Non-viral Co-transfection of Plasticity-inducing and β Cell Patterning Transcription Factors Mediates Pro-β Cell Reprogramming in Fibroblast Cultures
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Date
2024-03
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Abstract
Introduction: Previously, we have shown that plasmid DNA that encodes transcription factors for β cell patterning (e.g., Pdx1, Ngn3, Mafa) can drive the direct reprogramming of dermal fibroblasts (DFs) into induced β cells (iβCs) with potential to become an alternative method to treat type I diabetes. Recently, our objective has been to investigate whether plasmid DNA that encodes transcription factors for skin plasticity (e.g., Tcf3, Sox9, Trp63) can increase DFs multipotency and, therefore, β cell reprogramming efficiency.
Materials and Methods: We identified 3 transcription factors (3SP) for skin cell plasticity and 7 transcription factors (7βC) for β cell development. Bulk electroporation (BEP) was utilized for cell samples of ~1.2 million and nanoelectroporation (NEP) was utilized for cell samples of ~250,000. Combinations of plasmid DNA encoding for 3SP factors or pCMV6 (control plasmid DNA) were delivered to mouse embryonic fibroblasts (MEFs) in vitro using BEP. Combinations of plasmid DNA encoding for 3SP+7βC factors or pCMV6 (control plasmid DNA) were delivered MEFs in vitro using BEP and NEP. Gene and protein expressions were analyzed using qRT-PCR or immunohistology as appropriate. Data was collected and analyzed by blinded investigators using Prism GraphPad and ImageJ.
Results: The ability of 3SP to open the chromatin landscape was significant 7 days post-BEP. Furthermore, MEFs transfected with 3SP exhibited decreased fluorescent methylation markers 7 days post-BEP. MEFs transfected with 3SP+7βC transcription factors significantly increased gene expression for both insulin 1 and insulin 2 14 days post-BEP. This increased insulin 1/2 expression correlated with a small population ( < 1%) of transfected cells expressing insulin protein 14 days post-BEP and a larger population (>1%) expressing insulin protein 14 days post-NEP.
Conclusion: Our findings suggest that DFs transfected with plasmid DNA encoding for 3SP factors exhibit increased multipotency. Furthermore, DFs transfected with DNA encoding for 3SP+7βC factors have the potential to reprogram into iβCs. Current experimentation includes transcription factor reduction and transition to mouse models. Future development of this alternative cell source to treat and potentially cure type I diabetes could greatly improve quality of life for diabetic patients all over the world.
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Engineering and Technology (The Ohio State University Denman Undergraduate Research
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Keywords
Type 1 Diabetes, Non-viral Cell Reprogramming, Gene and Cell Therapy, β Cell Replacement