Generation of Xeno‐Free, cGMP‐Compliant Patient‐Specific iPSCs from Skin Biopsy

Abstract

This unit describes protocols for the generation of clinical-grade patient-specific induced pluripotent stem cell (iPSC)-derived retinal cells from patients with inherited retinal degenerative blindness. Specifically, we describe how using xeno-free reagents in an ISO class 5 environment, one can isolate and culture dermal fibroblasts, generate iPSCs and derive autologous retinal cells via 3D differentiation. The universal methods described herein for the isolation of dermal fibroblasts, and generation of iPSCs can be employed regardless of disease, tissue or cell type of interest.

Optimizing Donor Cellular Dissociation and Subretinal Injection Parameters for Stem Cell-Based Treatments

“…Our ultimate goal is to use these cells for loading of photoreceptor cell delivery scaffolds, which following further maturation in culture to induce photoreceptor precursor cell fate commitment, may be used for autologous cell replacement based treatment of patients with advanced retinal degenerative blindness. All steps of the 3D retinal differentiation protocol used in this study were described previously and performed in accordance with current GMP . Unlike earlier 2D differentiation protocols, in which it was difficult to separate retinal from nonretinal forebrain tissue, by using this 3D retinal differentiation approach we can readily generate and isolate RPCs in the absence of contaminating forebrain neurons (for method, refer ).…”

“…All steps of the 3D retinal differentiation protocol used in this study were described previously and performed in accordance with current GMP . Unlike earlier 2D differentiation protocols, in which it was difficult to separate retinal from nonretinal forebrain tissue, by using this 3D retinal differentiation approach we can readily generate and isolate RPCs in the absence of contaminating forebrain neurons (for method, refer ). In addition, by isolating cells from retinal organoids that are differentiated for 60 days, we are able to largely avoid contamination of inner retinal ganglion cells, which are born much earlier and largely lost by this time point due to lack of nutrient and oxygen exchange afforded to the innermost layers of the enlarged organoid .…”

“…Unlike earlier 2D differentiation protocols, in which it was difficult to separate retinal from nonretinal forebrain tissue, by using this 3D retinal differentiation approach we can readily generate and isolate RPCs in the absence of contaminating forebrain neurons (for method, refer ). In addition, by isolating cells from retinal organoids that are differentiated for 60 days, we are able to largely avoid contamination of inner retinal ganglion cells, which are born much earlier and largely lost by this time point due to lack of nutrient and oxygen exchange afforded to the innermost layers of the enlarged organoid . Following generation, we have described an effective dissociation protocol that allows us to consistently isolate RPCs for retinal cell graft production without compromising cell yield.…”

“…Three‐millimeter skin biopsies were obtained from patients with known inherited retinal disease. Fibroblast cells were isolated and expanded prior to being used for iPSC generation via overexpression of SOX2, C‐MYC, KLF4, and OCT4 as described previously . Passage 10 iPSCs were karyotyped and analyzed using the scorecard assay .…”

Feeder-free differentiation of cells exhibiting characteristics of corneal endothelium from human induced pluripotent stem cells

“…A 3 mm skin biopsy was obtained from the lower leg of a 45-year-old individual with normal ocular history and used for generation of patient-specific induced pluripotent stem cells (iPSCs) as described previously ( Sharma et al, 2017 ; Wiley et al, 2016b , 2017 ). Briefly, 250,000 patient-specific dermal fibroblasts were plated in one well of a six-well culture dish and transduced with non-integrating Sendai viral vectors driving expression of OCT4 , SOX2 , KLF4 and c-MYC at a multiplicity of infection of 3 (CytoTune-iPS Reprogramming Kit; Thermo Fisher Scientific).…”