CHAPTER 53

MICROARRAY ANALYSIS REVEALS RETINAL STEM CELL CHARACTERISTICS OF THE

ADULT HUMAN EYE

For contributed volumes

Brigitte Angénieux1, Lydia Michaut2, Daniel F. Schorderet3,Francis L. Munier1, Walter Gehring2, and Yvan Arsenijevic1,4

1. INTRODUCTION

In western countries, retinitis pigmentosa (RP) affects 1/3,500 individuals and agerelated macula degeneration (AMD) affects 1% to 3% of the population aged over 60. Invitro generation of retinal cells is thus a promising tool to screen protective drugs and toprovide an unlimited cell source for transplantation. However, one main limitation is theamount of cells available. Stem cells, that can generate unlimited quantity of cells, couldovercome this hurdle. Indeed, stem cells are defined by three characteristics: the ability toproduce a large population of cells (expansion) and the potency, to produce the differenti-ated cells composing the organ from which the stem cells are originated. They are also ableto self-renew indefinitely: for instance haematopoietic stem cells, located in the bonemarrow, can expand, divide and generate differentiated cells into the diverse lineagesthroughout the life, the stem cells conserving its status (Till et al, 1961). Intestinal stemcells also are able to regenerate the intestine all along life (Potten et al, 1975). The otherstem cells properties are the ability to produce a large population of cells (expansion) andas well as the differentiated cells composing the organ from which they originated.

Some species, such as the salamanders, can regenerate their retina (Haynes et al, 2004)which is not the case for the human retina. Whether this lack of regeneration is an evolu-tionary capacity loss, due to a blocking mechanism or a missing signal, is an importantquestion to address. Nevertheless, production of retinal cells can be observed even in species

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1 Oculogenetics Unit, Hôpital Ophtalmique Jules Gonin, Lausanne, Switzerland; 2 Dpt of Cell Biology, Biozentrum,Basel, Switzerland; 3 IRO, Institut de Recherche en Ophtalmologie, Sion, Switzerland; 4 Corresponding author: 15 avenue de France, Case Postale 133, CH-1000 Lausanne 7, Switzerland; Tel: +41 21 626 8260; Fax: +41 21 626 8888; yvan.arsenijevic@ophtal.vd.ch.

where no regeneration occurs. The eye of some fishes and amphibians continues growingduring adulthood due to the persistent activity of retinal stem cells (RSCs). In fishes, theRSCs are located in the ciliary margin zone (CMZ) at the periphery of the retina, at theproximity of the iris. Although the adult mammalian eye does not grow during adult life,Tropepe et al (2000) have shown that the adult mouse eye contains retinal stem cells in thehomologous zone (termed the ciliary margin zone), in the pigmented epithelium and not inthe neuroretina. We demonstrated that the adult human eye also contains retinal stem cellsin the same region (i.e. the pars plicata and the pars plana, Coles et al, 2004). These RSCsmeet the criteria of stem cells i.e. they can differentiate in vitro into all retinal cells, expandand self-renew. Here, we further characterize the human adult retinal stem cells to investi-gate whether the adult retinal stem cells share common characteristics with other stem cellpopulations.

2. ISOLATION OF HUMAN RETINAL STEM CELLS

Stem cells and human retinal progenitors were obtained from organ donors in accor-dance to their wishes or those of their families. The obtaining and the use of the tissuesagreed with the guidelines provided by the Ethical Committee of the Lausanne UniversitySchool of Medicine. The tissues were processed 10 to 24 hours post-mortem.

The pars plana (figure 53.1: white square) and the pars plicata (figure 53.1: greysquare) both gave rise to clonal spheres in vitro in the presence of either EGF, FGF-2 oronly with insulin after one week (Figure 53.2A sphere formed after one week in the pre-sence of EGF). Stem cells can be isolated regardless of the age of the donor (from 2 to 82years old, n = 21, figure 53.2B) suggesting that the stem cell pool is conserved throughoutlife. We expanded the cells by placing one clonal single sphere into a 24-wells plate in thepresence of EGF and 10% FBS. After 3 days, the cells began to spread out of the sphereleading to a monolayer culture in less than one month (Figure 53.2C). The monolayer culturecontains highly proliferative progenitor cells that can generate over 300 million cells withinone month (Figure 53.2D). During proliferation, 89 +/- 0,02% (n = 3) of the cells expressnestin (Figure 53.2E, n = 3), a marker of undifferentiated cells (Tohyama et al., 1992).

3. HUMAN RETINAL STEM CELLS CHARACTERIZATION

To further characterize this cell population, we performed an expression analysis usingthe Affymetrix® U133 Plus 2.0 GeneChip. We analyzed the global transcriptional expres-

378 B. ANGÉNIEUX ET AL.

Figure 53.1. Location of the pars plicata (white square) and plana (red square) in the human eye.

sion of expanded and differentiated hRPCs from two different donors (E and L). Duringexpansion (i.e. in the presence of EGF and 10% FBS), RSCs expressed markers present inneural and other stem cells: as evidenced by immunostaining (Figure 53.2E), nestin wasdetected at a high expression level during the expansion, validating the microarray approach.We also observed the expression of ABCG2 which is present in hematopoietic stem cells(Zhou et al, 2003), or Bmi1 which is required for hematopoietic stem cell (Park et al, 2003,Lessard et al, 2003) and neural stem cell renewal (Molofsky et al, 2003), or nucleosteminwhich is necessary for NSC renewal (Tsai and McKay, 2002). The expression of Bmi1 wasconfirmed by RT-PCR (data not shown). Thus, the highly proliferative cells derived fromthe adult human eye share common characteristics with other stem cells.

The ability of these cells to differentiate into the different cell types composing theorgan from which they have been isolated is one of the stem cell characteristics. Thus, toportray the progeny of the RSCs we also analysed their gene expression profile after dif-ferentiation (i.e. after withdrawal of FBS and stimulation by EGF). The cells express genesthat have different functions in neurons such as genes coding for the skeleton (Map2, Tau),for the synaptic vesicles proteins (SNAPAP). Moreover specific markers of retinal cells arealso expressed as shown by the presence of RPE65 (protein are expressed in retinal pig-mented cells), opsin, and peripherin (specific to photoreceptors). In one of the cell line, wealso detected by immunostaining, cells that have differentiated into retinal neurons andwhich expressed specific proteins such as calbindin for horizontal cells (Figure 53.3A), syn-

53. CHARACTERIZATION OF ADULT HUMAN RETINAL STEM CELLS 379

Figure 53.2. The adult human eye contains highly proliferative undifferentiated cells.

taxin for amacrine cells (Figure 53.3B), recoverin for photoreceptor and some bipolar cells(Figure 53.3C) and vimentin for glial cells (data not shown).

4. CONCLUSION

We showed that adult human eye contains cells that can easily be expanded (one cellgiving rise to more than 100 billion cells) and share common characteristics with other stemcell populations. Furthermore, after differentiation, those cells expressed markers of severalretinal cell types such as pigmented epithelium (RPE65), glia (vimentin) or retinal neurons(opsin, recoverin). The potential of generating retinal neurons in vitro is a promising tool.Indeed the human macula is composed of 200,000 cells which represent only 1/1,000,000of the total number of cells that we can generate from one RSC. Thus, RSCs could cir-cumvent the problem of the cell number limitation for transplantation studies and couldserve to dissect the mechanisms leading to the generation of neurons and their survival.

5. REFERENCES

Coles BL, Angenieux B, Inoue T, Del Rio-Tsonis K, Spence JR, McInnes RR, Arsenijevic Y, van der Kooy: Facileisolation and the characterization of human retinal stem cells. PNAS. 2004, 101(44).

Lessard J, Sauvageau G.: Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature2003, 423(6937).

Molofsky AV, Pardal R, Iwashita T, Park IK, Clarke MF, Morrison SJ.: Bmi-1 dependence distinguishes neuralstem cell self-renewal from progenitor proliferation. Nature 2003, 425(6961).

Park IK, Qian D, Kiel M, Becker MW, Pihalja M, Weissman IL, Morrison SJ, Clarke MF.: Bmi-1 is required formaintenance of adult self-renewing haematopoietic stem cells. Nature 2003, 423(6937).

Potten CS, Hendry JH.: Differential regeneration of intestinal proliferative cells and cryptogenic cells after irradi-ation, Int J Radiat Biol Relat Stud Phys Chem Med. 1975, 27(5).

Till J, and McCulloch E.: A direct measurement of the radiation sensitivity of normal mouse bone marrow cells.,Radiat. Res 1961, 14(213).

Tohyama T, Lee VM, Rorke LB, Marvin M, McKay RD, Trojanowski JQ.: Nestin expression in embryonic humanneuroepithelium and in human neuroepithelial tumor cells. Lab Invest 1992, 66(3).

Tsai RY, McKay RD.: A nucleolar mechanism controlling cell proliferation in stem cells and cancer cells. GenesDev., 2002 16(23).

Tropepe V, Coles BL, Chiasson BJ, Horsford DJ, Elia AJ, McInnes RR, van der Kooy D.: Retinal stem cells in theadult mammalian eye. Science 2000, 287(5460).

Zhou S, Schuetz JD, Bunting KD, Colapietro AM, Sampath J, Morris JJ, Lagutina I, Grosveld GC, Osawa M,Nakauchi H, Sorrentino BP.: The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cellsand is a molecular determinant of the side-population phenotype. Nat Med 2001, 7(9).

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recoverin

CB

syntaxincalbindin

A

Figure 53.3. The RSCs differentiate into retinal neurons.