Marco Onorati

Induced Pluripotent Stem Cells from Patients with Huntington’s Disease : Show CAG Repeat-Expansion-Associated Phenotypes

Huntingtons disease (HD) is an inherited neurodegenerative disorder caused by an expanded stretch of CAG trinucleotide repeats that results in neuronal dysfunction and death. Here, The HD Consortium reports the generation and characterization of 14 induced pluripotent stem cell (iPSC) lines from HD patients and controls. Microarray profiling revealed CAG-repeat-expansion-associated gene expression patterns that distinguish patient lines from controls, and early onset versus late onset HD. Differentiated HD neural cells showed disease-associated changes in electrophysiology, metabolism, cell adhesion, and ultimately cell death for lines with both medium and longer CAG repeat expansions. The longer repeat lines were however the most vulnerable to cellular stressors and BDNF withdrawal, as assessed using a range of assays across consortium laboratories. The HD iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in HD and provides a human stem cell platform for screening new candidate therapeutics.

Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32(+) medium-sized spiny neurons

Medium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington’s disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGFβ inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1+/GSX2+ telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1+/FOXP2+/CTIP2+/calbindin+/DARPP-32+ MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32+ neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.

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.

iPSC-derived neural precursors exert a neuroprotective role in immune-mediated demyelination via the secretion of LIF

The possibility of generating neural stem/precursor cells (NPCs) from induced pluripotent stem cells (iPSCs) has opened a new avenue of research that might nurture bench-to-bedside translation of cell transplantation protocols in central nervous system myelin disorders. Here we show that mouse iPSC-derived NPCs (miPSC-NPCs)-when intrathecally transplanted after disease onset-ameliorate clinical and pathological features of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Transplanted miPSC-NPCs exert the neuroprotective effect not through cell replacement, but through the secretion of leukaemia inhibitory factor that promotes survival, differentiation and the remyelination capacity of both endogenous oligodendrocyte precursors and mature oligodendrocytes. The early preservation of tissue integrity limits blood-brain barrier damage and central nervous system infiltration of blood-borne encephalitogenic leukocytes, ultimately responsible for demyelination and axonal damage. While proposing a novel mechanism of action, our results further expand the therapeutic potential of NPCs derived from iPSCs in myelin disorders.

EZ spheres: A stable and expandable culture system for the generation of pre-rosette multipotent stem cells from human ESCs and iPSCs

We have developed a simple method to generate and expand multipotent, self-renewing pre-rosette neural stem cells from both human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) without utilizing embryoid body formation, manual selection techniques, or complex combinations of small molecules. Human ESC and iPSC colonies were lifted and placed in a neural stem cell medium containing high concentrations of EGF and FGF-2. Cell aggregates (termed EZ spheres) could be expanded for long periods using a chopping method that maintained cell-cell contact. Early passage EZ spheres rapidly down-regulated OCT4 and up-regulated SOX2 and nestin expression. They retained the potential to form neural rosettes and consistently differentiated into a range of central and peripheral neural lineages. Thus, they represent a very early neural stem cell with greater differentiation flexibility than other previously described methods. As such, they will be useful for the rapidly expanding field of neurological development and disease modeling, high-content screening, and regenerative therapies based on pluripotent stem cell technology.

Neuropotent self-renewing neural stem (NS) cells derived from mouse induced pluripotent stem (iPS) cells

Neural stem (NS) cells are a homogenous population of stem cells that expands in monolayer under serum-free conditions while remaining highly neuropotent. Here, we generated NS cells from induced pluripotent stem (iPS) cells that were previously derived from mouse fibroblasts (NS-(f)iPS). We showed that NS-(f)iPS cells exhibit long-term expansion and express markers of neurogenic radial glia. Analyses of the regional markers expressed in NS-(f)iPS cells suggested a ventral-rhombencephalic identity. Upon exposure to differentiation protocols, NS-(f)iPS cells produce neurons, astrocytes, and oligodendrocytes with an efficiency similar to ES-derived NS cells. NS-(f)iPS cells represent a new tool for studying neural cell fate determination and terminal differentiation, providing an interesting resource for experimental transplantation. Comparative studies between NS cells derived from iPS cells, reprogrammed from different somatic sources, and from authentic ES cells are necessary to identify critical elements for multipotency acquisition.

Induced pluripotent stem cell lines from Huntington's disease mice undergo neuronal differentiation while showing alterations in the lysosomal pathway.

Huntingtons disease (HD) is an autosomal dominant neurodegenerative disorder caused by an excessive expansion of a CAG trinucleotide repeat in the gene encoding the protein huntingtin, resulting in an elongated stretch of glutamines near the N-terminus of the protein. Here we report the derivation of a collection of 11 induced pluripotent stem (iPS) cell lines generated through somatic reprogramming of fibroblasts obtained from the R6/2 transgenic HD mouse line. We show that CAG expansion has no effect on reprogramming efficiency, cell proliferation rate, brain-derived neurotrophic factor level, or neurogenic potential. However, genes involved in the cholesterol biosynthesis pathway, which is altered in HD, are also affected in HD-iPS cell lines. Furthermore, we found a lysosomal gene upregulation and an increase in lysosome number in HD-iPS cell lines. These observations suggest that iPS cells from HD mice replicate some but not all of the molecular phenotypes typically observed in the disease; additionally, they do not manifest increased cell death propensity either under self-renewal or differentiated conditions. More studies will be necessary to transform a revolutionary technology into a powerful platform for drug screening approaches.

Molecular and functional definition of the developing human striatum

The complexity of the human brain derives from the intricate interplay of molecular instructions during development. Here we systematically investigated gene expression changes in the prenatal human striatum and cerebral cortex during development from post-conception weeks 2 to 20. We identified tissue-specific gene coexpression networks, differentially expressed genes and a minimal set of bimodal genes, including those encoding transcription factors, that distinguished striatal from neocortical identities. Unexpected differences from mouse striatal development were discovered. We monitored 36 determinants at the protein level, revealing regional domains of expression and their refinement, during striatal development. We electrophysiologically profiled human striatal neurons differentiated in vitro and determined their refined molecular and functional properties. These results provide a resource and opportunity to gain global understanding of how transcriptional and functional processes converge to specify human striatal and neocortical neurons during development.

Preservation of positional identity in fetus-derived neural stem (NS) cells from different mouse central nervous system compartments

Neural stem (NS) cells are a self-renewing population of symmetrically dividing multipotent radial glia-like stem cells, characterized by homogeneous expansion in monolayer. Here we report that fetal NS cells isolated from different regions of the developing mouse nervous system behave in a similar manner with respect to self-renewal and neuropotency, but exhibit distinct positional identities. For example, NS cells from the neocortex maintain the expression of anterior transcription factors, including Otx2 and Foxg1, while Hoxb4 and Hoxb9 are uniquely found in spinal cord-derived NS cells. This molecular signature was stable for over 20 passages and was strictly linked to the developmental stage of the donor, because only NS cells derived from E14.5 cortex, and not those derived from E12.5 cortex, carried a consistent transcription factor profile. We also showed that traits of this positional code are maintained during neuronal differentiation, leading to the generation of electrophysiologically active neurons, even if they do not acquire a complete neurochemical identity.Neural stem (NS) cells are a self-renewing population of symmetrically dividing multipotent radial glia-like stem cells, characterized by homogeneous expansion in monolayer. Here we report that fetal NS cells isolated from different regions of the developing mouse nervous system behave in a similar manner with respect to self-renewal and neuropotency, but exhibit distinct positional identities. For example, NS cells from the neocortex maintain the expression of anterior transcription factors, including Otx2 and Foxg1, while Hoxb4 and Hoxb9 are uniquely found in spinal cord-derived NS cells. This molecular signature was stable for over 20 passages and was strictly linked to the developmental stage of the donor, because only NS cells derived from E14.5 cortex, and not those derived from E12.5 cortex, carried a consistent transcription factor profile. We also showed that traits of this positional code are maintained during neuronal differentiation, leading to the generation of electrophysiologically active neurons, even if they do not acquire a complete neurochemical identity.

Molecular and cellular reorganization of neural circuits in the human lineage

The makings of the primate brain Although nonhuman primate brains are similar to our own, the disparity between their and our cognitive abilities tells us that surface similarity is not the whole story. Sousa et al. overlaid transcriptome and histological analyses to see what makes human brains different from those of nonhuman primates. Various differentially expressed genes, such as those encoding transcription factors, could alter transcriptional programs. Others were associated with neuromodulatory systems. Furthermore, the dopaminergic interneurons found in the human neocortex were absent from the neocortex of nonhuman African apes. Such differences in neuronal transcriptional programs may underlie a variety of neurodevelopmental disorders. Science, this issue p. 1027 Comparing transcriptome and histology of human and nonhuman primate brains reveals changes that make humans unique. To better understand the molecular and cellular differences in brain organization between human and nonhuman primates, we performed transcriptome sequencing of 16 regions of adult human, chimpanzee, and macaque brains. Integration with human single-cell transcriptomic data revealed global, regional, and cell-type–specific species expression differences in genes representing distinct functional categories. We validated and further characterized the human specificity of genes enriched in distinct cell types through histological and functional analyses, including rare subpallial-derived interneurons expressing dopamine biosynthesis genes enriched in the human striatum and absent in the nonhuman African ape neocortex. Our integrated analysis of the generated data revealed diverse molecular and cellular features of the phylogenetic reorganization of the human brain across multiple levels, with relevance for brain function and disease.