Georgia Panagiotakos

Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage

Neural stem cells (NSCs) yield both neuronal and glial progeny, but their differentiation potential toward multiple region-specific neuron types remains remarkably poor. In contrast, embryonic stem cell (ESC) progeny readily yield region-specific neuronal fates in response to appropriate developmental signals. Here we demonstrate prospective and clonal isolation of neural rosette cells (termed R-NSCs), a novel NSC type with broad differentiation potential toward CNS and PNS fates and capable of in vivo engraftment. R-NSCs can be derived from human and mouse ESCs or from neural plate stage embryos. While R-NSCs express markers classically associated with NSC fate, we identified a set of genes that specifically mark the R-NSC state. Maintenance of R-NSCs is promoted by activation of SHH and Notch pathways. In the absence of these signals, R-NSCs rapidly lose rosette organization and progress to a more restricted NSC stage. We propose that R-NSCs represent the first characterized NSC stage capable of responding to patterning cues that direct differentiation toward region-specific neuronal fates. In addition, the R-NSC-specific genetic markers presented here offer new tools for harnessing the differentiation potential of human ESCs.

Isolation and directed differentiation of neural crest stem cells derived from human embryonic stem cells

Vertebrate neural crest development depends on pluripotent, migratory precursor cells. Although avian and murine neural crest stem (NCS) cells have been identified, the isolation of human NCS cells has remained elusive. Here we report the derivation of NCS cells from human embryonic stem cells at the neural rosette stage. We show that NCS cells plated at clonal density give rise to multiple neural crest lineages. The human NCS cells can be propagated in vitro and directed toward peripheral nervous system lineages (peripheral neurons, Schwann cells) and mesenchymal lineages (smooth muscle, adipogenic, osteogenic and chondrogenic cells). Transplantation of human NCS cells into the developing chick embryo and adult mouse hosts demonstrates survival, migration and differentiation compatible with neural crest identity. The availability of unlimited numbers of human NCS cells offers new opportunities for studies of neural crest development and for efforts to model and treat neural crest–related disorders.

Directed Differentiation and Transplantation of Human Embryonic Stem Cell-Derived Motoneurons

Motoneurons represent a specialized class of neurons essential for the control of body movement. Motoneuron loss is the cause of a wide range of neurological disorders including amyotrophic lateral sclerosis and spinal muscular atrophy. Embryonic stem cells are a promising cell source for the study and potential treatment of motoneuron diseases. Here, we present a novel in vitro protocol of the directed differentiation of human embryonic stem cells (hESCs) into engraftable motoneurons. Neural induction of hESCs was induced on MS5 stromal feeders, resulting in the formation of neural rosettes. In response to sonic hedgehog and retinoic acid, neural rosettes were efficiently directed into spinal motoneurons with appropriate in vitro morphological, physiological, and biochemical properties. Global gene expression analysis was used as an unbiased measure to confirm motoneuron identity and type. Transplantation of motoneuron progeny into the developing chick embryo resulted in robust engraftment, maintenance of motoneuron phenotype, and long‐distance axonal projections into peripheral host tissues. Transplantation into the adult rat spinal cord yielded neural grafts comprising a large number of human motoneurons with outgrowth of choline acetyltransferase positive fibers. These data provide evidence for in vivo survival of hESC‐derived motoneurons, a key requirement in the development of hESC‐based cell therapy in motoneuron disease.

Derivation of engraftable skeletal myoblasts from human embryonic stem cells

Human embryonic stem cells (hESCs) are a promising source for cell therapy in degenerative diseases. A key step in establishing the medical potential of hESCs is the development of techniques for the conversion of hESCs into tissue-restricted precursors suitable for transplantation. We recently described the derivation of multipotent mesenchymal precursors from hESCs. Nevertheless, our previous study was limited by the requirement for mouse feeders and the lack of in vivo data. Here we report a stroma-free induction system for deriving mesenchymal precursors. Selective culture conditions and fluorescence-activated cell sorting (FACS)-mediated purification yielded multipotent mesenchymal precursors and skeletal myoblasts. Skeletal muscle cells undergo in vitro maturation resulting in myotube formation and spontaneous twitching. We found that hESC-derived skeletal myoblasts were viable after transplantation into the tibialis anterior muscle of SCID/Beige mice, as assessed by bioluminescence imaging. Lack of teratoma formation and evidence of long-term myoblast engraftment suggests considerable potential for future therapeutic applications.

Migration and differentiation of neural precursors derived from human embryonic stem cells in the rat brain

Human embryonic stem (hES) cells provide a potentially unlimited cell source for regenerative medicine. Recently, differentiation strategies were developed to direct hES cells towards neural fates in vitro. However, the interaction of hES cell progeny with the adult brain environment remains unexplored. Here we report that hES cell–derived neural precursors differentiate into neurons, astrocytes and oligodendrocytes in the normal and lesioned brain of young adult rats and migrate extensively along white matter tracts. The differentiation and migration behavior of hES cell progeny was region specific. The hES cell–derived neural precursors integrated into the endogenous precursor pool in the subventricular zone, a site of persistent neurogenesis. Like adult neural stem cells, hES cell–derived precursors traveled along the rostral migratory stream to the olfactory bulb, where they contributed to neurogenesis. We found no evidence of cell fusion, suggesting that hES cell progeny are capable of responding appropriately to host cues in the subventricular zone.

Long-Term Impact of Radiation on the Stem Cell and Oligodendrocyte Precursors in the Brain

Background The cellular basis of long term radiation damage in the brain is not fully understood. Methods and Findings We administered a dose of 25Gy to adult rat brains while shielding the olfactory bulbs. Quantitative analyses were serially performed on different brain regions over 15 months. Our data reveal an immediate and permanent suppression of SVZ proliferation and neurogenesis. The olfactory bulb demonstrates a transient but remarkable SVZ-independent ability for compensation and maintenance of the calretinin interneuron population. The oligodendrocyte compartment exhibits a complex pattern of limited proliferation of NG2 progenitors but steady loss of the oligodendroglial antigen O4. As of nine months post radiation, diffuse demyelination starts in all irradiated brains. Counts of capillary segments and length demonstrate significant loss one day post radiation but swift and persistent recovery of the vasculature up to 15 months post XRT. MRI imaging confirms loss of volume of the corpus callosum and early signs of demyelination at 12 months. Ultrastructural analysis demonstrates progressive degradation of myelin sheaths with axonal preservation. Areas of focal necrosis appear beyond 15 months and are preceded by widespread demyelination. Human white matter specimens obtained post-radiation confirm early loss of oligodendrocyte progenitors and delayed onset of myelin sheath fragmentation with preserved capillaries. Conclusions This study demonstrates that long term radiation injury is associated with irreversible damage to the neural stem cell compartment in the rodent SVZ and loss of oligodendrocyte precursor cells in both rodent and human brain. Delayed onset demyelination precedes focal necrosis and is likely due to the loss of oligodendrocyte precursors and the inability of the stem cell compartment to compensate for this loss.

Assembly of functionally integrated human forebrain spheroids

The development of the nervous system involves a coordinated succession of events including the migration of GABAergic (γ-aminobutyric-acid-releasing) neurons from ventral to dorsal forebrain and their integration into cortical circuits. However, these interregional interactions have not yet been modelled with human cells. Here we generate three-dimensional spheroids from human pluripotent stem cells that resemble either the dorsal or ventral forebrain and contain cortical glutamatergic or GABAergic neurons. These subdomain-specific forebrain spheroids can be assembled in vitro to recapitulate the saltatory migration of interneurons observed in the fetal forebrain. Using this system, we find that in Timothy syndrome—a neurodevelopmental disorder that is caused by mutations in the CaV1.2 calcium channel—interneurons display abnormal migratory saltations. We also show that after migration, interneurons functionally integrate with glutamatergic neurons to form a microphysiological system. We anticipate that this approach will be useful for studying neural development and disease, and for deriving spheroids that resemble other brain regions to assemble circuits in vitro.

Cadherin-6 Mediates Axon-Target Matching in a Non-Image-Forming Visual Circuit

Neural circuits consist of highly precise connections among specific types of neurons that serve a common functional goal. How neurons distinguish among different synaptic targets to form functionally precise circuits remains largely unknown. Here, we show that during development, the adhesion molecule cadherin-6 (Cdh6) is expressed by a subset of retinal ganglion cells (RGCs) and also by their targets in the brain. All of the Cdh6-expressing retinorecipient nuclei mediate non-image-forming visual functions. A screen of mice expressing GFP in specific subsets of RGCs revealed that Cdh3-RGCs which also express Cdh6 selectively innervate Cdh6-expressing retinorecipient targets. Moreover, in Cdh6-deficient mice, the axons of Cdh3-RGCs fail to properly innervate their targets and instead project to other visual nuclei. These findings provide functional evidence that classical cadherins promote mammalian CNS circuit development by ensuring that axons of specific cell types connect to their appropriate synaptic targets.

Behavioral Abnormalities and Circuit Defects in the Basal Ganglia of a Mouse Model of 16p11.2 Deletion Syndrome

A deletion on human chromosome 16p11.2 is associated with autism spectrum disorders. We deleted the syntenic region on mouse chromosome 7F3. MRI and high-throughput single-cell transcriptomics revealed anatomical and cellular abnormalities, particularly in cortex and striatum of juvenile mutant mice (16p11(+/-)). We found elevated numbers of striatal medium spiny neurons (MSNs) expressing the dopamine D2 receptor (Drd2(+)) and fewer dopamine-sensitive (Drd1(+)) neurons in deep layers of cortex. Electrophysiological recordings of Drd2(+) MSN revealed synaptic defects, suggesting abnormal basal ganglia circuitry function in 16p11(+/-) mice. This is further supported by behavioral experiments showing hyperactivity, circling, and deficits in movement control. Strikingly, 16p11(+/-) mice showed a complete lack of habituation reminiscent of what is observed in some autistic individuals. Our findings unveil a fundamental role of genes affected by the 16p11.2 deletion in establishing the basal ganglia circuitry and provide insights in the pathophysiology of autism.

Acquisition of in vitro and in vivo functionality of Nurr1-induced dopamine neurons

Neural precursor cells provide an expandable source of neurons and glia for basic and translational applications. However, little progress has been made in directing naive neural precursors toward specific neuronal fates such as midbrain dopamine (DA) neurons. We have recently demonstrated that transgenic expression of the nuclear orphan receptor Nurr1 is sufficient to drive dopaminergic differentiation of forebrain embryonic rat neural precursors in vitro. However, Nurr1‐induced DA neurons exhibit immature neuronal morphologies and functional properties and are unable to induce behavioral recovery in rodent models of Parkinsons disease (PD). Here, we report on the identification of key genetic factors that drive morphological and functional differentiation of Nurr1‐derived DA neurons. We show that coexpression of Nurr1, Bcl‐XL, and Sonic hedgehog (SHH) or Nurr1 and the proneural bHLH factor Mash1 is sufficient to drive naive rat forebrain precursors into neurons exhibiting the biochemical, electrophysiological, and functional properties of DA neuron in vitro. On transplantation into the striatum of Parkinsonian rats, precursor cells engineered with Nurr1/SHH/Bcl‐XL or Nurr1/Mash1 survived in vivo and differentiated into mature DA neurons that can reverse the behavioral deficits in the grafted animals.—Park, C.‐H., Kang, J. S., Shin, Y. H., Chang, M.‐Y., Chung, S., Koh, H.‐C., Zhu, M. H., Oh, S. B., Lee, Y.‐S., Panagiotakos, G., Tabar, V., Studer, L., and Lee, S.‐H. Acquisition of in vitro and in vivo functionality of Nurr1‐induced dopamine neurons. FASEB J. 20, E1910‐E1923 (2006)