Title: iPSC derived Optic Vesicles (OVs)/Neural Retinal Progenitors (NRPs) – Production, Characterization and Cryopreservation
Legend: 1) Representative pictures of selected OVs derived from iPSCs grown on E8/Matrigel. iPSCs, cultured on E8/Matrigel, mTeSR1/Matrigel, and E8/vitronectin, were differentiated into OVs (Optic vesicle-like structures) composed of NRPCs (neuroretinal progenitor cells).
2) Characterization of OVs (60 days in culture)- a) OVs were dissociated and stained with DAPI, CRX and Recoverin. Cells expressing both CRX and Recoverin are photoreceptors whereas CRX is an early marker specific for photoreceptors. E8/Matrigel and mTeSR1/Matrigel conditions show >90% CRX+/Recoverin+ cells whereas E8/Vitronectin condition displays >90% cells expressing CRX only. The E8/Vitronectin condition may exhibit delayed maturation of photoreceptors. b) OVs were dissociated and viability was estimated. All the condition showed higher than 90% viability after 60 days in culture. 3) Cryopreservation of OVs- OVs were dissociated and cryopreserved in 4 different freezing medias. Post-thaw cells were stained with CRX and Recoverin to demonstrate viability of photoreceptors. All the cryopreservation medias were able to preserve photoreceptors. A representative picture is shown of post-thaw cells stained with CRX and Recoverin.
Description: Retinal degenerative diseases (RDDs), such as retinitis pigmentosa and Leber congenital amaurosis (LCA), are caused by disruption of photoreceptor function resulting in vision loss. Unfortunately, the vast majority of RDDs have no effective treatments. The ability to differentiate pluripotent stem cells into neural retinal progenitors (NRPs) provides a potential therapeutic route through replacement of the diseased photoreceptors. The Gamm Lab has developed a process for differentiating induced pluripotent stem cells (iPSCs) into NRP cells. In order to move this cell therapy into human clinical trials, cells must be manufactured and tested following current Good Manufacturing Practice (cGMP) guidelines – the FDA’s regulations for manufacturing and testing drugs. Waisman Biomanufacturing is collaborating with the Gamm Lab to establish a cGMP-compliant manufacturing process for human iPS cell-derived NRPs. Ideally a cryopreserved NRP cell therapy can be developed allowing cells to be thawed and injected into the subretinal space in order to improve vision in patients with inherited RDDs.
The first stage of this project was to adapt the current differentiation process from the Gamm Lab into a cGMP-compliant process that is suitable for production of cell therapeutics. An iPSC Master Cell Bank was produced and tested under cGMP to establish the starting material for the NRP differentiation process. A total of five trial production runs were successfully completed with three different iPSC lines. The current process yields approximately 200-500 Optic Vesicles (OVs) per 6-well plate or approximately 20-40 million cells per plate. Quality Control assays were developed to test the final NRP cells for expression of key markers (VSX2, PAX6, CRX, Recoverin) as well as negative markers for off-target contaminating cells and residual undifferentiated iPSCs. The second stage of the project involved evaluating formulation and cryopreservation methods for the Optic Vesicle (OV) intermediate structures as well as the dissociated final iPSC-NRP cell product. Initial studies have demonstrated some success with cryopreservation of the singularized iPSC-NRPs with excellent recovery of cells that are >90% CRX+/Recoverin+. The resulting iPSC-NRPs are currently being evaluated in non-human primate studies by the Gamm Lab.
About the Investigator: Derek Hei received a bachelor of science degree in chemical engineering from UW-Madison,graduating with honors in 1988. He completed his PhD in biochemical engineering in 1993 at the University of California, Berkeley. Following his PhD, Hei joined Genentech, in San Francisco, California, as a scientist in the Process Recovery R&D group. At Genentech, he was primarily involved in developing manufacturing processes and analytical methods for new protein biopharmaceuticals. Hei later joined Cerus Corp, in Concord, California, where he served as director of the biomedical engineering group. His group developed novel separation devices and systems for viral inactivation in cell therapeutics and biologics. In 2000, Hei joined the Waisman Center at UW-Madison to lead development of Waisman Biomanufacturing. As the director of Waisman Biomanufacturing, Hei interacts with project sponsors to address technical issues related to the development and production of cutting-edge biotherapeutics. Hei is also involved in the MS Biotechnology Program at UW-Madison where he is a co-instructor in biotech operations.