Human Neuroepithelial Stem Cells Research Articles

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.

An atlas of endogenous DNA double-strand breaks arising during human neural cell fate determination

Endogenous DNA double-strand breaks (DSBs) occurring in neural cells have been implicated in the pathogenesis of neurodevelopmental disorders (NDDs). Currently, a genomic map of endogenous DSBs arising during human neurogenesis is missing. Here, we applied in-suspension Breaks Labeling In Situ and Sequencing (sBLISS), RNA-Seq, and Hi-C to chart the genomic landscape of DSBs and relate it to gene expression and genome architecture in 2D cultures of human neuroepithelial stem cells (NES), neural progenitor cells (NPC), and post-mitotic neural cells (NEU). Endogenous DSBs were enriched at the promoter and along the gene body of transcriptionally active genes, at the borders of topologically associating domains (TADs), and around chromatin loop anchors. NDD risk genes harbored significantly more DSBs in comparison to other protein-coding genes, especially in NEU cells. We provide sBLISS, RNA-Seq, and Hi-C datasets for each differentiation stage, and all the scripts needed to reproduce our analyses. Our datasets and tools represent a unique resource that can be harnessed to investigate the role of genome fragility in the pathogenesis of NDDs.

Signal requirement for cortical potential of transplantable human neuroepithelial stem cells

The cerebral cortex develops from dorsal forebrain neuroepithelial progenitor cells. Following the initial expansion of the progenitor cell pool, these cells generate neurons of all the cortical layers and then astrocytes and oligodendrocytes. Yet, the regulatory pathways that control the expansion and maintenance of the progenitor cell pool are currently unknown. Here we define six basic pathway components that regulate proliferation of cortically specified human neuroepithelial stem cells (cNESCs) in vitro without the loss of cerebral cortex developmental potential. We show that activation of FGF and inhibition of BMP and ACTIVIN A signalling are required for long-term cNESC proliferation. We also demonstrate that cNESCs preserve dorsal telencephalon-specific potential when GSK3, AKT and nuclear CATENIN-β1 activity are low. Remarkably, regulation of these six pathway components supports the clonal expansion of cNESCs. Moreover, cNESCs differentiate into lower- and upper-layer cortical neurons in vitro and in vivo. The identification of mechanisms that drive the neuroepithelial stem cell self-renewal and differentiation and preserve this potential in vitro is key to developing regenerative and cell-based therapeutic approaches to treat neurological conditions.

Glyphosate-based herbicide induces long-lasting impairment in neuronal and glial differentiation.

Glyphosate‐based herbicides (GBH) are among the most sold pesticides in the world. There are several formulations based on the active ingredient glyphosate (GLY) used along with other chemicals to improve the absorption and penetration in plants. The final composition of commercial GBH may modify GLY toxicological profile, potentially enhancing its neurotoxic properties. The developing nervous system is particularly susceptible to insults occurring during the early phases of development, and exposure to chemicals in this period may lead to persistent impairments on neurogenesis and differentiation. The aim of this study was to evaluate the long‐lasting effects of a sub‐cytotoxic concentration, 2.5 parts per million of GBH and GLY, on the differentiation of human neuroepithelial stem cells (NES) derived from induced pluripotent stem cells (iPSC). We treated NES cells with each compound and evaluated the effects on key cellular processes, such as proliferation and differentiation in daughter cells never directly exposed to the toxicants. We found that GBH induced a more immature neuronal profile associated to increased PAX6, NESTIN and DCX expression, and a shift in the differentiation process toward glial cell fate at the expense of mature neurons, as shown by an increase in the glial markers GFAP, GLT1, GLAST and a decrease in MAP2. Such alterations were associated to dysregulation of key genes critically involved in neurogenesis, including PAX6, HES1, HES5, and DDK1. Altogether, the data indicate that subtoxic concentrations of GBH, but not of GLY, induce long‐lasting impairments on the differentiation potential of NES cells.

Recapitulation of Genetic Predisposition to Medulloblastoma in Human Neuroepithelial Stem Cells

Abstract INTRODUCTION Human neural stem cell cultures provide progenitor cells that are potential cells of origin for brain cancers. However, the extent to which genetic predisposition to tumor formation can be faithfully captured in stem cell lines is uncertain. Here, we evaluated neuroepithelial stem (NES) cells, long-term propagating hindbrain cells that are representative of the cerebellar primordium. METHODS First we transduced NES cells with N-MYC to test whether NES cells can be transformed to brain tumour initiating cells. In parallel, we generated induced pluripotent stem (iPS) cells from patient's with Gorlin syndrome (PTCH mutation) and subsequent differentiated these cells to NES cells to recapitulate the genetic predisposition to medulloblastoma formation. These NES cells were orthotopically transplanted into mouse cerebellum to test their tumor forming capacity. RESULTS Following orthotopic transplantation of NES cells transduced with N-MYC, we observed formation of medulloblastoma. Significantly, transcriptomes and patterns of DNA methylation from xenograft tumors were globally more representative of human medulloblastoma compared to a MYCN-driven genetically engineered mouse model. Orthotopic transplantation of NES cells generated from Gorlin syndrome also generated medulloblastoma. We engineered candidate cooperating mutations in Gorlin NES cells, with mutation of DDX3X or loss of GSE1 both accelerating tumorigenesis. CONCLUSION These findings demonstrate that human NES cells provide a potent experimental resource for dissecting genetic causation in medulloblastoma

NRXN1 Deletion and Exposure to Methylmercury Increase Astrocyte Differentiation by Different Notch-Dependent Transcriptional Mechanisms.

Controversial evidence points to a possible involvement of methylmercury (MeHg) in the etiopathogenesis of autism spectrum disorders (ASD). In the present study, we used human neuroepithelial stem cells from healthy donors and from an autistic patient bearing a bi-allelic deletion in the gene encoding for NRXN1 to evaluate whether MeHg would induce cellular changes comparable to those seen in cells derived from the ASD patient. In healthy cells, a subcytotoxic concentration of MeHg enhanced astroglial differentiation similarly to what observed in the diseased cells (N1), as shown by the number of GFAP positive cells and immunofluorescence signal intensity. In both healthy MeHg-treated and N1 untreated cells, aberrations in Notch pathway activity seemed to play a critical role in promoting the differentiation toward glia. Accordingly, treatment with the established Notch inhibitor DAPT reversed the altered differentiation. Although our data are not conclusive since only one of the genes involved in ASD is considered, the results provide novel evidence suggesting that developmental exposure to MeHg, even at subcytotoxic concentrations, induces alterations in astroglial differentiation similar to those observed in ASD.

Neural Stem Cells of Parkinson's Disease Patients Exhibit Aberrant Mitochondrial Morphology and Functionality.

SummaryEmerging evidence suggests that Parkinson's disease (PD), besides being an age-associated disorder, might also have a neurodevelopment component. Disruption of mitochondrial homeostasis has been highlighted as a crucial cofactor in its etiology. Here, we show that PD patient-specific human neuroepithelial stem cells (NESCs), carrying the LRRK2-G2019S mutation, recapitulate key mitochondrial defects previously described only in differentiated dopaminergic neurons. By combining high-content imaging approaches, 3D image analysis, and functional mitochondrial readouts we show that LRRK2-G2019S mutation causes aberrations in mitochondrial morphology and functionality compared with isogenic controls. LRRK2-G2019S NESCs display an increased number of mitochondria compared with isogenic control lines. However, these mitochondria are more fragmented and exhibit decreased membrane potential. Functional alterations in LRRK2-G2019S cultures are also accompanied by a reduced mitophagic clearance via lysosomes. These findings support the hypothesis that preceding mitochondrial developmental defects contribute to the manifestation of the PD pathology later in life.

Automated microfluidic cell culture of stem cell derived dopaminergic neurons

Parkinson’s disease is a slowly progressive neurodegenerative disease characterised by dysfunction and death of selectively vulnerable midbrain dopaminergic neurons and the development of human in vitro cellular models of the disease is a major challenge in Parkinson’s disease research. We constructed an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity. Using this system, we continuously monitored the differentiation of Parkinson’s disease patient derived human neuroepithelial stem cells into midbrain specific dopaminergic neurons. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol. This system is the first example of an automated Organ-on-a-Chip culture and has the potential to enable a versatile array of in vitro experiments for patient-specific disease modelling.

Human neuroepithelial stem cell regional specificity enables spinal cord repair through a relay circuit

Traumatic spinal cord injury results in persistent disability due to disconnection of surviving neural elements. Neural stem cell transplantation has been proposed as a therapeutic option, but optimal cell type and mechanistic aspects remain poorly defined. Here, we describe robust engraftment into lesioned immunodeficient mice of human neuroepithelial stem cells derived from the developing spinal cord and maintained in self-renewing adherent conditions for long periods. Extensive elongation of both graft and host axons occurs. Improved functional recovery after transplantation depends on neural relay function through the grafted neurons, requires the matching of neural identity to the anatomical site of injury, and is accompanied by expression of specific marker proteins. Thus, human neuroepithelial stem cells may provide an anatomically specific relay function for spinal cord injury recovery.

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia

The mechanisms underlying Zika virus (ZIKV)-related microcephaly and other neurodevelopment defects remain poorly understood. Here, we describe the derivation and characterization, including single-cell RNA-seq, of neocortical and spinal cord neuroepithelial stem (NES) cells to model early human neurodevelopment and ZIKV-related neuropathogenesis. By analyzing human NES cells, organotypic fetal brain slices, and a ZIKV-infected micrencephalic brain, we show that ZIKV infects both neocortical and spinal NES cells as well as their fetal homolog, radial glial cells (RGCs), causing disrupted mitoses, supernumerary centrosomes, structural disorganization, and cell death. ZIKV infection of NES cells and RGCs causes centrosomal depletion and mitochondrial sequestration of phospho-TBK1 during mitosis. We also found that nucleoside analogs inhibit ZIKV replication in NES cells, protecting them from ZIKV-induced pTBK1 relocalization and cell death. We established a model system of human neural stem cells to reveal cellular and molecular mechanisms underlying neurodevelopmental defects associated with ZIKV infection and its potential treatment.

Use of a hyaluronan and methylcellulose hydrogel to deliver neural stem cells to the stroke-injured brain

Event Abstract Back to Event Use of a hyaluronan and methylcellulose hydrogel to deliver neural stem cells to the stroke-injured brain Samantha L. Payne1, 2*, Michael J. Cooke2*, Priya Anandakumaran2, Balazs Varga3, Cindi Morshead4*, Andras Nagy3* and Molly S. Shoichet1, 2, 5* 1 University of Toronto, Chemical Engineering and Applied Chemistry, Canada 2 University of Toronto, Institute of Biomaterials and Biomedical Engineering, Canada 3 Mount Sinai Hospital, Lunenfeld-Tanenbaum Research Institute, Canada 4 University of Toronto, Institute of Medical Science, Canada 5 University of Toronto, Department of Chemistry, Canada Introduction: Worldwide 15 million people will suffer from a stroke each year, and up to two-thirds of survivors will experience life-long functional deficits. Despite the high prevalence of stroke, no clinical treatment exists that can replace lost cells and restore function. Recent regenerative medicine strategies have focused on the delivery of an exogenous source of cells to both directly replace lost neurons and glia as well as provide trophic support to endogenous cells. While cell transplantation is a promising strategy to regenerate stroke-injured tissues, current delivery techniques suffer from low cell survival. To increase cell survival following transplantation, our lab has developed a hydrogel composed of a physical blend of hyaluronan and methylcellulose, HAMC, and demonstrated its ability to improve cell delivery and increase survival in a model of spinal cord injury[1] and retinal regeneration[2]. We hypothesize that the use of HAMC to deliver mouse or human neural precursor cells will result in improved cell survival and functional recovery in a rat model of stroke. The goals of this work are to determine if the use of HAMC can 1) maintain cell viability prior to delivery, and 2) increase cell survival and functional recovery when transplanted in vivo. Materials and Methods: HAMC hydrogels were produced using a physical blend of 0.5%/0.5% w/v sterile methylcellulose and hyaluronan reconstituted in artificial cerebralspinal fluid (aCSF). First, the ability of HAMC to improve cell survival even before transplantation was tested by encapsulating mouse precursor cells (stem and progenitors) (mNPCs) and human neuroepithelial stem cells (hNECs)[3] in 3D HAMC and left on ice for 6 or 24 hours. Next, mNPCs were delivered to a mouse stroke model to determine if HAMC promotes the survival of cells after transplantation. Lastly, we wanted to translate the work with mNPCs towards a clinically-relevant model, and so tested the use of HAMC to deliver induced pluripotent stem cell-derived hNECs into the stroke-injured rat brain. hNECs were differentiated into neural precursor cells (hNPCs) and transplanted into the stroke-injured rat brain. Results and Discussion: HAMC significantly increased mNPC and hNECs survival when cells were encapsulated in HAMC and stored on ice after 6 hours compared to aCSF. This suggests that storing cells in HAMC prior to transplantation will reduce cell death and increase the number of live cells being delivered to the brain. In mice, HAMC increased cell survival of undifferentiated mNPCs and ultimately resulted in functional recovery after endothelin-1 stroke-injury[4]. In rats, HAMC also improved survival of hNPCs in an endothelin-1 stroke-injury. In ongoing studies, we are investigating behavioural recovery in rats. Conclusions: This study demonstrates the importance of the delivery vehicle on the success of cell transplantation and tissue regeneration in the stroke-injured brain using both mouse and rat models and mouse and human neural stem cells, respectively. This contributes to the broader field of regenerative medicine. We are grateful to CIHR and NSERC for funding

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia

Zika Virus Disrupts Phospho-TBK1 Localization and Mitosis in Human Neuroepithelial Stem Cells and Radial Glia