Poster Y17 - Non-viral Cell Reprogramming Methods Mediate Fibroblast to Pro-Pancreatic β Cell Conversions that Aim to Treat Type 1 Diabetes

Introduction: Type I diabetes is driven by the autoimmune destruction of pancreatic β cells, which results in decreased insulin production and loss of homeostatic glycemic control. To generate a functional cell-based therapy to treat type I diabetes, approximately 0.1 to 1 billion functional β cells are needed per patient. Allogeneic pancreatic islet transplantation is the current gold standard cell therapy to recover insulin production. However, its implementation is limited due to a lack of donor tissue, invasive procedural nature, and the need for already immunocompromised patients to take immunosuppressants for the remainder of their lives following treatment.

To address these limitations, studies have focused on developing alternative cell sources for insulin production using many cell reprogramming methods (e.g., stem cells, viruses) with different autologous endoderm cell types (e.g., hepatocytes, acinar cells). Many of these studies have shown that induced β cells (iβCs) can be derived from induced pluripotent stem cells (iPSCs). However, the translation potential of this iβC reprogramming method is limited due to lengthy “preprocessing steps” and the potential for tumorigenicity.

Thus, a practical cell-based method to treat type I diabetes that does not require immunosuppression or invasive surgery and can meet the required cell demand without inducing uncontrolled cell growth should be investigated and developed. Here, we continue addressing limitations and reporting on iβC production using direct cell reprogramming methods (i.e., converting one cell type directly into another without inducing pluripotency). We aim to derive β cells from fibroblasts via non-viral electroporation-based gene delivery.

Materials and

Methods: We identified 3 transcription factors (3SP) for skin cell plasticity and 7 transcription factors (7βC) for β cell development and encased them into plasmid vectors. Early experimentation characterized both 3SP and 7βC in many combinations and permutations, as well as together as 3SP+7βC. Bulk electroporation (BEP) and nanoelectroporation (NEP) were utilized for in vitro plasmid DNA delivery. Tissue nanotransfection (TNT) and implantable TNT (iTNT) were utilized for in vivo plasmid DNA delivery.

In vitro: 3SP+7βC factors or pCMV6 (control plasmid DNA) were delivered to INS1-EGFP mouse embryonic fibroblasts (MEFs) via BEP or NEP. Transfected MEFs were cultured to early (i.e., 1, 4 days) and later (i.e., 7, 14, 21 days) timepoints. Depending on the experimental parameters, INS1-EGFP MEFs were cultured to later time points in regular DMEM-based media or induction DMEM-based media and exposed to short-term low-concentration trypsin 3 days post-transfection.

In vivo: 3SP+7βC factors or pCMV6 were delivered to INS1-EGFP transgenic mice via TNT or iTNT. Participating INS1-EGFP mice were transfected at early and later time points. Depending on the experimental parameters, multiple transfections, protocol modifications, alternative tissue targeting, and weekly in vivo imaging system (IVIS) images were taken as needed.

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: We found that 3SP upregulated Pdx1 1 day post-BEP, despite it not containing Pdx1 (Figure 1A/B), suggesting 3SP’s ability to open the chromatin landscape. Insulin 1 and insulin 2 expression was found to be trending towards significance (Figure 1C/D), while glucagon was found to be significantly upregulated (Figure 1E), in INS1-EGFP MEFS by day 4 post-BEP with 3SP+7βC. EGFP was significantly upregulated in INS1-EGFP MEFs 4 days post-BEP with 3SP+7βC, suggesting appropriate insulin 1 promoter activation.

Later timepoint studies revealed significant insulin 1 and insulin 2 upregulation by 14 days, as well as significant glucagon upregulation by 7 days post-BEP with 3SP+7βC (Figure 2). These findings created studies focused on the day 14 time point.

Using the more precise NEP method, INS1-GFP MEFs were transfected with 3SP+7βC and fixed at 14 days. Positive immunoreactivity for the insulin protein was detected in 3SP+7βC transfected clusters (Figure 3A). Moreover, colocalization of positive insulin immunoreactivity and EGFP fluorescence was observed, indicating successful insulin 1 promoter activation along with appropriate protein expression (Figure 3B). This established our proof of concept that MEFs could be converted into iβCs.

To increase reprogramming efficiency, INS1-EGFP MEFs were transfected with 3SP+7βC, exposed to low-concentration trypsin for 2 minutes 3 days post-BEP to promote cluster formation, and fixed at 14 days post-BEP. EGFP-producing clusters were observed by 14 days, suggesting insulin 1 promoter activity, but no positive insulin immunoreactivity (Figure 4). Confirmational gene expression profiles for this study are ongoing.

Subcutaneously inserted iTNT devices loaded with 3SP+7βC have suggested β-like cell reprogramming by day 21 post-weekly transfection and IVIS imaging (Figure 5A). Positive IVIS EGFP signal was confirmed via histological analysis, which revealed EGFP fluorescence in tissue subcutaneously in contact with the iTNT reprogramming device (Figure 5B/C). Confirmational gene expression profiles for this study are ongoing.

Our findings are currently molding future studies, which include more in vivo characterization of iTNT and TNT’s ability to promote iβC conversions in parallel with in vitro mechanism models. Success here has the potential to lead to many great outcomes, but most importantly it could greatly improve diabetic patients' quality of life.

Acknowledgements (Optional): Thank you to all university and national-level funding sources listed below for supporting this work. A special thank you to my mentors Dr. Daniel Gallego-Perez and Dr. Luke Lemmerman, who continuously support me as a type 1 diabetic researcher and my dream of seeing a world without type 1 diabetes someday. To the entire Gallego-Perez and Higuita-Castro nanomedicine lab, I would not be able to do this work without everyone's support. To Dr. John Gunn, Steven Mousetes, and the entire biomedical science program at The Ohio State University College of Medicine, I am forever grateful for you all.

National
NIH DP1DK126199
NIH DP2EB028110
NIH Director's New Innovator Award

University
The President's Research Excellence Program: Catalyst Grant
The Ohio State University Wexner Medical Center
The Ohio State University College of Medicine
The Ohio State University STEP Program