Abstract
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. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12015-026-11091-y.