High-throughput screening of human induced pluripotent stem cell-derived brain organoids.

BACKGROUND: The need for scalable high-throughput screening (HTS) approaches for 3D human stem cell platforms remains a central challenge for disease modeling and drug discovery. We have developed a workflow to screen cortical organoids across platforms. NEW METHOD: We used serum-free embryoid bodies (SFEBs) derived from human induced pluripotent stem cells (hiPSCs) and employed high-content imaging (HCI) to assess neurite outgrowth and cellular composition within SFEBs. We multiplexed this screening assay with both multi-electrode arrays (MEAs) and single-cell calcium imaging. RESULTS: HCI was used to assess the number of excitatory neurons (VGlut+) in experimental replicates of hiPSC-derived SFEBs, demonstrating experiment-to-experiment consistency. Neurite detection using HCI was applied to assess neurite morphology. MEA analysis showed that firing and burst rates in SFEBs decreased with blockade of NMDARs and AMPARs and increased with GABAR blockade. We also demonstrate effective combination of both MEA and HCI to analyze VGlut+ populations surrounding electrodes within MEAs. HCI-based (Ca2+) transient analysis revealed firing in individual cells surrounding active MEA electrodes. COMPARISON WITH EXISTING METHODS: Current methods to generate neural organoids show high degrees of variability, and often require sectioning or special handling for analysis. The protocol outlined in this manuscript generates SFEBs with high degree of consistency making them amenable to complex assays combining HTS and electrophysiology allowing for an in-depth, unbiased analysis. CONCLUSIONS: SFEBs can be used in combination with HTS to compensate for experimental variability common in 3D cultures, while significantly decreasing processing speed, making this an efficient starting point for phenotypic drug screening.

Cortical interneuron function in autism spectrum condition.

Cortical interneurons (INs) are a diverse group of neurons that project locally and shape the function of neural networks throughout the brain. Multiple lines of evidence suggest that a proper balance of glutamate and GABA signaling is essential for both the proper function and development of the brain. Dysregulation of this system may lead to neurodevelopmental disorders, including autism spectrum condition (ASC). We evaluate the development and function of INs in rodent and human models and examine how neurodevelopmental dysfunction may produce core symptoms of ASC. Finding common physiological mechanisms that underlie neurodevelopmental disorders may lead to novel pharmacological targets and candidates that could improve the cognitive and emotional symptoms associated with ASC.

Convergent Pathways in Idiopathic Autism Revealed by Time Course Transcriptomic Analysis of Patient-Derived Neurons.

Potentially pathogenic alterations have been identified in individuals with autism spectrum disorders (ASDs) within a variety of key neurodevelopment genes. While this hints at a common ASD molecular etiology, gaps persist in our understanding of the neurodevelopmental mechanisms impacted by genetic variants enriched in ASD patients. Induced pluripotent stem cells (iPSCs) can model neurodevelopment in vitro, permitting the characterization of pathogenic mechanisms that manifest during corticogenesis. Taking this approach, we examined the transcriptional differences between iPSC-derived cortical neurons from patients with idiopathic ASD and unaffected controls over a 135-day course of neuronal differentiation. Our data show ASD-specific misregulation of genes involved in neuronal differentiation, axon guidance, cell migration, DNA and RNA metabolism, and neural region patterning. Furthermore, functional analysis revealed defects in neuronal migration and electrophysiological activity, providing compelling support for the transcriptome analysis data. This study reveals important and functionally validated insights into common processes altered in early neuronal development and corticogenesis and may contribute to ASD pathogenesis.

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays.

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system. Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Glial-Restricted Precursors Protect Neonatal Brain Slices from Hypoxic-Ischemic Cell Death Without Direct Tissue Contact.

Glial-Restricted Precursors (GRPs) are tripotential progenitors that have been shown to exhibit beneficial effects in several preclinical models of neurological disorders, including neonatal brain injury. The mechanisms of action of these cells, however, require further study, as do clinically relevant questions such as timing and route of cell administration. Here, we explored the effects of GRPs on neonatal hypoxia-ischemia during acute and subacute stages, using an in vitro transwell co-culture system with organotypic brain slices exposed to oxygen-glucose deprivation (OGD). OGD-exposed slices that were then co-cultured with GRPs without direct cell contact had decreased tissue injury and cortical cell death, as evaluated by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. This effect was more pronounced when cells were added during the subacute phase of the injury. Furthermore, GRPs reduced the amount of glutamate in the slice supernatant and changed the proliferation pattern of endogenous progenitor cells in brain slices. In summary, we show that GRPs exert a neuroprotective effect on neonatal hypoxia-ischemia without the need for direct cell-cell contact, thus confirming the rising view that beneficial actions of stem cells are more likely attributable to trophic or immunomodulatory support rather than to long-term integration.

Human Inducible Pluripotent Stem Cells and Autism Spectrum Disorder: Emerging Technologies.

Autism Spectrum Disorder (ASD) is a behaviorally defined neurodevelopmental condition. Symptoms of ASD cover the spectrum from mild qualitative differences in social interaction to severe communication and social and behavioral challenges that require lifelong support. Attempts at understanding the pathophysiology of ASD have been hampered by a multifactorial etiology that stretches the limits of current behavioral and cell based models. Recent progress has implicated numerous autism-risk genes but efforts to gain a better understanding of the underlying biological mechanisms have seen slow progress. This is in part due to lack of appropriate models for complete molecular and pharmacological studies. The advent of induced pluripotent stem cells (iPSC) has reinvigorated efforts to establish more complete model systems that more reliably identify molecular pathways and predict effective drug targets and candidates in ASD. iPSCs are particularly appealing because they can be derived from human patients and controls for research purposes and provide a technology for the development of a personalized treatment regimen for ASD patients. The pluripotency of iPSCs allow them to be reprogrammed into a number of CNS cell types and phenotypically screened across many patients. This quality is already being exploited in protocols to generate 2-dimensional (2-D) and three-dimensional (3-D) models of neurons and developing brain structures. iPSC models make powerful platforms that can be interrogated using electrophysiology, gene expression studies, and other cell-based quantitative assays. iPSC technology has limitations but when combined with other model systems has great potential for helping define the underlying pathophysiology of ASD. Autism Res 2016, 9: 513-535. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival.

OBJECTIVE: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI. METHODS: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5). At P22, intracallosal injections of either enhanced green fluorescent protein (eGFP) + GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at 4 and 8 weeks post-transplantation. RESULTS: GRP survival was comparable at 1 month but significantly lower at 2 months post-transplantation in NWMI mice compared with unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and prepulse inhibition (PPI) paradigms compared with unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared with saline-treated NWMI animals. INTERPRETATION: The reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral, and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes.

The potential for cell-based therapy in perinatal brain injuries.

Perinatal brain injuries are a leading cause of cerebral palsy worldwide. The potential of stem cell therapy to prevent or reduce these impairments has been widely discussed within the medical and scientific communities and an increasing amount of research is being conducted in this field. Animal studies support the idea that a number of stem cells types, including cord blood and mesenchymal stem cells have a neuroprotective effect in neonatal hypoxia-ischemia. Both these cell types are readily available in a clinical setting. The mechanisms of action appear to be diverse, including immunomodulation, activation of endogenous stem cells, release of growth factors, and anti-apoptotic effects. Here, we review the different types of stem cells and progenitor cells that are potential candidates for therapeutic strategies in perinatal brain injuries, and summarize recent preclinical and clinical studies.

Ischemia-induced neuroinflammation is associated with disrupted development of oligodendrocyte progenitors in a model of periventricular leukomalacia.

Microglial activation in crossing white matter tracts is a hallmark of noncystic periventricular leukomalacia (PVL), the leading pathology underlying cerebral palsy in prematurely born infants. Recent studies indicate that neuroinflammation within an early time window can produce long-lasting defects in oligodendroglial maturation, myelination deficit, as well as disruption of transcription factors important in oligodendroglial maturation. We recently reported an ischemic mouse model of PVL, induced by unilateral neonatal carotid artery ligation, leading to selective long-lasting bilateral myelination deficits, ipsilateral thinning of the corpus callosum, ventriculomegaly, as well as evidence of axonopathy. Here, we report that permanent unilateral carotid ligation on postnatal day 5 in CD-1 mice induces an inflammatory response, as defined by microglial activation and recruitment, as well as significant changes in cytokine expression (increased IL-1ß, IL-6, TGF-ß1, and TNF-α) following ischemia. Transient reduction in counts of oligodendrocyte progenitor cells (OPCs) at 24 and 48 h after ischemia, a shift in OPC cell size and morphology towards the more immature form, as well as likely migration of OPCs were found. These OPC changes were topographically associated with areas showing microglial activation, and OPC counts negatively correlated with increased microglial staining. The presented data show a striking neuroinflammatory response in an ischemia-induced model of PVL, associated with oligodendroglial injury. Future studies modulating the neuroinflammatory response in this model may contribute to a better understanding of the interaction between microglia and OPCs in PVL and open opportunities for future therapies.

Derivation of glial restricted precursors from E13 mice.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.

In vivo magnetization transfer MRI shows dysmyelination in an ischemic mouse model of periventricular leukomalacia.

Periventricular leukomalacia, PVL, is the leading cause of cerebral palsy in prematurely born infants, and therefore more effective interventions are required. The objective of this study was to develop an ischemic injury model of PVL in mice and to determine the feasibility of in vivo magnetization transfer (MT) magnetic resonance imaging (MRI) as a potential monitoring tool for the evaluation of disease severity and experimental therapeutics. Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal day 5 (P5); at P60, in vivo T2-weighted (T2w) and MT-MRI were performed and correlated with postmortem histopathology. In vivo T2w MRI showed thinning of the right corpus callosum, but no significant changes in hippocampal and hemispheric volumes. Magnetization transfer MRI revealed significant white matter abnormalities in the bilateral corpus callosum and internal capsule. These quantitative MT-MRI changes correlated highly with postmortem findings of reduced myelin basic protein in bilateral white matter tracts. Ventriculomegaly and persistent astrogliosis were observed on the ligated side, along with evidence of axonopathy and fewer oligodendrocytes in the corpus callosum. We present an ischemia-induced mouse model of PVL, which has pathologic abnormalities resembling autopsy reports in infants with PVL. We further validate in vivo MRI techniques as quantitative monitoring tools that highly correlate with postmortem histopathology.

Carboxypeptidase E knockout mice exhibit abnormal dendritic arborization and spine morphology in central nervous system neurons.

Carboxypeptidase E (CPE) is involved in maturation of neuropeptides and sorting of brain-derived neurotrophic factor (BDNF) to the regulated pathway for activity-dependent secretion from CNS neurons. CPE knockout (CPE-KO) mice have many neurological deficits, including deficits in learning and memory. Here, we analyzed the dendritic arborization and spine morphology of CPE-KO mice to determine a possible correlation of defects in such structures with the neurological deficits observed in these animals. Analysis of pyramidal neurons in layer V of cerebral cortex and in hippocampal CA1 region in 14-week-old CPE-KO mice showed more dendritic complexity compared with wild type (WT) mice. There were more dendritic intersections and more branch points in CPE-KO vs. WT neurons. Comparison of pyramidal cortical neurons in 6- vs. 14-week-old WT mice showed a decrease in dendritic arborization, reflecting the occurrence of normal dendritic pruning. However, this did not occur in CPE-KO neurons. Furthermore, analysis of spine morphology demonstrated a significant increase in the number of D-type spines regarded as nonfunctional in the cortical neurons of CPE-KO animals. Our findings suggest that CPE is an important, novel player in mediating appropriate dendritic patterning and spine formation in CNS neurons.

Platelet-derived growth factor-producing cells immortalized from rat mesencephalon with SV40 large T antigen transduced by an AAV vector.

PURPOSE: Adeno-associated virus (AAV) can infect a wide variety of mammalian cell types and is capable of infecting both dividing and non-dividing cell populations. Here we report the construction of a recombinant AAV vector which expresses the SV40 large T protein (AAV-T) and the use of this vector to immortalize primary cells from embryonic rat mesencephalon. METHODS: The AAV-T vector was constructed by introducing the BamH1 fragment of the pCMV/SVE/Neo plasmid containing T antigen and SV40 regulatory elements into the JM48 plasmid containing the inverted terminal repeats of AAV. Neuronal cultures from E-12 rat mesencephalon were grown in defined media supplemented with basic fibroblast growth factor. These cells were infected with the AAV-T vector. RESULTS: A cell line (designated RMAT) and six subclones were established from these cultures through multiple passages. This cell line was immunoreactive for SV40 large T antigen and the cytoskeletal proteins nestin and vimentin. Morphological differentiation and expression of neurofilament 160 kDa were induced by exposure to dibutyrl cyclic AMP. Immunoassays performed to measure endogenous production of growth factors showed that RMAT cells produced high levels of platelet-derived growth factor (PDGF). CONCLUSIONS: AAV may be a useful vector for the transduction of oncogenes to produce cell lines.