(I-357) Non-viral Co-transfection of Plasticity-inducing and β Cell Patterning Transcription Factors Mediates Pro-β Cell Reprogramming in Fibroblast Cultures

Introduction:: Type I diabetes is driven by autoimmune destruction of pancreatic β cells and results in decreased insulin production. In a search for insulin independence, studies have focused on developing alternative cell sources for insulin production using cell reprogramming methods (e.g., iPSCs, viruses). To generate a functional cell-based therapy to treat type I diabetes, approximately 0.1 to 1 billion functional β-like cells are needed per patient. Thus, to meet this cell demand, the ideal cell source used for reprogramming should be highly accessible, easily isolated, and easily expanded. One cell source that meets these criteria is skin dermal fibroblasts (DFs). However, current approaches to β cell derivation from DFs face several hurdles (e.g., tedious pre-processing steps and viral vector reliance). 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 DFs into induced β cells (iβCs). 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, Conclusions, and Discussions:: The ability of 3SP to open the chromatin landscape was significant 7 days post-BEP (Figure 1A). Furthermore, MEFs transfected with 3SP exhibited decreased fluorescent methylation markers 7 days post-BEP (Figure 1B). MEFs transfected with 3SP+7βC transcription factors significantly increased mRNA expression for both insulin 1 and insulin 2 14 days post-BEP (Figure 1C). This increased insulin 1/2 expression correlated with a small population (< 1%) of transfected cells expressing insulin protein 14 days post-BEP (Figure 1D) and a larger population ( >1%) expressing insulin protein 14 days post-NEP (Figure 1E).

Here we continue to report on development of a non-viral approach to multipotency induction of and β-cell derivation from DFs via electroporation-based gene delivery. Our findings suggest that DFs transfected with plasmid DNA encoding for 3SP factors exhibit increased multipotency. Our findings also suggest that DFs transfected with plasmid 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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