Characterization of Three-Dimensional Retinal Tissue Derived from Human Embryonic Stem Cells in Adherent Monolayer Cultures.

Stem cell-based therapy of retinal degenerative conditions is a promising modality to treat blindness, but requires new strategies to improve the number of functionally integrating cells. Grafting semidifferentiated retinal tissue rather than progenitors allows preservation of tissue structure and connectivity in retinal grafts, mandatory for vision restoration. Using human embryonic stem cells (hESCs), we derived retinal tissue growing in adherent conditions consisting of conjoined neural retina and retinal pigment epithelial (RPE) cells and evaluated cell fate determination and maturation in this tissue. We found that deriving such tissue in adherent conditions robustly induces all eye field genes (RX, PAX6, LHX2, SIX3, SIX6) and produces four layers of pure populations of retinal cells: RPE (expressing NHERF1, EZRIN, RPE65, DCT, TYR, TYRP, MITF, PMEL), early photoreceptors (PRs) (coexpressing CRX and RCVRN), inner nuclear layer neurons (expressing CALB2), and retinal ganglion cells [RGCs, expressing BRN3B and Neurofilament (NF) 200]. Furthermore, we found that retinal progenitors divide at the apical side of the hESC-derived retinal tissue (next to the RPE layer) and then migrate toward the basal side, similar to that found during embryonic retinogenesis. We detected synaptogenesis in hESC-derived retinal tissue, and found neurons containing many synaptophysin-positive boutons within the RGC and PR layers. We also observed long NF200-positive axons projected by RGCs toward the apical side. Whole-cell recordings demonstrated that putative amacrine and/or ganglion cells exhibited electrophysiological responses reminiscent of those in normal retinal neurons. These responses included voltage-gated Na(+) and K(+) currents, depolarization-induced spiking, and responses to neurotransmitter receptor agonists. Differentiation in adherent conditions allows generation of long and flexible pieces of 3D retinal tissue suitable for isolating transplantable slices of tissue for retinal replacement therapies.

Tgf-ß1 inhibits Cftr biogenesis and prevents functional rescue of ΔF508-Cftr in primary differentiated human bronchial epithelial cells.

CFTR is an integral transmembrane glycoprotein and a cAMP-activated Cl(-) channel. Mutations in the CFTR gene lead to Cystic Fibrosis (CF)-an autosomal recessive disease with majority of the morbidity and mortality resulting from airway infection, inflammation, and fibrosis. The most common disease-associated mutation in the CFTR gene-deletion of Phe508 (ΔF508) leads to a biosynthetic processing defect of CFTR. Correction of the defect and delivery of ΔF508-CFTR to the cell surface has been highly anticipated as a disease modifying therapy. Compared to promising results in cultured cell this approach was much less effective in CF patients in an early clinical trial. Although the cause of failure to rescue ΔF508-CFTR in the clinical trial has not been determined, presence of factor(s) that interfere with the rescue in vivo could be considered. The cytokine TGF-ß1 is frequently elevated in CF patients. TGF-ß1 has pleiotropic effects in different disease models and genetic backgrounds and little is known about TGF-ß1 effects on CFTR in human airway epithelial cells. Moreover, there are no published studies examining TGF-ß1 effects on the functional rescue of ΔF508-CFTR. Here we found that TGF-ß1 inhibits CFTR biogenesis by reducing mRNA levels and protein abundance in primary differentiated human bronchial epithelial (HBE) cells from non-CF individuals. TGF-ß1 inhibits CFTR biogenesis without compromising the epithelial phenotype or integrity of HBE cells. TGF-ß1 also inhibits biogenesis and impairs the functional rescue of ΔF508-CFTR in HBE cells from patients homozygous for the ΔF508 mutation. Our data indicate that activation of TGF-ß1 signaling may inhibit CFTR function in non-CF individuals and may interfere with therapies directed at correcting the processing defect of ΔF508-CFTR in CF patients.

Sox11 modulates brain-derived neurotrophic factor expression in an exon promoter-specific manner.

Sox11 is a high-mobility group (HMG)-containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity, and regeneration. BDNF transcription is elevated in the dorsal root ganglia (DRG) following nerve injury in parallel with Sox11, allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11, we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5'-AACAAAG-3' (I, IV, VII, and VIII) were increased at 1 or 3 days following axotomy. Exons 1 and IV showed the greatest increase, and only exon 1 remained elevated at 3 days. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription.

Sox11 transcription factor modulates peripheral nerve regeneration in adult mice.

The ability of adult peripheral sensory neurons to undergo functional and anatomical recovery following nerve injury is due in part to successful activation of transcriptional regulatory pathways. Previous in vitro evidence had suggested that the transcription factor Sox11, a HMG-domain containing protein that is highly expressed in developing sensory neurons, is an important component of this regenerative transcriptional control program. To further test the role of Sox11 in an in vivo system, we developed a new approach to specifically target small interfering RNAs (siRNAs) conjugated to the membrane permeable molecule Penetratin to injured sensory afferents. Injection of Sox11 siRNAs into the mouse saphenous nerve caused a transient knockdown of Sox11 mRNA that transiently inhibited in vivo regeneration. Electron microscopic level analysis of Sox11 RNAi-injected nerves showed that regeneration of myelinated and unmyelinated axons was inhibited. Nearly all neurons in ganglia of crushed nerves that were Sox11 immunopositive showed colabeling for the stress and injury-associated activating transcription factor 3 (ATF3). In addition, treatment with Sox11 siRNAs in vitro and in vivo caused a transcriptional and translational level reduction in ATF3 expression. These anatomical and expression data support an intrinsic role for Sox11 in events that underlie successful regeneration following peripheral nerve injury.

Artemin overexpression in skin enhances expression of TRPV1 and TRPA1 in cutaneous sensory neurons and leads to behavioral sensitivity to heat and cold.

Artemin, a neuronal survival factor in the glial cell line-derived neurotrophic factor family, binds the glycosylphosphatidylinositol-anchored protein GFRalpha3 and the receptor tyrosine kinase Ret. Expression of the GFRalpha3 receptor is primarily restricted to the peripheral nervous system and is found in a subpopulation of nociceptive sensory neurons of the dorsal root ganglia (DRGs) that coexpress the Ret and TrkA receptor tyrosine kinases and the thermosensitive channel TRPV1. To determine how artemin affects sensory neuron properties, transgenic mice that overexpress artemin in skin keratinocytes (ART-OE mice) were analyzed. Expression of artemin caused a 20.5% increase in DRG neuron number and increased the level of mRNA encoding GFRalpha3, TrkA, TRPV1, and the putative noxious cold-detecting channel TRPA1. Nearly all GFRalpha3-positive neurons expressed TRPV1 immunoreactivity, and most of these neurons were also positive for TRPA1. Interestingly, acid-sensing ion channel (ASIC) 1, 2a, 2b, and 3 mRNAs were decreased in the DRG, and this reduction was strongest in females. Analysis of sensory neuron physiological properties using an ex vivo preparation showed that cutaneous C-fiber nociceptors of ART-OE mice had reduced heat thresholds and increased firing rates in response to a heat ramp. No change in mechanical threshold was detected. Behavioral testing of ART-OE mice showed that they had increased sensitivity to both heat and noxious cold. These results indicate that the level of artemin in the skin modulates gene expression and response properties of afferents that project to the skin and that these changes lead to behavioral sensitivity to both hot and cold stimuli.

Distinct domains of the limbic system-associated membrane protein (LAMP) mediate discrete effects on neurite outgrowth.

The limbic system-associated membrane protein (LAMP) is a glycosylphosphatidylinositol-anchored glycoprotein with three immunoglobulin (Ig) domains that can either enhance or inhibit neurite outgrowth depending upon the neuronal population examined. In the present study, we investigate the domains responsible for these activities. Domain deletion revealed that the N-terminal IgI domain is necessary and sufficient for the neurite-promoting activity observed in hippocampal neurons. In contrast, inhibition of neurite outgrowth in SCG neurons, which is mediated by heterophilic interactions, requires full-length LAMP, although selective inhibition of the second Ig domain, but not the first or third domains, prevented the inhibitory effect. This indicates that the IgII domain of LAMP harbors the neurite-inhibiting activity, but only in the context of the full-length configuration. Covasphere-binding analyses demonstrate IgI/IgI interactions, but no interaction between IgII and any other domain, consistent with the biological activities that each domain mediates. The data suggest that LAMP may serve as a bifunctional guidance molecule, with distinct structural domains contributing to the promotion and inhibition of neurite outgrowth.