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Human neural progenitor cells promote photoreceptor survival in retinal explants

Human neural progenitor cells promote photoreceptor survival in retinal explants

Experimental Eye Research 90 (2010) 292e299 Contents lists available at ScienceDirect Experimental Eye Research journal homepage: www.elsevier.com/l...

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Experimental Eye Research 90 (2010) 292e299

Contents lists available at ScienceDirect

Experimental Eye Research journal homepage: www.elsevier.com/locate/yexer

Human neural progenitor cells promote photoreceptor survival in retinal explants Ulrica Englund-Johansson a,1, Camilla Mohlin b,1, Ingela Liljekvist-Soltic a, b, Per Ekström a, Kjell Johansson b, c, * a

Department of Ophthalmology, Clinical Sciences, Lund, University of Lund, Sweden School of Pure and Applied Natural Sciences, University of Kalmar, Sweden c Department of Clinical Medicine, School of Health and Medical Sciences, Örebro University, SE-701 82 Örebro, Sweden b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 8 June 2009 Accepted in revised form 10 November 2009 Available online 18 November 2009

Different types of progenitor and stem cells have been shown to provide neuroprotection in animal models of photoreceptor degeneration. The present study was conducted to investigate whether human neural progenitor cells (HNPCs) have neuroprotective properties on retinal explants models with calpainand caspase-3-dependent photoreceptor cell death. In the first experiments, HNPCs in a feeder layer were co-cultured for 6 days either with postnatal rd1 mouse or normal rat retinas. Retinal histological sections were used to determine outer nuclear layer (ONL) thickness, and to detect the number of photoreceptors with labeling for calpain activity, cleaved caspase-3 and TUNEL. The ONL thickness of co-cultured rat and rd1 retinas was found to be almost 10% and 40% thicker, respectively, compared to controls. Cell counts of calpain activity, cleaved caspase-3 and TUNEL labeled photoreceptors in both models revealed a 30e50% decrease when co-cultured with HNPCs. The results represent significant increases of photoreceptor survival in the co-cultured retinas. In the second experiments, for an identification of putative survival factors, or a combination of them, a growth factor profile was performed on conditioned medium. The relative levels of various growth factors were analyzed by densitometric measurements of growth factor array membranes. Following growth factors were identified as most potential survival factors; granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF), transforming growth factors (TGF-b1 and TGF-b2) and vascular endothelial growth factor (VEGF-D). HNPCs protect both against calpain- and caspase-3-dependent photoreceptor cell death in the rd1 mouse and against caspase-3-dependent photoreceptor cell death in normal rat retinas in vitro. The protective effect is possibly achieved by a variety of growth factors secreted from the HNPCs. Ó 2009 Elsevier Ltd. All rights reserved.

Keywords: retina photoreceptor apoptosis neuroprotection progenitor cells

1. Introduction A common feature of human retinal degenerative disorders, such as retinitis pigmentosa (RP), is decreased functionality resulting from the loss of photoreceptor cells. The disease is usually caused by an early loss of rod photoreceptors, preceding the slower secondary loss of cone photoreceptors. RP patients thus experience a progressive night blindness, followed by the loss of central vision (Phelan and Bok, 2000). A number of mutations have been associated with the disease (http://www.sph.uth.tmc.edu/retnet), and over 150 may relate to the rhodopsin gene. Other mutations may affect the signal transduction cascade or structural integrity within

* Corresponding author at: Department of Clinical Medicine, School of Health and Medical Sciences, Örebro University, SE-701 82 Örebro, Sweden. E-mail address: [email protected] (K. Johansson). 1 Equal contributors. 0014-4835/$ e see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.exer.2009.11.005

the photoreceptors. Despite the complexity of the underlying genetic mechanisms of inherited retinal degenerations, it is generally assumed that photoreceptor death is mediated via apoptosis (Remé et al., 1998). Studies of mouse and rat degeneration models for RP show that photoreceptor degeneration involves different death pathways including caspase- and calpain-dependent apoptosis (Doonan et al., 2005; Liu et al., 1999; Lohr et al., 2006; Wu et al., 2002). Irrespective of the death pathway, attenuation of apoptosis is a therapeutic strategy for RP and has received considerable attention. Molecules like brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), glial cell-line derived factor (GDNF) and vascular endothelial growth factor (VEGF) (Azadi et al., 2007; Liljekvist-Soltic et al., 2008; Ogilvie et al., 1999; Okoye et al., 2003; Saint-Geniez et al., 2008) represent neuroprotective factors, promoting survival of retinal neurons. In this respect, cell-based delivery of GDNF (Gamm et al., 2007) and CNTF (Sieving et al., 2006) in animal models and clinical trials, respectively, suggest

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a potential therapeutic utility in retinal degenerations. Although the usage of CNTF resulted in prolonged photoreceptor survival in degeneration models, sustained delivery also resulted in morphological alterations (Rhee et al., 2007) and reduced functionality (Bok et al., 2002; Schlichtenbrede et al., 2003). Interestingly, coadministration of BDNF and CNTF resulted in synergistic effect in rescuing photoreceptors in animal models of retinal degeneration (Azadi et al., 2007; Ogilvie et al., 1999; Okoye et al., 2003). It is possible that the complex retinal degenerative diseases require therapies that target multiple cellular pathways. An increasing number of studies have demonstrated that transplanted human neural progenitor cells (HNPCs) promote survival of degenerating neurons. Intracerebral grafted HNPCs show rescuing effects in models of brain diseases and disorders, including Parkinson's disease (Yasuhara et al., 2006), brain trauma (Hagan et al., 2003) as well as multiple sclerosis (Pluchino et al., 2003). Similarly, various models of retinal degeneration and photoreceptor apoptosis demonstrate that transplantation of intact embryonic retina as well as progenitor and/or stem cells may delay or inhibit the degeneration. A beneficial effect on photoreceptor survival in vitro and in vivo was observed with the usage of rodent embryonic progenitor (Liljekvist-Larsson and Johansson, 2005, 2007) and stem cells (Meyer et al., 2006; Wojciechowski et al., 2002), human umbilical cord cells (Lund et al., 2007), human neural progenitor cells (Gamm et al., 2007) and engraftment of embryonic retinal tissue (Ghosh et al., 2007). Enhanced host photoreceptor survival for 1.5e4 months post-operatively and the survival of grafted cells/tissue (Ghosh et al., 2007; Lund et al., 2007; Meyer et al., 2006; Wojciechowski et al., 2002), suggest that the grafts provide host photoreceptors or their microenvironment with beneficial cues. Recent data show that human neural progenitor cells (Gamm et al., 2007) and rat retinal progenitors (LiljekvistSoltic et al., 2008) produce different growth factors, which protect visual function in vivo and decrease caspase-3-dependent photoreceptor apoptosis in vitro, respectively. The current study examines possible protective effects of HNPCs in two retinal explant models exhibiting different photoreceptor degenerations; the rd1 mouse and normal rat retinas. Photoreceptor degeneration in rd1 mice is largely caspase-independent, and shows a distinguished calpain activity involvement (Paquet-Durand et al., 2006), whereas explantation of normal rat retinas is accompanied with a caspase-3-dependent photoreceptor cell death (LiljekvistLarsson and Johansson, 2005). We hypothesized that the progenitor cells secrete beneficial factors that provide protection to photoreceptors and attenuate their survival in the explanted retinas. Our results suggest that the HNPCs secreted mixture of growth factors is able to protect degenerating photoreceptors, irrespective of the apoptotic pathway. Subsequently, the ONL thickness of explanted retinas is preserved following co-culture with HNPCs. 2. Material and methods

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Sweden). Briefly, forebrain tissue was obtained from one 9-week (postconception) human embryo and isolated under compliance with the National Institute of Health guidelines, Swedish Government guidelines, and the local ethics committee. The HNPCs were cultured in defined DMEM-F12 medium (Invitrogen, Paisley, UK) supplemented with 2.0 mM L-glutamine (Sigma, St. Lois, MI), 0.6% glucose (Sigma), N2 supplement (Invitrogen), 2.0 mg/ml heparin (Sigma), and grown as free-floating neurospheres. Human basic fibroblast growth factor (20 ng/ml; Invitrogen), human epidermal growth factor (20 ng/ml; Invitrogen) and human leukemia inhibitory factor (10 ng/ml; Chemicon, Temecula, CA) were added every third day to the culture. The neurospheres were passaged by mechanical dissociation every 7e10 days and reseeded as single cells at a density of 1  105 cells/ ml. Cells from passage 33e35 times were used. 2.3. Co-culture of HNPCs and postnatal retinas HNPCs were cultured as recently described (Carpenter et al., 1999; Englund et al., 2002) and displayed stable growth kinetics. Separate pilot experiments showed that a seeding concentration of 2  105 cells/well was almost confluent after 1 week in vitro and displayed survival effects on photoreceptor survival in co-cultured retinas, and was thereby considered as optimal concentration for the purposes in the present study. Protocols for retinal explant culturing have been described previously (Azadi et al., 2007; Liljekvist-Larsson and Johansson, 2005), and were used here with some modifications (e.g. without most of the retinal pigment epithelium). Culture medium was composed of DMEM/F12 (Invitrogen) supplemented with 10% fetal calf serum (FCS) (Sigma), 1% penicillin/streptomycin, and 2 mM Lglutamine (Sigma). From each animal one retina was co-cultured with the HNPCs feeder layer (see below) and the contralateral retina was cultured in medium only as control. The retinas were explanted 24 h prior to co-culture and allowed to attach to the insert membrane. The specimens were cultured in DMEM/F12 (see above). Feeder layers of HNPCs were established two days after passage of 2  105 cells (small spheres)/well using the same medium as for explants (see above). After 24 h almost all cells were attached to the untreated well, and the co-culture started by placing an insert with attached retina into the well. The total medium volume was changed at this time, and every second day thereafter half of the medium was replaced with fresh medium. Conditioned medium from HNPCs/rd1 co-cultures, control medium from rd1 explants and HNPCs cultures without explant were stored at 20  C and used for growth factor analysis (see below). After 6 days of coculture, the explants were either fixed or directly frozen for histological evaluations. Feeder layers grown with rd1 retinas were also fixed. All cultures were kept at 37  C with 95% humidity and 5% CO2.

2.1. Animals

2.4. Immunohistochemistry

SpragueeDawley rats (Taconic, Copenhagen, Denmark) and CH3 rd1/rd1 mice from a colony kept in Lund were used throughout this study. Mouse and rat retinal tissues for explantation were taken from postnatal day 8 and 11e13 animals, respectively. Animal handling was in accordance with the approved national guidelines and the ARVO statement for animal care.

Inserts with explants were immersion fixed in 4% paraformaldehyde in phosphate buffer (PB) for 2 h at 4  C. After several rinses in PB, the specimens were cryoprotected (25% sucrose in PB) and cryosectioned at 10e12 mm. This fixative was also added for 10 min at 4  C to some HNPC feeder layers for immunocytochemical analysis. Specimens destined to in situ calpain activity assay were directly frozen in embedding medium and cryosectioned. Primary antibodies used are: rabbit anti-cleaved caspase-3 (1:1000; Cell Signaling Technology, Beverly, CA), mouse antirhodopsin (1:500; generously provided by Dr. R. Molday, University of British Columbia, Vancouver, Canada), rabbit anti-glial fibrillary

2.2. Generation and expansion of HNPCs The human neural progenitor cell line used for this study was generously provided by Prof. A Björklund (Lund University,

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acidic protein (1:500; GFAP; Dako, Glostrup, Denmark), mouse anti-microtubule associated protein-2 (1:200; MAP-2; Sigma). For the immunohistochemical staining, the specimens were incubated with phosphate buffered saline containing 0.25% Triton X (PBST) and 1% bovine serum albumin for 15 min at room temperature. The primary antibodies were applied overnight, and subsequently detected using appropriate FITC- or Texas Red-conjugated goat anti-mouse IgG Fab or donkey anti-rabbit IgG Fab (1:200; Jackson Laboratories, West Grove, PA). The sections were coverslipped using DAPI-containing Vectashield mounting medium (Vector Laboratories, Burlingame, CA) for counterstaining of nuclei. Both the primary and the secondary antibodies were diluted in PBST containing 1% bovine serum albumin (Sigma). Omission of the primary antibody was used as controls in all experiments. 2.5. Calpain activity assay Analysis of in situ calpain activity in unfixed retinal sections was performed as previously (Paquet-Durand et al., 2006). Briefly, sections of unfixed retina were incubated for 15 min in reaction buffer, consisting of 25 mM HEPES, 65 mM KCl, 2 mM MgCl2, 1.5 mM CaCl2, 2 mM dithiothreitol. The fluorescent calpain substrate 7-amino-4-chloromethylcoumarin, t-BOC-L-leu-cyl-Lmethionine amide (CMAC, t-BOC-Leu-Met, Invitrogen) was then added to the reaction buffer at a final concentration of 2 mM. Incubation was performed in darkness for 1 h at 35  C, followed by washes and mounting of the slides with Vectashield. 2.6. TUNEL assay A commercial TUNEL assay system with fluorescein-conjugated dUTP was used on fixed cryo-sections according to the instructions of the manufacturer (Roche, Mannheim, Germany). 2.7. Growth factor analysis To determine a growth factor profile of HNPCs/rd1 explant conditioned medium, media collected from rd1 explant cultures and a HNPCs culture without explants, a RayBio human growth factor antibody array I (Ray Biotech, Norrcross, GA) was used. The array consists of membranes coupled with 41 antibodies in duplicates and positive controls, which allows a comparison of the relative expression levels among different membranes. Briefly, the membranes were incubated in medium with blocking buffer 30 min followed by incubation in HNPCs/rd1 explant conditioned and the control media for 2 h at room temperature. Several washes in supplied wash buffers followed, where after the membranes were incubated with biotin-labeled antibodies at 4  C overnight. Visualization was achieved by ECL detection (Amersham, Buckinghamshire, UK). The protein concentrations of the media were determined and 20 mg protein/ml was used as optimal concentration. Densitometric analysis was performed using Image J software on scanned membranes from four independent HNPCs/rd1 explants conditioned and rd1 explant control medium samples. Triplicate measurement was performed on a batch of medium from a HNPCs culture without explants.

2.9. Data analysis The thickness of the ONL was measured at similar location of each DAPI-stained section. To avoid eccentricity-dependent effects, no measurements were made on the peripheral part of the explants. Counts of calpain, cleaved caspase-3 or TUNEL labeled cells were made on 2e3 areas per section and on 4e8 nonconsecutive sections. The measurements were averaged to a single value for each labeled retina. Each group (co-cultured and contralateral control retina) consisted of 4e8 animals. In HNPC feeder layers, the number of labeled cells was counted in micrographs taken at 20  magnification. One sample t-test (GraphPad; http:// graphscales.com/quickscales) was used to determine statistical significance. The values were normalized with respect to the average number of labeled cells, width of ONL or spot intensity within each experiment, and controls were set to 100%. All data are presented as mean  SEM, and p values less than 0.05 were considered statistically significant. 3. Results 3.1. HNPCs feeder layer characteristics HNPCs passaged between 33 and 35 times were used as feeder layers in the co-culture experiments. Since these late passages are known to produce cells with glial and neuronal phenotypes (Carpenter et al., 1999), appropriate cell markers were used to control the phenotypic composition of the HNPCs feeder layer after 7 days of differentiation in vitro. Fixed feeder layers co-cultured with rd1 explants were immunolabeled for GFAP and MAP-2 (Fig. 1) and glial and neuronal cells, respectively, were counted. The ratio of labeled cells relative to the total amount DAPI-stained cells was more than 65%, with 41.3  6.0% (n ¼ 6) being positive for GFAP and 25.3  3.3% (n ¼ 6) positive for MAP-2. This ratio of glial and neuronal cells in feeder layers of rd1 explant co-cultures, is in agreement with similar HNPC cell line expanded in vitro (Carpenter et al., 1999). As glial cells are known to produce growth factors (reviewed by Seth and Koul, 2008), a growth factor analysis of conditioned medium from HNPCs/rd1 explant co-cultures was compared to rd1 explant medium. The HNPCs/rd1 explant conditioned and rd1 explant control media showed similar growth factor profiles, and nine of the factors were consistently observed in at least 3 independent arrays (Fig. 1; Table 1). The relative levels of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GMCSF), insulin-like growth factor II (IGF-II), neurotrophic factor 3 (NT-3), placental growth factor (PIGF) and vascular endothelial growth factor (VEGF-D) were more than 25% higher compared to rd1 explant controls. The relative levels of transforming growth factors (TGF-b1 and TGF-b2) and fibroblast growth factor 4 (FGF-4) were less than 25%, but still significantly, higher relative to rd1 explant control medium (n ¼ 3 or 4). Triplicate arrays on medium from HNPCs culture without explant, confirmed the secretion of the growth factors mentioned above (Fig. 1E). 3.2. HNPCs reduce photoreceptor cell death

2.8. Microscopy Sections were examined using epifluorescence microscope (Nikon, Tokyo, Japan) equipped with appropriate filters. Images were captured with digital acquisition systems (Nikon DS-U1 or DEI750, Optronics, Goletta, GA). Image brightness/contrast adjustment was done using Adobe PhotoShop (Adobe, San José, CA).

The protective effect of rat retinal progenitor cells on caspase3-dependent photoreceptor apoptosis in rat retinal explants (Liljekvist-Soltic et al., 2008), prompted us to investigate a possible effect of HNPCs on various photoreceptor death pathways. For this purpose, the densities of calpain and cleaved caspase-3 immunostained photoreceptors were determined in the ONL of control and

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Fig. 1. Phenotypic characterization of HNPCs and increased secretion of growth factors as determined by membrane-based array. (AeB) Micrographs of feeder layer after 6 days of co-culture showing subsets of GFAP (red) stained glial cells and MAP-2 (green) stained neurons. Nuclei are DAPI (blue) counterstained. (C) Growth factor array membranes with all identified growth factors in medium conditioned by HNPCs and rd1 explants. (D) High magnification of arrays. Nine of the identified factors (boxed in C) in 3 or more independent arrays had mean intensities that were relatively higher than growth factor levels in control medium from rd1 retinal explant cultures. The mean intensity of the growth factor levels in conditioned medium are denoted in Table 1. (E) Growth factor array of HNPCs conditioned medium without explants.

in experimental rd1 mouse and normal rat retinas (cleaved caspase-3 only), respectively. The inherited degeneration in rd1 mouse retinas explanted at postnatal day 8 resulted in a considerable loss of photoreceptor cell death, with 1114  115 cells/mm2 in the ONL being positive for calpain activity after 7 days in vitro (Fig. 2). Co-culture with HNPC for 6 days significantly reduced this value to less than half (471  52 cells/ mm2, p < 0.0001; n ¼ 8). Cleaved caspase-3 immunostaining of rd1 retinas revealed a moderate number (392  60 cells/mm2; n ¼ 6) of labeled cells in the ONL. The density of photoreceptors labeled for cleaved caspase-3 decreased by 30% to 282  58 cells/mm2 (p ¼ 0.0045; n ¼ 6) when exposed to HNPCs (Fig. 2).

Table 1 Relative increases of growth factor levels in conditioned medium from HNPCs/rd1 explant co-cultures (% increase compared to control medium from rd1 retinal explant cultures). Growth factor

mean  SEM

FGF-4 G-CSF GMCSF IGF-II NT-3 PIGF TGF-b TGF-b2 VEGF-D

18.4 27.8 36.1 28.8 26.7 27.1 16.4 9.2 25.0

        

8.8 14.7 15.3 15.7 5.91 6.7 11.1 6.53 9.7

p 0.012; 0.016; 0.025; 0.020; 0.001; 0.008; 0.017; 0.005; 0.016;

n n n n n n n n n

¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼ ¼

3 4 4 4 4 3 3 3 3

Explantation of postnatal day 11e13 rat retinas resulted in photoreceptor degeneration, with 1237  225 cells/mm2 in the ONL being immunostained for cleaved caspase-3 after 7 days in vitro (Fig. 3). When co-cultured with HNPCs for 6 days, the density of cleaved caspase-3 positive photoreceptors declined significantly to 628  104 cells/mm2 (p ¼ 0.0017; n ¼ 8). Collectively, these findings suggest that HNPCs may exert protection by suppressing calpainand caspase-3-dependent cell death. 3.3. HNPCs and preservation of retinal structure To assess structural protection, histological evaluation was conducted on specimens co-cultured with HNPCs and on control retinas. Sections were counterstained with DAPI to emphasize retinal layering as well as ONL structure and thickness (Figs. 2 and 3). All retinas showed normal layering and a peripheral thinning, and control retinas showed a tendency to develop folds. Rhodopsin expression was observed in all cultured specimens and was localized in photoreceptor outer segments and in the ONL (Figs. 2 and 3). The ONL in rat retinas also displayed a central thinning, which appeared to be associated with small clusters of remaining and activated retinal pigment epithelium (RPE) cells (see LiljekvistLarsson et al., 2003). To assess structural neuroprotection, histologic analysis and TUNEL was conducted on retinal samples after 6 days in culture with HNPC feeder layers (Figs. 2 and 3). Retinas exposed to HNPCs possessed several rows of photoreceptors, and the ONL was

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Fig. 2. Photoreceptor cell death and morphology of rd1 mouse retinal explants. Effects of HNPCs were evaluated by counting the number of labeled cells in the ONL. (A) Calpain staining (blue; arrows) on sections of control (top) and co-cultured (middle) retinas. (bottom) Effects of HNPCs against calpain-dependent cell death. Data are shown as per cent of control (***p < 0.0001; n ¼ 8). (B) Cleaved caspase-3 (cc-3) staining (red) on sections of control (top) and co-cultured (middle) retinas. (bottom) Effects of HNPCs against caspase-3dependent cell death, shown as per cent of control (**p ¼ 0.0045; n ¼ 6). (C) TUNEL labeling (green) demonstrating photoreceptor cell death in sections of control (top) and cocultured (middle) retinas. (bottom) Effects of HNPCs on overall cell death, shown as per cent of control (**p ¼ 0.0053; n ¼ 4). (D) Rhodopsin immunostaining (red) on DAPI-stained sections of control (top) and co-cultured (middle) retinas. (bottom) Effects of HNPCs on ONL thickness is shown as per cent of control (***p ¼ 0.0006; n ¼ 7). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars 20 mm.

significantly thicker than contralateral controls (Fig. 2). The mean width of the ONL in control rd1 mice was 35.2  2.9 mm and almost 40% thicker in co-cultured retinas (48.8  2.6 mm, p ¼ 0.0006; n ¼ 7) (Fig. 2). Counts of TUNEL-positive cells in the ONL of rd1 mice showed significant differences between control and experimental retinas. On average, 2021  76 TUNEL-positive cells/mm2 were observed in controls. When incubated with HNPCs this value was significantly reduced to 1015  156 cells/mm2 (p ¼ 0.005; n ¼ 4). Similar result was also observed using rat retinal explants (Fig. 3). The mean ONL was slightly thicker in co-cultures (52.7  1.3 mm) compared to mean ONL width in contralateral controls (48.7  0.8 mm, p ¼ 0.003; n ¼ 8). TUNEL revealed 2548  556 cells/mm2 in the ONL of controls. Co-culture with HNPCs revealed a decrease by 40% to 1300  218 TUNEL-positive cells/mm2 (p ¼ 0.043; n ¼ 8). These findings suggest that protective effects on photoreceptors result in ONL preservation in mouse and rat retinal explants. 4. Discussion By the use of organotypic rd1 mouse and normal rat retinal explants, we show that co-cultured HNPCs efficiently enhance photoreceptor survival and preserve retinal structure in situations that lead to photoreceptor degeneration. Furthermore, analysis of the medium conditioned by the co-culture of HNPC feeder layers and retinal explants showed the presence of growth factors, some of which are established effectors on photoreceptors. Our findings suggest HNPCs may secrete several growth factors that promote photoreceptor survival.

Retinal explants cultures have been frequently used to study different aspects of retinal neuroscience including photoreceptor neuroprotection and stem cell transplantation, and provide a convenient way of testing the biological effects of various cells and molecules (Azadi et al., 2007; Johnson and Martin, 2008; Liljekvist-Soltic et al., 2008; Ogilvie et al., 1999; Pinzón-Duarte et al., 2004). For both the mouse and rat explants used here, the HNPCs reduced the caspase-3 activation seen in the degenerating photoreceptors, and for the rd1 explants the HNPCs treatment also robustly reduced the much higher number of cells with activated calpain. The effect of HNPCs on calpain-dependent apoptosis was not tested in the rat retina because pharmacological data (Perche et al., 2007, 2008) indicate calpain-independent photoreceptor degeneration in this species. Our data suggest that calpaindependent photoreceptor degeneration dominates over that of caspase-3-dependent during photoreceptor cell death in the rd1 mouse, which goes well with other reports on the relative lack of caspase-3 importance in this model (Doonan et al., 2005; Zeiss et al., 2004). By contrast, caspase-3 appears to be the major mediator of photoreceptor apoptosis in explanted rat retinas. There are some questions of our findings. First, are there upstream general signal initiators of calpain- and caspase-3-dependent photoreceptor apoptosis that respond to the growth factors? A common feature is that the HNPCs appear to secrete several growth factors, and they may have multiple effects that collectively promote photoreceptor homeostasis. It has recently been demonstrated that retinal detachment resulted in rapid alterations of cytokine and growth factor homeostasis, which itself may initiate

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Fig. 3. Photoreceptor cell death and morphology of explanted normal rat retina. Effects of HNPCs were evaluated by counting the number of labeled cells in the ONL. (A) Cleaved caspase-3 (cc-3) staining (red) on sections of control (top) and co-cultured (middle) retinas. (bottom) Effects of HNPCs against caspase-3-dependent cell death. Data are shown as per cent of control (**p ¼ 0.0017; n ¼ 8). (B) TUNEL labeling (green) demonstrating photoreceptor cell death in sections of control (top) and co-cultured (middle) retinas. (bottom) Effects of HNPCs on overall cell death, shown as per cent of control (*p ¼ 0.043; n ¼ 8). (C) Rhodopsin immunostaining (green) on DAPI-stained sections of control (top) and cocultured (middle) retinas. (bottom) Effects of HNPCs on ONL thickness is shown as per cent of control (**p ¼ 0.003; n ¼ 8). GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bars 20 mm.

apoptosis (Nakazawa et al., 2006). Other responses in RP models include activation of matrix metalloproteinases (Ahuja et al., 2006) as well as nitric oxide production and oxidative stress (Komeima et al., 2007, 2008; Sanz et al., 2007). Thus, photoreceptor degenerations like other neuronal degenerations likely have a complex pathophysiology and multiple mechanisms that are active simultaneously. The factors produced by the HNPCs may target different mechanisms that together balance essential survival system(s). Another question is whether two or more apoptotic pathways exist in the photoreceptors and do they operate independently? This study demonstrates that both calpain activity and caspase-3 immunolabeling in the rd1 mouse retina are affected by the HNPCs, but that calpain may be the major and dominating death pathway. However, the density of photoreceptors positive for TUNEL outnumbers the sum of calpain and caspase-3 labeled photoreceptor. While this could result from differential sensitivities of the assays used, it may also indicate the presence of additional death pathway(s) including endoplasmic reticulum stress players such as caspase-12 (Sanges et al., 2006). Similarly, the HNPCs preserve ONL integrity and halt caspase-3-dependent photoreceptor degeneration in the rat retina but TUNEL suggests also here the presence of other death pathways. Recent studies on cultured rat retinas (Liljekvist-Soltic et al., 2008) and rd1 mouse retina (Sanges et al., 2006)

have demonstrated the presence of caspase-12 immunoreactivity in degenerating photoreceptor cells. In contrast to the caspase-3dependent photoreceptor apoptosis in rat retinal explants, caspase12 appeared to be less affected by rescue factors (Liljekvist-Soltic et al., 2008). Similar to findings following light-induced retinal degeneration, identified and yet unidentified death pathways are therefore likely to contribute to photoreceptor apoptosis in vitro (Hao et al., 2002). The protective effect of HNPCs within the rd1 and normal rat retina appears to be related to growth factor production. In a recent study it was shown that HNPCs produced small amounts of IGF-1 and FGF-2 in vitro, and transplantation of HNPCs into the degenerating rat retina showed both functional and morphological improvement (Gamm et al., 2007). By growth factor analysis of HNPCs conditioned medium we were able to demonstrate relative increases for at least nine factors. Of these factors, FGF, IGF and VEGF belong to growth factor families that are well known to promote photoreceptor survival (Gamm et al., 2007; LiljekvistSoltic et al., 2008), whereas GMCSF is known to be involved in retinal microglial function (John et al., 2003) and in neuroprotection in ischemic cerebral injury (Schäbitz et al., 2008). NT-3 is an established neuroprotector in retinal degenerations (LaVail et al., 2008) whereas such an effect was recently described for

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G-CSF (Oishi et al., 2008). Similar to our data, a complex composition of growth factor-related proteins has also been observed in other glial cell-derived photoreceptor protecting media (Hauck et al., 2008). One interpretation of our data is that a broad mixture of growth factors has rescue effects on photoreceptor degeneration that is experimentally induced in rat retina and innate to the photoreceptors in the rd1 mouse retina. If this means that a broad mixture of growth factors targeting different apoptotic pathways is the most successful therapy for photoreceptor rescue has to be established. An important question in this context is whether growth factors from the HNPCs act directly on photoreceptors or indirectly via Müller cells. One prevailing theory is that Müller cells protect photoreceptors from apoptosis by secretion of growth factors and neurotrophic factors (Harada et al., 2002; Hauck et al., 2008; Wahlin et al., 2000, 2001). Of the identified growth factors here, several have been shown to increase their expression in activated Müller cells. For instance, TGF-b2 expression increases after experimental detachment and may act on TGF-b2 receptors located on the Müller cells (Guérin et al., 2001). TGF-b2 is also a potent inducer of VEGF secretion in retinal pigment epithelium (RPE) cells (Nagineni et al., 2003). VEGF is required for the maintenance of photoreceptors and their function, and acts on VEGF receptors localized to the Müller cells (Liljekvist-Soltic et al., 2008; SaintGeniez et al., 2008). Sustained levels but not bolus injections of NT3 provide photoreceptor protection to damaging light (LaVail et al., 2008). This effect may be mediated by high-affinity TrkC receptors on Müller cells. G-CSF is secreted in relatively high levels from the HNPCs, and was recently shown to protect photoreceptors against light-induced damage (Oishi et al., 2008). The study also demonstrated a general G-CSF receptor expression in the retina including Müller cells and photoreceptors. Thus, it is possible that the beneficial effect of HNPCs-derived factors described in the present study may converge onto the Müller cells. However, the point and morphological correlates that indicate the difference between beneficial and detrimental effects are yet to be determined, as is the possibility that the conditioned medium contains yet other efficient factors, not included in the array used here. Furthermore, although the array is designed for recognition of human factors, it cannot be completely excluded that some of the measured entities were produced by the explants themselves, as a response to unique HNPCs-derived factors. 5. Conclusion Our results show that co-cultured HNPCs attenuate both calpainand caspase-3-dependent photoreceptor degeneration in explanted rd1 mouse and normal rat retinas. It is also suggested that the HNPCs secrete a mixture of growth factors, which collectively target different death pathways promoting photoreceptor survival. Acknowledgements Supported by the Faculty of Natural Sciences at Kalmar University (KJ), Signhild Engkvist Foundation (KJ), Synfrämjandet (KJ), Foundation Fighting Blindness (PE via T v Veen), VR-M (PE), Ragnar och Torsten Söderbergs Foundation (UEJ, PE), Lars Hiertas Fund (UEJ), Magnus Bergvalls Fund (UEJ), Elsa och Torsten Segerfalk Fund (UEJ), Lund University (UEJ), the Medical Faculty at Lund University (UEJ) and the Crown Princess Margaretas Committee for the Blind (KJ, UEJ, PE). Dr Francois Paquet-Durand is acknowledged for advice on the calpain activity staining protocol. The authors thank Birgitta Klefbohm and Hodan Abdalle for excellent technical assistance.

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