Background: Induced pluripotent stem cells (iPSCs)-derived β cells are promising candidates for the cell therapy of type 1 diabetes. However, cellular reprogramming generates a variable number of iPSC clones with unpredictable differentiation potential. The aim of this study is the identification of a gene signature for the selection of the best β cell differentiation-prone iPSC clone from a patient.

Methods: Eleven iPSC clones were generated from the same donor. iPSC clones were stabilized and differentiated in vitro into cells of the definitive endoderm (DE) stage. The presence of the pluripotency marker OCT4 and the definitive endoderm marker CXCR4 were evaluated by flow cytometry analysis. A gene expression analysis was then performed on the 11 clones at the iPSC and DE stages through Nanostring technology. The expression of 770 genes involved in stemness and trilineage specification was determined and the differential gene expression was correlated with differentiation efficiency. Finally, four iPSC clones were selected and differentiated into mature β cells.

Results: Flow cytometry analysis showed that the 11 iPSC clones, starting with a homogeneous level of pluripotency (Oct4 ≥ 95%), have a heterogeneous differentiation potential at the DE stage (45% ≤ Cxcr4 ≥ 96%). Gene expression analysis, at the iPSC stage, revealed 129 differentially expressed genes (P-Adj <0.05), which compartmentalize the 11 iPSC clones into two distinct clusters. The differentiation into β cells of 4 selected iPSC clones belonging to the 2 clusters showed a distinct differentiation efficiency, as mirrored by the insulin-positive cells at the final stage (P <0.01). Furthermore, the analysis of the identified genes, such as ZFHX3, BMP7, KLF4, ROR2, and WNT3, involved in the stem cell pluripotency, Hippo, Wnt, and TGF-β pathways, directly correlated with the differentiation efficiency. Validation of the gene signature on new batches of iPSC from other patients and at the protein level is ongoing.

Conclusions: This study enabled the identification of genes differentially expressed at the pluripotency stage suitable for the selection of the best β cell differentiation-prone iPSC clones from a donor, for future cell therapy.