Hydrogel-based milliwell arrays for standardized and scalable retinal organoid cultures.

The development of improved methods to culture retinal organoids is relevant for the investigation of mechanisms of retinal development under pathophysiological conditions, for screening of neuroprotective compounds, and for providing a cellular source for clinical transplantation. We report a tissue-engineering approach to accelerate and standardize the production of retinal organoids by culturing mouse embryonic stem cells (mESC) in optimal physico-chemical microenvironments. Arrayed round-bottom milliwells composed of biomimetic hydrogels, combined with an optimized medium formulation, promoted the rapid generation of retina-like tissue from mESC aggregates in a highly efficient and stereotypical manner: ∼93% of the aggregates contained retinal organoid structures. 26 day-old retinal organoids were composed of ∼80% of photoreceptors, of which ∼22% are GNAT2-positive cones, an important and rare sensory cell type that is difficult to study in rodent models. The compartmentalization of retinal organoids into predefined locations on a two-dimensional array not only allowed us to derive almost all aggregates into retinal organoids, but also to reliably capture the dynamics of individual organoids, an advantageous requirement for high-throughput experimentation. Our improved retinal organoid culture system should be useful for applications that require scalability and single-organoid traceability.

Hyperactivation of retina by light in mice leads to photoreceptor cell death mediated by VEGF and retinal pigment epithelium permeability.

Light toxicity is suspected to enhance certain retinal degenerative processes such as age-related macular degeneration. Death of photoreceptors can be induced by their exposure to the visible light, and although cellular processes within photoreceptors have been characterized extensively, the role of the retinal pigment epithelium (RPE) in this model is less well understood. We demonstrate that exposition to intense light causes the immediate breakdown of the outer blood-retinal barrier (BRB). In a molecular level, we observed the slackening of adherens junctions tying up the RPE and massive leakage of albumin into the neural retina. Retinal pigment epithelial cells normally secrete vascular endothelial growth factor (VEGF) at their basolateral side; light damage in contrast leads to VEGF increase on the apical side--that is, in the neuroretina. Blocking VEGF, by means of lentiviral gene transfer to express an anti-VEGF antibody in RPE cells, inhibits outer BRB breakdown and retinal degeneration, as illustrated by functional, behavioral and morphometric analysis. Our data show that exposure to high levels of visible light induces hyperpermeability of the RPE, likely involving VEGF signaling. The resulting retinal edema contributes to irreversible damage to photoreceptors. These data suggest that anti-VEGF compounds are of therapeutic interest when the outer BRB is altered by retinal stresses.

Use of human MAR elements to improve retroviral vector production.

Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors.

Efficient lentiviral gene transfer into corneal stroma cells using a femtosecond laser.

We investigated a new procedure for gene transfer into the stroma of pig cornea for the delivery of therapeutic factors. A delimited space was created at 110 mum depth with a LDV femtosecond laser in pig corneas, and a HIV1-derived lentiviral vector expressing green fluorescent protein (GFP) (LV-CMV-GFP) was injected into the pocket. Corneas were subsequently dissected and kept in culture as explants. After 5 days, histological analysis of the explants revealed that the corneal pockets had closed and that the gene transfer procedure was efficient over the whole pocket area. Almost all the keratocytes were transduced in this area. Vector diffusion at right angles to the pocket's plane encompasses four (endothelium side) to 10 (epithelium side) layers of keratocytes. After 21 days, the level of transduction was similar to the results obtained after 5 days. The femtosecond laser technique allows a reliable injection and diffusion of lentiviral vectors to efficiently transduce stromal cells in a delimited area. Showing the efficacy of this procedure in vivo could represent an important step toward treatment or prevention of recurrent angiogenesis of the corneal stroma.

Lentiviral vector-mediated gene transfer in adult mouse photoreceptors is impaired by the presence of a physical barrier.

Gene transfer offers a substantial promise for the therapy of degenerative ocular diseases. Lentiviral vectors have the ability to efficiently transduce murine photoreceptors during the first days of life, but they are poorly effective on photoreceptors during adulthood. Here, we studied whether a physical barrier was responsible for this impairment. Previous studies have described the capacity of enzymes, such as chondroitinase ABC and neuraminidase X, to modify the structure of the interphotoreceptor matrix (IPM) when subretinally injected. Considering the IPM as a physical barrier that may decrease photoreceptor transduction, we injected different enzymes into the subretinal space of the adult mouse simultaneously with the lentiviral vector preparation, to increase viral transduction by fragilizing the IPM. Subretinal injection of neuraminidase X and chondroitinase ABC induces modifications in the IPM by, respectively, revealing or decreasing peanut agglutinin sites on photoreceptors. The simultaneous subretinal injection of neuraminidase X with a lentiviral vector driving the expression of a reporter gene in the photoreceptors increases the number of transduced cells significantly (around five-fold). After the enzyme treatment, the diffusion of the vector between the pigmented epithelium and the photoreceptors appears to facilitate the lentiviral vector transduction. Such approach targeting the IPM may help to design new strategies to improve gene delivery into the adult photoreceptors.

Activity analysis of housekeeping promoters using self-inactivating lentiviral vector delivery into the mouse retina.

For most retinal degeneration disorders, no efficient treatment exists to preserve photoreceptors (PRs) and, consequently, to maintain vision. Gene transfer appears to be a promising approach to prevent PR loss. In order to design adequate vectors to target specific retinal cell types, we have analyzed the expression pattern of three different promoters (mouse phosphoglycerate kinase 1 (PGK), elongation factor-1 (EFS), rhodopsin (Rho)) in newborn and adult DBA/2 mice retinas using self-inactivating lentiviral vectors. At 7 days after intraocular injection and in optimal conditions, cell transduction was observed up to 1.5 mm from the injection site. PGK promoter expression was predominant in the retinal pigment epithelium (RPE), especially in adult mice, whereas the EFS promoter allowed a broad expression in the retina. Finally, as expected, the Rho promoter was specifically expressed in PRs. Differences in the cell types transduced and in transduction efficiency were observed between newborn and adult injected eyes emphasizing the importance of such basic studies for further gene therapy approaches as well as for understanding the transcriptional changes during retinal maturation. Thus, for future attempts to slow or rescue retinal degeneration by lentiviral delivery, PGK and EFS are more suitable to control the expression of a supporting secreted factor, PGK being mainly expressed in RPE and EFS in different cell types throughout the entire retina, whereas Rho should allow to specifically deliver the therapeutic gene to PRs.

Insulin-like growth factor-I is necessary for neural stem cell proliferation and demonstrates distinct actions of epidermal growth factor and fibroblast growth factor-2.

Neural stem cells (NSCs), when stimulated with epidermal growth factor (EGF) or fibroblast growth factor-2 (FGF-2), have the capacity to renew, expand, and produce precursors for neurons, astrocytes, and oligodendrocytes. We postulated that the early appearance of insulin-like growth factor (IGF-I) receptors during mouse striatum development implies a role in NSC regulation. Thus, we tested in vitro the action of IGF-I on the proliferation of striatal NSCs. In the absence of IGF-I, neither EGF nor FGF-2 was able to induce the proliferation of E14 mouse striatal cells. However, addition of IGF-I generated large proliferative clusters, termed spheres, in a dose-dependent manner. The newly generated spheres were multipotent, and clonal analysis revealed that EGF or FGF-2, in the presence of IGF-I, acted directly on NSCs. The actions of IGF-I suggest distinct modes of action of EGF or FGF-2 on NSCs. First, continuous versus delayed administration of these neurotrophic factors showed that neither IGF-I nor EGF had an effect on NSC survival, whereas FGF-2 promoted the survival or maintenance of the stem cell state of 50% of NSCs for 6 d. Second, short-term exposure to IGF-I induced the proliferation of NSCs in the presence of EGF, but not of FGF-2, through an autocrine secretion of IGF-I. These findings suggest that IGF-I is a key factor in the regulation of NSC activation and that EGF and FGF-2 control striatal NSC proliferation, in part, through distinct intracellular mechanisms.

Isolation of multipotent neural precursors residing in the cortex of the adult human brain.

Multipotent precursors able to generate neurons, astrocytes, and oligodendrocytes have previously been isolated from human brain embryos and recently from neurogenic regions of the adult human brains. The isolation of multipotent neural precursors from adult human should open new perspectives to study adult neurogenesis and for brain repair. The present study describes the in vitro isolation from adult human brains of a progenitor responsive to both epidermal and basic fibroblast growth factors that forms spheres as it proliferates. Single spheres derived from various regions of the brain generate in vitro neurons, astrocytes, and oligodendrocytes. The clonal origin of the spheres was revealed by genomic viral insertion using lentiviral vector. Interestingly, this vector appears to be a potent tool for gene transfer into human neural progeny. Ninety-six percent of the spheres investigated were multipotent. Multipotent precursors were isolated from all brain regions studied, including the temporal and the frontal cortex, the amygdala, the hippocampus, and the ventricular zone. This study is the first evidence that primitive precursors such as multipotent precursors exist in the adult human cortex and can reside far from the ventricles. Neurogenesis derived from adult human progenitors differ to murine neurogenesis by the requirement of laminin for oligodendrocyte generation and by the action of basic-fibroblast growth factor and platelet derived growth factor that prevented the formation of oligodendrocytes and neurons. Moreover, the differentiation of human adult precursors seems to differ from fetal ones: adult precursors do not necessitate the removal of mitogen for differentiation. These results indicate that the study of adult multipotent precursors is a new platform to study adult human neurogenesis, potentially generate neural cells for transplantation, and design protocols for in vivo stimulation.

Differential roles of tumor necrosis factor-alpha and interferon-gamma in mouse hypermetabolic and anorectic responses induced by LPS.

Lipopolysaccharide (LPS)-induced effects on energy balance are characterized by alterations in energy expenditure (hypermetabolism) and food intake (anorexia). To study the role of tumour necrosis factor alpha (TNF-alpha) on some of these metabolic responses to endotoxin, we have used transgenic mice expressing soluble tumour necrosis factor receptor-1 IgG fusion protein (TNFR1-IgG) as well as TNF-alpha knockout (KO), lymphotoxin-alpha (LT-alpha) KO, and interferon-gamma receptor (IFN-gamma R) KO mice. The results from TNFR1-IgG transgenic mice suggest that the hypermetabolic and anorectic responses induced by LPS are independently regulated since, in the absence of TNF-alpha or LT-alpha, the LPS-induced hypermetabolism is almost prevented but not the anorexia. The anorectic response shows the strongest association with IFN-gamma since both IFN-gamma R KO mice and mice treated with anti-IFN-gamma antibody showed marked reduction in the LPS-induced anorexia compared to other mice. IFN-gamma R KO mice also have an attenuated thermogenic response to endotoxin. Anti-Asialo GM1 antibody treatment attenuated both the hypermetabolic and anorectic responses to LPS, to an extent comparable to that observed in IFN-gamma R KO mice. This finding suggests that natural killer cells (lymphocytic subsets) may be involved in IFN-gamma production and play an important role in the metabolic alterations induced by LPS. We also showed that the hypermetabolic response of control mice is associated with an upregulation of cytokine expression within the brain and an increase in permeability of the blood brain barrier. LPS-induced anorexia appears to involve peripheral cytokine expression.

Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

A role for the POU-III transcription factor Brn-4 in the regulation of striatal neuron precursor differentiation.

Both insulin-like growth factor-I (IGF-I) and brain-derived neurotrophic factor (BDNF) induce the differentiation of post-mitotic neuronal precursors, derived from embryonic day 14 (E14) mouse striatal multipotent stem cells. Here we ask whether this differentiation is mediated by a member of the POU-III class of neural transcription factors. Exposure of stem cell progeny to either IGF-I or BDNF resulted in a rapid upregulation of Brn-4 mRNA and protein. Indirect immunocytochemistry with Brn-4 antiserum showed that the protein was expressed in newly generated neurons. Other POU-III genes, such as Brn-1 and Brn-2, did not exhibit this upregulation. Basic FGF, a mitogen for these neuronal precursors, did not stimulate Brn-4 expression. In the E14 mouse striatum, Brn-4-immunoreactive cells formed a boundary between the nestin-immunoreactive cells of the ventricular zone and the beta-tubulin-immunoreactive neurons migrating into the mantle zone. Loss of Brn-4 function during the differentiation of stem cell-derived or primary E14 striatal neuron precursors, by inclusion of antisense oligonucleotides, caused a reduction in the number of beta-tubulin-immunoreactive neurons. These findings suggest that Brn-4 mediates, at least in part, the actions of epigenetic signals that induce striatal neuron-precursor differentiation.

Region-specific effect of testosterone on oxytocin receptor binding in the brain of the aged rat.

We reported previously a significant reduction of oxytocin (OT) receptor binding in the brain of 20-month old rats relative to 3-month old ones. The present study shows that testosterone treatment of aging rats restores normal adult levels of OT receptor binding in the olfactory tubercle and in the hypothalamic ventromedial nucleus, but not in the caudate-putamen. These data indicate that the reduced plasma testosterone found in 20-month old rats is responsible for the loss of OT receptors in the olfactory tubercle and hypothalamic ventromedial nucleus, whereas other aging-related mechanisms may account for the decrease of OT receptor binding in the caudate-putamen.

Insulin-like growth factor-I is a differentiation factor for postmitotic CNS stem cell-derived neuronal precursors: distinct actions from those of brain-derived neurotrophic factor.

Insulin-like growth factor-I (IGF-I) has been reported previously to promote the proliferation, survival, and maturation of sympathetic neuroblasts, the genesis of retinal neurons, and the survival of CNS projection and motor neurons. Here we asked whether IGF-I could promote the in vitro differentiation of postmitotic mammalian CNS neuronal precursors derived from multipotent epidermal growth factor (EGF)-responsive stem cells. In the absence of IGF-I, virtually no neurons were present in cultured stem cell progeny, whereas IGF-I increased neuron number by eight- to 40-fold. Brief exposures (2 hr) to IGF-I were sufficient to allow for neuronal differentiation without affecting proliferation or survival. IGF-I actions could be mimicked by insulin and IGF-II at concentrations that correspond to the pharmacology of the IGF-I receptor, the latter for which the mRNA was detected in undifferentiated stem cell progeny. Although ineffectual alone at low concentrations (10 nM) that would activate its own receptor, insulin was able to potentiate the actions of IGF-I by acting on mitotically active neural precursors. When neuronal precursor differentiation by IGF-I was examined in relation to brain-derived neurotrophic factor (BDNF), two important observations were made: (1) BDNF could potentiate the differentiating actions of IGF-I plus insulin, and (2) BDNF could act on a separate population of precursors that did not require IGF-I plus insulin for differentiation. Taken together, these results suggest that IGF-I and BDNF may act together or sequentially to promote neuronal precursor differentiation.

Vasopressin binding sites in the central nervous system: distribution and regulation.

High affinity binding sites for vasopressin (VP) are widely distributed within the rat brain and spinal cord. Since their presence is associated with neuronal sensitivity to VP application, their anatomical distribution maps structures which could be activated by endogenous VP. Interestingly, marked species-related differences of the VP receptor distribution have been revealed. Some evidence has also been provided that mechanisms of receptor regulation may vary among species. In the rat, the expression of VP binding sites in some motor nuclei shows remarkable plasticity, in particular up-regulation after axotomy. These data suggest that VP may, in addition to affecting motoneuronal excitability, act as a trophic factor onto motoneurones.

Distribution of vasopressin and oxytocin receptors in the rat spinal cord: sex-related differences and effect of castration in pudendal motor nuclei.

The distribution of vasopressin and oxytocin receptors was established by in vitro autoradiography in the spinal cord of adult rats of either sex, as well as in male castrates. In both males and females, high concentrations of vasopressin binding sites were found in a few groups of somatic motoneurons: the large lateral group at the cervicothoracic junction in segments C8 and Th1; the small medial group in segments L3-L5; and the pudendal and retrodorsolateral nuclei in segments L5-L6. The extension and intensity of labelling in pudendal nuclei were markedly lower in females than in males, in particular in the dorsomedial nucleus, where binding was either not or hardly detectable. Gonadectomy in males resulted in a significant reduction of binding in pudendal nuclei, but not in other labelled motor nuclei. Moderate amounts of vasopressin binding sites were also found evenly distributed throughout the central gray at all segmental levels. Oxytocin binding sites were detectable in all spinal segments, but in low amounts and restricted to the superficial layers of the dorsal horn. The abundance of vasopressin binding sites in the central gray suggests that vasopressin may be involved in most spinal functions. The permanent expression of vasopressin binding sites in pudendal motor nuclei of is particular interest with regard to the known plasticity of pudendal motoneurons.

Vasopressin potentiation of the melatonin synthetic pathway via specific V1a receptors in the rat pineal gland.

The pineal gland releases the "time-keeping' hormone melatonin following a rhythmic sympathetic input which translates light information. The aim of this work was to study the role and mechanism of action of the central vasopressinergic input on pineal cAMP-dependent melatonin synthesis in the rat. The pineal was found to display vasopressin receptors of the V1a subtype, as the V1a antagonist [125I]HO-LVA bound in a saturable manner to pineal membranes with a high affinity (kd = 10 pM) and a maximal binding capacity (B(max)) of 13 fmol/mg protein. Vasopressin was able to displace [125I]HO-LVA binding in a dose-dependent manner (k(i) = 1.9 nM). Vasopressin had no effect on the basal cAMP level and melatonin secretion in cultured rat pinealocytes. However, it clearly and dose-dependently (EC50 = 7 nM) potentiated by 2-3 times cAMP accumulation and by 1.5-2.5 times melatonin secretion induced by moderate noradrenergic stimulation. On strongly stimulated pinealocytes, however, vasopressin could potentiate cAMP accumulation, but not melatonin secretion. The potentiatory effect of vasopressin was inhibited in the presence of the V1a antagonist. These results indicate that vasopressin is a potent modulator of rat pineal synthetic activity.

Reduced binding of oxytocin in the rat brain during aging.

Central oxytocin (OT) receptors were labelled in 3-month-old and 20-month-old rats with an iodinated OT antagonist. Comparison of the autoradiograms by quantitative image analysis revealed in the old animals a significant reduction of binding in three regions; the number of labelled OT receptors was decreased by 90% in the head of the caudate putamen, by 68% in the olfactory tubercle, and by 41% in the ventromedial hypothalamic nucleus. Previous studies had shown that the expression of OT receptors in the olfactory tubercle and in the ventromedial hypothalamic nucleus was dependent upon gonadal steroids. Therefore we hypothesize that the reduced number of OT receptors in the latter two structures of aged rats was the consequence of the 4-fold decrease of plasma testosterone that we found in this age. Another mechanism may be responsible for the marked reduction of OT receptors in the caudate putamen.

Characterization of a novel, linear radioiodinated vasopressin antagonist: an excellent radioligand for vasopressin V1a receptors.

We report on the pharmacological properties of a potent and selective linear vasopressin (AVP) V1a receptor antagonist HO-Phenylacetyl1-D-Tyr(Me)2-Phe3-Gln4-Asn5-Arg6-Pro7-Arg8-NH2 (HO-LVA). Iodinated on the phenolic substituent at position 1, [125I]-HO-LVA displayed the highest affinity for rat liver V1a receptors (8 pM) ever reported. Furthermore, affinities of HO-LVA and I-HO-LVA for V1b, V2 and oxytocin (OT) receptors was 400- to 1,000-fold lower than for V1a receptors, rendering it a highly selective ligand. Both HO-LVA and its iodinated derivative are V1 antagonists, they potently inhibited AVP-induced inositol-phosphate accumulation in WRK1 cells, and also, although with a much lower potency, the AVP-induced ACTH release from freshly prepared pituitary cells. Using autoradiography [125I]-HO-LVA appeared to be the first radioligand to successfully identify and localize the presence of V1a receptors in rat liver and blood vessel walls. Moreover, several new brain regions expressing V1a receptors could be identified, in addition to those brain regions that were previously identified with other radiolabelled AVP analogues.

Axotomy induces the expression of vasopressin receptors in cranial and spinal motor nuclei in the adult rat.

8-L-Arginine vasopressin ([Arg8]VP) receptors are expressed transiently in the rat facial nucleus during the perinatal period. Electrophysiological studies suggest that at least part of these receptors is located on facial motoneurones. In the present study we report that, in the adult rat, unilateral section of a facial nerve results in a massive and transient reexpression of [Arg8]VP receptors in the deeferented facial nucleus. Data were obtained by quantitative film autoradiography. During the first 2 postoperative weeks, binding of an iodinated ligand selective for V1a-type receptors increased about 10-fold. Maximal levels of binding were maintained for 1-2 weeks and then started to decrease. Binding was not strictly restricted to the facial nucleus but included the neuropile between motoneuronal pools and the perifacial area, which may indicate a dendritic localization of [Arg8]VP receptors. To investigate whether other motor nuclei also react to axotomy by up-regulating [Arg8]VP receptors, we sectioned either a hypoglossal nerve or a sciatic nerve. Two weeks after surgery, the hypoglossal nucleus or sciatic motoneuronal pools ipsilateral to the lesion were intensely labeled with the iodinated ligand. In contrast, nerve section had no effect on oxytocin binding sites in facial, hypoglossal, or sciatic motor nuclei. The results suggest that [Arg8]VP receptor expression in motor nuclei may depend upon neuromuscular contacts and, thus, that [Arg8]VP may be involved in the establishment of neuromuscular connections during development and in their reestablishment after nerve injury.