Neuronal differentiation of adult rat hippocampus-derived neural stem cells transplanted into embryonic rat explanted retinas with retinoic acid pretreatment

Brain Research 954 (2002) 286–293 www.elsevier.com / locate / bres

Research report

Neuronal differentiation of adult rat hippocampus-derived neural stem cells transplanted into embryonic rat explanted retinas with retinoic acid pretreatment Joe Akita a , Masayo Takahashi a , *, Masato Hojo b , Akihiro Nishida a , Masatoshi Haruta a , Yoshihito Honda a a

Department of Ophthalmology and Visual Sciences, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606 -8507, Japan b Department of Neurosurgery, Kyoto University, Kyoto University Graduate School of Medicine, Kyoto 606 -8507, Japan Accepted 20 June 2002

Abstract The purpose of this study was to evaluate the effects of the retinal environment and retinoic acid (RA) pretreatment on the differentiation of transplanted adult rat hippocampus-derived neural stem cells (AHSCs). AHSCs were transplanted into embryonic (E18) or neonatal (P6) rat retinal explants and the mixture was cultured for 2 weeks. Other AHSCs were stimulated by 0.5 mM all-trans RA for 6 days before transplantation. Immunofluorescent double staining showed that a larger number of AHSCs became b-tubulin III-positive neurons in the E18 than in P6 retinas. In addition, many AHSCs became MAP2ab-positive and MAP5-positive neurons following RA pretreatment and transplantation. Only a few AHSCs became HPC-1-, calbindin-, PKC- or rhodopsin-positive cells under these conditions. We conclude that the microenvironment supplied by embryonic retinas is conductive to neuronal differentiation in general. RA stimulation before transplantation was also effective in stimulating differentiation.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Cell differentiation and migration Keywords: Neural stem cell; Transplantation; Organ culture; Retina; Retinoic acid; Differentiation

1. Introduction The retina is part of the central nervous system, and as such, retinal neurons will not replicate to replace dead neurons. Because neuronal death in the retina causes severe loss of visual function, human embryonic or adult retinas have been transplanted into eyes to try to rescue and / or to replace the retinal neurons [11,13,18,21,30]. However, acquiring a large number of embryonic retinal cells is difficult, and stem cell transplantation has been developed as a new technique for retinal regeneration. Neural stem cells have been determined to be present in various parts of the adult central nervous system *Corresponding author. Tel.: 181-75-751-4721; fax: 181-75-7514731. E-mail address: [email protected] (M. Takahashi).

[14,15,17,20,36,49]. The stem cells have multipotency of differentiation and self-renewability, and they can proliferate in serum-free medium. Most interestingly, they can differentiate into site-specific lineage under the influence of the microenvironment [9,34,37,41,47]. Thus, transplantation therapy with stem cells is expected to be important for neural regeneration. This therapy has already succeeded to some extent in animal models of Parkinson’s disease [40,42], Huntington’s disease [6], and spinal cord injury [8,27]. We have been investigating the possibility of using neural stem cell transplantation into the retina for retinal regeneration. We have used adult rat hippocampus-derived neural stem cells (AHSCs) that have been proven to have multipotency and self-renewability by clonal analyses [29]. AHSCs can expand in serum-free medium and proliferate in response to basic fibroblast growth factor (bFGF). In

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0006-8993( 02 )03356-5

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previous in vitro experiments, bFGF withdrawal and retinoic acid (RA) stimulation for AHSCs can trigger differentiation into neurons [29]. In vivo, AHSCs without any pretreatment can differentiate into olfactory bulb neurons when implanted into the rostral migratory passway, and will express tyrosine hydroxylase that is expressed in olfactory bulb neurons but not in hippocampal neurons [41]. We have found that AHSCs will integrate into neonatal rat retinas after transplantation into the vitreous space. ¨ They adopt the morphologies and positions of Muller, amacrine, bipolar, horizontal, and photoreceptor cells [43]. We also found that AHSCs transplanted into mechanicallyinjured adult rat retinas were incorporated and subsequently differentiated into neuronal and glial lineage. Immunoelectron microscopic analyses revealed that these cells made graft–host contacts such as puncta adhaerentia and synapse-like structures [28]. In other experiments brainderived neural stem cells were transplanted into retinas [24,48], but they failed to differentiate into photoreceptors. In this study, we have investigated the influence of the microenvironment provided by embryonic retinas on AHSC differentiation where extrinsic factors and cell–cell interactions are most suitable for retinal cell differentiation. In addition, we have investigated whether pretreatment with RA before transplantation can alter the differentiation of AHSCs into more mature neurons.

2. Materials and methods

2.1. Experimental animals The use of animals in this study was in accordance with the Guideline for Animal Experiments of Kyoto University. Pregnant and neonatal Fischer-344 rats were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). Pregnant rats were anesthetized with an intramuscular injection of a 1:1 mixture of xylazine hydrochloride (4 mg / kg) and ketamine hydrochloride (10 mg / kg). The uterine horns were immediately excised and embryos were removed in cold Dulbecco’s phosphate-buffered saline (D-PBS, Gibco BRL, Rockville, MD). Neonatal rats were anesthetized on crushed ice and killed by decapitation.

2.2. Cell cultures The donor cells were LacZ-labeled clonal adult rat hippocampus-derived neural stem cells (AHSCs, clone PZ5, kindly provided by F.H. Gage, Salk Institute, La Jolla, CA). AHSCs were cultured on poly-L-ornithinecoated dishes containing Dulbecco’s modified Eagle’s medium / Ham’s F12 (DMEM / F12, Gibco BRL) supplemented with N2 (Gibco BRL) and 20 ng / ml basic fibroblast growth factor (bFGF, Genzyme, Cambridge,

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MA), and incubated at 37 8C in a humidified 5% CO 2 in air. The medium was changed every 3 days. After subculture for 2 weeks to 3 months, AHSCs were harvested for transplanting with 0.05% trypsin in DMEM / F12, washed with 0.01% trypsin inhibitor (Wako) in DMEM / F12, and suspended at a density of 30 000 cells / ml in high-glucose Dulbecco’s phosphate-buffered saline (D-PBS, Gibco BRL) containing 20 ng / ml bFGF. To pretreat AHSCs with RA, the medium was replaced with DMEM / F12 (Gibco BRL) supplemented with N2 (Gibco BRL) containing 0.5 mM all-trans retinoic acid (RA, Wako, Osaka, Japan) and 0.5% fatal bovine serum without bFGF, and cultured under the same condition. The RA-pretreated AHSCs were suspended in the same manner as described for transplantation 6 days after the start of stimulation. For the differentiation experiments, monolayer culture as cell culture groups RA-pretreated AHSCs were cultured in N2 medium containing 0.5% FBS.

2.3. Retinal organ culture Retinal organ culture was performed, as described [10,38,39,45] with minor modification. Briefly, eyes were enucleated from rat embryos or neonates and transferred to cold phosphate-buffered saline (PBS, Gibco BRL). The retinas without the retinal pigment epithelium were isolated from other eye parts in Millicell-CM membrane culture inserts (Millipore: diameter 30 mm, pore size 0.4 mm) and placed onto the membrane with the ganglion cell layer upward. The inserts with neural retina were placed in six-well plates containing approximately 1 ml / well of medium. The culture medium contained 50% minimum essential medium with 25 mM HEPES (Gibco BRL), 25% Hank’s solution (Gibco BRL), 25% heat-inactivated horse serum (Gibco BRL), 200 mM L-glutamine (Gibco BRL), and 6.75 mg / ml glucose (Gibco BRL). Organ cultures were maintained at 34 8C, 5% CO 2 , and fed every 2 or 3 days by replacing 0.8 ml of the medium.

2.4. Transplantation The host retinas were explanted from embryonic day 18 (E18) or postnatal day 6 (P6) Fischer-344 rats. Cell suspension containing clonal AHSCs (3 ml of 3310 4 cells / ml) with or without RA pretreatment was added to the retinas with a Hamilton syringe fitted with a 30-gauge needle immediately after isolation of the host retinas. For controls, 3 ml of the vehicle were added to the retina. The explanted retinas were cultured for 14 days.

2.5. Histochemistry The retinas were fixed 14 days after transplantation in 4% paraformaldehyde (Wako) in PBS for 10 min at room temperature and then infiltrated with 25% sucrose in PBS

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for 30 min at room temperature while still attached to the substrate membrane. Subsequently, the retinas were embedded in OCT compound (Miles, Elkhart, IN), and cryostat sections of 14 mm thickness were cut perpendicular to the plane of the substratum and mounted on silanized slides (Dako, Kyoto, Japan) for histochemical analysis. For X-gal staining, the sections were rinsed with PBS and incubated in PBS containing 2 mM magnesium chloride (MgCl 2 , Wako) and 0.005% saponin (Sigma, St. Louis, MO) for 10 min at 4 8C. Subsequently they were incubated in PBS containing 1 mg / ml 5-bromo-4-chloro-3indolyl-b-D-galactopyranoside (X-gal, Wako), 5 mM potassium ferricyanide (Wako), 5 mM potassium ferrocyanide (Wako), 2 mM MgCl 2 , and 0.005% saponin for 12 h at 32 8C. They were then immersed in 50 mg / ml carmalum solution (Wako) for counter staining. The negative controls, which did not have transplanted AHSCs, were treated in the same way. After dehydration, they were mounted with Mount-Quick (Daido-Sangyo, Saitama, Japan) containing 60% xylene and observed with an inverted light microscope. For immunofluorescent staining, retinal sections or monolayer culture cells were fixed in 4% paraformaldehyde (Wako) in PBS for 30 min at 4 8C, rinsed with PBS, and blocked with 20% BlockAce (Dainihon-Seiyaku, Osaka, Japan) in PBS containing 0.005% saponin (Sigma) for 30 min. After removing the blocking solution, the sections were incubated with primary antibodies for 24 h at 4 8C. Primary antibodies were used at the following concentrations: mouse monoclonal anti-b-galactosidase (bgal, 1:1000; Promega, Madison, WI) and rabbit polyclonal anti-b-gal (1:5000; Chemicon, Temecula, CA) as AHSC donor cell marker; mouse monoclonal anti-nestin (1:1000; Pharmingen, San Diego, CA) as neural stem cell marker; mouse monoclonal anti-b-tubulin isotype III (1:500; Sigma), mouse monoclonal anti-microtubule associated protein (MAP) 5 (1:1000; Chemicon), and mouse monoclonal anti-MAP (2a12b) (1:100; Sigma) as neural cell markers; rabbit polyclonal anti-glial fibrillary acidic protein (GFAP) (1:1000; Dako) as glial cell marker; mouse monoclonal anti-HPC-1 (1:1000; Sigma) as amacrine cell marker; mouse monoclonal anti-calbindin (1:500; Sigma) as horizontal cell marker; mouse monoclonal anti-protein kinase C (1:500; Sigma) as bipolar cell marker; rabbit polyclonal anti-rhodopsin (1:1000; LSL, Tokyo, Japan) as rod photoreceptor cell marker. After washing with PBS, the sections were incubated with the appropriate secondary antibodies for 90 min at room temperature. Secondary antibodies were used at the following concentrations: fluorescein isothiocyanate (FITC)-conjugated sheep antimouse immunoglobulin (Ig) (1:100; Amersham, Buckinghamshire, UK); FITC-conjugated donkey anti-rabbit Ig (1:100; Amersham); Texas Red-conjugated sheep antimouse immunoglobulin (Ig) (1:100; Amersham); Texas Red-conjugated donkey anti-rabbit Ig (1:100; Amersham).

Cell nuclei were counterstained with 49,69-diamino-2phenylindole, dihydrochloride (DAPI) (1 mg / ml; Molecular Probes, Eugene, OR) supplemented in the secondary antibody solution. Negative control section was treated in the same way with omission of the primary antibody. All antibodies were diluted in PBS containing 0.005% saponin and 5% BlockAce. The sections were then washed with PBS, mounted with glycerol / PBS (1:1). Confocal microscopy was performed with a laser-scanning confocal microscope (model 1024; Bio-Rad, Hercules, CA) mounted on an inverted microscope (Axiovert; Zeiss, Oberkochen, Germany).

2.6. Quantification and statistical analysis For quantitative analyses of the transplantation experiments, at least three photographs from the equatorial part of the retina where the retinal layer architecture are well differentiated were taken from at least three randomly selected retinal sections for every condition. For differentiation experiments using monolayer cultures, at least three photographs were taken for each condition and repeated three times on randomly selected retinal sections. The photographs were taken with a single wavelength excitation and appropriate filter blocks for both FITC and TexasRed were used. Two or three color channels were handled separately to ensure no bleed-through signal was obtained. Color images were generated with Adobe Photoshop, Version 5.0 (Adobe Systems, Mountain View, CA). The percentages6standard deviations (S.D.) of double- or triple-labeled cells were estimated by counting the cells in high-power fields (363) under fluorescence. Statistical significance was analyzed by nonparametric Mann–Whitney U-test conducted with the statistical programs StatView, Version 4.1 (Abacus Concepts, Berkley, CA).

3. Results We first determined that cultured neonatal and embryonic retinas developed in a normal manner as described earlier [10,38]. Histological examination of E18 retinas showed that the cells consisted of retinoblasts and the architectural retinal layering had not occurred. The rod photoreceptors had still not differentiated. After 14 days of culture, E18 and P6 retinas both developed well. Outer nuclear layer, outer plexiform layer, inner nuclear layer and inner plexiform layer could be differentiated (Fig. 1). Rod photoreceptors could also detect by immunohistochemistry. As in our previous study [43], many transplanted AHSCs migrated into various layers of the explanted retinas without destroying the layered architecture of the host (Fig. 1).

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Fig. 1. Light micrographs of 2 week-cultured retinas from E18 (top) after transplantation of RA-stimulated AHSCs and P6 (bottom) after transplantation of non-treated AHSCs. AHSCs are labeled with X-gal (blue). Note that the transplanted AHSCs are well integrated into the retina, especially in the inner nuclear layer, without destroying normal retinal architecture of the host retina. ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, retinal ganglion cell layer. Bar550 mm.

3.1. AHSCs transplantation into E18 or P6 explanted retina From the immunofluorescent stained sections, we

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counted the number of double-labeled cells that integrated into the explants. The percentage of double-labeled AHSCs that were b-tubulin III-positive was 16.662.0%, MAP5-positive was 18.562.7%, and MAP2ab-positive was 3.863.6% in the E18 explanted retinas. The comparable percentages for the P6 retinas were 9.063.8% for b-tubulin III-positive, 16.665.3% for MAP5-positive and 2.662.4% for MAP2ab-positive. The higher number of AHSCs that differentiated into b-tubulin III-positive neurons in embryonic retinas than in neonatal retinas was significant (P,0.05, Mann–Whitney U-test). The other differences were not significant (Fig. 2). None of the transplanted AHSCs was positive for HPC1 (amacrine cell marker), calbindin, (horizontal cell marker), and PKC (bipolar cell marker) in either E18 or P6 retinas. In addition, none of the transplanted AHSCs was rhodopsin-positive in either E18 or P6 retinas. In the integrated AHSCs, 17.966.4% were GFAP-positive glial cells compared to 10.561.4% in the host P6 retinas. Although more AHSCs tended to differentiate into glial lineage in E18 retinas than in P6 retinas, the difference was not significant (Fig. 2).

3.2. Effect of RA pretreatment on differentiation To try to increase the differentiated AHSCs into more mature neurons, the AHSCs were stimulated with 0.5 mM RA for 6 days before transplanting into the retinal explants. In the RA pretreated group, 13.564.4% of the integrated cells differentiated into MAP2ab-positive mature neurons compared to 3.863.6% in the non-treated

Fig. 2. Percentages of double-labeled cells to all integrated AHSCs under the following conditions; green, in P6 after transplantation of non-treated AHSCs; white, in E18 after transplantation of non-treated AHSCs; red, in E18 after transplantation of pretreated AHSCs with RA; blue, in cell cultured condition of pretreated AHSCs with RA. Both the average and the absolute number6standard deviation of immunofluorescent cells were counted in high power fields of confocal microscopic images. *P,0.05, Mann–Whitney U-test.

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group (P,0.05: Mann–Whitney U-test), and 14.462.1% of the integrated cells differentiated into b-tubulin IIIpositive neurons compared to 16.662.0% in the nontreated groups (Fig. 2). Although these results appear somewhat inconsistent, we suggest that RA pretreatment induced AHSCs to differentiate into more mature neurons because b-tubulin III is expressed in immature neurons and MAP2ab in relatively mature neurons. On the other hand, each percentage of GFAP-positive or

nestin-positive cells in RA pretreated groups was lower than in the non-treated groups. In the RA pretreated groups, 15.565.5% of integrated cells differentiated into GFAP-positive glial cells compared to 17.966.4% in the non-treated groups. Nestin-positive cells, a neural stem cell marker, were 16.662.7% of the integrated cells in RA pretreated groups compared to 35.162.9% in non-treated groups (Fig. 2). These results also suggest that RA pretreatment had inducted AHSCs to differentiate into neuronal lineage. Retina-specific marker-positive cells were essentially absent and none of the donor cells was rhodopsin-positive even in the RA pretreated cells. Typical images of immunofluorescent double-stained cells that were pretreated by RA are shown in Fig. 3.

3.3. Transplantation of pretreated AHSCs To confirm that embryonic retinas provide a more suitable condition than the standard culture medium for AHSCs differentiation into neurons, we compared the differentiation percentages under the two conditions. Typical images of immunofluorescent-stained cells are shown in Fig. 4, and the percentages of positive cells to all of AHSCs are shown in Fig. 2. In cell culture groups, 11.665.5% of pretreated AHSCs differentiated into MAP5-positive neurons compared to 23.062.2% in the transplanted group (P,0.05: Mann–Whitney U-test). HPC-1, calbindin, PKC, and rhodopsin-positive AHSCs were not found in the cell culture groups.

4. Discussion

Fig. 3. Immunofluorescent double-stained images of transplanted retina. Retinal sections were stained with anti-b-gal antibodies (left, green) as AHSCs marker and other antibodies (middle, red). Right column is merged image of middle and left column. Arrows point to double-labeled AHSCs. These confocal microscopic images were obtained from outer (sections stained with anti-rhodopsin antibody) and inner nuclear layer (sections stained with other antibodies) of the host retinas. Sections of cultured retinas were taken from E18 after transplantation of AHSCs pretreated with RA. Bar520 mm.

As for the host retinas, Our results confirmed that cultured neonatal and embryonic retinas develop in a normal manner that closely mimics retinal development in vivo [10,38]. Reh et al. have reported that cell–cell interactions determine neuronal phenotypes in rodent retinal cultures [32], and this organ culture system has the advantage of keeping the cell–cell interactions intact which may play an important role in cell survival and differentiation. Embryonic retinas also produce some extrinsic factors that affect proliferation and differentiation of retinal precursor cells. Previous studies have shown that extrinsic factors such as taurine [3], transforming growth factor (TGF)-a, epidermal growth factor [5], TGF-b [4] and activin A [12] play an essential role in the determination of retinal cell phenotypes. In this study the explanted retinas have normally developed and differentiated into all types of retinal cells including rod photoreceptors. Thus extrinsic factors that are necessary for retinal precursors to develop into mature retinal neurons were present in the explanted retinas. Explanted embryonic retinas therefore seem to provide the most suitable microenvironment for transplanted neural stem cells to

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Fig. 4. Differentiated AHSCs (arrows) in monolayer-culture condition. They express b-III-tubulin, MAP-5, MAP-2ab, and GFAP. They were stained 2 weeks after pretreatment with RA. Bar510 mm.

differentiate into mature retinal cells. However, none of the AHSC-derived authentic retinal cell, especially rod photoreceptor, was obtained in this study. Other extrinsic factors that are unknown and necessary for AHSCs to develop into retinal precursors might be absent. The absence of retinal pigment epithelium (RPE) which help regulate rod photoreceptor cell proliferation by modulating mitosis and apoptosis [31,35], may also prevent AHSCs from differentiating into authentic retinal cells. An additional benefit of this culture system in comparison with the retina in vivo is that the donor cells have better access to the host retina at the time of transplantation than in vivo experiments. Actually, it is extremely difficult to inject donor cells into the vitreous cavity consistently in embryonic and early neonatal rat eyes. In contrast, transplantation into an explanted retina is easy and reliable. Thus, this wellestablished retinal organ culture system is quite useful for the study of stem cell transplant experiments. Concerning the donor, we have previously shown that AHSCs transplanted into injured adult retina formed synapse-like structures between host and transplanted AHSCs by immunoelectron microscopic examination [28]. Toda et al. have reported that differentiated AHSCs formed functional glutamatergic and GABAergic synapses in vitro by physiological examination using patch-clamp methods [44]. These studies suggest that AHSCs have the potential

to develop functional synapses. RA has an essential role for retinal development by modulating proliferation, differentiation, and survival [19,22,23,26]. On the other hand, some intrinsic factors, such as Pax-6 [7], Prox1 [7], Crx [16] and Chx10 [25] of homeobox genes, or NeuroD [1] and Nrl [33] of transcription factors regulate retinal development. To convert AHSCs into authentic retinal neurons RA pretreatment alone was not sufficient, and AHSCs may need additional intrinsic factors to respond to the cue of RA and differentiate into retinal neurons. However, AHSCs have many advantages as donor cells for retinal transplantation because they are a cell line which has been well characterized can be expanded through numerous passages in vitro and can be frozen for storage [17,29]. During culture, they can be easily modified by pretreatment with growth factors, RA, or by gene transfer before they are transplanted. In addition, there are less ethical problems clinically in the transplantation of AHSCs as donor cells than donor cells derived from embryonic or neonatal tissue. Recently, it was shown that retinal progenitor cells are present in adult rodents [2,46]. As these cells differentiate into authentic retinal cells in vitro and in vivo, they are expected to apply for retinal regeneration. However, they have not been shown to have multipotency and selfrenewability by clonal analyses as yet. Additionally, only a

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small population of them differentiated into authentic retinal neurons that might be insufficient for clinical use. In summary, we were able to obtain a higher percentage of mature neurons by transplanting AHSCs into embryonic retina with RA pretreatment; however, none of the cells could be shown to be authentic retinal neurons with or without RA pretreatment. Our results also showed that RA pretreatment was effective in inducing more neurons from grafted neural stem cells. Because the explanted retinal progenitor cells differentiated into photoreceptor cells, it appears that hippocampus-derived neural stem cells lack some intrinsic factors that are necessary for photoreceptor differentiation. Thus, some ex vivo manipulation such as gene transfer and / or growth factor stimulation will be necessary.

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