Scaffolds from Induced Pluripotent Stem Cell-Derived Fibroblast Matrix for Diabetic Foot Ulcer Treatment

Diabetic foot ulcers (DFU) are highly prevalent amongst diabetics (60 M) and a major economic burden (760 billion USD). DFU are chronic wounds refractory to surgical debridement and therapeutic dressings, with15-20% of cases requiring limb amputation. Tissue engineering solutions such as Omnigraft®, a collagen-glycosaminoglycan (CG) scaffold, resolved 50% of DFUs; thus, there is still an unmet clinical need. In this thesis, human inducible pluripotent stem cells (iPSC) derived fibroblasts are explored, as a source of material for scaffold fabrication. These fibroblasts have been described as rejuvenated fibroblasts capable of increased extracellular matrix (ECM) production. Their ECM is enriched in elements such as collagen III, fibronectin (similarly to what is found in foetal wounds, which heal scar free). Thus, the aim of this thesis is to harness the potential of iPSC-derived fibroblast ECM, to fabricate a freeze-dried scaffold. This (i) combines the iPSC ECM with the porous scaffold fabrication technique, (ii) facilitates rejuvenated autograft transplant fabrication and (iii) avoids the challenges of direct cell therapy.

The study presented in Chapter 2 describes the fabrication of scaffolds comprising a 1:1 mixture of collagen I and ECM from a well-studied cell line iPSC derived fibroblasts (post-iPSF). The results showed that the new scaffolds (i) supported DFU fibroblast growth, (ii) showed higher presence of collagen III and IV versus CG scaffolds with (iii) comparable vascularization potential.

Using the same technique from Chapter 2, scaffolds were generated using iPSC-reprogrammed fibroblasts that were derived from biopsied DFU patients in the clinic (i-DFU) in Chapter 3. These scaffolds facilitated DFU growth (i.e., the patient-matched cells pre-reprogramming) and yielded more Collagen I, III and laminin presence vs CG controls seeded with the same cells. These scaffolds had a minimal effect on normal patient fibroblasts

An alternative scaffold fabrication process was developed in Chapter 4, by allowing cell line post-iPSF to deposit ECM in situ on CG scaffolds. After decellularization, DFU were seeded for 3 weeks exhibiting: (i) increased GAG presence, matching control levels, (ii) showing higher Collagen IV presence versus DFU seeded CG, while (iii) keeping same vascularization potential as CG control. Of note, Post-iPSF seeded CG exhibited low friction during handling (interesting for chondrogenic repair)

In chapter 5, the decellularization technique from chapter 4 was applied to iPSC-reprogrammed fibroblasts that were derived from biopsied DFU patients in the clinic. These scaffolds allowed patient-matched DFU growth and GAG deposition to the same extent as DFU seeded on CG control. Specifically, Collagen I and III levels were found elevated versus DFU CG control. Collagen I and III presence was observed at higher level in DFU (i.e., the patient-matched cells pre-reprogramming) seeded scaffolds versus normal patient fibroblasts (from a different patient), resembling effect from chapter 3. At the same time, i-DFU decellularized scaffolds had higher average vascularization over CG, and no adverse innate immune response was observed in vitro.

A lubricating effect was observed for the scaffolds produced in Chapter 4, which motivated the further investigation of these scaffolds for translation to chondrogenic repair (Chapter 6). Lubrication appeared after 3 weeks in culture. Inclusion of post-iPSF ECM in scaffolds (as per chapter 2) was seen to outperform CG scaffolds soaked with lubricin. Patient biopsy-derived cells on CG had high lubrication, but did not show a strong change in their lubricating properties following iPSC reprogramming. This lubricating effect shows promise to enhance the initial lubrication properties of chondrogenic scaffolds.

Collectively, this thesis investigates different approaches to exploit iPSC-derived fibroblast ECM, and for the first time demonstrates its use as a material for scaffold fabrication. The results motivate the in-vivo testing to fully unveil their benefits for DFU healing. Additionally, the thesis highlights a possible “ECM-memory” mechanism, improving DFU performance on patient-matched scaffolds. Finally, this material can be translated to cartilage research for its lubricating properties.