Characterization CNTF-NS and control-NS cells six weeks after intravitreal transplantation into <i>nclf</i> mice.

<p>Six weeks after intravitreal transplantation, CNTF-NS (a-c) and control-NS cells (d-f) were identified in the host eyes by their expression of the fluorescent reporter proteins Venus (a) and tdTomato (d), respectively. Both, Venus-positive CNTF-NS cells and tdTomato-positive control-NS cells were attached to the posterior poles of the lenses where they were mainly differentiated into GFAP-positive astrocytes (b, e). Expression of CNTF was detectable in astrocytes derived from CNTF-NS cells (c), but not in astrocytes derived from control-NS cells (f). CNTF, ciliary neurotrophic factor; DAPI, 4’,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein. Bar in f (for a-f): 50 μm.</p

Sustained Neural Stem Cell-Based Intraocular Delivery of CNTF Attenuates Photoreceptor Loss in the <i>nclf</i> Mouse Model of Neuronal Ceroid Lipofuscinosis

<div><p>A sustained intraocular administration of neurotrophic factors is among the strategies aimed at establishing treatments for currently untreatable degenerative retinal disorders. In the present study we have analyzed the neuroprotective effects of a continuous neural stem (NS) cell-based intraocular delivery of ciliary neurotrophic factor (CNTF) on photoreceptor cells in the <i>nclf</i> mouse, an animal model of the neurodegenerative lysosomal storage disorder variant late infantile neuronal ceroid lipofuscinosis (vLINCL). To this aim, we genetically modified adherently cultivated NS cells with a polycistronic lentiviral vector encoding a secretable variant of CNTF together with a Venus reporter gene (CNTF-NS cells). NS cells for control experiments (control-NS cells) were modified with a vector encoding the reporter gene tdTomato. Clonal CNTF-NS and control-NS cell lines were established using fluorescent activated cell sorting and intravitreally grafted into 14 days old <i>nclf</i> mice at the onset of retinal degeneration. The grafted cells preferentially differentiated into astrocytes that were attached to the posterior side of the lenses and the vitreal side of the retinas and stably expressed the transgenes for at least six weeks, the latest post-transplantation time point analyzed. Integration of donor cells into host retinas, ongoing proliferation of grafted cells or adverse effects of the donor cells on the morphology of the host eyes were not observed. Quantitative analyses of host retinas two, four and six weeks after cell transplantation revealed the presence of significantly more photoreceptor cells in eyes with grafted CNTF-NS cells than in eyes with grafted control-NS cells. This is the first demonstration that a continuous intraocular administration of a neurotrophic factor attenuates retinal degeneration in an animal model of neuronal ceroid lipofuscinosis.</p></div

<i>In vivo</i> myelin-formation by pre-differentiated ‘RSCs’.

<p>Analysis of retinas four weeks after intraretinal transplantation of oligo-primed <i>actin-dsRed</i>-‘RSCs’ (red) into adult mice. Many donor cells identified by dsRed expression (A) were located on the vitreal side of the retina (the edges of a flat mounted retina are marked by the dashed white line) and some formed elongated structures radiating towards the optic disc (white star)(some are labeled by arrows in AII; AII is an enlarged view of the boxed area in AI) that are positive for MBP (green, A). Following transplantation of oligo-primed <i>actin-dsRed</i> expressing ‘RSCs’ (red, B), donor cells integrated into the GCL and IPL (red, B) of the host retina. Co-localization of MBP immunoreactivity (green, B) and dsRed fluorescence was restricted to the GCL (B, nuclear DAPI staining (blue) is additionally present in the merged image). Histological analysis of a semi-thin section revealed myelinated axons in the nerve fiber layer of an experimental retina (C; arrows). Transmission electron microscopy confirmed the presence of compact myelin around many RGC axons (some labeled by arrows) in retinas transplanted with ‘RSCs’ (D). Note the increased diameter of myelinated in comparison to unmyelinated axons (D; some labeled by red stars). Scale bars: 200 µm (AI), 50 µm (AII, B), 10 µm (C), 2500 nm (D). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; ONL, outer nuclear layer.</p

Differentiation of primary retinal cells and <i>in vitro</i> expanded ‘RSCs’ isolated from rhoEGFP reporter mice.

<p>Primary cells isolated at PN2 from retinas of <i>rhoEGFP</i> transgenic mice and cultured for 7 days <i>in vitro</i> (A) showed expression of GFP (green - identifying rod photoreceptors), β-III-tubulin (red - identifying neurons), or GFAP (white - identifying glial cells; merged images additionally contain nuclear DAPI staining). ‘RSCs’ generated from <i>rhoEGFP</i> transgenic mice and propagated <i>in vitro</i> for 6 passages were subjected to priming (B) or Notch inhibition with DAPT (D; C represents the DMSO control) differentiation conditions for 10 days. ‘RSCs’ differentiated into β-III-tubulin-positive neurons (red) and GFAP-positive glia (white), but did not show GFP expression indicative for photoreceptor differentiation (B, C, D). Scale bars: 20 um.</p

Oligo-differentiated ‘RSCs’ show slight elevated levels of photoreceptor-specific genes, but fail to generate photoreceptors.

<p>Cultivated peripheral ‘RSCs’ subjected to the full oligodendrocyte differentiation protocol showed a slight increase in the expression of the retina-specific genes <i>Chx10</i>, <i>Rax</i>, <i>Otx2</i>, <i>Crx</i>, <i>rhodopsin</i> and <i>RXRgamma</i> as analysed by Q-PCR albeit at very low absolute levels (A). Immunocytochemical analysis of ‘RSCs’ subjected to oligodendrocyte differentiation <i>in vitro</i> did not reveal positive signals for rhodopsin (red) or recoverin (white) proteins and therefore no evidence for generation of photoreceptors (B). Also following transplantation of oligo-primed ‘RSCs’ (C; green) into the subretinal space of degenerative P347S mice (C) donor cells (green) did not show immunopositivity for the photoreceptor marker recoverin (red). Nuclear DAPI staining is shown in blue, rhodopsin localization in red, recoverin localization in white (B). Scale bars: 50 µm (B). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole.</p

Immunocytochemistry, RT-PCR and Q-PCR analyses of neonatal ‘RSCs’ during <i>in vitro</i> cultivation.

<p>Following expansion in the presence of growth factors ‘RSCs’ show immunoreactivity for <i>nestin</i>, <i>Sox2</i> and <i>Pax6</i> at the protein (A) and mRNA levels (B, C). Further gene expression examination using semi-quantitative RT-PCR revealed that throughout all passages analyzed ‘RSCs’ express components of the notch signaling pathway (<i>Notch1</i> receptor, <i>Hes1</i> and <i>Hes5</i>). Levels of transcription factors crucial for eye and retina development (primary retina, PN1) were highly variable: <i>Six3</i> expression was stable up to P20, <i>Lhx2</i> and <i>Six6</i> levels decreased with increasing passage number, and <i>Rax</i> and <i>Chx10</i> were no longer detectable beginning with passage 3 (B). In comparison, NSCs isolated from E14.5 spinal cord or striatum and cultured for 10 or 5 passages, respectively, showed expression of <i>nestin</i>, <i>Sox2</i>, <i>Pax6</i>, and notch pathway components, but were negative for <i>Rax</i>, <i>Chx10</i>, <i>Six3</i>, and <i>Six6</i>. Adult and PN1 primary retina served as a positive or negative control (B). Q-PCR analysis performed on peripheral ‘RSCs’ from P3 and on their primary counterparts (peripheral retinal cells from PN0) confirmed the above RT-PCR results: expanded cells from low passages expressed <i>Nes</i>, <i>Sox2</i>, <i>Pax6</i>, <i>Notch1</i>, <i>Hes1</i>, <i>Hes5</i>, <i>Six3</i> and <i>Six6</i> (C). Although <i>Lhx2</i> level in P3 ‘RSCs’ was as high as in primary retinal cells, <i>Rax</i> and <i>Chx10</i> genes were undetectable (C). Gene expression levels are related to the mean expression levels of housekeeping genes. Scale bar: 50 µm. Abbreviations: E, embryonic day; exp, expanded; NSC, neural stem cells; P, passage; PN, postnatal day; ‘RSCs’, retinal stem cells; spcord, spinal cord.</p

Intravitreally grafted CNTF-NS cells attenuate photoreceptor degeneration in <i>nclf</i> mice.

<p>A CNTF-NS cell clone was grafted into one (a, c, e) and a control-NS cell clone into the contralateral eye (b, d, f) of 14 days old <i>nclf</i> mice. Central retinal sections were stained with anti-recoverin antibodies and DAPI two (a, b), four (c, d) and six (e, f) weeks after transplantation. Note the thicker outer nuclear layer (onl) of CNTF-treated retinas when compared to control retinas at all post-transplantation time points. DAPI, 4’,6-diamidino-2-phenylindole; onl, outer nuclear layer. Bar in f (for a-f): 50 μm.</p

Expression of CNTF and the reporter genes in clonal CNTF-NS and control-NS cell lines.

<p>All cells in the clonal CNTF-NS cell line expressed the reporter gene Venus (a) and showed CNTF-immunoreactivity in the perinuclear region (b). Control-NS cells, in comparison, expressed the reporter gene tdTomato (c) but lacked detectable expression of the cytokine (d). CNTF, ciliary neurotrophic factor; DAPI, 4’,6-diamidino-2-phenylindole. Bar in d (for a-d): 20 μm.</p

Lentiviral vectors and immunoblot analyses of culture supernatants from clonal CNTF-NS and control-NS cell lines.

<p>A lentiviral vector encoding a secretable variant of mouse ciliary neurotrophic factor (CNTF), an internal ribosome entry site (IRES) sequence of the encephalomyocarditis virus and a Venus reporter and a zeocin (ZEO) resistance gene separated by a P2A sequence of porcine teschovirus-1 (2A) under regulatory control of the cytomegalovirus enhancer/chicken ß-actin (CAG) promoter (a) was used to generate CNTF-secreting NS cells. NS cells for control experiments were transduced with a vector containing the CAG promoter, an IRES sequence and a tdTomato (tdTom) reporter gene fused to a blasticidin (BSD) resistance gene (b). Immunoblot analysis (c) of culture supernatants from the newly established CNTF-NS cell clone (clone 2) revealed elevated secretion levels of CNTF when compared to the original clonal CNTF-NS cell line (clone 1). Supernatants from control-NS cell clones (control) lacked detectable levels of the cytokine (c). Recombinant mouse CNTF (rmCNTF) was loaded as a reference. Ψ, packaging signal; cPPT, central polypurine tract; LoxP, recognition site of Cre recombinase; RRE, rev-responsive element; SIN-LTR, self-inactivating long-terminal repeat; wPRE, woodchuck hepatitis virus posttranscriptional regulatory element.</p

Photoreceptor numbers in eyes of <i>nclf</i> mice with grafted CNTF-NS or control-NS cells at different post-transplantation time points.

<p>A CNTF-NS and a control-NS cell line were intravitreally grafted into 14 days old <i>nclf</i> mice and photoreceptor numbers were determined in central retinal sections at six defined positions two, four and six weeks after transplantation. Note that CNTF-treated eyes contained significantly more photoreceptors (filled bars) than the contralateral eyes with grafted control-NS cells (open bars) at all post-transplantation time points. Each bar represents the mean value (±SEM) from six retinas. ***, p<0.001 (Newman-Keuls post hoc test after the mixed two-way ANOVA).</p