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.

A proposal for an international Code of Conduct for data sharing in genomics.

As genomic research becomes commonplace across the world, there is an increased need to coordinate practices among researchers, especially with regard to data sharing. One such way is an international code of conduct. In September 2020, an expert panel consisting of representatives from various fields convened to discuss a draft proposal formed via a synthesis of existing professional codes and other recommendations. This article presents an overview and analysis of the main issues related to international genomic research that were discussed by the expert panel, and the results of the discussion and follow up responses by the experts. As a result, the article presents as an annex a proposal for an international code of conduct for data sharing in genomics that is meant to establish best practices.

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.

Single cell analysis of autism patient with bi-allelic NRXN1-alpha deletion reveals skewed fate choice in neural progenitors and impaired neuronal functionality.

We generated human iPS derived neural stem cells and differentiated cells from healthy control individuals and an individual with autism spectrum disorder carrying bi-allelic NRXN1-alpha deletion. We investigated the expression of NRXN1-alpha during neural induction and neural differentiation and observed a pivotal role for NRXN1-alpha during early neural induction and neuronal differentiation. Single cell RNA-seq pinpointed neural stem cells carrying NRXN1-alpha deletion shifting towards radial glia-like cell identity and revealed higher proportion of differentiated astroglia. Furthermore, neuronal cells carrying NRXN1-alpha deletion were identified as immature by single cell RNA-seq analysis, displayed significant depression in calcium signaling activity and presented impaired maturation action potential profile in neurons investigated with electrophysiology. Our observations propose NRXN1-alpha plays an important role for the efficient establishment of neural stem cells, in neuronal differentiation and in maturation of functional excitatory neuronal cells.

SQSTM1/p62-Directed Metabolic Reprogramming Is Essential for Normal Neurodifferentiation.

Neurodegenerative disorders are an increasingly common and irreversible burden on society, often affecting the aging population, but their etiology and disease mechanisms are poorly understood. Studying monogenic neurodegenerative diseases with known genetic cause provides an opportunity to understand cellular mechanisms also affected in more complex disorders. We recently reported that loss-of-function mutations in the autophagy adaptor protein SQSTM1/p62 lead to a slowly progressive neurodegenerative disease presenting in childhood. To further elucidate the neuronal involvement, we studied the cellular consequences of loss of p62 in a neuroepithelial stem cell (NESC) model and differentiated neurons derived from reprogrammed p62 patient cells or by CRISPR/Cas9-directed gene editing in NESCs. Transcriptomic and proteomic analyses suggest that p62 is essential for neuronal differentiation by controlling the metabolic shift from aerobic glycolysis to oxidative phosphorylation required for neuronal maturation. This shift is blocked by the failure to sufficiently downregulate lactate dehydrogenase expression due to the loss of p62, possibly through impaired Hif-1α downregulation and increased sensitivity to oxidative stress. The findings imply an important role for p62 in neuronal energy metabolism and particularly in the regulation of the shift between glycolysis and oxidative phosphorylation required for normal neurodifferentiation.

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Derivation of human iPS cell lines from monozygotic twins in defined and xeno free conditions.

Human induced pluripotent stem (hiPS) cell lines CTRL-9-II and CTRL-10-I were derived from healthy monozygotic twin donors using non-integrating RNA based Sendai virus reprogramming and cultured in a xeno-free chemically defined condition. The established hiPS cell lines, CTRL-9-II and CTRL-10-I, are karyotypically normal, free from reprogramming vectors, display endogenously expression of pluripotency factors at levels similar to embryonic stem cells. The generated iPS cell lines demonstrate pluripotency by passing bioinformatics assay PluriTest and by embryonic body assay.