cGMP production of patient-specific iPSCs and photoreceptor precursor cells to treat retinal degenerative blindness

Abstract

Immunologically-matched, induced pluripotent stem cell (iPSC)-derived photoreceptor precursor cells have the potential to restore vision to patients with retinal degenerative diseases like retinitis pigmentosa. The purpose of this study was to develop clinically-compatible methods for manufacturing photoreceptor precursor cells from adult skin in a non-profit cGMP environment. Biopsies were obtained from 35 adult patients with inherited retinal degeneration and fibroblast lines were established under ISO class 5 cGMP conditions. Patient-specific iPSCs were then generated, clonally expanded and validated. Post-mitotic photoreceptor precursor cells were generated using a stepwise cGMP-compliant 3D differentiation protocol. The recapitulation of the enhanced S-cone phenotype in retinal organoids generated from a patient with NR2E3 mutations demonstrated the fidelity of these protocols. Transplantation into immune compromised animals revealed no evidence of abnormal proliferation or tumor formation. These studies will enable clinical trials to test the safety and efficiency of patient-specific photoreceptor cell replacement in humans.

Two-photon polymerization for production of human iPSC-derived retinal cell grafts

“…Assuming that the relationship between scaffold volume and printing time is linear, we can use the time-optimized printing parameters to create a full-size scaffold (roughly 5 mm in diameter) in less than 2 days. This period is reasonable given that it takes much longer than this to differentiate the cells needed to seed such a graft [25–27]. Furthermore, our results indicate that at these conditions, large scaffolds will have intact horizontal pores and that tuning the size of vertical pores will not significantly affect the margin of error for pore size.…”

“…At the end of this time period, we added 1 mL fresh neural retina media 2 [2% B27 supplement (Life Technologies, Thermo Fisher Scientific), 1 mg/mL Primocin (Invivogen, San Diego, CA), 1 mM sodium pyruvate (Life Technologies), 2 mM GlutaMAX (Gibco), 5 μg/mL insulin (Sigma-Aldrich), 100 μg/mL transferrin (Sigma-Aldrich), 100 μg/mL human serum albumin (Sigma-Aldrich), 60 ng/mL progesterone (Sigma-Aldrich), 16 μg/mL putrescine (Sigma-Aldrich), 40 ng/mL sodium selenite (Sigma-Aldrich), 40 ng/mL thyroxine (Sigma-Aldrich), 40 ng/mL tri-iodothyronine (Sigma-Aldrich), 50 ng/mL BDNF (Life Technologies), 10 ng/mL CNTF (Life Technologies), 10 ng/mL hbFGF (EMD Millipore), 5 μM forskolin (Sigma-Aldrich) and 1% human serum (Sigma-Aldrich) in neurobasal media (Life Technologies)] to inactivate the papain, then we centrifuged the cells at 100g for 2 minutes, discarded the supernatant and resuspended the dissociated pellet in 100 μL fresh neural retina media 2 (yielding about 10,000 cells/μL). Our previous work showed that after thirty days in culture, retinal organoids contain a large fraction of cells that express the early retinal markers SOX2, PAX6, OTX2 and VSX2 [25]. Thus, for the purpose of this work, we assumed that cells dissociated from 30-day retinal organoids were retinal progenitor cells and assumed that they maintained this phenotype during the brief culture time described here.…”

“…To generate clinical-grade retinal progenitor cells we used our recently developed 3D-cGMP-differentiation protocol [25]. Briefly, we passaged, dissociated and resuspended individual iPSC colonies derived from a donor with normal ocular history in 3D differentiation medium [DMEM (Gibco, Thermo Fisher Scientific, Grand Island, NY), 10% heat-inactivated human serum (Innovative Research, Novi, MI), 20% CTS KnockOut Serum Replacement XenoFree medium (Gibco), 0.1 mM MEM non-essential amino acids (Gibco), 1 mM Sodium Pyruvate (Gibco), 0.1 mM 2-Mercaptoethanol (Gibco), 1% ECM (human type 1 and type 3 collagen/vitronectin/fibronectin, Advanced BioMatrix, Carlsbad, CA), 20 mM Y-27632 ROCK Inhibitor (EMD Milipore, Billerica, MA), 3 nMIWR1e (Cayman Chemical, Ann Arbor, MI), 3 M StemMACS CHIR (Miltenyi Biotec Inc., San Diego, CA), 100 nM SAG (Enzo Life Sciences, Farmingdale, NY)], and then plated the cells in a 96-well sphere forming plate (Corning) at 1×10 4 cells/well.…”

Rebuilding the Missing Part—A Review on Photoreceptor Transplantation

“…However, given the high variability still observed in the differentiation outcome of human PSCs further detailed knowledge of retina and photoreceptor development for targeted modification of molecular pathways will be essential for developing pipelines that allow clinical grade photoreceptor transplant production according to good manufacturing practice (GMP) guidelines (Wiley et al, 2016). Furthermore, the extended time periods for the generation of human photoreceptors, particularly rods, with more than 100 days in vitro , represents a major technical challenge for robust clinical grade cell production.…”

The CRB1 Complex: Following the Trail of Crumbs to a Feasible Gene Therapy Strategy

“…Generating retina-like organoids from human embryonic stem cells and iPSCs is relatively autonomous, although neural induction requires the addition of extrinsic factors such as B-27 and N-2 supplements. However, providing additional factors such as retinoic acid and Notch inhibitors can accelerate neuronal development and maturation (Wiley et al, 2016). The use of in vitro disease models using human iPSCs has begun to overtake the use of human embryonic stem cells, due in large part to ethical concerns and technical issues (Zacharias et al, 2011).…”