ABSTRACT
: Induced pluripotent stem cells (iPSCs) can be efficiently differentiated into retinal pigment epithelium (RPE), offering the possibility of autologous cell replacement therapy for retinal degeneration stemming from RPE loss. The generation and maintenance of epithelial apical-basolateral polarity is fundamental for iPSC-derived RPE (iPSC-RPE) to recapitulate native RPE structure and function. Presently, no criteria have been established to determine clonal or donor based heterogeneity in the polarization and maturation state of iPSC-RPE. We provide an unbiased structural, molecular, and physiological evaluation of 15 iPSC-RPE that have been derived from distinct tissues from several different donors. We assessed the intact RPE monolayer in terms of an ATP-dependent signaling pathway that drives critical aspects of RPE function, including calcium and electrophysiological responses, as well as steady-state fluid transport. These responses have key in vivo counterparts that together help determine the homeostasis of the distal retina. We characterized the donor and clonal variation and found that iPSC-RPE function was more significantly affected by the genetic differences between different donors than the epigenetic differences associated with different starting tissues. This study provides a reference dataset to authenticate genetically diverse iPSC-RPE derived for clinical applications. SIGNIFICANCE: The retinal pigment epithelium (RPE) is essential for maintaining visual function. RPE derived from human induced pluripotent stem cells (iPSC-RPE) offer a promising cell-based transplantation therapy for slowing or rescuing RPE-induced visual function loss. For effective treatment, iPSC-RPE must recapitulate the physiology of native human RPE. A set of physiologically relevant functional assays are provided that assess the polarized functional activity and maturation state of the intact RPE monolayer. The present data show that donor-to-donor variability exceeds the tissue-to-tissue variability for a given donor and provides, for the first time, criteria necessary to identify iPSC-RPE most suitable for clinical application.
ABSTRACT
We describe the structural and functional properties of three-dimensional (3D) nerve guides fabricated from poly-ε-caprolactone (PCL) using the air gap electrospinning process. This process makes it possible to deposit nano-to-micron diameter fibers into linear bundles that are aligned in parallel with the long axis of a cylindrical construct. By varying starting electrospinning conditions it is possible to modulate scaffold material properties and void space volume. The architecture of these constructs provides thousands of potential channels to direct axon growth. In cell culture functional assays, scaffolds composed of individual PCL fibers ranging from 400 to 1500 nm supported the penetration and growth of axons from rat dorsal root ganglion. To test the efficacy of our guide design we reconstructed 10mm lesions in the rodent sciatic nerve with scaffolds that had fibers 1 µm in average diameter and void volumes >90%. Seven weeks post implantation, microscopic examination of the regenerating tissue revealed dense, parallel arrays of myelinated and non-myelinated axons. Functional blood vessels were scattered throughout the implant. We speculate that end organ targeting might be improved in nerve injuries if axons can be directed to regenerate along specific tissue planes by a guide composed of 3D fiber arrays.
