stepwise induction protocol with CHIR99021 and FGF2. After 35&#xa0;days, cells were transferred to α-MEM medium containing 2% horse serum (HS) to create a monolayer skeletal muscle progenitor sheet in a temperature-responsive culture dish. To evaluate myotube formation from myogenic progenitor cells under various conditions using compounds; DAPT, SB, and DAPT + SB combination, myosin heavy chain expression was assessed by immunofluorescence staining.</p> Results <p>Using α-MEM medium containing 2% HS, we could create a monolayer skeletal muscle progenitor sheet derived from human iPSCs. Compared to individual treatments, the most effective myotube formation was observed using a DAPT + SB combination.</p> Conclusions <p>We created, for the first time, bioengineered skeletal muscle progenitor sheet derived from human iPSCs. Combinatorial treatment with DAPT and SB was the most effective to promote myotube formation in the bioengineered sheet. iPSC-derived skeletal muscle progenitor sheets may be useful for tissue-engineered diaphragmatic replacement in the future.</p>

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Human induced pluripotent stem cell-derived bioengineered skeletal muscle progenitor sheet

  • 期刊论文

  • 发表日期：

    2025年11月21日

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  • Nozomi Aoki,
  • Yoshikazu Matsuoka,
  • Hiroshi Yamakawa,
  • Rina Tanaka,
  • Sakiko Yoshimoto,
  • Tokiko Okunobo,
  • Ryosuke Satake,
  • Hiroki Nakamura,
  • Hirofumi Hitomi,
  • Takashi Doi

摘要

Introduction

Neonates with congenital diaphragmatic hernia (CDH) often require surgical closure of the large diaphragmatic defect with a patch; however, artificial materials are associated with complications such as foreign body reaction, infection, and recurrence. Regenerative medicine is a promising alternative for CDH patch repair, particularly through the application of stem cells and tissue engineering techniques. Despite recent research focusing on amniotic fluid stem cells, no research has focused on the use of human induced pluripotent stem cells (iPSCs) to create a skeletal muscle sheet for diaphragmatic replacement. DAPT and SB431542 (SB) have been described to promote hypertrophy and fusion of myoblasts lines for myotube formation. In this study, we generated an iPSC-derived skeletal muscle progenitor sheet and evaluated its viability for diaphragmatic replacement in CDH.

Methods

Human iPSCs (HiPS-RIKEN-2F) were cultured in StemFit AK02N medium on iMatrix511-coated dishes and differentiated into skeletal myogenic progenitor cells using a stepwise induction protocol with CHIR99021 and FGF2. After 35 days, cells were transferred to α-MEM medium containing 2% horse serum (HS) to create a monolayer skeletal muscle progenitor sheet in a temperature-responsive culture dish. To evaluate myotube formation from myogenic progenitor cells under various conditions using compounds; DAPT, SB, and DAPT + SB combination, myosin heavy chain expression was assessed by immunofluorescence staining.

Results

Using α-MEM medium containing 2% HS, we could create a monolayer skeletal muscle progenitor sheet derived from human iPSCs. Compared to individual treatments, the most effective myotube formation was observed using a DAPT + SB combination.

Conclusions

We created, for the first time, bioengineered skeletal muscle progenitor sheet derived from human iPSCs. Combinatorial treatment with DAPT and SB was the most effective to promote myotube formation in the bioengineered sheet. iPSC-derived skeletal muscle progenitor sheets may be useful for tissue-engineered diaphragmatic replacement in the future.