ELSEVIER
Molecular and Cellular Endocrinology
journal homepage: www.elsevier.com/locate/mce
Med-dar ynd Celular Endocrinology
Differentiation of mesenchymal stem cells and embryonic stem cells into steroidogenic cells using steroidogenic factor-1 and liver receptor homolog-1*
Takashi Yazawaa,*, Shinya Kawabeª, Yoshihiko Inaokaa, Reiko Okadaª, Tetsuya Mizutania, Yoshitaka Imamichia, Yunfeng Juª, Yukiko Yamazakia, Yoko Usamia, Mayu Kuribayashia, Akihiro Umezawa b, Kaoru Miyamotoa
a Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki 23, Matsuoka, Eiheiji-cho, Fukui 910-1193, Japan
b National Research Institute for Child Health and Development, Tokyo 157-8535, Japan
ARTICLE INFO
Article history: Received 31 August 2010 Received in revised form 23 November 2010 Accepted 23 November 2010
Keywords: Steroidogenic factor-1 Liver receptor homolog-1 Steroid hormone Stem cells
ABSTRACT
Previously, we have demonstrated that mesenchymal stem cells could be differentiated into steroido- genic cells through steroidogenic factor-1 and 8bromo-cAMP treatment. Use of liver receptor homolog-1, another of the nuclear receptor 5A family nuclear receptors, with 8bromo-cAMP also resulted in the differ- entiation of human mesenchymal stem cells into steroid hormone-producing cells. The same approaches could not be applied to other undifferentiated cells such as embryonic stem cells or embryonal carcinoma cells, because the over-expression of the nuclear receptor 5A family is cytotoxic to these cells. We estab- lished embryonic stem cells carrying tetracycline-regulated steroidogenic factor-1 gene at the ROSA26 locus. The embryonic stem cells were first differentiated into a mesenchymal cell lineage by culturing on collagen IV-coated dishes and treating with pulse exposures of retinoic acid before expression of steroido- genic factor-1. Although the untreated embryonic stem cells could not be converted into steroidogenic cells by expression of steroidogenic factor-1 in the absence of leukemia inhibitory factor due to inability of the cells to survive, the differentiated cells could be successfully converted into steroidogenic cells when expression of steroidogenic factor-1 was induced. They exhibited characteristics of adrenocortical-like cells and produced a large amount of corticosterone. These results indicated that pluripotent stem cells could be differentiated into steroidogenic cells by the nuclear receptor 5A family of protein via the mes- enchymal cell lineage. This approach may provide a source of cells for future gene therapy for diseases caused by steroidogenesis deficiencies.
@ 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Steroidogenic factor-1 (SF-1), also known as Ad4BP, and liver receptor homolog-1 (LRH-1) belong to the NR5A subfamily of nuclear receptors (Krylova et al., 2005). SF-1 is essential for adrenal and gonadal development, and SF-1 knockout mice exhibit adrenal and gonadal agenesis and impaired gonadotropin expression, resulting in postnatal death due to severe adrenal insufficiency (Luo et al., 1994; Sadovsky et al., 1995). SF-1 is expressed in the adrenal cortex, testicular Leydig and Sertoli cells, ovarian theca and gran- ulosa cells, pituitary gonadotropes and hypothalamus (Parker and
Schimmer, 1997; Morohashi, 1999; Schimmer and White, 2010). It regulates the cell-specific expression of a variety of different genes involved in steroidogenesis, including a number of steroid hydrox- ylases (Lala et al., 1992; Morohashi et al., 1992). With the aid of cAMP, it can induce the differentiation of bone marrow-derived mesenchymal stem cells (MSCs) into steroidogenic cells such as testicular Leydig cells and adrenocortical cells (Yazawa et al., 2006, 2008). However, the same approaches were inappropriate for other undifferentiated cells such as embryonic stem (ES) cells or embry- onal carcinomas, because they barely survived after expression of SF-1. LRH-1 is mainly expressed in tissues of endodermal origin in adults (Fayard et al., 2004; Lee and Moore, 2008). Recently, elevated expression of LRH-1 has been demonstrated in gonads, suggesting the involvement of LRH-1 in steroidogenesis (Volle et al., 2007; Duggavathi et al., 2008).
In this study, we showed the differentiation of steroidogenic cells from MSCs and ES cells by SF-1 and LRH-1. Treatment with LRH-1 and 8br-cAMP resulted in the differentiation of human MSCs (hMSCs) into steroidogenic cells, with similar results exhibited using SF-1. This method could not be applied to ES cells. In this
Abbreviations: SF-1, steroidogenic factor-1; LRH-1, liver receptor homolog-1; MSC, mesenchymal stem cell; ES cells, embryonic stem cells; Tc, tetracycline; RA, retinoic acid; Gapdh, glyceraldehyde-3-phosphate dehydrogenase.
* This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Smoking Research Foun- dation.
* Corresponding author. Tel .: +81 776 61 8316; fax: +81 776 61 8102. E-mail address: yazawa@u-fukui.ac.jp (T. Yazawa).
A
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StAR-1.3kb
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CYP11A1-2.3kb
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fold induction
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study we developed a method for differentiation of ES cells into steroidogenic cells using a tetracycline (Tc)-regulated gene expres- sion system for SF-1 (Masui et al., 2005).
2. Materials and methods
2.1. Cell culture, transfection and luciferase assays
The hMSCs (Okamoto et al., 2002; Mori et al., 2005), HEK293 and Phoenix cells were cultured in DMEM with 10% fetal calf serum (FCS). HEK293 cells were transfected using Lipofectamine plus (Invitrogen, Carlsbad, CA, USA). Luciferase assays were performed as described previously (Yazawa et al., 2003). Each data point represents the mean of at least four independent experiments. The murine ES cells, EBRTcH3, were cultured as described before (Masui et al., 2005). Briefly, they were cultured in the absence of feeder cells in Glasgow minimal essential medium (GMEM; Sigma-Aldrich; St. Louis, MO, USA) supplemented with 10% FCS, 1 mM sodium pyruvate (Invitrogen), 10-4 M 2-mercaptoethanol (Nacalai Tesque; Kyoto, Japan), 1 x nonessential amino acids (Invitrogen) and 1000 U/ml of leukemia inhibitory factor (LIF) on gelatin-coated dishes. Culture methods for the induction of MSCs have been described elsewhere (Takashima et al., 2007). Culture media were collected for the measurement of steroid hormone production by enzyme- linked immunosorbent (ELISA) assays (Cayman Chemical Co., Ann Arbor, MI, USA) as described previously (Yazawa et al., 2008, 2009, 2010).
2.2. Plasmids
The pGL2-StAR1.3kb vector was kindly provided by Dr. Teruo Sugawara (Hokkaido University Graduate School of Medicine, Sapporo, Japan). To introduce the SF-1 gene into ROSA-TET locus, the exchange vector was created by the inser- tion of the XhoI-NotI fragment of human SF-1 cDNA into pPthC (Masui et al., 2005), which had been cleaved by Xhol-Notl. Exchange vector is necessary for introduction of genes of interest into mouse genomic ROSA-TET locus by homologous recombi- nation. The pCAGGS-Cre plasmid has been described elsewhere (Araki et al., 1997). Each vector containing the entire coding region for SF-1 and LRH-1 was generated by RT-PCR and subcloned into pQCXIP (Clontech, Palo Alto, CA, supplied by Takara Bio Inc., Shiga, Japan). The other vectors have been described before (Yazawa et al., 2009, 2010).
2.3. RT-PCR
Total RNA from the cultured cells was extracted using Trizol reagent (Invit- rogen). RT-PCR was performed as described previously (Yazawa et al., 2006). The RT-PCR products were subjected to electrophoresis on 1.5% (w/v) agarose gels, and the resulting bands were visualized by staining with ethidium bromide. The primers used have been described previously (Yazawa et al., 2006, 2009; Takashima et al., 2007).
2.4. Retrovirus preparation and infection
The Phoenix packaging cell line was transiently transfected with the retroviral plasmids using the Lipofectamine Plus reagent (Invitrogen). The supernatant was concentrated by centrifugation and the virus solution stored at -80 ℃ until required. MSCs were infected with the retrovirus in the presence of 8 µg/ml polybrene (Sigma) for 48 h. The cells were then replated and selected using puromycin.
2.5. Exchange reaction of the Tc-regulated unit
EBRTcH3 cells were seeded onto gelatin-coated 6-well plates in medium con- taining 1 µg/ml Tc (Tc+ medium; Sigma-Aldrich). The circular plasmid DNA of the exchange vector, pCAGGS-Cre and Lipofectamine 2000 (Invitrogen) was sepa- rately mixed with GMEM and combined to make the transfection mixture. This was added and incubated for 3-5 h, and then re-plated onto 10 cm dishes containing Tc+ medium. After two days, the medium was changed to Tc+ medium with 1.5 µg/ml puromycin (Sigma).
2.6. Western blot analysis
The extraction of protein from cultured cells and subsequent quantification was performed as described previously (Yazawa et al., 2003, 2008). Equal amounts of protein (50 µg) were analyzed by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blot analyses of SF-1 and glyceraldehyde-3- phosphate dehydrogenase (GAPDH) were carried out with antiserum directed against Ad4BP (kindly provided by Dr. Ken-ichirou Morohashi, University of Kyushu, Fukuoka, Japan) and GAPDH (6C5; Santa Cruz Biotechnology, Santa Cruz, CA). ECL Western blot reagents (Amersham Pharmacia Biotech, Piscataway, NJ) were used to visualize protein bands.
3. Results
3.1. Differentiation of MSCs into steroidogenic cells using SF-1 and LRH-1
We investigated the effects of SF-1 and LRH-1 on activation of steroidogenesis-related gene promoters in HEK293 cells (Fig. 1). Consistent with many previous reports, SF-1 activated the pro- moters of steroidogenesis-related genes, such as StAR, CYP11A1, HSD3B2 and CYP17, in a dose-dependent manner. LRH-1 could also activate these promoters, although the extent was much lower than that of SF-1 except in the case of HSD3B2.
To examine abilities of SF-1 and LRH-1 to induce the differenti- ation of hMSCs into steroidogenic cells, hMSCs were transduced with SF-1 or LRH-1 by retrovirus-mediated transfection. Trans-
duction of SF-1 into hMSCs induced expression of the type II 3ß-hydroxysteroid dehydrogenase (HSD3B2) gene (Fig. 2A), with these cells also producing progesterone (Fig. 2B). As we reported previously (Yazawa et al., 2006), 8br-cAMP treatment further induced various steroidogenic enzymes. Concomitantly, 8br-cAMP treatment markedly increased the production of progesterone. Similar results were obtained in LRH-1-transduced hMSCs. Expres- sion of SF-1 was never induced in LRH-1-transduced cells, and vice versa. These results demonstrate that LRH-1 had similar potential as SF-1 for the induction of MSC differentiation into steroidogenic cells.
3.2. Differentiation of ES cells into steroidogenic cells using tetracycline
Our studies clearly indicate that the NR5A family can direct the differentiation of stem cells into steroidogenic cells. However, this approach was not appropriate for pluripotent stem cells such as ES cells (Yazawa et al., 2006), as they barely survive the expression of the NR5A family in the absence of LIF. To circumvent these prob- lems, we used the ES cell line EBRTcH3, carrying a Tc-repressible transgene at the ROSA26 locus (Masui et al., 2005). The SF-1 cDNA along with a gene encoding the yellow fluorescent protein, Venus, was integrated into the ROSA-TET locus by a knock-in method, and puromycin resistant clones were selected (Fig. 3A). Induc- tion of the genes from the ROSA-TET locus was checked by RT-PCR and fluorescence of the reporter protein, Venus. Withdrawal of Tc from the culture medium resulted in the induction of Venus flu- orescence in virtually all cells within 48 h (Fig. 3B), whereas no fluorescence was detected in cells cultured in the presence of Tc. Although SF-1 mRNA and proteins were also induced in ES cells in
A
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₿-actin
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| Cells | 8BrcAMP(-) | 8BrcAMP(+) | (fm/cell/hour) |
|---|---|---|---|
| GFP-hMSC | N.D. | N.D. | |
| SF-1-hMSC | 0.04±0.01 | 1.17±0.26 | |
| LRH-1-hMSC | 0.07±0.02 | 1.31±0.17 |
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Fig. 3. Induction of SF-1 expression in the ROSA-TET system. (A) Schematic representation of the ROSA-TET locus exchanged with the SF-1-expression cassette in EBRTcH3 cells. (B) Induction of Venus fluorescence. Cells were observed before (panels a and b) or 48 h after induction (panels c-f) cultured with or without Tc. Bright field (panels a, c and e) and fluorescence images (panels b, d and f) are presented. (C and D) Induction of SF-1 expression in SF-1-EBRTcH3 cells. The mRNA (C) and protein (D) samples were prepared 48 h after the removal of Tc.
the absence of Tc, the cells were maintained in the undifferentiated state and never expressed any steroidogenic marker genes, includ- ing Cyp11a1 (Fig. 3Cand D). On the other hand, ES cells ceased to proliferate and died after several days when LIF was removed from culture medium. These results were in agreement with our previ- ous observations in which steroidogenic cells could not be induced directly from ES cells (Yazawa et al., 2006).
We induced the expression of SF-1 after differentiation of the ES cells into MSCs. For differentiation of ES cells into MSCs, they were cultured on collagen IV-coated dishes and treated with pulse expo- sures to RA as described by Nishikawa and colleagues (Takashima et al., 2007) (Fig. 4A). Consistent with previous reports, molecular markers of the mesenchymal cell lineage such as PDGFR&, PDGFRØ and OB-CAD were robustly induced by RA treatment (Fig. 4B), indicating that the ES cells were successfully differentiated into mesenchymal cells. The cells were further cultured in the absence of RA and Tc for three days. In contrast to undifferentiated ES cells, the differentiated cells were able to survive following SF-1 expression in the absence of LIF.
As shown in Fig. 5A, expression of SF-1 in the differentiated cells resulted in the expression of various steroidogenesis-related genes, such as Cyp11a1, Hsd3b1, Cyp17, Cyp21 Cyp11b1 and Acthr. The gene expression pattern was quite similar to that in adrenocortical cells, especially fasciculata cells. Consistent with the gene expres- sion profile, corticosterone was the most secreted steroid hormone from these cells (Fig. 5B), with Cyp17 expression barely detectable in the adult murine adrenal gland. Cortisol was also produced in these cells, although it was markedly lesser than corticosterone. These results indicate that ES cells could also be differentiated into steroidogenic cells by SF-1 via the mesenchymal cell lineage.
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4. Discussion
SF-1 has been clearly demonstrated to be a master regulator of steroidogenic organs. SF-1 knockout mice show agenesis of the primary steroidogenic organs, including the adrenal glands and gonads (Parker and Schimmer, 1997; Morohashi, 1999). SF-1 can induce the differentiation of MSCs into steroidogenic cells (Yazawa et al., 2006, 2008). In this study, we demonstrated that LRH-1 also has the capability to differentiate MSCs into steroidogenic cells. Consistent with our results, it has been reported that LRH-1 and SF-
A
SF-1-EBRTcH3
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Testis
Adrenal
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StAR
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Cyp21
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Acthr
Lhr
Gapdh
B
| Cells (Tet 1µg/ml) | progesterone | testosterone | estradiol | corticosterone | aldosterone | (ng/ml) |
|---|---|---|---|---|---|---|
| SF-1-EBRTcH3(+) | N.D. | N.D. | N.D. | N.D. | N.D. | |
| SF-1-EBRTcH3-MSCs(+) | N.D. | N.D. | N.D. | N.D. | N.D. | |
| SF-1-EBRTcH3-MSCs(-) | 638.2±65.98 | 65.1±5.33 | 0.05±0.04 | 896.7±90.25 | 12.4±2.19 |
1. N.D. means for no detectable values.
2. Data are means and SEM values of at least two independent experiments.
Fig. 5. Differentiation of ES cells into adrenocortical-like cells. (A) RT-PCR analysis of each gene in SF-1 induced (-) and uninduced (+) cells. Testis and adrenal represent a testis and an adrenal gland from an adult mouse. (B) Production of steroid hormones by each treatment. Means and SEM values of at least two independent experiments. N.D. means no detectable values.
1 could play similar roles in steroidogenesis in certain cells (Wang et al., 2001; Saxena et al., 2007; Yazawa et al., 2010). On the other hand, differentiated cells such as HEK293 cells stably transformed with SF-1 or LRH-1 did not express steroidogenic enzymes nor did they produce steroid hormones, suggesting that the expression of these genes in situ are controlled by additional factors.
LRH-1 is abundantly expressed in ES cells and is necessary for Oct-3/4 expression (an essential gene for maintenance of the inner cell mass and pluripotency of ES cells) at the epiblast stage (Gu et al., 2005). In addition, Oct-3/4 can be replaced by LRH-1 for the reprogramming of murine somatic cells into induced pluripotent stem (iPS) cells (Heng et al., 2010). As the differentiation of MSCs into steroidogenic cells, SF-1 and LRH-1 have similar potential for the regulation of Oct-3/4 expression (Barnea and Bergman, 2000; Gu et al., 2005), and therefore the same potential for induction of somatic cells into iPS cells (Heng et al., 2010). Niwa et al. (2000) demonstrated that quantitative expression of Oct-3/4 defines the fate of ES cells. A less than twofold increase in Oct-3/4 expres- sion causes differentiation of ES cells into primitive endoderm and mesoderm, whereas repression of Oct-3/4 expression induces loss of pluripotency and causes de-differentiation of cells into the trophectoderm. It has been shown that DAX-1, a common transcrip- tional inhibitor of Oct-3/4, SF-1 and LRH-1 are also important for the pluripotency and survival of ES cells (Yu et al., 1998; Sun et al., 2009; Khalfallah et al., 2009). DAX-1 expression is detectable in ES cells and its expression is reduced upon differentiation of the cells into each germ layer. DAX-1 knockdown induces loss of pluripotency even under culture conditions for maintaining the undifferentiated state (Sun et al., 2009; Khalfallah et al., 2009), whereas complete deletion of DAX-1 by gene targeting results in cell death (Yu et al., 1998). Over-expression of the NR5A family was also cytotoxic to ES cells. These facts strongly suggest that regulated and coordinated expression of NR5A genes is essential for the pluripotency and sur- vival of ES cells. These properties of the NR5A family are likely to cause difficulties in the induction of steroidogenic cells by NR5A members directly from ES cells.
In a previous study (Crawford et al., 1997), stable expression of SF-1 was shown to direct ES cells towards the steroidogenic lin- eage. However, the steroidogenic capacity of the cells was limited since a membrane-permeable substrate, 20x-hydroxycholesterol, was necessary to produce progesterone, the only steroid produced from the cells (Crawford et al., 1997). We demonstrated that regu- lated expression of SF-1 by the ROSA-TET system made it possible to derive steroidogenic cells from ES cells, with a capacity for autonomously secreting various steroid hormones.
It was reported that ES cells cultured on collagen-IV coated plates and treated with RA undergo differentiation into the mes- enchymal cell lineage including MSCs, and that steroidogenic cells could be induced by SF-1 from MSCs (Takashima et al., 2007; Yazawa et al., 2006, 2008). Although we did not fully characterize the differentiated cells derived from the ES cells by RA treatment in this study, it is conceivable that the ES cell-derived steroidogenic cells must be produced via multipotent MSCs. Steroidogenic cells could be induced by SF-1 from MSCs, but not from differentiated cells such as fibroblasts, preadipocytes and HEK293 cells (Yazawa et al., 2006; Yanase et al., 2006). In support of this hypothesis, the ES cells could not be converted into steroidogenic cells via Tc- induced expression of SF-1 under culture conditions that induced differentiation of ES cells into preadipocytes, which also expressed PDGFR& as MSCs (data not shown). However, the exact origin of ES cell-derived steroidogenic cells should further investigated.
The ES cells-derived steroidogenic cells exhibited the very sim- ilar gene expression patterns to that of adrenocortical cells and produced a large amount of corticosterone, despite with Cyp17 expression was detectable. As in the case of other steroid hydrox- ylases, it is well-known that the expression of the CYP17/Cyp17
gene is regulated by SF-1 and LRH-1 (Zhang and Mellon, 1996; Lin et al., 2001; Yazawa et al., 2009). Hence, it is conceivable that the human CYP17 gene is expressed in both in gonadal and adrenal steroidogenic cells. In contrast, the murine Cyp17 gene is expressed only in gonadal cells in adults. However, it was shown that Cyp17 is expressed in the murine fetal adrenal gland (Heikkila et al., 2002). Therefore, it is possible that steroidogenic cells derived from murine ES cells might reflect the fetal adrenal phenotype. Further studies are necessary for the determination of steroidogenic lineage and the regulation of Cyp17 expression.
In summary, we have shown that, as in the case of SF-1, LRH- 1 could drive the differentiation of MSCs into steroidogenic cells. In addition, ES cells could also be differentiated into steroidogenic cells through the regulated expression of SF-1 using the ROSA- TET system. This approach might also provide the opportunity, through the use of MSCs, for the development of cell and gene ther- apy treatments in steroidogenesis deficiencies. Additionally, this approach could be a powerful tool for studies on the differentiation of steroidogenic cell lineages.
Acknowledgments
We are grateful to Drs. K. Morohashi (University of Kyushu), T. Sugawara (University of Hokkaido), H. Niwa (Riken, BRC) and K. Araki (University of Kumamoto) for providing reagents. We also thank Ms. Y. Inoue, K. Matsuura and H. Fujii for technical assistance.
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