| First Author | Izadifar M | Year | 2024 |
| Journal | Stem Cell Res Ther | Volume | 15 |
| Issue | 1 | Pages | 486 |
| PubMed ID | 39696686 | Mgi Jnum | J:373650 |
| Mgi Id | MGI:8248799 | Doi | 10.1186/s13287-024-04059-7 |
| Citation | Izadifar M, et al. (2024) Microfluidic chip systems for characterizing glucose-responsive insulin-secreting cells equipped with FailSafe kill-switch. Stem Cell Res Ther 15(1):486 |
| abstractText | BACKGROUND: Pluripotent cell-derived islet replacement therapy offers promise for treating Type 1 diabetes (T1D), but concerns about uncontrolled cell proliferation and tumorigenicity present significant safety challenges. To address the safety concern, this study aims to establish a proof-of-concept for a glucose-responsive, insulin-secreting cell line integrated with a built-in FailSafe kill-switch. METHOD: We generated beta cell-induced progenitor-like cells (betaiPLCs) from primary mouse pancreatic beta cells through interrupted reprogramming. Then, we transcriptionally linked our FailSafe (FS) kill-switch, HSV-thymidine kinase (TK), to Cdk1 gene using a CRISPR/Cas9 knock-in strategy, resulting in a FailSafe betaiPLC line, designated as FSbetaiPLCs. Subsequently we evaluated and confirmed the functionality of the drug-inducible kill-switch in FSbetaiPLCs at different ganciclovir (GCV) concentrations using our PDMS-based transcapillary microfluidic system. Finally, we assessed the functionality of FSbetaiPLCs by characterizing the dynamics of insulin secretion in response to changes in glucose concentration using our microfluidic perfusion glucose-stimulated insulin secretion (GSIS) assay-on- chip. RESULTS: The betaiPLCs exhibited Ins1, Pdx1 and Nkx6.1 expression, and glucose responsive insulin secretion, the essential properties of pancreatic beta cells. The betaiPLCs were amenable to genome editing which allowed for the insertion of the kill-switch into the 3'UTR of Cdk1, confirmed by PCR genotyping. Our transcapillary microfluidic system confirmed the functionality of the drug-inducible kill-switch in FSbetaiPLCs, showing an effective cell ablation of dividing cells from a heterogeneous cell population at different ganciclovir (GCV) concentrations. The Ki67 expression assessment further confirmed that slow- or non-dividing cells in the FSbetaiPLC population were resistant to GCV. Our perfusion glucose-stimulated insulin secretion (GSIS) assay-on-chip revealed that the resistant non-dividing FSbetaiPLCs exhibited higher levels of insulin secretion and glucose responsiveness compared to their proliferating counterparts. CONCLUSIONS: This study establishes a proof-of-concept for the integration of a FailSafe kill-switch system into a glucose-responsive, insulin-secreting cell line to address the safety concerns in stem cell-derived cell replacement treatment for T1D. The microfluidic systems provided valuable insights into the functionality and safety of these engineered cells, demonstrating the potential of the kill-switch to reduce the risk of tumorigenicity in pluripotent cell-derived insulin-secreting cells. |
