3D Poly(Lactic Acid) Scaffolds Promote Different Behaviors on Endothelial Progenitors and Adipose-Derived Stromal Cells in Comparison With Standard 2D Cultures

Tissue engineering is a branch of regenerative medicine, which comprises the combination of biomaterials, cells and other bioactive molecules to regenerate tissues. Biomaterial scaffolds act as substrate and as physical support for cells and they can also reproduce the extracellular matrix cues. Although tissue engineering applications in cellular therapy tend to focus on the use of specialized cells from particular tissues or stem cells, little attention has been paid to endothelial progenitors, an important cell type in tissue regeneration. We combined 3D printed poly(lactic acid) scaffolds comprising two different pore sizes with human adipose-derived stromal cells (hASCs) and expanded CD133+ cells to evaluate how these two cell types respond to the different architectures. hASCs represent an ideal source of cells for tissue engineering applications due to their low immunogenicity, paracrine activity and ability to differentiate. Expanded CD133+ cells were isolated from umbilical cord blood and represent a source of endothelial-like cells with angiogenic potential. Fluorescence microscopy and scanning electron microscopy showed that both cell types were able to adhere to the scaffolds and maintain their characteristic morphologies. The porous PLA scaffolds stimulated cell cycle progression of hASCs but led to an arrest in the G1 phase and reduced proliferation of expanded CD133+ cells. Also, while hASCs maintained their undifferentiated profile after 7 days of culture on the scaffolds, expanded CD133+ cells presented a reduction of the von Willebrand factor (vWF), which affected the cells’ angiogenic potential. We did not observe changes in cell behavior for any of the parameters analyzed between the scaffolds with different pore sizes, but the 3D environment created by the scaffolds had different effects on the cell types tested. Unlike the extensively used mesenchymal stem cell types, the 3D PLA scaffolds led to opposite behaviors of the expanded CD133+ cells in terms of cytotoxicity, proliferation and immunophenotype. The results obtained reinforce the importance of studying how different cell types respond to 3D culture systems when considering the scaffold approach for tissue engineering.

Regenerative capacity of the corneal transition zone for endothelial cell therapy

The corneal endothelium located on the posterior corneal surface is responsible for regulating stromal hydration. This is contributed by a monolayer of corneal endothelial cells (CECs), which are metabolically active in a continuous fluid-coupled efflux of ions from the corneal stroma into the aqueous humor, preventing stromal over-hydration and preserving the orderly arrangement of stromal collagen fibrils, which is essential for corneal transparency. Mature CECs do not have regenerative capacity and cell loss due to aging and diseases results in irreversible stromal edema and a loss of corneal clarity. The current gold standard of treatment for this worldwide blindness caused by corneal endothelial failure is the corneal transplantation using cadaveric donor corneas. The top indication is Fuchs corneal endothelial dystrophy/degeneration, which represents 39% of all corneal transplants performed. However, the global shortage of transplantable donor corneas has restricted the treatment outcomes, hence instigating a need to research for alternative therapies. One such avenue is the CEC regeneration from endothelial progenitors, which have been identified in the peripheral endothelium and the adjacent transition zone. This review examines the evidence supporting the existence of endothelial progenitors in the posterior limbus and summarizes the existing knowledge on the microanatomy of the transitional zone. We give an overview of the isolation and ex vivo propagation of human endothelial progenitors in the transition zone, and their growth and differentiation capacity to the corneal endothelium. Transplanting these bioengineered constructs into in vivo models of corneal endothelial degeneration will prove the efficacy and viability, and the long-term maintenance of functional endothelium. This will develop a novel regenerative therapy for the management of corneal endothelial diseases.

ETV2 (Ets Variant Transcription Factor 2)- Rhoj Cascade Regulates Endothelial Progenitor Cell Migration During Embryogenesis

Objective: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. The regulatory mechanisms that govern their migration are not completely defined. Here, we describe a novel role for ETV2 (Ets variant transcription factor 2) in cell migration and provide evidence for an ETV2 -Rhoj network as a mechanism responsible for this process. Approach and Results: Analysis of RNAseq datasets showed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 chromatin immunoprecipitation-seq and assay for transposase accessible chromatin-seq datasets, which showed enrichment of chromatin immunoprecipitation-seq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Migratory assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells, embryoid bodies, and mouse embryonic fibroblasts. Knockout of Etv2 led to migratory defects of Etv2-EYFP + angioblasts to their predefined regions of developing embryos relative to wild-type controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds the promoter region of Rhoj serving as an upstream regulator of cell migration. Single-cell RNAseq analysis of Etv2-EYFP + sorted cells revealed coexpression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both embryoid bodies and mouse embryonic fibroblasts, whereas, its expression was abolished in the Etv2 knockout embryoid bodies. Finally, shRNA-mediated knockdown of Rhoj resulted in migration defects, which were partially rescued by overexpression of ETV2. Conclusions: These results define an ETV2 -Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration.

Abstract 16812: ETV2- Rhoj Cascade Regulates Endothelial Progenitor Cell Migration During Embryogenesis

Background: Endothelial progenitors migrate early during embryogenesis to form the primary vascular plexus. ETV2, an Ets-transcription factor, governs the specification of the earliest hemato-endothelial progenitors during embryogenesis. The regulatory mechanisms that govern their migration are undefined. In the present study, we describe a novel role for ETV2 in cell migration and provide evidence for an ETV2 -Rhoj network as a mechanism responsible for this process. Approach and Results: We analyzed our RNAseq datasets, which revealed robust enrichment of migratory/motility pathways following overexpression of ETV2 during mesodermal differentiation. We then analyzed ETV2 ChIPseq and ATACseq datasets, which showed enrichment of ChIPseq peaks with increased chromatin accessibility in migratory genes following overexpression of ETV2. Additionally, scratch and sprouting assays showed that overexpression of ETV2 enhanced cell migration in mouse embryonic stem cells (ESCs), embryoid bodies (EBs) and mouse embryonic fibroblasts (MEFs). Knockout of Etv2 led to migratory defects of Etv2-EYFP + angioblasts to their pre-defined regions of developing embryos relative to wildtype controls at embryonic day (E) 8.5, supporting its role during migration. Mechanistically, we showed that ETV2 binds to the promoter region of Rhoj serving as an upstream regulator of cell migration. Single cell RNAseq analysis of Etv2-EYFP + sorted cells revealed co-expression of Etv2 and Rhoj in endothelial progenitors at E7.75 and E8.25. Overexpression of ETV2 led to a robust increase in Rhoj in both EBs and MEFs, whereas, its expression was abolished in the Etv2 knockout EBs. Finally, shRNA-mediated knockdown of Rhoj resulted in migratory defects which were rescued by overexpression of ETV2. Conclusions: These results define an ETV2 -Rhoj cascade, which is important for the regulation of endothelial progenitor cell migration during embryogenesis.