Generation of Induced Pluripotent Stem Cells from Diabetic Foot Ulcer Fibroblasts Using a Nonintegrative Sendai Virus

Abstract

Diabetic foot ulcers (DFUs) are nonhealing chronic wounds that are a serious complication of diabetes. Since induced pluripotent stem cells (iPSCs) may offer a potent source of autologous cells to heal these wounds, we studied if repair-deficient fibroblasts, derived from DFU patients and age-and site-matched control fibroblasts, could be reprogrammed to iPSCs. To establish this, we used Sendai virus to successfully reprogram six primary fibroblast cell lines derived from ulcerated skin of two DFU patients (DFU8, DFU25), nonulcerated foot skin from two diabetic patients (DFF24, DFF9), and healthy foot skin from two nondiabetic patients (NFF12, NFF14). We confirmed reprogramming to a pluripotent state through three independent criteria: immunofluorescent staining for SSEA-4 and TRA-1-81, formation of embryoid bodies with differentiation potential to all three embryonic germ layers in vitro, and formation of teratomas in vivo. All iPSC lines showed normal karyotypes and typical, nonmethylated CpG sites for OCT4 and NANOG. iPSCs derived from DFUs were similar to those derived from site-matched nonulcerated skin from both diabetic and nondiabetic patients. These results have established for the first time that multiple, DFU-derived fibroblast cell lines can be reprogrammed with efficiencies similar to control fibroblasts, thus demonstrating their utility for future regenerative therapy of DFUs.

Differentiation of diabetic foot ulcer–derived induced pluripotent stem cells reveals distinct cellular and tissue phenotypes

“…We characterized the cellular features of iPSC-derived fibroblasts by comparing them with age-and locationmatched fibroblasts of nondiabetic and diabetic origin from which they were initially reprogrammed. Primary fibroblasts were isolated from the foot skin of patients without diabetes [nondiabetic foot fibroblasts (NFFs)], foot skin of patients with type 2 diabetes [diabetic foot fibroblasts (DFFs)], and DFUFs from patients with type 2 diabetes (8,9,31) and were reprogrammed into iPSCs that met all criteria of pluripotency (32). Using a differentiation protocol that we previously developed ( Fig.…”

“…We have recently reported the generation of iPSCs from primary, DFU-derived fibroblasts (32). In the current study, we have characterized the cellular and 3D tissue phenotypes associated with iPSC-derived fibroblasts that were initially reprogrammed from DFUs, by comparing them to site-and age-matched fibroblasts from patients with diabetes but no ulcers and from nondiabetic patients.…”

Stem Cells for Diabetes Complications: A Future Potential Cure

“…Remarkably, these newly induced fibroblasts from iPSCs from diabetic fibroblasts were similar to those from non-diabetic fibroblasts. 30 This provides evidence for the potential use of iPSCs to improve wound healing in diabetic patients. Indeed, a study by Khamaisi et al demonstrated that iPSCs in diabetic models exhibited more efficient wound healing and secreted higher levels of pro-angiogenic factors.…”

The potential and limitations of induced pluripotent stem cells to achieve wound healing

“…Despite their great promise, the use of pluripotent stem cells whether induced or embryonic is limited by their tumorigenic potential [71][72][73]. Because iPSC and human embryonic cells are capable of differentiating into cells from any of the three germ layers, they also carry the potential to form teratomas in the undifferentiated state.…”