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 3-D 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. © 2017 by John Wiley & Sons, Inc.

Keywords: cGMP; current Good Manufacturing Practice; fibroblasts; induced pluripotent stem cells; photoreceptor precursor cells; retina; xeno-free.

Figure 1. Generation of patient-specific fibroblasts, iPSCs and iPSC-derived retinal cells within an FDA-registered, cGMP-compatible non-profit facility

A) Image acquired from a secure live-feed camera within the Dezii Translational Vision Research Facility at the Stephen A. Wynn Institute at the University of Iowa showing one of two working suites that include a BioSpherix Xvivo Closed Incubation System and a cellular technician feeding iPSCs through protective arm inserts. B) Light micrograph of dermal fibroblasts migrating and growing from a piece of a skin biopsy. C) Confluent patient-specific dermal fibroblasts. D) Patient fibroblasts 3 days post-Sendai viral transduction. Transduced cells are beginning to abandon the spindle appearance that is typical of fibroblasts (D′: high magnification inset). E) Developing iPSC colonies 25 days post-transduction (i.e., 21 days following passage from the 6-well plate into a 100 mm LN521-coated culture dish). Large distinct colonies can be readily identified at this stage. Note that cultures still contain un-reprogrammed patient-specific dermal fibroblasts (E′: high magnification inset). F) iPSC colonies at 24hrs post-manual isolation and passage from the 100 mm dish into a 12-well LN521-coated culture plate (note absence of un-reprogrammed dermal fibroblasts; F′: high magnification inset). G) Typical patient-specific iPSC colonies at passage 10 post-reprogramming. Cells within densely packed colonies display a classic iPSC morphology, including a high nuclear-to-cytoplasm ratio (G′). H) Example of an early iPSC-derived retinal organoid featuring evaginating loops of neural epithelium (arrowheads) that, following mechanical dissection (along red dotted lines) and further culture in neural retinal medium, will go on to develop cells of the retinal lineage. I) Retinal organoids at 30 days post-differentiation displaying clusters of cells that express the retinal progenitor-cell transcription factors, SOX2 (green), PAX6 (red) and OTX2 (gray). J) At 70 days post-differentiation developing organoids possess independent layers of cells that express the inner retinal-specific marker, HuC/D (green) and photoreceptor precursor cells that robustly express PAX6 (red). K) At 150 days post-differentiation the outer most layer is largely made up of neural rosettes (K′), which contain cells that express the photoreceptor-specific markers NR2E3 (green), Recoverin (RCVRN, red) and CRX (grey). Inset (K′) shows high magnification of a recoverin-positive photoreceptor-like cell with extended neural process. Small adjacent panels (I-K) are of individual fluorophores. Nuclei in I and K were counterstained with DAPI. Scale bars = 1000 μm in B-D and F, 400 μm in G-H and 100 μm in I-K.