Isolation, characterization, and in vitro and in vivo differentiation of putative thecal stem cells Arata Honda*, Michiko Hirose*, Kenshiro Hara†, Shogo Matoba†, Kimiko Inoue*, Himomi Miki*, Hitoshi Hiura‡, Mito Kanatsu-Shinohara§, Yoshiakira Kanai†, Tomohiro Kono‡, Takashi Shinohara§, and Atsuo Ogura*¶ *BioResource Center, RIKEN, Tsukuba, Ibaraki 305-0074, Japan; †Department of Veterinary Anatomy, University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan; ‡Department of BioScience, Tokyo University of Agriculture, Setagaya-ku, Tokyo 156-8502, Japan; and §Department of Molecular Genetics, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan Edited by R. Michael Roberts, University of Missouri, Columbia, MO, and approved June 7, 2007 (received for review April 26, 2007) Although ovarian theca cells play an indispensable role in follicu- clues about the mechanisms of formation and maintenance of logenesis by providing follicular structural integrity and steroid the Leydig cell population and their biochemical characteristics. substrates for estrogen production, little information is available Initially, we undertook this study to isolate putative ‘‘female about their recruitment, growth, and differentiation because their germ-line stem (GS) cells,’’ the presence of which is still highly immature forms have not been identified. We have isolated puta- controversial among reproductive biologists and stem cell re- tive thecal stem cells with the ability to self-renew and differen- searchers (6–8). We initially expected that the isolation proce- tiate in vivo and in vitro. They are similar to fibroblasts in mor- dure optimized to male GS cells from neonatal testes (9, 10) phology and proliferate in vitro as round colonies with a might be applicable to putative female counterparts in neonatal homogenous cell population. They were induced to differentiate ovaries. Under our experimental conditions, we were able to into early precursors and steroidogenic cells in a stepwise manner generate round spherical colonies that produced a number of after treatment with serum, luteinizing hormone, and paracrine small primitive oocytes as if oogenesis had proceeded inside. factors from granulosa cells. At each differentiation step, these However, detailed biochemical analysis revealed that most of the cells displayed appropriate gene expression and morphological cell types comprising the colonies were somatic cells and not markers and later secreted androstenedione. The fully mature germ-line cells. These cells were later identified as putative morphology was achieved by coculture with isolated granulosa ovarian thecal stem cells. cells. When transplanted into the ovaries, the putative thecal stem A fundamental property of stem and progenitor cell division is cells colonized exclusively in the ovarian interstitium and the thecal the capacity to retain the proliferative state or to generate differ- layer of follicles as differentiated cells. Thus, thecal stem cells entiated daughter cells. These putative thecal stem cells were appear to be present in neonatal ovaries and can be isolated, purified easily in vitro, formed characteristic anchor-independent purified, and induced to differentiate in vitro. Thecal stem cells round colonies, and, after stimulation, started to differentiate and show characteristic signs of steroidogenesis. They also colonized the could provide an invaluable in vitro experimental system to study BIOLOGY interactions among the oocytes, granulosa cells, and theca cells host ovarian tissue after transplantation in a manner similar to that DEVELOPMENTAL during normal folliculogenesis and to study ovarian pathology of Leydig stem cells in mice and rats (4, 5). Our study provides caused by theca cell dysfunction. evidence for the presence of putative thecal stem cells in the neonatal mouse ovary. Interestingly, unlike Leydig stem cells, follicle ͉ oocyte ͉ ovary putative thecal stem cells can be purified in vitro simply by opti- mizing the culture conditions without any cell-sorting procedures. Thus, these isolated putative thecal stem cells may provide an uring folliculogenesis in mammals, oocytes grow while invaluable and reproducible experimental system to study their surrounded by an increasing number of granulosa cell D biochemical characteristics. This system would include gene expres- layers. From the preantral stage onward, theca cells differentiate sion and signaling cascades, which are essential for normal follicu- as the outer layer of the follicle. These elements provide logenesis in mammals. structural integrity and the androgen substrate for granulosa cell estrogen production, thus playing an indispensable role in fol- Results licular development (1). Mature theca cells can be distinguished Isolation of Neonatal Ovarian Cells by Selective Culture. Because the by their morphology and location in situ and can be isolated original purpose of this study was to establish cell lines of female easily in a crude suspension for in vitro analysis. Much informa- GS cells (6), cells were cultured from newborn mouse ovaries tion has been reported about the factors and mechanisms according to a protocol for male GS cells with slight modifica- regulating theca cell steroidogenesis in several mammalian spe- tions (9, 10). The culture medium was essentially a serum-free cies (2). However, our knowledge about theca cell recruitment GS medium (GSM-K) containing growth factors [see Materials and growth is limited because of the lack of in vitro experimental systems to isolate, culture, and differentiate immature theca cells. Generally, it is difficult to distinguish immature theca cells Author contributions: A.H., M.H., T.K., and A.O. designed research; A.H., M.H., K.H., S.M., from ovarian stroma cells based on their morphology (3). K.I., H.M., H.H., M.K.-S., Y.K., T.K., T.S., and A.O. performed research; M.K.-S. and T.S. contributed new reagents/analytic tools; A.H., M.H., K.H., S.M., K.I., and Y.K. analyzed data; In contrast to theca cells, their male counterparts, Leydig cells, and A.H., Y.K., and A.O. wrote the paper. have been studied in detail because their putative stem cells can The authors declare no conflict of interest. be isolated from mouse and rat testes by flow cytometric sorting This article is a PNAS Direct Submission. (4, 5). Leydig cells can remain undifferentiated in vitro for Abbreviations: GS, germ-line stem; GSM, GS medium; GSM-K, serum-free GSM; GSM-S, months and can respond to growth factors to differentiate into serum-containing GSM; ER, endoplasmic reticulum; LH, luteinizing hormone; MVH, mouse steroidogenic precursor cells (5). After transplantation into the vasa homologue. testes of Leydig cell-deficient mice, these stem cells recolonize ¶To whom correspondence should be addressed. E-mail: [email protected]. normally in the host testicular interstitium and restore the serum This article contains supporting information online at www.pnas.org/cgi/content/full/ testosterone concentration (4). These in vitro and in vivo ap- 0703787104/DC1. proaches using isolated Leydig stem cells may provide further © 2007 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0703787104 PNAS ͉ July 24, 2007 ͉ vol. 104 ͉ no. 30 ͉ 12389–12394 Downloaded by guest on September 29, 2021 Fig. 2. Cytological and gene expression analysis of thecal stem cell colonies. (A) Paraffin-embedded section of colonies stained for BrdU (blue) and MVH (brown). Small cells were generally positive for BrdU but not for MVH. Grow- ing oocytes (arrows) were positive for MVH but not for BrdU, indicating that the oocytes came from preexisting cells in the colonies. (Scale bar: 50 ␮m.) (B) Gene expression analysis (RT-PCR) of the colonies. After the third passage (P3), oocytes disappeared from the colonies, as demonstrated by the absence of oocyte-specific markers (zona pellucida proteins Zp1, Zp2, and Zp3). MEF, Fig. 1. Development of thecal stem cell colonies in vitro.(A) The size of the mouse embryonic fibroblast. (C) Electron micrographs of the colonies. Each colonies increased rapidly during the first several days and gradually reached cell in the colonies had a relatively small cytoplasm that was rich in ribosomes the maximum size of Ϸ120 ␮m in diameter. Many oocytes (Ϸ20 ␮min and rough ER (arrowheads) (Upper). Cell-to-cell connections were sparse, and diameter) continued to protrude from the surface of the colonies (arrows). their intercellular space was filled with basement membrane (BM) and colla- (Scale bars: Left, 100 ␮m; Right,50␮m.) (B) The colonies are weakly positive for gen fibers (CF) (Upper). A thecal stem cell (T) directly surrounds the zona alkaline phosphatase staining. (C) Effect of growth factors on colony size pellucida (ZP) of an oocyte (O), but unlike typical granulosa cells, it does not during the first 14 days of culture. Single growth factors, or some combination protrude foot processes to the oocyte surface through the zona (Lower). thereof, were added to the basic GSM. , P Ͻ 0.05; , P Ͻ 0.01 [compared with * ** Arrowheads indicate microvilli from the oocyte. (Scale bars: 2 ␮m.) growth-factor-free GSM (three replicates, one-way ANOVA)]. and Methods and supporting information (SI) Table 1]. For the a 14-day culture period. Four growth factors, bFGF, EGF, LIF, primary cell preparation, fibroblast cells were allowed to attach and IGF1, exhibited significant effects on colony growth, al- onto the bottom of the gelatin-coated culture plate, but only though they did not seem to prolong the