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

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

CNTF and reporter gene expression in neural cell types derived from CNTF-NS cells and control-NS cells <i>in vitro</i>.

<p>CNTF-NS (a-c, g-i) and control-NS cells (d-f, j-l) were differentiated into neurons (a-f) or astrocytes (g-l). Note that all MAP-2-positive neurons (b) and GFAP-positive astrocytes (h) derived from CNTF-NS cells co-expressed the reporter gene Venus (a, g) and CNTF (c, i). Neurons (e) and astrocytes (k) derived from control-NS cells, in comparison, expressed the reporter gene tdTomato (d, j) but no detectable levels of the cytokine (f, l). CNTF, ciliary neurotrophic factor; DAPI, 4’,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2. Bar in l (for a-l): 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

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

<i>In vivo</i> myelin-formation by pre-differentiated ‘RSCs’ derived from peripheral regions of the developing retina.

<p>Analysis of wild-type retinas four weeks after transplantation of oligo-primed <i>actin-EGFP</i>-‘RSCs’ (green) from P3 into adult mice. Many GFP-expressing donor cells form MBP-positive elongated structures on the vitreal side of the retina (red; some are labeled by arrows in B; B is an enlarged view of the boxed area in A) showing MBP-positive fibers radiating towards the optic disc (white star). Scale bars: 100 µm (A), 50 µm (B).</p

Increased neuronal differentiation of ‘RSCs’ by priming and notch inhibition.

<p>Generation efficiency of neuronal cell types by expanded ’RSCs’ is dependent on differentiation conditions. ’RSC’ cultures subjected to different differentiation conditions detailed in (A) and immunolabeled with antibodies directed against GFAP (C, green) and β-III-tubulin (C, red) contain different percentages of neurons in dependence of the applied protocol (B, C). The percentage of β-III-tubulin -positive neurons is significantly increased by ’neuronal-priming’ from <17% to <32% when compared to differentiation conditions where mitogens are replaced by NCS and can be further increased to <76% by inhibition of notch-signaling using DAPT (B). DMSO represents the DAPT control experiment with neuron numbers (<37%) corresponding to ’priming’ (B, C). Scale bars: 50 µm. Abbreviations: d, days; DAPI, 4,6-diamidino-2-phenylindole; DAPT, [N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butylester; DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein. **p<0.01, ***p<0.001.</p

Oligodendrocyte differentiation of ‘RSCs’ <i>in vitro</i>.

<p>Whereas primary cells isolated at E14.5 from different regions of the CNS, i.e. spinal cord, striatum, and cortex, showed strong MBP expression (B) when subjected to the oligodendroglial differentiation protocol detailed in (A), primary retinal cells remained MBP-negative (B). However, after <i>in vitro</i> expansion central and peripheral ‘RSC’ (P3) cultures generated by appropriate dissection procedures (C) responded to the oligodendrocyte differentiation protocol with expression of MBP as shown by immunocytochemistry (D) and RT-PCR performed on RNA isolated from undifferentiated, oligo-primed and oligo-differentiated ‘RSCs’ and NSCs from P3 (E). Expanded peripheral ‘RSCs’ (P3) as well as their primary counterparts (peripheral retinal cells from PN0) were subjected to the oligo-differentiation protocol <i>in vitro</i> and their gene expression profile was investigated in detail using Q-PCR array for oligodendrocyte-related gene expression (F). Primary cells expressed moderate levels of <i>Olig1/2</i> genes, whereas expanded ‘RSCs’ contained very high levels of these transcripts. While primary cells subjected to oligo-differentiation conditions did not respond to the treatment with an increase in the expression of oligodendrocyte-related genes, expanded ‘RSCs’ exhibited a significant increase in <i>MBP</i> transcript levels along with other oligodendrocyte-related genes that were analyzed (<i>Vcan</i>, <i>Nkx6.2</i>, <i>Sox10</i>). Generation of oligodendrocytes from expanded ‘RSCs’ was Shh-independent since we could not detect endogenous <i>Shh</i> nor its downstream target <i>Gli1</i> (F). Scale bars: 100 µm (B) and 50 µm (D). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; E, embryonic day; MBP, myelin basic protein; Oligo1/1+2, oligodendroglial differentiation step 1/1+2; PN, postnatal day; T3, 3,3,5-triodothyronine; Rho, rhodopsin; Rxrg, retinoid X receptor gamma; Rcvrn - recoverin.</p

Differentiation of ‘RSCs’ <i>in vitro</i>.

<p>Following differentiation in 1% newborn calf serum expanded ‘RSCs’ showed immunoreactivity for the pan-neuronal markers β-III-tubulin (A, red) and MAP2 (B, red) or the glial marker GFAP (A, green). A subfraction of cells expressed the interneuron markers calretinin (B, green) or calbindin (B, red) or, after prolonged maintenance in differentiation conditions, the mature neuron marker NeuN (B, red). In contrast to primary neonatal retinal cells (D) subjected to the same differentiation conditions, expanded ‘RSC’ cultures are devoid of recoverin (red) or rhodopsin (green) expressing photoreceptors (C). Scale bars: 50 µm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein 2; NeuN, neuron-specific nuclear antigen.</p