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Background <p>Induced pluripotent stem cell (iPSC)-derived β-like cells hold great promise for cell replacement therapy in type 1 diabetes. However, the reprogramming process generates iPSC clones with variable differentiation capacity, hindering the selection of optimal cell lines. This study aimed to identify an early-stage transcriptional signature capable of predicting the β cell differentiation potential of donor-matched iPSC clones.</p> Methods <p>Eleven iPSC clones derived from a single donor were differentiated to the definitive endoderm (DE) stage; six were further driven toward pancreatic progenitors (PP) and insulin-producing cells. Differentiation efficiency was evaluated by flow cytometry and qPCR at iPSC, DE, PP, and β cell stages. At the pluripotent stage, expression profiling of 770 genes related to pluripotency and trilineage specification was performed to identify predictive molecular markers.</p> Results <p>Transcriptomic analysis segregated the clones into two groups (Gr1 and Gr2) with significantly different differentiation outcomes. Gr2 clones exhibited superior DE efficiency (Cxcr4⁺: 90.1 ± 5.6% vs. 79.8 ± 3.6%; <i>P</i> = 0.027) and higher expression of PP markers (Pdx1⁺, Nkx6.1⁺, and double-positive cells; <i>P</i> ≤ 0.05). At the β cell stage, Gr2 clones showed increased frequencies of Pdx1⁺/Ins⁺ and Nkx6.1⁺/Ins⁺ cells (<i>P</i> ≤ 0.05), along with enhanced glucose-stimulated insulin secretion. A set of 73 differentially expressed genes, enriched in pathways related to naïve/primed pluripotency, endoderm commitment, and metabolism, was identified. From this, a ten-gene signature validated by qPCR strongly correlated with pancreatic marker expression at all stages.</p> Conclusions <p>An early gene expression signature at the pluripotent stage predicts the pancreatic endocrine differentiation potential of iPSC clones. This molecular screening approach may enable rapid preselection of high-performing clones, thereby accelerating the development of personalized stem cell–based therapies for diabetes.&#xa0;</p> Summary <p> Cellular reprogramming is a fundamental tool in regenerative medicine but often produces iPSC clones with heterogeneous differentiation potential. Identifying the most suitable clones typically requires time-consuming assays and prolonged in vitro testing. This study presents a streamlined transcriptomic approach to predict, at the pluripotent stage, the differentiation efficiency of iPSC clones into pancreatic endoderm and insulin-producing cells, enabling early selection of high-performing lines for the development of diabetes cell therapy.</p>

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Gene Expression at the Pluripotency Stage Predicts Pancreatic Endocrine Differentiation in iPSC Clones

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月4日

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  • Valentina Zamarian,
  • Laura Monaco,
  • Manuela Marras,
  • Chiara Ceriani,
  • Silvia Pellegrini,
  • Lorenzo Piemonti,
  • Valeria Sordi

摘要

Background

Induced pluripotent stem cell (iPSC)-derived β-like cells hold great promise for cell replacement therapy in type 1 diabetes. However, the reprogramming process generates iPSC clones with variable differentiation capacity, hindering the selection of optimal cell lines. This study aimed to identify an early-stage transcriptional signature capable of predicting the β cell differentiation potential of donor-matched iPSC clones.

Methods

Eleven iPSC clones derived from a single donor were differentiated to the definitive endoderm (DE) stage; six were further driven toward pancreatic progenitors (PP) and insulin-producing cells. Differentiation efficiency was evaluated by flow cytometry and qPCR at iPSC, DE, PP, and β cell stages. At the pluripotent stage, expression profiling of 770 genes related to pluripotency and trilineage specification was performed to identify predictive molecular markers.

Results

Transcriptomic analysis segregated the clones into two groups (Gr1 and Gr2) with significantly different differentiation outcomes. Gr2 clones exhibited superior DE efficiency (Cxcr4⁺: 90.1 ± 5.6% vs. 79.8 ± 3.6%; P = 0.027) and higher expression of PP markers (Pdx1⁺, Nkx6.1⁺, and double-positive cells; P ≤ 0.05). At the β cell stage, Gr2 clones showed increased frequencies of Pdx1⁺/Ins⁺ and Nkx6.1⁺/Ins⁺ cells (P ≤ 0.05), along with enhanced glucose-stimulated insulin secretion. A set of 73 differentially expressed genes, enriched in pathways related to naïve/primed pluripotency, endoderm commitment, and metabolism, was identified. From this, a ten-gene signature validated by qPCR strongly correlated with pancreatic marker expression at all stages.

Conclusions

An early gene expression signature at the pluripotent stage predicts the pancreatic endocrine differentiation potential of iPSC clones. This molecular screening approach may enable rapid preselection of high-performing clones, thereby accelerating the development of personalized stem cell–based therapies for diabetes. 

Summary

Cellular reprogramming is a fundamental tool in regenerative medicine but often produces iPSC clones with heterogeneous differentiation potential. Identifying the most suitable clones typically requires time-consuming assays and prolonged in vitro testing. This study presents a streamlined transcriptomic approach to predict, at the pluripotent stage, the differentiation efficiency of iPSC clones into pancreatic endoderm and insulin-producing cells, enabling early selection of high-performing lines for the development of diabetes cell therapy.