ORIGINAL PAPER

Med Mol Morphol (2010) 43:139–144 © The Japanese Society for Clinical Molecular Morphology 2010DOI 10.1007/s00795-009-0486-3

Naoki Yamamoto · Atsuhiro Tanikawa Masayuki Horiguchi

Basic study of retinal stem/progenitor cell separation from mouse iris tissue

Abstract We described the possibility of retinal regenera-tion using a novel and effi cient technique for culturing and separating retinal stem/progenitor cells from iris tissue. Immunohistochemical staining of adult agouti mouse iris tissue revealed the presence of nestin/low-affi nity neuro-trophin receptor p75 (p75NTR)-positive cells on the endothe-lium camerae anterioris side. Cultured mouse iris-derived cells contained little or no melanin and were found to be positive for nestin. Most nestin-positive cells were analyzed for the coexpression of p75NTR as a cell membrane protein. When the p75NTR was used as a marker to sort the cells, we obtained a dense population of nestin-positive cells. Fur-thermore, the nestin/p75NTR-positive cells were able to dif-ferentiate into neural retina cells. Thus, this culture and separation technique is useful for obtaining retinal stem/progenitor cells from adult mouse iris tissue and for the effi cient production of neural retina cells.

Key words Retinal stem/progenitor cells · Low-affi nity neu-rotrophin receptor p75 (p75NTR) · Nestin · Iris tissue · Neural retina cell · Cell sorting

Introduction

No current therapeutic modality allows complete recovery of vision in patients with retinal diseases such as retinal pigment degeneration and age-related macular degenera-tion, probably because the retina is a collective entity of neurons, and mammalian neural retina cells cannot self-repair once they are injured. On the other hand, self-renewal of the injured retina has been documented in newts.1,2 In

N. Yamamoto (*)Laboratory of Molecular Biology & Histochemistry, Fujita Health University Joint Research Laboratory, 1-98 Dengakugakubo, Kutsukake-cho, Toyoake, Aichi 470-1192, JapanTel. +81-562-93-2317; Fax +81-562-92-5382e-mail: naokiy@fujita-hu.ac.jp

A. Tanikawa · M. HoriguchiDepartment of Ophthalmology, Fujita Health University School of Medicine, Aichi, Japan

this regeneration process, tissue stem (multipotent stem)/progenitor cells found in various tissues in adults have attracted attention and have been used to generate mam-malian retina.3–7 The use of eyeball tissues, including imma-ture retinal tissue,8,9 the ciliary body,10 and the iris,11–13 has also been reported. Considering their possible clinical appli-cation, we have been investigating retinal regeneration using autologous tissues and have focused on iris tissue, which embryologically belongs to the optic cup inner layer, as does the neural retina, and allows the collection of autol-ogous tissue in a safe manner. We describe a novel tech-nique developed in our laboratory that uses a cell marker of the low-affi nity neurotrophin receptor p75 (p75NTR; p75 nerve growth factor receptor; CD271) to obtain retinal stem/progenitor cells that can differentiate into neural retina cells.

Materials and methods

Animals

We used male DBA/2 Cr wild-type agouti mice (Japan SLC, Shizuoka, Japan). All procedures were performed according to the ARVO Statement for the Use of Animals in Ophthal-mic and Vision Research and were approved by the Educa-tion and Research Center for Animal Models of Human Diseases of Fujita Health University.

Cell isolation and culturing of iris tissue

The iris tissue was obtained and processed according to a previously described protocol.13,14 Briefl y, DBA/2 Cr mice were decapitated on postnatal day 30, and their eyes were removed. The excision of iris tissue was performed using an ophthalmic micro-scissor without including the ciliary body (Fig. 1a). The extracted iris tissue was treated with 0.2% collagenase (Nitta Gelatin, Osaka, Japan) and washed twice with phosphate-buffered saline solution (PBS; Sigma, St. Louis, MO, USA). The isolated cells were cultured in

Received: October 6, 2009 / Accepted: November 17, 2009

140

Dulbecco’s modifi ed Eagle’s medium/Ham’s F12 (DMEM/F12; Sigma) supplemented with 5% fetal bovine serum (FBS; JRH Biosciences, Adelaide, Australia), 1 ng/ml basic fi broblastic growth factor (b-FGF; Sigma), 1 ng/ml epider-mal growth factor (EGF; Sigma), 100 nM TAT-Pep5 (the p75NTR signaling inhibitor, only for the beginning of the culture; Calbiochem, San Diego, CA, USA), and 1% penicil-lin/streptomycin (Sigma) in a 35-mm culture dish coated with collagen (TOYOBO, Osaka, Japan) at 37°C in a 5% CO2 humidifi ed incubator.

Immunohistochemistry of iris tissue

The DBA2/Cr mouse eyes were fi xed in SUPER FIX rapid fi xative solution (Kurabo Industries, Osaka, Japan).15 The eyeballs were fi xed within approximately 60 min, and 3-μm paraffi n sections were prepared from the fi xed mouse eye-balls in the usual manner and incubated with anti-p75NTR antibody (1:200; Chemicon International, Temecula, CA,

USA) and anti-nestin antibody (1:100; Chemicon) for 1 h at 37°C. The secondary antibodies and working dilutions were as follows: Alexa Fluor 488 labeled anti-mouse goat IgG antibody (for nestin, 1:1000; Invitrogen, Carlsbad, CA, USA) and Alexa Fluor 594 labeled anti-rabbit goat IgG antibody (for p75NTR, 1:1000; Invitrogen), which were incu-bated with the sections for 1 h at 37°C. DAPI (VECTA-SHIELD H-1200; Vector Laboratories, Burlingame, CA, USA) was used for nuclear staining. A fl uorescence micro-scope (Power BX-51; Olympus, Tokyo, Japan) was used for observation.

Spherical culture of iris-derived cells

Single cells were cultured from isolated mouse iris tissue in a super low-adhesion dish (HydroCell; CellSeed, Tokyo, Japan) in DMEM/F12 medium supplemented with N2-sup-plement (Invitrogen) and growth factors (20 ng/ml) as per the neurosphere method. After 5 days, the total number of

iris

lens ciliary body

a p75NTR

Nestin p75NTR

Recoverin Calbindin

retina

cornea

lens

iris

optic nerve

cilliarybody

b c d

Nestin Mergee fNestin Mergee f

g i j k

l m n o p

*

Corneal endothelium side

lens side

Passage 1 Passage 3

h

Fig. 1. The differentiation of retinal stem/progenitor cells from mouse iris tissue into neural retina cells. Rigorous elimination of contaminat-ing cells from the ciliary body was achieved by carefully making an incision along the iris circumference. The ciliary body is distinguishable as a pleated structure after separation from the iris and cornea (a). A section of a whole eyeball from a 4-week-old mouse stained with hema-toxylin and eosin (b). The marked area (*) indicates high-magnifi cation views of iris tissue differential interference (in c), iris tissue staining with various antibodies related to markers of retinal stem/progenitor, p75NTR (in d), nestin (in e), and a merged image showing both p75NTR

and nestin staining (in f). The iris-derived culture cells were found to be nonpigmented or slightly pigmented cells (g). In the neurosphere method, the single cells isolated from iris tissue were cultured at the 1st passage (h) and 3rd passage (i). The number of spheres was decreased after the subculture. The sphere formed cells that were stained for nestin (j) and p75NTR (k). The iris-derived culture nonsorted p75NTR (l), p75NTR-positive (m), or p75NTR-negative (n) cells were dif-ferentiated into neural cells. The cells that differentiated from p75NTR-positive cells were stained with recoverin (o) and calbindin (p). Bars b 500 μm; c–f 50 μm; g 100 μm; j, k 20 μm; l–n 100 μm; o, p 50 μm

141

spheres was counted, and all cells that had dissociated from the spheres were subcultured (3 times passage). The cells that had dissociated from the spheres were fi xed in 4% (w/v) paraformaldehyde (PFA; Sigma) and stained with anti-nestin and anti-p75NTR antibody.

Flow cytometric analysis and cell sorting

The iris-derived culture cells were subcultured using TrypLE Select (Invitrogen), washed twice with PBS, and fi xed in 4% (w/v) PFA for 15 min at 4°C. Then, the cells were washed twice with PBS, suspended in PBS containing 0.1 mg/ml saponin (MP Biomedicals, Illkrich, France) and 5% FBS, and allowed to stand for 10 min at room temperature. Thereafter, anti-nestin (1:100) and anti-p75NTR (1:200) were added and allowed to incubate at 4°C for 30 min. The cells were washed with PBS and incubated with the Alexa Fluor 488 labeled anti-mouse goat IgG (for nestin, 1:500; Invitro-gen) and PE-Cy5 labeled anti-rabbit goat IgG (for p75NTR, 1:200; Santa Cruz Biotechnology) at 4°C for 30 min. Next, the cells were washed with PBS, and fl ow cytometry [fl uo-rescence-activated cell sorting (FACS), FACSCan; BD Bio-sciences, San Jose, CA, USA] was performed three times independently.

As described for the fl ow cytometry analysis, the iris-derived culture cells were labeled with p75NTR. The p75NTR-Alexa Fluor 488 labeled cells were sorted (FACSVantage SE; BD Biosciences) so as to select p75NTR-positive cells.

Neural retina induction of p75NTR-positive cells

To induce neural differentiation, the subcultured p75NTR-positive or p75NTR-negative sorted cells and nonsorted p75NTR cells were cultured on laminin-coated dishes in 1 μM retinoic acid (RA; Sigma), 10 ng/ml bFGF, and FBS for 10 days. The cells that were induced to differentiate were fi xed in 4% (w/v) PFA and stained with anti-recoverin antibody (1:500; Chemicon) and calbindin (1:100; Chemicon). RNA from the p75NTR-positive and -negative cells was prepared using the TaqMan Gene Expression Cell-to-Ct Kit (Applied Biosystems, Foster City, CA, USA) at set intervals, and a quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed three times independently using PRISM-7900 HT (Applied Biosys-tems). The assay ID of the primers and TaqMan probe (Applied Biosystems) used were as follows: recoverin: Mm00501325_m1 (NW_009038), calbindin: Mm00486645_m1 (NW_009788), and GAPDH: Mm99999915_g1 (NM_001001303) as the internal control.

Results

Detection of neural retina stem/progenitor cells in mouse iris tissue

Paraffi n sections of the whole eyeballs were fi xed with SUPER FIX solution and stained with hematoxylin and

eosin (Fig. 1b). SUPER FIX solution penetrated into the tissue more quickly than in the usual preparations, which was useful because the structures of different tissues, such as the cornea, iris, lens, retina, and optic nerve, were well preserved.15

Paraffi n sections of the mouse iris were double-stained with p75NTR and nestin, which is a marker of neural retina stem/progenitor cells.7,10 p75NTR-positive cells were detected on the corneal endothelium side (Fig. 1d). Many of these p75NTR-positive cells contained little or no melanin (Fig. 1c). Most nestin-positive cells were p75NTR-positive (Fig. 1e,f). In contrast, no p75NTR-positive cells were detected on the iris pigmented side of the lens.

Adhesive and spherical culture of iris-derived cells

Mouse iris tissue was isolated by treatment with collagenase adhered to the culture dish. During culture for 7 days, slightly pigmented or nonpigmented cells proliferated (Fig. 1g). On the other hand, the single cells isolated from iris tissue were cultured by the neurosphere method, and the formed spheres gradually lost their pigment during prolif-eration, their peripheries became hypopigmented, and number of spheres was reduced. Whenever we subcultured the spheres, few cells contained the pigment, and few formed spheres (Figs. 1h,i, 2).10,13,16 Most of the sphere-forming cells were positive for nestin17 (Fig. 1j) and p75NTR (Fig. 1k).

350

Sphere Number

Pigment Pigment Pigment

54

250

300

159

150

200

45

16100

150

8759

42

15

0

50

1 2 3Passage Number

Fig. 2. Single cells isolated from iris tissue were cultured by the neu-rosphere method. The spheres were subcultured from the 1st passage to 3rd passage. The formed spheres gradually lost their pigment, and the number of spheres was reduced. Cells subjected to the neurosphere method cannot be subcultured more than three times, and the cells in the core of the sphere were not viable

142

Cell analysis and sorting of iris-derived culture cells

The iris-derived culture cells showed that the nestin-posi-tive cells accounted for 69.6% ± 6.46%, the p75NTR-positive cells for 78.7% ± 5.41%, and the nestin/p75NTR-positive cells for 65.7% ± 6.41%, respectively (Fig. 3a). When the nestin/p75NTR-positive cells were back-gated to dot plots of the forward scatter, which is a parameter of cell size, and the side scatter (SSC), which is a parameter of intracellular structure, the majority of cells were in an area with a small SSC parameter value (data not shown). Because cells with a small parameter value are characterized by less SSC light, the cells containing little or no melanin were considered to be nestin/p75NTR-positive cells.

As the results of FACS indicated that most nestin-posi-tive cells were also p75NTR positive, we sorted the iris-derived culture cells by p75NTR so as to obtain a high number of nestin-positive cells (Fig. 3b). Repeated analysis of the sorted cells showed that p75NTR-positive cells accounted for more than 98% of the cells (data not shown).

Analysis of p75NTR-positive cell induction

When the iris-derived nonsorted culture cells were induced to undergo neural retina differentiation, some of them fi rst developed a nerve cell-like morphology, but the remaining cells were not changed to nerve-like cells (Fig. 1l). Thus, the iris-derived culture cells were a mixture of cells with nerve-like and non-nerve-like morphology. Nearly all the sorted p75NTR-positive cells differentiated into nerve-like cells (Fig. 1m), but the many sorted p75NTR-negative cells were not changed into nerve-like cells (Fig. 1n).

We characterized the p75NTR-positive cells as neural retina cells. The sorted p75NTR-positive cells were cultured in the presence of RA differentiated markers recoverin (characteristic of photoreceptor cells) and calbindin (char-acteristic of horizontal cells) (Fig. 1o,p). Neural retina cell markers were detected on the differentiated p75NTR-positive cells cultured with RA.

The recoverin and calbindin mRNA expression levels were higher in the cells that were differentiated from p75NTR-positive cells than in the cells differentiated from p75NTR-negative cells according to qRT-PCR (Fig. 4). Com-pared to the cells differentiated from p75NTR-negative cells, the levels of recoverin-mRNA and calbindin-mRNA in the cells differentiated from p75NTR-positive cells were increased by 36.6 ± 3.10 and 20.5 ± 2.02 fold, respectively.

Discussion

The low-affi nity neurotrophin receptor p75 (p75NTR) is one of various markers of self-renewing tissue stem cells,18–20 and

Nestin AlexaFluor® 488

p75N

TR

PE

-Cy5

100

101

102

103

104

100

101

102

103

104

64.7 %

100

101

102

103

104

G1

p75NTR AlexaFluor® 488

0

20

40

60

80

100

% o

f Max

baFig. 3. Cell analysis and the sorting of iris-derived culture cells. Of the iris-derived culture cells, 65.7% ± 6.41% showed double-positive staining of nestin and p75NTR cells in fl uorescence-activated cell sorting (FACS) analysis (a). The p75NTR-Alexa Fluor 488-labeled iris-derived culture cells were selected (Gate 1: G1) to obtain a high number of nestin-positive cells using a cell sorter (b)

40

30

40

Recoverin Calbindin5N

TRne

gativ

e ce

ll

36.620

expr

essi

on fr

om p

75

20.510

ncre

ase

in m

RN

A e

1.0 1.0

0

Fold

in

p75NTR negative cells p75NTR positive cells

Fig. 4. Comparison of neural retina induction derived from p75NTR-positive or p75NTR-negative cells. The mRNA expression levels of the neural retina cell markers recoverin and calbindin were higher in the cells differentiated from p75NTR-positive cells than in the cells differen-tiated from p75NTR-negative cells according to quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Using the cells differentiated from p75NTR negative as a standard, recoverin mRNA and calbindin mRNA were detected 36.6 and 20.5 times, respectively, in the cells differentiated from p75NTR-positive cells

143

p75NTR-positive cells isolated from human esophageal epi-thelial cells show several phenotypic traits characteristic of tissue stem cells, such as slow-cycling cells and relatively immature keratinocytes.21

It was reported that after surgical resection the lens from newts is able to regenerate from the dorsal iris. This fi nding suggested that iris cells have a high potential for transdif-ferentiation.22–24 In some studies on cells derived from the adult rat iris, photoreceptors were successfully generated by ectopic expression of the Crx gene, which is the homeobox gene involved in the differentiation and maintenance of photoreceptors.11,25–27 Moreover, using iris pigment epithe-lial cells from white leghorn chicks, it was demonstrated that cells in spheres derived from these epithelial cells were able to differentiate into retinal cells.12 These studies commonly used the neurosphere method to generate neural retina stem/progenitor cells. The neurosphere method involves the use of a medium containing bFGF and/or EGF, as well as N2 or B27, instead of serum.28 This method allows the selec-tion of cells positive for nestin.29,30 Cells cultured by the neurosphere method proliferate in suspension and appear as aggregates.

However, cells derived from mouse iris tissue cultured by the neurosphere method cannot be subcultured more than three times, and the cells in the core of the sphere are not viable. Thus, we searched for culture condi-tions appropriate for the proliferation of nestin-positive cells and cell-surface markers useful for selecting nestin-positive cells with a cell sorter. After extensive investiga-tion, we found that nestin-positive cells were also positive for p75NTR, which is a receptor common to neurotrophins that is also called the low-affi nity neurotrophin receptor p75 (p75NTR). The neurotrophin family consists of four members: nerve growth factor (NGF), brain-derived neu-rotrophic factor (BDNF), neurotrophin-3 (NT-3), and neu-rotrophin-4/5 (NT-4/5), and NT-3 and NT-4/5 have a sequence homology of at least 50%. A neurotrophin is a kind of cytokine that is involved in the viability of nerves,31 the promotion axis regeneration,32 and the growth and development of the regeneration-promoting nervous system.33 The receptors for this neurotrophin family are molecules of the Trk family of tyrosine kinase receptors: NGF has a high affi nity for Trk A, BNDF and NT-4/5 have high affi nities for Trk B, and NT-3 has a high affi nity for Trk C. p75NTR can bind to all four of these neurotroph-ins.34–36 p75NTR has lately been renamed CD271. Thus, it is also possible to select nestin-positive cells using members of the Trk A neurotrophin family (data not shown). In addition, cultured cells derived from mouse iris tissue during cryopreservation are able to provide proliferating cells.

Finally, because nestin is a cytoplasm protein, no method that allowed the easy isolation of nestin-positive cells has been developed until now. We analyzed the proteins that developed in the cell membrane of nestin-positive cells and found p75NTR. As a result, we established a method capable of isolating nestin-positive cells with a cell sorter. This method may greatly contribute to the progress of neurosci-ences as well as retina studies.

Acknowledgments The authors thank Prof. K. Taniguchi, Prof. H. Akamatsu, Prof. K. Matsunaga, and Prof. H. Yamada (Fujita Health University, Japan) for their helpful support. This work was supported by a grant-in-aid for the High-Tech Frontier Project (2002–2006), a grant-in-aid for Collaboration with the Local Communities Project, and grants-in-aid for young scientists KAKENHI 17700341 and 19700324 from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. This work is now under examination by the Japa-nese Patent Offi ce with the aim of obtaining a technical patent (appli-cation of published patent No. 2008-92891).

References

1. Reh TA, Tully T (1986) Regulation of tyrosine hydroxylase-containing amacrine cell number in larval frog retina. Dev Biol 114:463–469

2. Reh TA, Levine EM (1998) Multipotential stem cells and progeni-tors in the vertebrate retina. J Neurobiol 36:206–220

3. Takahashi M, Palmer TD, Takahashi J, Gage FH (1998) Widespread integration and survival of adult-derived neural progenitor cells in the developing optic retina. Mol Cell Neurosci 12:340–348

4. Nishida A, Takahashi M, Tanihara H, Nakano I, Takahashi JB, Mizoguchi A, Ide C, Honda Y (2000) Incorporation and differen-tiation of hippocampus-derived neural stem cells transplanted in injured adult rat retina. Invest Ophthalmol Vis Sci 41:4268–4274

5. Kurimoto Y, Shibuki H, Kaneko Y, Ichikawa M, Kurokawa T, Takahashi M, Yoshimura N (2001) Transplantation of adult rat hippocampus-derived neural stem cells into retina injured by tran-sient ischemia. Neurosci Lett 306:57–60

6. Akita J, Takahashi M, Hojo M, Nishida A, Haruta M, Honda Y (2002) Neuronal differentiation of adult rat hippocampus-derived neural stem cells transplanted into embryonic rat explanted retinas with retinoic acid pretreatment. Brain Res 954:286–293

7. Suzuki T, Ooto S, Akagi T, Amemiya K, Igarashi R, Mizushima Y, Takahashi M (2003) Effects of prolonged delivery of brain-derived neurotrophic factor on the fate of neural stem cells transplanted into the developing rat retina. Biochem Biophys Res Commun 309:843–847

8. Turner DL, Cepko CL (1987) A common progenitor for neurons and glia persists in rat retina late in development. Nature (Lond) 328:131–136

9. Ahmad I, Dooley CM, Thoreson WB, Rogers JA, Afi at S (1999) In vitro analysis of a mammalian retinal progenitor that gives rise to neurons and glia. Brain Res 831:1–10

10. Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, van der Kooy D (2000) Retinal stem cells in the adult mam-malian eye. Science 287:2032–2036

11. Haruta M, Kosaka M, Kanegae Y, Saito I, Inoue T, Kageyama R, Nishida A, Honda Y, Takahashi M (2001) Induction of photorecep-tor-specifi c phenotypes in adult mammalian iris tissue. Nat Neuro-sci 4:1163–1164

12. Yamamoto N, Marunouchi T (2005) Studies for the regenerative therapy of neural retinal cells (in Japanese). Tissue Cult Res Commun 24:101–106

13. Sun G, Asami M, Ohta H, Kosaka J, Kosaka M (2006) Retinal stem/progenitor properties of iris pigment epithelial cells. Dev Biol 289:243–252

14. Yamamoto N (2009) Regenerative medicine using the tissue stem cell: separation and differentiation of tissue stem cell (in Japanese). BullFujita-Gakuen Med Soc 33:11–21

15. Yamamoto N, Majima K, Marunouchi T (2008) A study of the proliferating activity in lens epithelium and the identifi cation of tissue-type stem cells. Med Mol Morphol 41:83–91

16. Asami M, Sun G, Yamaguchi M, Kosaka M (2007) Multipotent cells from mammalian iris pigment epithelium. Dev Biol 304:433–446

17. Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate fi lament protein. Cell 60:585–595

18. Kunimura C, Kikuchi K, Ahmed N, Shimizu A, Yasumoto S (1998) Telomerase activity in a specifi c cell subset co-expressing integrin beta1/EGFR but not p75NGFR/bcl2/integrin beta4 in normal human epithelial cells. Oncogene 17:187–197

144

19. Hahn CG, Han LY, Rawson NE, Mirza N, Borgmann-Winter K, Lenox RH, Arnold SE (2005) In vivo and in vitro neurogenesis in human olfactory epithelium. J Comp Neurol 483:154–163

20. Yamamoto N, Akamatsu H, Hasegawa S, Yamada T, Nakata S, Ohkuma M, Miyachi E, Marunouchi T, Matsunaga K (2007) Isola-tion of multipotent stem cells from mouse adipose tissue. Dermatol Sci 48:43–52

21. Okumura T, Shimada Y, Imamura M, Yasumoto S (2003) Neuro-trophin receptor p75 (NTR) characterizes human esophageal kera-tinocyte stem cells in vitro. Oncogene 22:4017–4026

22. Eguchi G, Abe SI, Watanabe K (1974) Differentiation of lens-like structures from newt iris epithelial cells in vitro. Proc Natl Acad Sci U S A 71:5052–5056

23. Kodama R, Eguchi G (1995) From lens regeneration in the newt to in-vitro transdifferentiation of vertebrate pigmented epithelial cells. Semin Cell Biol 6:143–149

24. Kosaka M, Kodama R, Eguchi G (1998) In vitro culture system for iris-pigmented epithelial cells for molecular analysis of transdif-ferentiation. Exp Cell Res 245:245–251

25. Furukawa T, Morrow EM, Cepko CL (1997) Crx, a novel otx-like homeobox gene, shows photoreceptor-specifi c expression and reg-ulates photoreceptor differentiation. Cell 91:531–541

26. Chen S, Wang QL, Nie Z, Sun H, Lennon G, Copeland NG, Gilbert DJ, Jenkins NA, Zack DJ (1997) Crx, a novel Otx-like paired-homeodomain protein, binds to and transactivates photoreceptor cell-specifi c genes. Neuron 19:1017–1030

27. Freund CL, Gregory-Evans CY, Furukawa T, Papaioannou M, Looser J, Ploder L, Bellingham J, Ng D, Herbrick JA, Duncan A, Scherer SW, Tsui LC, Loutradis-Anagnostou A, Jacobson SG,

Cepko CL, Bhattacharya SS, McInnes RR (1997) Cone-rod dystro-phy due to mutations in a novel photoreceptor-specifi c homeobox gene (CRX) essential for maintenance of the photoreceptor. Cell 91:543–553

28. Okano H (2002) Neural stem cells: progression of basic research and perspective for clinical application. Keio J Med 51:115–128

29. Hockfi eld S, McKay RD (1985) Identifi cation of major cell classes in the developing mammalian nervous system. J Neurosci 5:3310–3328

30. Lendahl U, Zimmerman LB, McKay RD (1990) CNS stem cells express a new class of intermediate fi lament protein. Cell 60:585–595

31. Johansson A, Lundborg M, Sköld CM, Lundahl J, Tornling G, Eklund A, Camner P (1997) Functional morphological and pheno-typical differences between rat alveolar and interstitial macro-phages. Am J Res Cell Mol Biol 16:582–588

32. Hollowell JP, Villadiego A, Rich KM (1990) Sciatic nerve regenera-tion across gaps within silicone chambers: long-term effects of NGF and consideration of axonal branching. Exp Neurol 110:45–51

33. Levi-Montalcini R, Booker B (1960) Destruction of the sympa-thetic ganglia in mammals by an antiserum to a nerve growth protein. Proc Natl Acad Sci U S A 46:384–391

34. Bothwell M (1995) Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci 18:223–253

35. Bibel M, Hoppe E, Barde YA (1999) Biochemical and functional interactions between the neurotrophin receptors trk and p75NTR. EMBO J 18:616–622

36. Dechant G (2001) Molecular interactions between neurotrophin receptors. Cell Tissue Res 305:229–238