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Somatic stem cells of the ovary and their relationship to human ovarian cancers Henry L. Chang 1 , David T. MacLaughlin 1 and Patricia K. Donahoe 1,§ , 1 Pediatric Surgical Research Laboratories, Pediatric Surgical Services, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA 02114 Table of Contents 1. Introduction ............................................................................... 2 2. Ovarian development ....................................................................... 2 2.1. Germ cells ......................................................................... 2 2.2. Ovarian somatic tissue and ovarian surface epithelium .................................... 3 2.3. Genetic regulation of ovarian development .............................................. 4 3. Granulosa and theca cells ................................................................... 4 3.1. Granulosa cells ..................................................................... 4 3.2. Theca cells ......................................................................... 5 4. Granulosa cell tumors and thecomas .......................................................... 5 5. Ovarian surface epithelium and related malignancies ............................................. 5 5.1. Ovarian surface epithelium ........................................................... 5 5.2. Epithelial ovarian carcinoma .......................................................... 7 6. Acknowledgements ....................................................................... 11 7. References ............................................................................... 11 Abstract Mammalian ovaries undergo considerable remodeling during the lifetime of the organism, leading to the supposition that somatic stem cells account for or contribute to this cyclic regeneration. While much of ovarian stem cell research has been focused on germ cells, recent interest in normal somatic stem cells has been driven by their possible links to ovarian cancer stem cells. While evidence for stem cell biology with regards to granulosa cells is scant, recent work has isolated potential somatic stem cells for the theca and ovarian surface epithelium. Additionally, evidence for potential cancer initiating cells for ovarian epithelial carcinomas continues to mount. *Edited by Haifan Lin. Last revised April 02, 2009. Published April 30, 2009. This chapter should be cited as: Chang, H.L., MacLaughlin, D.T., and Donahoe, P.K., Somatic stem cells of the ovary and their relationship to human ovarian cancers (April 30, 2009), StemBook, ed. The Stem Cell Research Community, StemBook, doi/10.3824/stembook.1.43.1, http://www.stembook.org. Copyright: C 2009 Henry L. Chang, David T. MacLaughlin, and Patricia K. Donahoe. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. § To whom correspondence should be addressed. E-mail: [email protected]. 1 stembook.org

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Somatic stem cells of the ovaryand their relationship tohuman ovarian cancers∗

Henry L. Chang1, David T. MacLaughlin1 and Patricia K. Donahoe1,§,1Pediatric Surgical Research Laboratories, Pediatric Surgical Services,Massachusetts General Hospital, Harvard Medical School, Boston, MA,USA 02114

Table of Contents1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Ovarian development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. Germ cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2. Ovarian somatic tissue and ovarian surface epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.3. Genetic regulation of ovarian development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

3. Granulosa and theca cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.1. Granulosa cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43.2. Theca cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

4. Granulosa cell tumors and thecomas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55. Ovarian surface epithelium and related malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

5.1. Ovarian surface epithelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55.2. Epithelial ovarian carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

6. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Abstract

Mammalian ovaries undergo considerable remodeling during the lifetime of the organism, leading to thesupposition that somatic stem cells account for or contribute to this cyclic regeneration. While much of ovarianstem cell research has been focused on germ cells, recent interest in normal somatic stem cells has been driven bytheir possible links to ovarian cancer stem cells. While evidence for stem cell biology with regards to granulosacells is scant, recent work has isolated potential somatic stem cells for the theca and ovarian surface epithelium.Additionally, evidence for potential cancer initiating cells for ovarian epithelial carcinomas continues to mount.

*Edited by Haifan Lin. Last revised April 02, 2009. Published April 30, 2009. This chapter should be cited as: Chang, H.L., MacLaughlin, D.T.,and Donahoe, P.K., Somatic stem cells of the ovary and their relationship to human ovarian cancers (April 30, 2009), StemBook, ed. The Stem CellResearch Community, StemBook, doi/10.3824/stembook.1.43.1, http://www.stembook.org.

Copyright: C© 2009 Henry L. Chang, David T. MacLaughlin, and Patricia K. Donahoe. This is an open-access article distributed under the termsof the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalwork is properly cited.§To whom correspondence should be addressed. E-mail: [email protected].

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Somatic stem cells of the ovary and their relationship to human ovarian cancers

Figure 1. Schematic summary of current evidence for ovarian somatic stem cells. Of the three primary functional somatic cell types of the ovary, theovarian surface epithelium and the theca cells currently have evidence supporting stem/progenitor cell biology (briefly stated in figure) whereas there is nospecific evidence for stem cell activity associated with granulosa cells.

1. Introduction

Reproductive organs undergo considerable remodeling during the lifetime of the mammalian organism, leadingto the supposition that somatic stem cells account for or contribute to this cyclic regeneration. While much of ovarianstem cell research has been focused on germ cells, recent interest in normal somatic stem cells has been driven bytheir possible links to ovarian cancer stem cells. Given that the ovarian surface epithelium is the postulated sourcefor 90% of human ovarian cancers (Gondos, 1975; Herbst, 1994; Auersperg et al., 1998), understanding presumptivestem/progenitor function in this less well understood component of the ovary may reveal mechanisms of tumorprogression with resultant important clinical implications. The current status of our understanding of stem cell biologyin the somatic components of the ovary is schematized in Figure 1.

2. Ovarian development

An understanding of the salient features of normal ovarian development is necessary before delving into stemcell functions of its somatic compartments and begins with the formation, migration, and organogenesis of the germcells (see Loffler and Koopman, 2002 and Oktem and Oktay, 2008 for comprehensive reviews).

2.1. Germ cells

Primordial germ cells (PGCs) form at E6.5 in the proximal epiblast where as few as six Blimp-1 expressing cellsare detected (Ohinata et al., 2005). Blimp-1 appears to initiate lineage specificity by repressing Hox and other somatic

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genes while extra-embryonic ectoderm BMP 2, 4, and 8b signaling (Molyneaux and Wylie, 2004; Lawson et al., 1999;Ying et al., 2000) expands this population which expresses tissue-specific alkaline phosphatase and Stella (Saitou et al.,2002) at embryonic day 7.2 (E7.2) in the mouse (Ginsburg et al., 1990) and as early as week 3 in the endoderm of theyolk sac wall of the human (Hacker and Moore, 1992). PGC migration to the mesonephros occurs between E8-E12in the mouse (Loffler and Koopman, 2002) and week 8 of gestation in the human (Hacker and Moore, 1992). Mouseprimordial germ cells undergo proliferation and imprint erasure as they traverse from the proximal primitive streak atthe base of the allantois to the hindgut endoderm (E8.5–9.5) and then to the genital ridges. Progression of the PGCsto the developing urogenital ridges appears to involve largely uncharacterized chemotactic signals (De Felici et al.,2005; Molyneaux et al., 2003; Godin et al., 1990), integrins (Anderson et al., 1999), and C-kit signaling (Buehr et al.,1993). As they migrate, PGCs proliferate 170-fold by mitotic division (Tam and Snow, 1981) and lose imprintingimposed by DNA methylation (E10.5–12.5; Hajkova et al., 2002; Lee et al., 2002; Reik and Walter, 2001) and complexchromatin modification by methylation or acetylation of histones on lysine and arginine residues (Hajkova et al.,2008; Seki et al., 2007; Seki et al., 2005; Kimmins and Sassone-Corsi, 2005). This erasure is presumably necessary toreset epigenetic memory in germ cells and its complex regulation remains a continuing challenge for somatic nucleartransfer. The transmembrane protein Fragilis induces the germ cell gene Stella which represses the developmentalhomeobox genes, while Oct4, Sox2, and Nanog, preserve the pleuripotency of the PGCs (Saitou et al., 2002). ThePGCs lose their migratory phenotype at E12.5 (Ginsburg et al., 1990) and at E13.5 female germ cells undergo meiosisin an anterior to posterior wave which denotes ovarian differentiation to become oogonia, a process mediated by Stra8which is stimulated by retinoic acid. In contrast to the embryonic ovary, the embryonic testes expresses CYP26b1which degrades retinoic acid so Stra8 is not expressed (Bowles et al., 2006; Koubova et al., 2006).

Normal migration and colonization of the PGCs are necessary for further ovarian development, as ovariandysgenesis with degeneration of ovarian somatic cells occurs in germ cell deficient mice (Merchant-Larios andCenteno, 1981; Behringer et al., 1990; Hashimoto et al., 1990). Death of germ cells occurs when E12.5 ovaries areplaced ectopically beneath the renal capsule, an event followed by formation of testicular tubules, again indicating theimportant role of the germ cells in ovarian development (Taketo et al., 1993). Furthermore, there is evidence to suggestdevelopmental germ cell tumors may result from incomplete migration of the PGCs to the developing ovary (Gobelet al., 2000; Schneider et al., 2001).

2.2. Ovarian somatic tissue and ovarian surface epithelium

Figure 2 gives a schematic overview of ovarian somatic development. The development of the somatic gonadbegins on E10 in the mouse (4 weeks gestation in the human) as a thickening of the coelomic epithelium on the

Figure 2. Schematic representation of ovarian embryonic development. (A) Cross-section through the dorsal part of a 13-mm human embryo. (B)Sequential changes in the gonadal ridge, which is covered by modified coelomic epithelium (shaded). The epithelium proliferates and forms cords thatpenetrate into the ovarian cortex and give rise to the granulosa cells of the primordial follicles. The follicles become separated from the overlying ovariansurface epithelium (OSE) by stroma. The Mullerian ducts (Mul. Duct) develop as invaginations of the coelomic epithelium dorsolaterally from the gonadalridges. (Adapted with permission from Auersperg, N., Wong, A.S., Choi, K.C., Kang, S.K., Leung, P.C. (2001). Ovarian surface epithelium: biology,endocrinology, and pathology. Endocr Rev 22, 255–288. Figure 1. Copyright 2001, The Endocrine Society).

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ventro-medial side of the mesoderm (Swain and Lovell-Badge, 1999). The indifferent gonad is essentially a mass ofblastema (the primordial mesenchymal cell mass) surrounded by the coelomic-epithelium derived surface epithelium.This mesenchymal cell mass contains elements which are destined to become the supportive (granulosa), steroidogenic(theca and granulosa), or structural (stroma) cells of the ovary. After the primordial germ cells arrive in the developinggonad (∼E12–13.5), an arrangement of loose cords called the ovigerous cords begins to form around clusters of germcells (Odor and Blandau, 1969; Konishi et al., 1986). Developing somatic cells (presumed pre-granulosa cells) thenseparate the germ cell clusters into individual oocytes surrounded by a monolayer of granulosa cells, forming theprimordial follicle (Pepling and Spralding, 1998; Merchant-Larios and Chimal-Monroy, 1989). While the embryonicorigins of the granulosa cells are still a matter of debate, there is evidence suggesting that the ovarian surface epitheliumis at least a partial source of granulosa cells (Sawyer et al., 2002).

2.3. Genetic regulation of ovarian development

Compared to the genetic regulation of testicular development, the genes responsible for ovarian developmentare relatively unknown and it was once thought the ovaries developed passively as a result of the absence of testiculardetermining genes. Only a handful of genes required for the formation of the ovary have been identified and studied,almost exclusively in the mouse. Of these, Wnt-4 is the gene most clearly associated with ovarian development, withhomozygous mutant males exhibiting normal testicular development while their female counterparts are virilized withabsence of the Mullerian duct and morphologically masculinzed gonads with subsequent degeneration of meiotic-stageoocytes (Vainio et al., 1999). Follistatin is a downstream component of Wnt-4 signaling which has also been associatedwith regulating normal ovarian organogenesis (Yao et al., 2004). Recent data has shown that Wnt-4 activation isregulated by Respondin-1 mediation of the canonical β-catenin pathway and that β-catenin stabilization in the XYgonad is sufficient to cause male-to-female sex reversal (Chassot et al., 2008; Maatouk et al., 2008). Additionally, amember of the forkhead transcription factors, Foxl2, has been identified as a gene that appears to repress the malegenetic program and insure normal granulosa cell development around growing oocytes, allowing for normal ovariandevelopment (Ottolenghi et al., 2005; Schmidt et al., 2004; Uda et al., 2004). While work continues on the geneticregulation of ovarian development, it is interesting to note that prenatal ovarian development occurs independent ofsteroid hormone action (Couse et al., 1999).

3. Granulosa and theca cells

The granulosa and theca cells of the ovary serve to support the germ cells within the developing follicle.Initially indistinguishable from the ovarian stroma, theca cells surround the developing follicle, form the two layersknown as the theca externa and interna, and produce the androgens which are ultimately converted to estradiol by thegranulosa cells. While converting theca-produced androgens into estradiol via aromatase, the granulosa cells form themultilayered cumulus oophorus and later the Graafian or pre-ovulatory follicle which surrounds the germ cells. Afterovulation, both the granulosa and theca cells contribute to the corpus luteum which is responsible for producing theestrogen and progesterone necessary to support a developing pregnancy. While the complex biology of these cellssuggests an obvious somatic stem cell-mediated process, substantive evidence to this end is lacking for the granulosacells but is just recently being elucidated for the theca cells.

3.1. Granulosa cells

The origin of the pre-granulosa cells (the flattened somatic cells of the primordial follicle surrounding theoocyte) is not known but evidence supports three proposed sources: the developing ovarian blastema (Pinkerton et al.,1961), the mesonephric cells of the rete ovarii (Byskov and Lintern-Moore, 1973), and the developing ovarian surfaceepithelium (Gondos, 1975; Sawyer et al., 2002). Until recently, most would have agreed that the cells that give rise tothe granulosa cells (and their derived structures) during folliculogenesis have already segregated with each primordialfollicle, and separate each germ cell from the surrounding ovarian stroma by a basal lamina where they lie dormantprior to follicular recruitment. While Bukovsky (1995; 2004; 2008) suggests that the tunica albuginea immediatelyunderlying the ovarian surface epithelium gives rise to both post-natal oocytes and their associated pre-granulosa cells(Bukovsky et al., 2004), the existence of germline stem cells and the possibility of post-natal oogenesis and theirfurther development remains to be determined definitively (Johnson et al., 2005; Eggan et al., 2006, see Tilly et al.,2008 for a comprehensive review).

While granulosa cell growth and differentiation during folliculogenesis is a complex and interesting interplayof paracrine and endocrine factors, definitive evidence that the development of the granulosa-derived structures of thefollicle is a stem cell-mediated process has yet to be produced. It could be argued that given the complex changes

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that occur during folliculogenesis, the pre-granulosa cells residing in the primordial follicle should be labeled fate-determined progenitor cells capable of forming the various structures of the developing follicle. However, there hasbeen a lack of evidence so far to support the presence of asymmetric division, pluri- or multi- potency, or indefiniteself-renewal in granulosa cells that characterizes stem cell biology.

3.2. Theca cells

While the presence of probable theca cell precursors in the ovarian stroma and interstitium was proposedby Hirshfield in 1991, identification and isolation of these cells has proven elusive. Recent work by Honda andhis colleagues (Honda et al., 2007) led to isolation of ‘putative thecal stem cells’ after enzymatic and mechanicaldissociation of newborn mice ovaries and growth of the resulting cell suspension in serum-free germline stem cell(GS) media. Non-adherent anchorage independent spheres exhibited the morphology of ovarian interstitial somaticcells and expressed gene profiles suggestive of theca cells, not germ or granulosa cells. By supplementing the mediawith serum, luteinizing hormone, insulin-like growth factor-1, stem cell factor, and granulosa cell-conditioned mediain a stepwise manner, they were able to induce subcultures of these cells to differentiate into lipid producing, androgensecreting cells which morphologically resembled theca cells. Furthermore, transplantation of these cells isolated fromwhole-body green fluorescence protein-expressing transgenic mice into the ovaries of wild-type recipients showedscattered interstitial GFP with aggregation of GFP cells immediately adjacent to developing follicles and subsequentGFP expression in both theca interna and externa during folliculogenesis (see Figure 3). While there are still questionsto be answered such as the exact location of these thecal precursors in vivo, the cell surface marker profile of thesecells, and the niche in which these cells reside, the ability to isolate and characterize these cells represents a significantstep towards understanding follicular development.

4. Granulosa cell tumors and thecomas

Adult granulosa cell tumors (GCT), the most common ovarian stromal tumor, account for approximately 2–5%of all ovarian cancers. Juvenile GCTs are 20 to 50 times more rare (Schumer and Cannistra, 2003; Colombo et al.,2007). Unlike the epithelial ovarian cancers, sex-cord stromal ovarian tumors (including granulosa cell tumors andthecomas) do not seem to have a demonstrable hereditary component. In addition, reports of oncogene involvementare inconclusive (Semczuk et al., 2004; Shen et al., 1996; Enomoto et al., 1991), though there is data to suggestdysregulation of the canonical Wnt/β-catenin pathway may play a role in the development of granulosa cell tumors(Boerboom et al., 2005). Additionally, an imbalance in chromosomes 4, 9, and 12 have been reported repeatedly inthecomas, suggesting that genes in these regions may contribute to the development of these tumors (Streblow et al.,2007; Liang et al., 2001; Shashi et al., 1994). Evidence for stem cells in these tumors is, however, circumspect atbest. Based solely upon morphological and histological factors, reports have implicated putative somatic stem cellinvolvement in certain ovarian stromal tumor subtypes, such as sertoli-leydig cell tumors in women; however, evidencethat would definitively confirm these observations is currently lacking.

5. Ovarian surface epithelium and related malignancies

5.1. Ovarian surface epithelium

The simple squamous-to-cuboid single-layered epithelial cell structure of the normal human ovarian surfaceepithelium (OSE) belies its complex biology. Several studies have shown that rather than being a passive structureduring ovulation, the OSE plays an active role in both follicular rupture and subsequent ovarian remodeling. The factthat OSE can transition back and forth between epithelial and mesenchymal phenotypes has been well-established(Kruk and Auersperg, 1992; Auersperg et al., 1999; Salamanca et al., 2004; reviewed in Ahmed et al., 2007) and thisepithelial-mesenchymal transition is believed to be part of the normal process of post-ovulatory ovarian remodeling.Additionally, the OSE has been shown to contribute to repairing the ovarian stroma after ovulation by producing andremodeling components of the extracellular matrix (Kruk and Auersperg, 1992; Kruk and Auersperg, 1994; Auersperget al., 2001; Salamanca et al., 2004).

Given its ability to differentiate between two cell types and its role in the cyclical disruption and repair thatoccurs with ovulation, OSE biology seems an intuitive candidate to study in order to understand stem cell mediatedprocesses. We studied the normal OSE as a somatic stem cell source given that repeated ovulation is thought topredispose the OSE, the postulated origin of 90% of ovarian carcinomas, to malignant transformation (Mahdavi et al.,2006). Szotek and colleagues have recently identified a putative somatic stem/progenitor cell in the ovarian surfaceepithelium (Szotek et al., 2008). A transgenic mouse model of doxycycline inducible green fluorescence protein

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A

B

C

D

Figure 3. Intraovarian transplantation of the thecal stem cells. (A) A host ovary from a mature mouse (left). Thecal stem cell colonies (arrows in right)were transplanted into the ovary by a glass pipette. An air bubble placed for controllable transfer (arrowhead). (Scale bar: 2 mm) (B) Donor thecal cells(EGFP-positive) surrounding two large follicles were clearly visible by fluorescence microscopy (arrows). (C) Frozen section of a large follicular area.The donor thecal stem cells differentiated into large cells and were located in both the inner (I) and outer (O) thecal layers. They were also present in theinterstitial area of small cells (arrowheads). (Scale bar: 50 mm) (D) Frozen section of a small primary follicle. A few small, probably less differentiated, thecacells were present around the follicle (arrowheads). (Scale bar: 50 mm) (B-D right) Corresponding fields observed by fluorescent microscopy are shown.(Adapted with permission from Honda, A., Hirose, M., Hara, K., Matoba, S., Inoue, K., Miki, H., Hiura, H., Kanatsu-Shinohara, M., Kanai, Y., Kono, T. et al.(2007). Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells. Proc Natl Acad Sci USA 104, 12389–12394. Figure5. Copyright 2007 National Academy of Sciences, U.S.A).

tagged histones (Tet-on-H2B-GFP) was used; after an initial prolonged pulse, GFP expression or fluorescence can befollowed or chased (Tumbar et al., 2004; Brennand et al., 2007). After a chase period of several months, we identifiedslowly-cycling or quiescent cells in the OSE by retention of label (see Figure 4A), while mitotically active cells whichshould, by diluting their GFP label with each division, be unlabelled. Using quiescence and label retention as evidencefor asymmetric division of the OSE, we then characterized the OSE LRCs by expression of the epithelial markers(cytokeratin-8 and E-cadherin) and the mesenchymal marker, Vimentin, and enrichment in the cytoprotective ABCtransporter-associated Hoescht dye-excluding side population (SP), which has been associated with stem/progenitorcells in a variety of tissues and malignancies (Goodell et al., 1996; Jonker et al., 2005; Szotek et al., 2006; Ono et al.,2007; Rossi et al., 2008). When examined for mitotic activity before and after ovulation by iodo-deoxy-uracil (IdU)incorporation, these GFP LRCs were induced to proliferate after ovulation, indicating responsiveness to the estrouscycle (see Figure 4B). Finally, these LRCs showed increased growth potential compared to their non-GFP counterparts

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Figure 4. Identification and functional characterization of label retaining cells in the ovarian surface epithelium. (A) Three month chase of H2B-GFPovaries demonstrate label retaining cells (LRCs) in the coelomic epithelium (CE) of the ovary. (B) CE LRCs were observed to colocalize with IdU (arrowheads)on either sides of the re-epithelializing ovulation wound, indicating mitotic activity in this area. (C) H2B-GFP 4 month chase CE cells sorted for GFP showedincreased colony formation by well surface area percentage compared to non-GFP cells (35% versus 14%, P<0.05, n = 3). Representative Giemsa-stainednon-GFP and GFP wells (C, inset) shown above their respective graph bars. (Adapted with permission from Szotek, P.P, Chang, H.L., Brennand, K., Fujino,A., Pieretti-Vanmarcke, R., Lo Ceslo, C., Dombkowski, D., Preffer, F., Cohen, K.S., Teixeira, J., Donahoe, P.K. (2008). Normal ovarian surface epitheliallabel-retaining cells exhibit stem/progenitor cell characteristics. Proc Natl Acad Sci USA 105, 12469–12473. Figures 1C, 3B, and 4D. Copyright 2008National Academy of Sciences, U.S.A)

in in vitro colony formation assays (see Figure 4C). Furthermore, we observed that these LRCs were consistentlyfound adjacent to CD31+ vascular endothelial cells (Movie; Szotek, unpublished observations). These assays andcharacteristics collectively point to these cells as the putative somatic stem/progenitor cell of the OSE (Szotek et al.,2008).

Another property that has been attributed to the OSE is the ability post-natally to contribute new follicles tothe pool of primordial follicles. Studies published by Bukovsky and colleagues present data in which they contendthe tunica albuginea of the post-natal ovary immediately underlying the OSE is capable of producing new primordialfollicles (1995; 2004; 2008). Recent studies have reported the isolation of cells from OSE of postmenopausal womenand women with premature ovarian failure which express developmental embryonic markers including Oct-4, VASA,Nanog, c-Kit and Sox-2 (Virant-Klun et al., 2008a). Furthermore, the authors claim that these cells are capable ofdeveloping embryoid body-, oocyte- and blast-like structures in culture (Virant-Klun et al., 2008b). Though there hasbeen some evidence to suggest the existence of germ line stem cells and post-natal oogenesis, such cells do not appearto ovulate, are not found in the Fallopian tube, and do not contribute to pregnancies (Johnson et al., 2005; Eggan et al.,2006, reviewed in Tilly et al., 2008).

5.2. Epithelial ovarian carcinoma

It is estimated that over 21,000 women will be diagnosed with ovarian carcinoma in 2008 and of these, approxi-mately 15,000 will succumb to their disease (NCI SEER database: see http://seer.cancer.gov/statfacts/html/ovary.html).

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Survival is directly related to stage, with 5-year survival rates of 92.7% for those diagnosed with localized disease,to 30.6% for those with distant disease at diagnosis (NCI SEER). Unfortunately, two-thirds of patients already haveevidence of distant disease at diagnosis (NCI SEER). While the majority of patients respond to initial therapy, mostrecur with chemoresistant disease. Hence re-evaluation of the current treatment paradigms of ovarian cancers isneeded.

Cancer stem cells (CSC), or tumor-initiating cells, are postulated to be specialized cells within tumors whichare responsible for propagating cancer growth (Al-Hajj and Clarke, 2004). CSCs are thought to have the ability togive rise to daughter non-tumorigenic cancer cells while retaining their ability to self-renew and form tumors (Al-Hajj and Clarke, 2004). Small populations of clonogenic cells capable of tumorigenesis, self-renewal, differentiation,and chemoresistance in vitro and in vivo have been identified as CSCs in a variety of solid tumors (Al-Hajj et al.,2003; Collins et al., 2005; Dalerba et al., 2007; Li et al., 2007). There are several characteristics of epithelial ovariancarcinomas (EOCs) that indicate that it may be a stem cell-driven disease. Firstly, though the OSE is the postulatedsource of EOCs (Gondos, 1975; Herbst, 1994; Auersperg et al., 1998), EOCs can generate differentiated subtypesthat recapitulate the histology of other normal gynecologic tissues (Landen et al., 2008). Secondly, the high rate ofchemoresistant recurrence after initial treatment success suggests that there are cells within the cancer population whichare 1) capable of repopulating then entire tumor burden from a small number of cells and 2) exhibit cytoprotectivemechanisms thought to exist on somatic stem cells.

Less speculative, experimental evidence for the existence of ovarian cancer stem cells was first reported with theidentification and isolation a single tumorigenic clone by anchorage independent spheroid formation from the ascitesof a patient with advanced disease (Bapat et al., 2005). The authors then went on to characterize this clone and provideimmunohistologic evidence that suggested differentiation along epithelial, granulosa, and germ cell lineages. Theseclones were shown to form tumors and metastasize in nude mice and retained tumorigenic ability with sequentialtransplant (Bapat et al., 2005). The authors concluded that their findings suggest that stem/progenitor cell-drivenbiology may contribute to the aggressive behavior of EOCs.

In a step further, by using the murine transgenic epithelial ovarian cancer cell line MOVCAR-7, producedwhen the large T antigen is driven by the Mullerian Inhibiting Substance type II receptor promoter (Connolly et al.,2003), Szotek and colleagues identified a putative cancer stem cell within this cell line by using the dye effluxmarker SP (Szotek et al., 2006). This subset of cells were found to form palpable tumors when injected into thedorsal fat pad of nude mice, faster and at fewer inoculated numbers than the non-SP cells (see Figure 5A). TheSP cells also were cell-cycle arrested and exhibited resistance to conventional chemotherapeutic agents whereasthe non-SP cells were and did not (see Figure 5B). We also observed that Mullerian Inhibiting Substance, theprotein responsible for the regression of the Mullerian duct during development, was able to inhibit the growthof these SP cells in vitro (see Figure 5C). Additionally, verapamil-sensitive SPs were identified in human ovariancancer cell lines and in the ascites of a small number of patient, suggesting that the SP could be used to iden-tify the CSC of ovarian cancers in patients though these human cells were not functionally characterized in thisstudy.

In the human, a recent study has identified a subpopulation of putative CSCs from primary human ovarian tumors(Zhang et al., 2008). In this study, ovarian serous adenocarcinomas were disaggregated and grown in conditionsselecting for anchorage independent spheroid formation (see Figure 6A). After several passages, purified sphere-forming cells were isolated and found to express various stem cell markers (stem cell factor, Notch-1, Nanog, ABCG2,and Oct-4), demonstrate chemoresistance to ovarian cancer therapeutics, and form palpable tumors in athymic nudemice with inoculation of as few as 100 purified cells (see Figure 6B) compared to no growth with injection of 1 × 106

non-spheroid cells. Further characterization of these cells identified an enrichment for the hyaluronate receptor CD44and CD117 (c-kit) in the spheroid cells (see Figure 6C). The authors found that, similar to spheroid cells, CD44(+)CD117(+) cells isolated from primary human tumors were able to serially propagate the original tumor at injectionsof only 100 cells whereas up to 1×105 CD44(-) CD117(-) cells formed no tumors. These findings lead the authors toassert that EOCs are derived from a CD44(+) CD117(+) CSC population.

While the initial studies identifying and isolating CSCs in ovarian cancer have yielded valuable insight intothe biology of this devastating disease, the therapeutic implications of these findings have yet to be realized. Furtherstudies characterizing the CSC population in a wider population of patients with correlations made to stage at diagnosis,response to treatment, and, ultimately, survival are necessary to take the next step to design therapies targeting thesespecialized cells. In order to achieve sustained response and convert ovarian cancer to a manageable disease, it isapparent that treatment will need to be patient specific, cancer specific and stem cell specific.

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Figure 5. Characterization of MOVCAR 7 side population cells. (A) Measurable tumors were detected in three of three SP-injected (at 5 × 105 cells)animals at 10 weeks after implantation, whereas zero of three NSP-injected animals demonstrated tumors at the first appearance of SP tumors. (B) SPcells showed only 30% inhibition by MTT assay (P< 4.2 × 10−4) while NSP cells were inhibited 81% (∗∗P<5.1 × 10−10) by doxorubicin versus vehiclecontrols (shorter bars indicate more inhibition of cell growth). In contrast, (C) the proliferation of MOVCAR 7 SP and NSP cells also analyzed by MTTassay demonstrated inhibition of both SP (∗86%) and NSP (∗∗93%) cells by MIS versus vehicle control (shorter bars indicate more inhibition of cell growth).(Adapted with permission from Szotek, P.P., Pieretti-Vanmarcke, R., Masiakos, P.T., Dinulescu, D.M., Connolly, D., Foster, R., Dombkowski, D., Preffer, F.,MacLaughlin, D.T., Donahoe, P.K. (2006). Ovarian cancer side population defines cells with stem cell-like characteristics and Mullerian Inhibiting Substanceresponsiveness. Proc Natl Acad Sci USA 103, 11154–11159. Figures 3A, 4B, and 5E. Copyright 2006 National Academy of Sciences, U.S.A).

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A

C

B

Figure 6. Isolation and characterization of candidate human ovarian tumor cancer initiating cells form self-renewing, anchorage-independentspheroids under stem cell–selective conditions. (A) Cell suspensions form small, non-adherent clusters 1 wk after plating (top left). After ∼10 passages,1% of spheres persist as larger, symmetric, prototypical spheroids (top right). Typical spheroids contained ∼100 cells and could be serially passaged for>6 mo (bottom left). Under differentiating conditions, sphere-forming cells adhere to plates and form symmetric holoclones (bottom right). (B) Injectionof ∼100 OCICs per mouse from patient tumor 1 (left) or patient tumor 2 (right) dissociated spheroids generated xenograft tumors with 2/2 efficiency. (C)Staining of anti-CD44 monoclonal antibodies (red) in ovarian tumor spheroid (top left); immunofluorescence staining of anti-CD117 monoclonal antibodies(green) in ovarian tumor sphere cells (top right); CD44+ sphere cells colocalize with CD117+ cells (orange overlay, bottom left) and additionally stainedwith DAPI (blue; bottom right). Magnification, 200x. (Adapted with permission from Zhang, S., Balch, C., Chan, M.W., Lai, H.C., Matei, D., Schilder, J.M.,Yan, P.S., Huang, T.H., Nephew, K.P. (2008). Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res68, 4311–4320. Figures 1A, 3A and C.).

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Movie 1. Ovarian label retaining cells adjacent to CD31+ vascular endothelium. Ovarian surface label retaining cells (green) were consistently foundimmediately adjacent to CD31+ vascular endothelial cells (red) after several months of chase (movie courtesy of Szotek, unpublished observation).

6. Acknowledgements

The authors would like to thank Dr. Jose Teixeira for his critical reading of this text, Dr. Paul Szotek for the useof unpublished observations, and Ms. Caroline Coletti for her editorial expertise.

HLC was supported by NIH/MGH T32 in Cancer Biology # 2T32CA071345-11. DTM is supported by NIH/NCIGrant 5R01CA017393-30, the McBride Family Fund, the Commons Development Group, and the Ovarian CancerResearch Fund (New York). PKD is supported by Harvard Stem Cell Institute Grant DP-0010-07-00, Brigham andWomen’s SPORE Grant 5P50CA105009-03, and NIH/NCI Grant 5R01CA017393-33.

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