- Email: [email protected]
Different characteristics of rat retinal progenitor cells from different culture periods
Neuroscience Letters 341 (2003) 213–216 www.elsevier.com/locate/neulet
Different characteristics of rat retinal progenitor cells from different culture periods Tadamichi Akagia, Masatoshi Harutab, Joe Akitac, Akihiro Nishidad, Yoshihito Hondaa, Masayo Takahashib,* a
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japan c Department of Ophthalmology, Tenri Yorozu Hospital, Nara 632-8552, Japan d Developmental Biology Department, Osaka Bioscience Institute, Osaka 565-0874, Japan
b
Received 22 January 2003; accepted 3 February 2003
Abstract Embryonic retina is one of the possible cell sources that will repair degenerated retina such as retinitis pigmentosa. Retinal progenitor cells isolated from embryonic rats could be cultured and expanded in serum free medium with both epidermal growth factor and basic fibroblast growth factor. We analyzed the properties of two different retinal progenitor cells in terms of culture periods. Retinal progenitor cells from embryonic retina could be expanded keeping immature cell properties and had the ability to migrate into degenerated adult retina from subretinal space after transplantation. They differentiated into neurons and glias, even into photoreceptor cells both in vitro and in vivo. However, they appeared to lose their tissue specificity after a long-term culture. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Retinal cell culture; Retinal transplantation; Photoreceptor cells; Retinal progenitor; Retinal degeneration; Culture period
The isolation of neural stem cells evoked a great deal of interest as a possible therapy for neural degenerative diseases. Recently, several types of neural cell lines or progenitor cells, including stem cells, have been used for retinal transplantation. Adult rat hippocampus-derived neural stem cells efficiently integrated into the retina to adopt the morphology and position of retina-specific cells [15]. These cells, however, do not exhibit retina-specific markers. It has been reported that embryonic retinal progenitor cells both survive and differentiate into retinal neurons, including the photoreceptor subtype of cells, following subretinal transplantation [5]. These cells, however, remained as a cluster in the subretinal space, without any migration into the retina. In this study, we tested two different populations of retinal progenitor cells, third-passaged and tenth-passaged ones. Neural progenitor cells derived from embryonic retinas retain the potential to migrate into the retina and to express neuronal or glial markers, including a photoreceptor * Corresponding author. Tel.: þ 81-75-751-4721; fax: þ81-75-751-4731. E-mail address: [email protected] (M. Takahashi).
cell-specific marker. These cells possess the properties of immature cells, but lost retina-specific characteristics after long term culture. For experiment, embryonic day 18 (E18) Fisher rat retinas, without retinal pigment epithelium, were explanted and mechanically dissociated into small retinal pieces. These pieces were plated on laminin (Gibco, Rockville, MD)-coated dishes in Dulbecco’s modified Eagle’s medium-Ham’s F12 (Gibco) containing B27 supplement (Gibco), 20 ng/ml basic fibroblast growth factor (bFGF; Genzyme/Techne, Menneapolis, Minnesota), and 20 ng/ml epidermal growth factors (EGF; Genzyme/Techne). Cells were mechanically dissociated and passaged approximately every 10 days. We examined retinal progenitor cells after either three passages, approximately 40 days after primary culture, or ten passages, after about 3 months of culture. At these time points, some of the cells from primary culture had attached to the dishes, while others remained floating. Both populations of cells could form neurospheres. From the attached neurospheres, isolated cells, characterized by round cell bodies with processes, migrated out from the spheres (Fig. 1a). The majority of the cells in neurospheres
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00177-0
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T. Akagi et al. / Neuroscience Letters 341 (2003) 213–216
Fig. 1. Retinal progenitor cells could differentiate into neurons and glial cells in vitro. Tenth-passaged (a –d) and third-passaged (e) retinal progenitor cells. (a) Phase-contrast micrographs demonstrate that a subset of floating and attached cells grew into neurosphere-like colonies with isolated cells migrating out from the spheres. (b) Neural spheres prior to the induction of differentiation were double labeled for nestin (green) and DAPI (blue). Most cells exhibited nestin-like immunoreactivity (c,d). Retinal progenitor cells were double labeled for GFAP (red) and either b III tubulin (c) or MAP5 (d) (green) after induction of differentiation. The white arrowhead indicates a MAP5-positive cell. (e) a population of the third-passaged retinal progenitor cells differentiated into rhodopsin(red)- and/or recoverin(green)-immunoreactive cells after induction of differentiation. The white arrows indicate double-immunoreactive cells. Scale bars, 20 mm.
were positive for nestin, a marker for neural progenitor cells (Fig. 1b). As the proliferative capacity of these cells increased after the third passage in comparison to just after isolation, the passage interval became shorter (3 – 5 days). We could expand neural progenitor cells under these culture conditions for at least 3 months without losing immunoreactivity for nestin. To induce differentiation, medium without bFGF and EGF was supplemented with retinoic acid and 0.5% fetal bovine serum. After 2 weeks of culture under differentiation conditions, the cells were analyzed immunocytochemically. Migrated cells from attached neurospheres expressed neuronal or glial markers following differentiation, regardless of the number of passages (Fig. 1c,d). Approximately 6 , 7% of the cells from third-passaged spheres could also express a photoreceptor-specific marker, recoverin (Fig. 1e). In addition, small numbers of cells from third-passaged spheres demonstrated rhodopsin immunoreactivity. Tenthpassaged cells, however, could not express either recoverin or rhodopsin following identical differentiation conditions. Eight-week-old male Fisher rats were used as recipients for retinal transplantation. One week prior to transplantation, photoreceptor degeneration was induced by intraperitoneal injection of 75 mg/kg N-methyl-N-nitrosourea (MNU) as described previously [11,12,18]. Three days prior to transplantation, the gene encoding green fluorescent protein (GFP) was transferred into the retinal progenitor cells in vitro using a replicationdeficient adenovirus at multiplicity of infection of 35. Following infection approximately 90% of the cells expressed GFP (data not shown) [7]. Third-passaged and tenth-passaged retinal progenitor cells were harvested for grafting and separated mechanically into pieces, including small spheres. Before transplantation, cells were resuspended at a density of 30 000 cells/ml in high-glucose Dulbecco’s phosphate-buffered saline (Gibco), containing 20 ng/ml bFGF and 20 ng/ml EGF. Using a microscope, we performed two different techniques for subretinal transplantation, a vitreous approach under direct observation with a plano-concave
contact lens [13] and a scleral approach [15]. Three microliters of the cell suspension was slowly injected into the subretinal space using a microsyringe fitted with a 30-gauge blunt needle. Twenty-four eyes per cell type were examined for either third-passaged or tenthpassaged retinal progenitor cells. Animals were sacrificed 1, 2, and 4 weeks after transplantation. After the frozen sample was sectioned into 16 mm thick in a cryostat, we performed immunostaining with the following primary antibodies: anti-nestin (PharMingen, San Diego, CA), anti-glial fibrillary acidic protein (GFAP; Chemicon, Temecula, CA), anti-b III tubulin (Sigma, St. Louis, MO), anti-microtubule associated protein (MAP) 5 (Chemicon), anti-neurofilament 200 antibody (Sigma), anti-rhodopsin antibody RET-P1 (Sigma), and antirecoverin antibody (kind gift of J.F. McGinnis and R.J. Elins). Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; 1mg/ml; Molecular Probes, Eugene, OR). Sections were observed with a laser-scanning confocal microscope. Both types of grafted retinal progenitor cells expressing GFP migrated into the retina from the subretinal space and extended multiple processes (Fig. 2a,e). A population of these cells differentiated morphologically into neurons (Fig. 2f), measured by their de novo expression of neuronal or glial markers (Fig. 2b,c,g – i). While a portion of the thirdpassaged retinal progenitor cells expressed photoreceptorspecific markers, the tenth-passaged cells could not, confirming our in vitro results (Fig. 2j). The tenth-passaged retinal progenitor cells showed more extensive migration ratio of grafted cells into the retina than the third-passaged ones (Fig. 3), although both cell populations could produce neurons or glia within the retina. We could expand retinal progenitor cells in vitro retaining immature progenitor cell properties for as long as 6 months in serum free medium with bFGF and EGF. This continuous proliferation while retaining an immature status is critical to obtaining enough donor cells for therapeutic use. These cells could migrate into the degenerated retinas of adult rats and then differentiate into
T. Akagi et al. / Neuroscience Letters 341 (2003) 213–216
215
Fig. 3. Quantification of the migration of GFP-expressing graft cells. Cell numbers were counted in sections derived from four independent retinas. The total resident cells were 617 in the tenth-passaged samples and 424 in the third-passaged samples. The retina was classified into three parts, the subretinal space, inner nuclear layer (INL) and ganglion nuclear layer (GCL). The outer nuclear layer (ONL) was absent due to MNU treatment. The ratio of migration into retina, which was estimated by existing at INL or GCL per remaining at subretinal space, for tenth-passaged retinal progenitor cells was higher than that for third-passaged ones. Furthermore, tenth-passaged retinal progenitor cells migrated farther, into the GCL from the subretinal space, while the third-passaged cells only reached the INL. Standard deviation *P , 0:025, Mann-Whitney U-test.
Fig. 2. Retinal progenitor cells could migrate effectively into adult rat retina and express neuronal and glial markers. Tenth-passaged (a –d) and thirdpassaged (e –j) retinal progenitor cells. Confocal images of the retina 1 (a), 2 (b,e,h,i), and 4 (c,d,f,g,j) weeks after cell transplantation. (a,e,f) Many transplanted cells migrated into the retina from the subretinal space, gaining multiple neuron-like processes. (b– d,g–j) Double-label immunofluorescence. Green: GFP; red: b III tubulin (b), GFAP (c,i), PKC (d) NF200 (g), MAP5 (h), and recoverin (j) staining. The white arrowheads indicate double-immunoreactive grafted cells. The white arrow indicates a PKCnegative grafted cell whose shape is similar to the surrounding PKCpositive cells. Scale bars, 20 mm.
both neurons and glia within these adult tissues. Furthermore, the grafted cells from minimally passaged cultures could also express photoreceptor-specific markers. Adult stem cells derived from various tissues have recently been reported to differentiate into neurons beyond their germ layer [3]. For example, bone marrow stromal cells [4,10], skin stem cells [16], and amnionic cells [14] can produce neurons. Embryonic stem (ES) cells and neural stem cells from various regions of central nervous system also produce neurons. All of these sources are possible candidates for retinal cell transplantation therapy. Only a few types of cells among these possibilities, however, can generate retina-specific cells needed for the treatment of retinal diseases. Retinal stem cells are reported to exist in the ciliary epithelium isolated from the adult rat eye [2,17] and the retina of the embryonic rat eye [1]. Transfection of iris tissue from the adult rat with the Crx gene can produce cells that express photoreceptor-specific markers [6]. ES cells can differentiate into retinal pigment epithelial cells [8]. It is still difficult, however, to obtain sufficient
photoreceptor cells for transplantation in retinal degenerative diseases such as retinitis pigmentosa. Our study in the rat system generating many photoreceptor cells suggests that progenitor cells from embryonic retinas could still be a choice for retinal transplantation if expanded significantly for the treatment of many patients. We tested the effect of culture periods on retinal progenitor cell potential in this report. We observed differences between third-passaged and tenth-passaged retinal progenitor cells in their ability to differentiate into retinal cells. Third-passaged retinal progenitor cells could differentiate into photoreceptor cells both in vitro and in vivo. While they could produce neurons and glia, tenthpassaged cells could not generate expression of recoverin or rhodopsin, photoreceptor-specific markers [9]. Thus, the multipotent characteristics of these retinal progenitor cells changed after long-term culture with many passages. The cells had not transformed, however, as they stopped proliferating once bFGF and EGF were removed from the medium and as they did not form tumors in vivo. One of the possible explanation of these results is that more immature progenitors survive and become dominant after long-term culture. Other is that the photoreceptor progenitor cells may have become more primitive in serum free medium with bFGF and EGF, such that they would lose the intrinsic factors needed for photoreceptor differentiation. It is reported that long term culture with bFGF and EGF may reprogram and/or transdifferentiate organ-specific stem cells by overcoming their intrinsic restrictions [3], causing the retinal progenitor cells to return to a more
216
T. Akagi et al. / Neuroscience Letters 341 (2003) 213–216
undifferentiated states. If this hypothesis is correct, we should change extrinsic factors to induce a more efficient differentiation of retinal progenitor cells into photoreceptor cells. The length of culture prior to transplantation also determined the migratory ability of the transplanted retinal progenitor cells. Tenth-passaged retinal progenitor cells migrated farther into the retina from the subretinal space and extended more processes than third-passaged cells. The majority of the third-passaged progenitor cells remained in the subretinal space or outer part of inner nuclear layer with short neuron-like processes after subretinal transplantation. This tendency suggests that retinal progenitor cells gain increasing migratory ability after long culture and many passages. In conclusion, our data demonstrated that progenitor cells from embryonic retina could be expanded while retaining immature cell properties. These cells possessed the ability to migrate into degenerated adult retina from the subretinal space. Progenitor cells differentiated into neurons, glia, and even photoreceptor cells. Although, the cells appeared to lose their tissue specificity after long-term culture. For cell transplantation therapy, it is important to obtain a specific cell type that is required for treatment of the disease. Moreover, it is necessary to harvest large enough cells to recover retinal function after transplantation In the future, we should evaluate the most suitable culture conditions for retinal progenitor cells to optimize their efficiency as donor cells for retinal transplantation.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements [14]
We would like to thank Dr Kanegae and Dr Saitoh for providing the adenovirus stocks expressing GFP.
[15]
References
[16]
[1] I. Ahmad, C.M. Dooley, W.B. Thoreson, J.A. Rogers, S. Afiat, In vitro analysis of a mammalian retinal progenitor that gives rise to neurons and glia, Brain Res. 831 (1999) 1–10. [2] I. Ahmad, L. Tang, H. Pham, Identification of neural progenitors in the adult mammalian eye, Biochem. Biophys. Res. Commun. 270 (2000) 517–521. [3] D.J. Anderson, F.H. Gage, I.L. Weissman, Can stem cells cross lineage boundaries?, Nat. Med. 7 (2001) 393 –395. [4] T.R. Brazelton, F.M. Rossi, G.I. Keshet, H.M. Blau, From marrow to
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brain: expression of neuronal phenotypes in adult mice, Science 290 (2000) 1775–1779. D.M. Chacko, J.A. Rogers, J.E. Turner, I. Ahmad, Survival and differentiation of cultured retinal progenitors transplanted in the subretinal space of the rat, Biochem. Biophys. Res. Commun. 268 (2000) 842 –846. M. Haruta, M. Kosaka, Y. Kanegae, I. Saito, T. Inoue, R. Kageyama, A. Nishida, Y. Honda, M. Takahashi, Induction of photoreceptorspecific phenotypes in adult mammalian iris tissue, Nat. Neurosci. 4 (2001) 1163–1164. Y. Kanegae, M. Makimura, I. Saito, A simple and efficient method for purification of infectious recombinant adenovirus, Jpn. J. Med. Sci. Biol. 47 (1994) 157– 166. H. Kawasaki, H. Suemori, K. Mizuseki, K. Watanabe, F. Urano, H. Ichinose, M. Haruta, M. Takahashi, K. Yoshikawa, S. Nishikawa, N. Nakatsuji, Y. Sasai, Generation of dopaminergic neurons and pigmented epithelia from primate ES cells by stromal cell-derived inducing activity, Proc. Natl. Acad. Sci. USA 99 (2002) 1580– 1585. J.F. McGinnis, P.L. Stepanik, W. Chen, R. Elias, W. Cao, V. Lerious, Unique retina cell phenotypes revealed by immunological analysis of recoverin expression in rat retina cells, J. Neurosci. Res. 55 (1999) 252 –260. E. Mezey, K.J. Chandross, G. Harta, R.A. Maki, S.R. McKercher, Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow, Science 290 (2000) 1779– 1782. M. Nakajima, K. Yuge, H. Senzaki, N. Shikata, H. Miki, M. Uyama, A. Tsubura, Photoreceptor apoptosis induced by a single systemic administration of N-methyl-N-nitrosourea in the rat retina, Am. J. Pathol. 148 (1996) 631 –641. H. Nambu, K. Yoshizawa, J. Yang, D. Yamamoto, A. Tsubura, Agespecific and dose-dependent retinal dysplasia and degeneration induced by a single intraperitoneal administration of N-methyl-Nnitrosourea to Rats, Toxicol. Pathol. 11 (1998) 127 –131. A. Nishida, M. Takahashi, H. Tanihara, I. Nakano, J.B. Takahashi, A. Mizoguchi, C. Ide, Y. Honda, Incorporation and differentiation of hippocampus-derived neural stem cells transplanted in injured adult rat retina, Invest. Ophthalmol. Vis. Sci. 41 (2000) 4268–4274. N. Sakuragawa, R. Thangavel, M. Mizuguchi, M. Hirasawa, I. Kamo, Expression of markers for both neuronal and glial cells in human amniotic epithelial cells, Neurosci. Lett. 209 (1996) 9–12. M. Takahashi, T.D. Palmer, J. Takahashi, F.H. Gage, Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina, Mol. Cell Neurosci. 12 (1998) 340 –348. J.G. Toma, M. Akhavan, K.J. Fernandes, F. Barnabe-Heider, A. Sadikot, D.R. Kaplan, F.D. Miller, Isolation of multipotent adult stem cells from the dermis of mammalian skin, Nat. Cell Biol. 3 (2001) 778 –784. V. Tropepe, B.L. Coles, B.J. Chiasson, D.J. Horsford, A.J. Elia, R.R. McInnes, D. van der Kooy, Retinal stem cells in the adult mammalian eye, Science 287 (2000) 2032–2036. K. Yoshizawa, H. Nambu, J. Yang, Y. Oishi, H. Senzaki, N. Shikata, H. Miki, A. Tsubura, Mechanisms of photoreceptor cell apoptosis induced by N-methyl-N- nitrosourea in Sprague– Dawley rats, Lab. Invest. 79 (1999) 1359–1367.
Different characteristics of rat retinal progenitor cells from different culture periods Tadamichi Akagia, Masatoshi Harutab, Joe Akitac, Akihiro Nishidad, Yoshihito Hondaa, Masayo Takahashib,* a
Department of Ophthalmology and Visual Sciences, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan Department of Experimental Therapeutics, Translational Research Center, Kyoto University Hospital, Kyoto 606-8507, Japan c Department of Ophthalmology, Tenri Yorozu Hospital, Nara 632-8552, Japan d Developmental Biology Department, Osaka Bioscience Institute, Osaka 565-0874, Japan
b
Received 22 January 2003; accepted 3 February 2003
Abstract Embryonic retina is one of the possible cell sources that will repair degenerated retina such as retinitis pigmentosa. Retinal progenitor cells isolated from embryonic rats could be cultured and expanded in serum free medium with both epidermal growth factor and basic fibroblast growth factor. We analyzed the properties of two different retinal progenitor cells in terms of culture periods. Retinal progenitor cells from embryonic retina could be expanded keeping immature cell properties and had the ability to migrate into degenerated adult retina from subretinal space after transplantation. They differentiated into neurons and glias, even into photoreceptor cells both in vitro and in vivo. However, they appeared to lose their tissue specificity after a long-term culture. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Retinal cell culture; Retinal transplantation; Photoreceptor cells; Retinal progenitor; Retinal degeneration; Culture period
The isolation of neural stem cells evoked a great deal of interest as a possible therapy for neural degenerative diseases. Recently, several types of neural cell lines or progenitor cells, including stem cells, have been used for retinal transplantation. Adult rat hippocampus-derived neural stem cells efficiently integrated into the retina to adopt the morphology and position of retina-specific cells [15]. These cells, however, do not exhibit retina-specific markers. It has been reported that embryonic retinal progenitor cells both survive and differentiate into retinal neurons, including the photoreceptor subtype of cells, following subretinal transplantation [5]. These cells, however, remained as a cluster in the subretinal space, without any migration into the retina. In this study, we tested two different populations of retinal progenitor cells, third-passaged and tenth-passaged ones. Neural progenitor cells derived from embryonic retinas retain the potential to migrate into the retina and to express neuronal or glial markers, including a photoreceptor * Corresponding author. Tel.: þ 81-75-751-4721; fax: þ81-75-751-4731. E-mail address: [email protected] (M. Takahashi).
cell-specific marker. These cells possess the properties of immature cells, but lost retina-specific characteristics after long term culture. For experiment, embryonic day 18 (E18) Fisher rat retinas, without retinal pigment epithelium, were explanted and mechanically dissociated into small retinal pieces. These pieces were plated on laminin (Gibco, Rockville, MD)-coated dishes in Dulbecco’s modified Eagle’s medium-Ham’s F12 (Gibco) containing B27 supplement (Gibco), 20 ng/ml basic fibroblast growth factor (bFGF; Genzyme/Techne, Menneapolis, Minnesota), and 20 ng/ml epidermal growth factors (EGF; Genzyme/Techne). Cells were mechanically dissociated and passaged approximately every 10 days. We examined retinal progenitor cells after either three passages, approximately 40 days after primary culture, or ten passages, after about 3 months of culture. At these time points, some of the cells from primary culture had attached to the dishes, while others remained floating. Both populations of cells could form neurospheres. From the attached neurospheres, isolated cells, characterized by round cell bodies with processes, migrated out from the spheres (Fig. 1a). The majority of the cells in neurospheres
0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00177-0
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Fig. 1. Retinal progenitor cells could differentiate into neurons and glial cells in vitro. Tenth-passaged (a –d) and third-passaged (e) retinal progenitor cells. (a) Phase-contrast micrographs demonstrate that a subset of floating and attached cells grew into neurosphere-like colonies with isolated cells migrating out from the spheres. (b) Neural spheres prior to the induction of differentiation were double labeled for nestin (green) and DAPI (blue). Most cells exhibited nestin-like immunoreactivity (c,d). Retinal progenitor cells were double labeled for GFAP (red) and either b III tubulin (c) or MAP5 (d) (green) after induction of differentiation. The white arrowhead indicates a MAP5-positive cell. (e) a population of the third-passaged retinal progenitor cells differentiated into rhodopsin(red)- and/or recoverin(green)-immunoreactive cells after induction of differentiation. The white arrows indicate double-immunoreactive cells. Scale bars, 20 mm.
were positive for nestin, a marker for neural progenitor cells (Fig. 1b). As the proliferative capacity of these cells increased after the third passage in comparison to just after isolation, the passage interval became shorter (3 – 5 days). We could expand neural progenitor cells under these culture conditions for at least 3 months without losing immunoreactivity for nestin. To induce differentiation, medium without bFGF and EGF was supplemented with retinoic acid and 0.5% fetal bovine serum. After 2 weeks of culture under differentiation conditions, the cells were analyzed immunocytochemically. Migrated cells from attached neurospheres expressed neuronal or glial markers following differentiation, regardless of the number of passages (Fig. 1c,d). Approximately 6 , 7% of the cells from third-passaged spheres could also express a photoreceptor-specific marker, recoverin (Fig. 1e). In addition, small numbers of cells from third-passaged spheres demonstrated rhodopsin immunoreactivity. Tenthpassaged cells, however, could not express either recoverin or rhodopsin following identical differentiation conditions. Eight-week-old male Fisher rats were used as recipients for retinal transplantation. One week prior to transplantation, photoreceptor degeneration was induced by intraperitoneal injection of 75 mg/kg N-methyl-N-nitrosourea (MNU) as described previously [11,12,18]. Three days prior to transplantation, the gene encoding green fluorescent protein (GFP) was transferred into the retinal progenitor cells in vitro using a replicationdeficient adenovirus at multiplicity of infection of 35. Following infection approximately 90% of the cells expressed GFP (data not shown) [7]. Third-passaged and tenth-passaged retinal progenitor cells were harvested for grafting and separated mechanically into pieces, including small spheres. Before transplantation, cells were resuspended at a density of 30 000 cells/ml in high-glucose Dulbecco’s phosphate-buffered saline (Gibco), containing 20 ng/ml bFGF and 20 ng/ml EGF. Using a microscope, we performed two different techniques for subretinal transplantation, a vitreous approach under direct observation with a plano-concave
contact lens [13] and a scleral approach [15]. Three microliters of the cell suspension was slowly injected into the subretinal space using a microsyringe fitted with a 30-gauge blunt needle. Twenty-four eyes per cell type were examined for either third-passaged or tenthpassaged retinal progenitor cells. Animals were sacrificed 1, 2, and 4 weeks after transplantation. After the frozen sample was sectioned into 16 mm thick in a cryostat, we performed immunostaining with the following primary antibodies: anti-nestin (PharMingen, San Diego, CA), anti-glial fibrillary acidic protein (GFAP; Chemicon, Temecula, CA), anti-b III tubulin (Sigma, St. Louis, MO), anti-microtubule associated protein (MAP) 5 (Chemicon), anti-neurofilament 200 antibody (Sigma), anti-rhodopsin antibody RET-P1 (Sigma), and antirecoverin antibody (kind gift of J.F. McGinnis and R.J. Elins). Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; 1mg/ml; Molecular Probes, Eugene, OR). Sections were observed with a laser-scanning confocal microscope. Both types of grafted retinal progenitor cells expressing GFP migrated into the retina from the subretinal space and extended multiple processes (Fig. 2a,e). A population of these cells differentiated morphologically into neurons (Fig. 2f), measured by their de novo expression of neuronal or glial markers (Fig. 2b,c,g – i). While a portion of the thirdpassaged retinal progenitor cells expressed photoreceptorspecific markers, the tenth-passaged cells could not, confirming our in vitro results (Fig. 2j). The tenth-passaged retinal progenitor cells showed more extensive migration ratio of grafted cells into the retina than the third-passaged ones (Fig. 3), although both cell populations could produce neurons or glia within the retina. We could expand retinal progenitor cells in vitro retaining immature progenitor cell properties for as long as 6 months in serum free medium with bFGF and EGF. This continuous proliferation while retaining an immature status is critical to obtaining enough donor cells for therapeutic use. These cells could migrate into the degenerated retinas of adult rats and then differentiate into
T. Akagi et al. / Neuroscience Letters 341 (2003) 213–216
215
Fig. 3. Quantification of the migration of GFP-expressing graft cells. Cell numbers were counted in sections derived from four independent retinas. The total resident cells were 617 in the tenth-passaged samples and 424 in the third-passaged samples. The retina was classified into three parts, the subretinal space, inner nuclear layer (INL) and ganglion nuclear layer (GCL). The outer nuclear layer (ONL) was absent due to MNU treatment. The ratio of migration into retina, which was estimated by existing at INL or GCL per remaining at subretinal space, for tenth-passaged retinal progenitor cells was higher than that for third-passaged ones. Furthermore, tenth-passaged retinal progenitor cells migrated farther, into the GCL from the subretinal space, while the third-passaged cells only reached the INL. Standard deviation *P , 0:025, Mann-Whitney U-test.
Fig. 2. Retinal progenitor cells could migrate effectively into adult rat retina and express neuronal and glial markers. Tenth-passaged (a –d) and thirdpassaged (e –j) retinal progenitor cells. Confocal images of the retina 1 (a), 2 (b,e,h,i), and 4 (c,d,f,g,j) weeks after cell transplantation. (a,e,f) Many transplanted cells migrated into the retina from the subretinal space, gaining multiple neuron-like processes. (b– d,g–j) Double-label immunofluorescence. Green: GFP; red: b III tubulin (b), GFAP (c,i), PKC (d) NF200 (g), MAP5 (h), and recoverin (j) staining. The white arrowheads indicate double-immunoreactive grafted cells. The white arrow indicates a PKCnegative grafted cell whose shape is similar to the surrounding PKCpositive cells. Scale bars, 20 mm.
both neurons and glia within these adult tissues. Furthermore, the grafted cells from minimally passaged cultures could also express photoreceptor-specific markers. Adult stem cells derived from various tissues have recently been reported to differentiate into neurons beyond their germ layer [3]. For example, bone marrow stromal cells [4,10], skin stem cells [16], and amnionic cells [14] can produce neurons. Embryonic stem (ES) cells and neural stem cells from various regions of central nervous system also produce neurons. All of these sources are possible candidates for retinal cell transplantation therapy. Only a few types of cells among these possibilities, however, can generate retina-specific cells needed for the treatment of retinal diseases. Retinal stem cells are reported to exist in the ciliary epithelium isolated from the adult rat eye [2,17] and the retina of the embryonic rat eye [1]. Transfection of iris tissue from the adult rat with the Crx gene can produce cells that express photoreceptor-specific markers [6]. ES cells can differentiate into retinal pigment epithelial cells [8]. It is still difficult, however, to obtain sufficient
photoreceptor cells for transplantation in retinal degenerative diseases such as retinitis pigmentosa. Our study in the rat system generating many photoreceptor cells suggests that progenitor cells from embryonic retinas could still be a choice for retinal transplantation if expanded significantly for the treatment of many patients. We tested the effect of culture periods on retinal progenitor cell potential in this report. We observed differences between third-passaged and tenth-passaged retinal progenitor cells in their ability to differentiate into retinal cells. Third-passaged retinal progenitor cells could differentiate into photoreceptor cells both in vitro and in vivo. While they could produce neurons and glia, tenthpassaged cells could not generate expression of recoverin or rhodopsin, photoreceptor-specific markers [9]. Thus, the multipotent characteristics of these retinal progenitor cells changed after long-term culture with many passages. The cells had not transformed, however, as they stopped proliferating once bFGF and EGF were removed from the medium and as they did not form tumors in vivo. One of the possible explanation of these results is that more immature progenitors survive and become dominant after long-term culture. Other is that the photoreceptor progenitor cells may have become more primitive in serum free medium with bFGF and EGF, such that they would lose the intrinsic factors needed for photoreceptor differentiation. It is reported that long term culture with bFGF and EGF may reprogram and/or transdifferentiate organ-specific stem cells by overcoming their intrinsic restrictions [3], causing the retinal progenitor cells to return to a more
216
T. Akagi et al. / Neuroscience Letters 341 (2003) 213–216
undifferentiated states. If this hypothesis is correct, we should change extrinsic factors to induce a more efficient differentiation of retinal progenitor cells into photoreceptor cells. The length of culture prior to transplantation also determined the migratory ability of the transplanted retinal progenitor cells. Tenth-passaged retinal progenitor cells migrated farther into the retina from the subretinal space and extended more processes than third-passaged cells. The majority of the third-passaged progenitor cells remained in the subretinal space or outer part of inner nuclear layer with short neuron-like processes after subretinal transplantation. This tendency suggests that retinal progenitor cells gain increasing migratory ability after long culture and many passages. In conclusion, our data demonstrated that progenitor cells from embryonic retina could be expanded while retaining immature cell properties. These cells possessed the ability to migrate into degenerated adult retina from the subretinal space. Progenitor cells differentiated into neurons, glia, and even photoreceptor cells. Although, the cells appeared to lose their tissue specificity after long-term culture. For cell transplantation therapy, it is important to obtain a specific cell type that is required for treatment of the disease. Moreover, it is necessary to harvest large enough cells to recover retinal function after transplantation In the future, we should evaluate the most suitable culture conditions for retinal progenitor cells to optimize their efficiency as donor cells for retinal transplantation.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgements [14]
We would like to thank Dr Kanegae and Dr Saitoh for providing the adenovirus stocks expressing GFP.
[15]
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