Pre-clinical evaluation of clinically relevant iPS cell derived neuroepithelial stem cells as an off-the-shelf cell therapy for spinal cord injury.

Preclinical transplantations using human neuroepithelial stem (NES) cells in spinal cord injury models have exhibited promising results and demonstrated cell integration and functional improvement in transplanted animals. Previous studies have relied on the generation of research grade cell lines in continuous culture. Using fresh cells presents logistic hurdles for clinical transition regarding time and resources for maintaining high quality standards. In this study, we generated a good manufacturing practice (GMP) compliant human iPS cell line in GMP clean rooms alongside a research grade iPS cell line which was produced using standardized protocols with GMP compliant chemicals. These two iPS cell lines were differentiated into human NES cells, from which six batches of cell therapy doses were produced. The doses were cryopreserved, thawed on demand and grafted in a rat spinal cord injury model. Our findings demonstrate that NES cells can be directly grafted post-thaw with high cell viability, maintaining their cell identity and differentiation capacity. This opens the possibility of manufacturing off-the-shelf cell therapy products. Moreover, our manufacturing process yields stable cell doses with minimal batch-to-batch variability, characterized by consistent expression of identity markers as well as similar viability of cells across the two iPS cell lines. These cryopreserved cell doses exhibit sustained viability, functionality, and quality for at least 2 years. Our results provide proof of concept that cryopreserved NES cells present a viable alternative to transplanting freshly cultured cells in future cell therapies and exemplify a platform from which cell formulation can be optimized and facilitate the transition to clinical trials.

Multiple therapeutic effects of human neural stem cells derived from induced pluripotent stem cells in a rat model of post-traumatic syringomyelia.

BACKGROUND: Post-traumatic syringomyelia (PTS) affects patients with chronic spinal cord injury (SCI) and is characterized by progressive deterioration of neurological symptoms. To improve surgical treatment, we studied the therapeutic effects of neuroepithelial-like stem cells (NESCs) derived from induced pluripotent stem cells (iPSCs) in a rat model of PTS. To facilitate clinical translation, we studied NESCs derived from Good Manufacturing Practice (GMP)-compliant iPSCs. METHODS: Human GMP-compliant iPSCs were used to derive NESCs. Cryo-preserved NESCs were used off-the-shelf for intraspinal implantation to PTS rats 1 or 10 weeks post-injury, and rats were sacrificed 10 weeks later. In vivo cyst volumes were measured with micro-MRI. Phenotypes of differentiated NESCs and host responses were analyzed by immunohistochemistry. FINDINGS: Off-the-shelf NESCs transplanted to PTS rats 10 weeks post-injury reduced cyst volume. The grafted NESCs differentiated mainly into glial cells. Importantly, NESCs also stimulated tissue repair. They reduced the density of glial scars and neurite-inhibiting chondroitin sulfate proteoglycan 4 (CSPG4), stimulated host oligodendrocyte precursor cells to migrate and proliferate, reduced active microglia/macrophages, and promoted axonal regrowth after subacute as well as chronic transplantation. INTERPRETATION: Significant neural repair promoted by NESCs demonstrated that human NESCs could be used as a complement to standard surgery in PTS. We envisage that future PTS patients transplanted with NESCs will benefit both from eliminating the symptoms of PTS, as well as a long-term improvement of the neurological symptoms of SCI. FUNDING: This work was supported by Vinnova (2016-04134), Karolinska Institutet StratRegen, and the Chinese Scholarship Council.

Protocol for the derivation, culturing, and differentiation of human iPS-cell-derived neuroepithelial stem cells to study neural differentiation in vitro.

Here, we present a revised protocol to derive neuroepithelial stem (NES) cells from human induced pluripotent stem cells. NES cells can be further differentiated into a culture of neurons (90%) and glia (10%). We describe how to derive and maintain NES cells in culture and how to differentiate them. In addition, we show the potential use of NES cells to study the role of reactive oxygen species in neuronal differentiation and a guideline for NES cell transfection. For complete details on the use and execution of this protocol, please refer to Calvo-Garrido et al. (2019); Falk et al. (2012).