Functional maturation of human iPSC-derived pyramidal neurons in vivo is dependent on proximity with the host tissue.

Human induced pluripotent stem cells (hiPSCs) have been used extensively in vitro to model early events in neurodevelopment. Because of a number of shortcomings, previous work has established a potential to use these cells in vivo after transplantation into the mouse brain. Here, we describe a systematic approach for the analysis of transplanted hiPSC-derived neurons and glial cells over time in the mouse brain. Using functional two-photon imaging of GCaMP6f- expressing human neural cells, we define and quantify the embryonic-like features of their spontaneous activity. This is substantiated by detailed electron microscopy (EM) of the graft. We relate this to the synaptic development the neurons undergo up to 7 months in vivo. This system can now be used further for the genetic or experimental manipulation of developing hiPSC-derived cells addressing neurodevelopmental diseases like schizophrenia or Autism Spectrum Disorder.

Long-term development of human iPSC-derived pyramidal neurons quantified after transplantation into the neonatal mouse cortex.

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".

Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.

Tobacco smoking is a public health problem, with ∼5 million deaths per year, representing a heavy burden for many countries. No effective therapeutic strategies are currently available for nicotine addiction, and it is therefore crucial to understand the etiological and pathophysiological factors contributing to this addiction. The neuronal α5 nicotinic acetylcholine receptor (nAChR) subunit is critically involved in nicotine dependence. In particular, the human polymorphism α5D398N corresponds to the strongest correlation with nicotine dependence risk found to date in occidental populations, according to meta-analysis of genome-wide association studies. To understand the specific contribution of this subunit in the context of nicotine addiction, an efficient screening system for native human nAChRs is needed. We have differentiated human induced pluripotent stem (iPS) cells into midbrain dopaminergic (DA) neurons and obtained a comprehensive characterization of these neurons by quantitative RT-PCR. The functional properties of nAChRs expressed in these human DA neurons, with or without the polymorphism in the α5 subunit, were studied with the patch-clamp electrophysiological technique. Our results in human DA neurons carrying the polymorphism in the α5 subunit showed an increase in EC50, indicating that, in the presence of the polymorphism, more nicotine or acetylcholine chloride is necessary to obtain the same effect. This human cell culturing system can now be used in drug discovery approaches to screen for compounds that interact specifically with human native and polymorphic nAChRs.-Deflorio, C., Blanchard, S., Carisì, M. C., Bohl, D., Maskos, U. Human polymorphisms in nicotinic receptors: a functional analysis in iPS-derived dopaminergic neurons.

Immunohistochemical toolkit for tracking and quantifying xenotransplanted human stem cells.

AIM: Biomarker-based tracking of human stem cells xenotransplanted into animal models is crucial for studying their fate in the field of cell therapy or tumor xenografting. MATERIALS & METHODS: Using immunohistochemistry and in situ hybridization, we analyzed the expression of three human-specific biomarkers: Ku80, human mitochondria (hMito) and Alu. RESULTS: We showed that Ku80, hMito and Alu biomarkers are broadly expressed in human tissues with no or low cross-reactivity toward rat, mouse or pig tissues. In vitro, we demonstrated that their expression is stable over time and does not change along the differentiation of human-derived induced pluripotent stem cells or human glial-restricted precursors. We tracked in vivo these cell populations after transplantation in rodent spinal cords using aforementioned biomarkers and human-specific antibodies detecting apoptotic, proliferating or neural-committed cells. CONCLUSION: This study assesses the human-species specificity of Ku80, hMito and Alu, and proposes useful biomarkers for characterizing human stem cells in xenotransplantation paradigms.

Combination of hypoglossal-facial nerve surgical reconstruction and neurotrophin-3 gene therapy for facial palsy.

OBJECT: Facial nerve injury results in facial palsy that has great impact on the psychosocial conditions of affected patients. Reconstruction of the facial nerve to restore facial symmetry and expression is still a significant surgical challenge. In this study, the authors assessed a hypoglossal-facial nerve anastomosis method combined with neurotrophic factor gene therapy to treat facial palsy in adult rats after facial nerve injury. METHODS: Surgery consisted of the interposition of a predegenerated nerve graft (PNG) that was anastomosed with the hypoglossal and facial nerves at each of its extremities. The hypoglossal nerve was cut approximately 50% for this anastomosis to conserve partial hypoglossal function. Before their transplantation, the PNGs were genetically engineered using lentiviral vectors to induce overexpression of the neurotrophic factor neurotrophin-3 (NT-3) to improve axonal regrowth in the reconstructed nerve pathway. Reconstruction was performed after facial nerve injury, either immediately or after a delay of 9 weeks. The rats were followed up for 4 months postoperatively, and treatment outcomes were then assessed. RESULTS: Compared with the functional innervation in control rats that underwent facial nerve injury without subsequent treatment, functional innervation of the paralyzed whisker pad by hypoglossal motoneurons in rats treated 4 months after nerve reconstruction was evidenced by the retrograde transport of neuronal tracers, the recording of muscle action potentials conducted by the PNG, and the recovery of facial symmetry. Although a better outcome was observed when reconstruction was performed immediately after facial nerve injury, reconstruction with NT3-treated PNGs significantly improved functional reinnervation of the paralyzed whisker pad even when implantation occurred 9 weeks posttrauma. CONCLUSIONS: Results demonstrated that hypoglossal-facial nerve anastomosis facilitates innervation of paralyzed facial muscle via hypoglossal motoneurons without sacrificing ipsilateral hemitongue function. Neurotrophin-3 treatment through gene therapy could effectively improve such innervation, even after delayed reconstruction. These findings suggest that the combination of surgical reconstruction and NT-3 gene therapy is promising for its potential application in treating facial palsy in humans.

Combination of microsurgery and gene therapy for spinal dorsal root injury repair.

Brachial plexus injury is frequent after traffic accident in adults or shoulder dystocia in newborns. Whereas surgery can restore arm movements, therapeutic options are missing for sensory defects. Dorsal root (DR) ganglion neurons convey sensory information to the central nervous system (CNS) through a peripheral and a central axon. Central axons severed through DR section or avulsion during brachial plexus injury inefficiently regenerate and do not reenter the spinal cord. We show that a combination of microsurgery and gene therapy circumvented the functional barrier to axonal regrowth at the peripheral and CNS interface. After cervical DR section in rats, microsurgery restored anatomical continuity through a nerve graft that laterally connected the injured DR to an intact DR. Gene transfer to cells in the nerve graft induced the local release of neurotrophin-3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) and stimulated axonal regrowth. Central DR ganglion axons efficiently regenerated and invaded appropriate areas of the spinal cord dorsal horn, leading to partial recovery of nociception and proprioception. Microsurgery created conditions for functional restoration of DR ganglion central axons, which were improved in combination with gene therapy. This combination treatment provides means to reduce disability due to somatosensory defects after brachial plexus injury.

Directed evolution of motor neurons from genetically engineered neural precursors.

Stem cell-based therapies hold therapeutic promise for degenerative motor neuron diseases, such as amyotrophic lateral sclerosis, and for spinal cord injury. Fetal neural progenitors present less risk of tumor formation than embryonic stem cells but inefficiently differentiate into motor neurons, in line with their low expression of motor neuron-specific transcription factors and poor response to soluble external factors. To overcome this limitation, we genetically engineered fetal rat spinal cord neurospheres to express the transcription factors HB9, Nkx6.1, and Neurogenin2. Enforced expression of the three factors rendered neural precursors responsive to Sonic hedgehog and retinoic acid and directed their differentiation into cholinergic motor neurons that projected axons and formed contacts with cocultured myotubes. When transplanted in the injured adult rat spinal cord, a model of acute motor neuron degeneration, the engineered precursors transiently proliferated, colonized the ventral horn, expressed motor neuron-specific differentiation markers, and projected cholinergic axons in the ventral root. We conclude that genetic engineering can drive the differentiation of fetal neural precursors into motor neurons that efficiently engraft in the spinal cord. The strategy thus holds promise for cell replacement in motor neuron and related diseases. Disclosure of potential conflicts of interest is found at the end of this article.

Forced expression of the motor neuron determinant HB9 in neural stem cells affects neurogenesis.

In contrast to mouse embryonic stem cells and in spite of overlapping gene expression profiles, neural stem cells (NSCs) isolated from the embryonic spinal cord do not respond to physiological morphogenetic stimuli provided by Sonic hedgehog and retinoic acid and do not generate motor neurons upon differentiation. Transcription factors expressed in motor neuron progenitors during embryogenesis include Pax6, Ngn2, Nkx6.1 and Olig2, whose expression precedes that of factors specifying motor neuron fate, including HB9, Islet1 and LIM3. We showed that all these factors were present in neural progenitors derived from mouse ES cells, whereas NSCs derived from the rat embryonic spinal cord expressed neither HB9 nor Islet1 and contained low levels of Nkx6.1 and LIM3. We constructed a lentivirus vector to express HB9 and GFP in NSCs and examined the consequences of HB9 expression on other transcription factors and cell differentiation. Compared to cell expressing GFP alone, NSCs expressing GFP and HB9 cycled less rapidly, downregulated Pax6 and Ngn2 mRNA levels, produced higher proportions of neurons in vitro and lower numbers of neurons after transplantation in the spinal cord of recipient rats. Oligodendrocytic and astrocytic differentiations were not affected. HB9 expressing NSCs did not express Islet1 or upregulate LIM3. They neither responded to Sonic hedgehog and retinoic acid nor produced cholinergic neurons. We concluded that forced HB9 expression affected neurogenesis but was not sufficient to confer motor neuron fate to NSCs.

PHR1, an integral membrane protein of the inner ear sensory cells, directly interacts with myosin 1c and myosin VIIa.

By using the yeast two-hybrid technique, we identified a candidate protein ligand of the myosin 1c tail, PHR1, and found that this protein can also bind to the myosin VIIa tail. PHR1 is an integral membrane protein that contains a pleckstrin homology (PH) domain. Myosin 1c and myosin VIIa are two unconventional myosins present in the inner ear sensory cells. We showed that PHR1 immunoprecipitates with either myosin tail by using protein extracts from cotransfected HEK293 cells. In vitro binding assays confirmed that PHR1 directly interacts with these two myosins. In both cases the binding involves the PH domain. In vitro interactions between PHR1 and the myosin tails were not affected by the presence or absence of Ca2+ and calmodulin. Finally, we found that PHR1 is able to dimerise. As PHR1 is expressed in the vestibular and cochlear sensory cells, its direct interactions with the myosin 1c and VIIa tails are likely to play a role in anchoring the actin cytoskeleton to the plasma membrane of these cells. Moreover, as both myosins have been implicated in the mechanotransduction slow adaptation process that takes place in the hair bundles, we propose that PHR1 is also involved in this process.

Myosin VIIa, harmonin and cadherin 23, three Usher I gene products that cooperate to shape the sensory hair cell bundle.

Deaf-blindness in three distinct genetic forms of Usher type I syndrome (USH1) is caused by defects in myosin VIIa, harmonin and cadherin 23. Despite being critical for hearing, the functions of these proteins in the inner ear remain elusive. Here we show that harmonin, a PDZ domain-containing protein, and cadherin 23 are both present in the growing stereocilia and that they bind to each other. Moreover, we demonstrate that harmonin b is an F-actin-bundling protein, which is thus likely to anchor cadherin 23 to the stereocilia microfilaments, thereby identifying a novel anchorage mode of the cadherins to the actin cytoskeleton. Moreover, harmonin b interacts directly with myosin VIIa, and is absent from the disorganized hair bundles of myosin VIIa mutant mice, suggesting that myosin VIIa conveys harmonin b along the actin core of the developing stereocilia. We propose that the shaping of the hair bundle relies on a functional unit composed of myosin VIIa, harmonin b and cadherin 23 that is essential to ensure the cohesion of the stereocilia.

MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes.

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa-defective mice.