What does this research mean for the field?

Mesenchymal-like off-target cells in hPSC-derived pancreatic islet clusters originate from mesodermal contamination during definitive endoderm differentiation and can be eliminated by genetically restricting mesoderm specification. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

What question did this study set out to answer?

The aim was to characterize mesenchymal off-target cells during the differentiation of human pluripotent stem cells into pancreatic islet-like clusters.

June 7, 2026

3025-LB: Characterization and Elimination of Mesenchymal Off-Target Cells during Differentiation of Human Pluripotent Stem Cells to Pancreatic Islet-Like Clusters

Key Points

The aim was to characterize mesenchymal off-target cells during the differentiation of human pluripotent stem cells into pancreatic islet-like clusters.
Differentiation of hPSC into islet-like clusters using a suspension-based protocol.
Analysis via flow cytometry, single-cell RNA sequencing, and spatial transcriptomics.
Development of an assay for selective expansion of mesenchymal-like cells.
Islet-like clusters demonstrated robust endocrine function in vitro and in vivo.
Mesenchymal-like off-target cells were detected using the new sensitive assay.
Genetic engineering of hPSC effectively eliminated mesenchymal-like cells from the final clusters.

Abstract

Introduction and Objective: Differentiation of human pluripotent stem cells (hPSC) into the pancreatic endocrine lineage is a promising approach for type 1 diabetes therapy. However, off-target cell types arising during differentiation raise safety concerns. We aimed to characterize these off-target populations, determine their developmental origins, and develop strategies to detect and eliminate them. Methods: Islet-like clusters were generated using a suspension-based hPSC differentiation protocol and analyzed by flow cytometry, histology, single-cell RNA sequencing, and spatial transcriptomics. Functional assessment was performed in vitro and after transplantation into diabetic and non-diabetic mice. To improve sensitivity for detecting non-endocrine contaminants, we developed an assay enabling selective expansion of mesenchymal-like cells. scRNA-seq was used to trace the lineage origins of off-target populations, and hPSC were genetically engineered to limit mesoderm formation during definitive endoderm (DE) differentiation. Results: The resulting islet-like clusters showed robust endocrine function in vitro and in vivo. Nonetheless, mesenchymal-like off-target cells were present, and conventional assays failed to detect them reliably or predict their emergence after transplantation. The newly developed assay enabled sensitive detection of mesenchymal-like cells by promoting their in vitro expansion. scRNA-seq revealed that these cells originated from minor mesodermal contamination during DE differentiation. Engineering hPSC to restrict mesoderm specification eliminated mesenchymal-like cells from the final clusters. Conclusion: Comprehensive characterization of off-target populations is essential for generating safe hPSC-derived islet-like clusters. Our detection assay and lineage-restriction strategy effectively remove mesenchymal-like contaminants, supporting the development of safer cell therapy products. Disclosure C. Honore: Employee; Current; Novo Nordisk A/S. Stock/Shareholder; Current; Novo Nordisk A/S.

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