Scaffolds Functionalized with Matrix from Induced Pluripotent Stem Cell Fibroblasts for Diabetic Wound Healing

Abstract

Diabetic foot ulcers (DFUs) are chronic wounds, with 20% of cases resulting in amputation, despite intervention. A recently approved tissue engineering product—a cell‐free collagen‐glycosaminoglycan (GAG) scaffold—demonstrates 50% success, motivating its functionalization with extracellular matrix (ECM). Induced pluripotent stem cell (iPSC) technology reprograms somatic cells into an embryonic‐like state. Recent findings describe how iPSCs‐derived fibroblasts (“post‐iPSF”) are proangiogenic, produce more ECM than their somatic precursors (“pre‐iPSF”), and their ECM has characteristics of foetal ECM (a wound regeneration advantage, as fetuses heal scar‐free). ECM production is 45% higher from post‐iPSF and has favorable components (e.g., Collagen I and III, and fibronectin). Herein, a freeze‐dried scaffold using ECM grown by post‐iPSF cells (Post‐iPSF Coll) is developed and tested vs precursors ECM‐activated scaffolds (Pre‐iPSF Coll). When seeded with healthy or DFU fibroblasts, both ECM‐derived scaffolds have more diverse ECM and more robust immune responses to cues. Post‐iPSF‐Coll had higher GAG, higher cell content, higher Vascular Endothelial Growth Factor (VEGF) in DFUs, and higher Interleukin‐1‐receptor antagonist (IL‐1ra) vs. pre‐iPSF Coll. This work constitutes the first step in exploiting ECM from iPSF for tissue engineering scaffolds.

Optimization of extracellular matrix production from human induced pluripotent stem cell‐derived fibroblasts for scaffold fabrication for application in wound healing

“…Our work showed that scaffolds made of iPSFs ECM when seeded with healthy or diabetic foot ulcers fibroblast, enhanced the production of key ECM components and VEGF release in both types of fibroblasts and induced robust immune response to cues. In addition, iPSF scaffolds induced higher GAG, VEGF and higher cell content in diabetic foot ulcers fibroblasts when compared to scaffolds made of source fibroblasts 10 …”

“…In addition to Omnigraft, other bioengineered skin substitutes such as Apligraft® and Dermagraf® have been developed and implemented in clinics, although with similar success rates 9 . Recently we functionalized collagen‐based scaffolds with ECM grown by induced pluripotent stem (iPS) derived fibroblasts (iPSF) and tested its potential in wound healing 10 . The motivation for the inclusion of iPS‐derived fibroblast ECM was due to its previous demonstration of increased ECM production compared to source fibroblasts 10,11 .…”

“…One of the limitations of using cell‐derived matrix is achieving sufficient in vitro production of ECM for scaffold fabrication within an acceptable timeframe. In order to scale‐up ECM production, we have tested several cell culture conditions and identified optimal cell density and media composition that increased matrix deposition twofold without adversely affecting the ratio of different matrix components 10 . Nonetheless, in traditional 2D cell culturing methods, cells are seeded on a plastic surface with specific cell media that is quite different from physiological conditions 13 .…”

“…Most studies, to our knowledge, that implemented macromolecular crowders to increase collagen deposition were done in a short time period of 2–7 days, 21,22 and did not exceed 14 days 23,24 . However, our prior observations suggest that in order to produce reasonable amounts of matrix for scaffold fabrication, a much longer cell culturing time of at least 3–5 weeks is necessary 10 …”

Cellular reprogramming of diabetic foot ulcer fibroblasts triggers pro‐healing miRNA‐mediated epigenetic signature

“…*=p ≤ 0.05, **=p ≤ 0.01, ***=p ≤ 0.001 by healing-promoting phenotype in vitro and in vivo murine models of diabetic wound healing when compared to parental fibroblasts, and we have characterized their gene expression profiles postreprogramming. 5,10,36 Our previous studies also identified an aberrant miRNA and DNA methylation signature of primary human fibroblasts derived from DFU. 2,37 Thus, the current work focused on identifying miR driven epigenetic changes in iPSC-derived DFU fibroblasts that have regained healthy cellular functions upon reprogramming.…”