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

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

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

Macrophages modulate migration and invasion of human tongue squamous cell carcinoma

Oral tongue squamous cell carcinoma (OTSCC) has a high mortality rate and the incidence is rising worldwide. Despite advances in treatment, the disease lacks specific prognostic markers and treatment modality. The spreading of OTSCC is dependent on the tumor microenvironment and involves tumor-associated macrophages (TAMs). Although the presence of TAMs is associated with poor prognosis in OTSCC, the specific mechanisms underlying this are still unknown. The aim here was to investigate the effect of macrophages (Mfs) on HSC-3 tongue carcinoma cells and NF-kappaB activity. We polarized THP-1 cells to M1 (inflammatory), M2 (TAM-like) and R848 (imidazoquinoline-treated) type Mfs. We then investigated the effect of Mfs on HSC-3 cell migration and NF-kappaB activity, cytokine production and invasion using several different in vitro migration models, a human 3D tissue invasion model, antibody arrays, confocal microscopy, immunohistochemistry and a mouse invasion model. We found that in co-culture studies all types of Mfs fused with HSC-3 cells, a process which was partially due to efferocytosis. HSC-3 cells induced expression of epidermal growth factor and transforming growth factor-beta in co-cultures with M2 Mfs. Direct cell-cell contact between M2 Mfs and HSC-3 cells induced migration and invasion of HSC-3 cells while M1 Mfs reduced HSC-3 cell invasion. M2 Mfs had an excess of NF-kappaB p50 subunit and a lack of p65 subunits both in the presence and absence of HSC-3 cells, indicating dysregulation and pro-tumorigenic NF-kappaB activation. TAM-like cells were abundantly present in close vicinity to carcinoma cells in OTSCC patient samples. We conclude that M2 Mfs/TAMs have an important role in OTSCC regulating adhesion, migration, invasion and cytokine production of carcinoma cells favouring tumor growth. These results demonstrate that OTSCC patients could benefit from therapies targeting TAMs, polarizing TAM-like M2 Mfs to inflammatory macrophages and modulating NF-kappaB activity

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

Summary: 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. : SQSTM1/p62 is a known autophagy adaptor that, if lost, causes childhood-onset neurodegeneration. Data from Wredenberg et al. show that loss of p62 in a neuronal stem cell model does not affect mitophagy but instead leads to impaired differentiation. The authors suggest p62 finely tunes LDHA expression and thus controls the metabolic shift to OXPHOS required for proper differentiation. Keywords: SQSTM1, p62, hypoxia, mitochondria, neurodifferentiation, neuroepithelial-like stem cells, neuronal development, oxidative stress, mitophagy, neurodegeneratio

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

Expression of Mf-markers and NF-κB subunits in HSC-3 and M2 Mf co-culture.

<p>Vybrant CM-Dil labeled HSC-3 cells (red) and unlabeled M2 Mfs were incubated with DMSO or 10 ng/ml TNF-α for 30 min where after cells were fixed for immunofluorescence with antibodies for CD68, NF-κB p50 and p65 subunits. Some samples were pre-incubated with 10 μM BAY 11-7082 prior to TNF-α activation. AlexaFluor488-conjugated secondary antibody was used for visualization. Samples were mounted with DAPI- mountain medium to visualize nuclei (blue). Samples were photographed with a Leica Confocal microscope with 63x oil immersion objective. Red arrows indicate HSC-3 cells and green arrows Mfs. Scale bars 50 μm.</p

Efferocytosis in HSC-3 and Mf co-cultures.

<p>Vybrant CM-Dil labeled HSC-3 (red) and Vybrant DiO-labeled M2 Mfs (green) were co-cultured, treated with 3 mM efferocytosis-inhibitor amiloride (AMI) or 10 μM NF-κB inhibitor BAY 11-7082. Co-cultures of HSC-3 cells and M2 Mfs were photographed with an EVOS FL Cell Imaging System microscope. Pictures presented are from day 2 (n = 4). Fusion of membranes was observed between HSC-3 cells and M2 Mfs in DMSO (vehicle)- and BAY-treated cultures (white arrows in Overlay-section). Scale bars 200 μm.</p

Co-culture of HSC-3 cells and Mfs induce invasion in an organotypic 3D tissue model.

<p>(A) HSC-3 cells were treated with Mf-CM and/or 10 μM BAY 11-7082 (n = 6) and adhesion to fibronectin-coated wells was analysed after 2 hours with Crystal violet-staining. Stained wells were analysed by measuring absorbance at 540 nm. (B) Labeled HSC-3 cells were cultured on top of Matrigel and Mfs, HSC-3 or THP-1 cells were cultured in the lower chambers (n = 6). Invaded cells were analyzed from the membrane by measuring fluorescence. HSC-3 cells and Mfs were co-cultured on top of human myoma tissue (C,D, n = 3) for 10 days where after tissues were fixed and processed for immunohistochemistry. Pan-cytokeratin stained sections (in C, scale bars: 500 μm) were photographed and invasion areas and invasion depths were analysed with the Leica Qwin3 software. Conditioned medium was collected at day 4 and 8 from myomas and medium containing 0.5 (Mf CMs) or 15 μg (HSC-3) protein were subjected to gelatin zymography (E, day 4). Samples were done in triplicate, representative picture of samples is shown in E. Recombinant MMP-2 and -9 were used as positive controls. Relative intensity of MMP-9 bands were analyzed with the ImageJ-software (F).</p