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Endocrinology Vol. 147, No. 9 4104-4111
Copyright © 2006 by The Endocrine Society

Differentiation of Adult Stem Cells Derived from Bone Marrow Stroma into Leydig or Adrenocortical Cells

Takashi Yazawa, Tetsuya Mizutani, Kazuya Yamada, Hiroko Kawata, Toshio Sekiguchi, Miki Yoshino, Takashi Kajitani, Zhangfei Shou, Akihiro Umezawa and Kaoru Miyamoto

Department of Biochemistry (T.Y., T.M., K.Y., H.K., T.S., M.Y., T.K., Z.S., K.M.), Faculty of Medical Sciences, University of Fukui, Fukui 910-1193, Japan; Core Research for Evolutional Science and Technology (T.Y., T.M., K.Y., H.K., T.S., M.Y., T.K., Z.S., K.M.), Japan Science and Technology Agency, Saitama 332-0012, Japan; and National Research Institute for Child Health and Development (A.U.), Tokyo 157-8535, Japan

Address all correspondence and requests for reprints to: Kaoru Miyamoto, Department of Biochemistry, Faculty of Medical Sciences, University of Fukui, Shimoaizuki, Matsuoka-cho, Fukui 910-1193, Japan. E-mail: kmiyamot{at}fmsrsa.fukui-med.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Adult stem cells from bone marrow, referred to as mesenchymal stem cells or marrow stromal cells (MSCs), are defined as pluripotent cells and have the ability to differentiate into multiple mesodermal cells. In this study, we investigated whether MSCs from rat, mouse, and human are able to differentiate into steroidogenic cells. When transplanted into immature rat testes, adherent marrow-derived cells (including MSCs) were found to be engrafted and differentiate into steroidogenic cells that were indistinguishable from Leydig cells. Isolated murine MSCs transfected with green fluorescence protein driven by the promoter of P450 side-chain cleaving enzyme gene (CYP11A), a steroidogenic cell-specific gene, were used to detect steroidogenic cell production in vitro. During in vitro differentiation, green fluorescence protein-positive cells, which had characteristics similar to those of Leydig cells, were found. Stable transfection of murine MSCs with a transcription factor, steroidogenic factor-1, followed by treatment with cAMP almost recapitulated the properties of Leydig cells, including the production of testosterone. Transfection of human MSCs with steroidogenic factor-1 also led to their conversion to steroidogenic cells, but they appeared to be glucocorticoid- rather than testosterone-producing cells. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEM CELLS ARE self-renewing elements with the capacity to generate multiple distinct cell lineages. They exist in various tissues, even in adults, and have been isolated from a variety of differentiated tissues, including bone marrow, umbilical blood, brain, and fat (1, 2, 3, 4, 5, 6). Among these, bone marrow-derived mesenchymal stem cells (MSCs), also known as marrow stromal cells, are defined as pluripotent cells and have been shown to differentiate into adipocytes, chondrocytes, osteoblasts, and hematopoietic-supporting stroma both in vivo and ex vivo (1, 2, 3). Furthermore, they are able to generate cells of all three germ layers (7, 8). In addition to their multipotency for differentiation, MSCs have attracted considerable interest for use in cell and gene therapy because these cells can easily be obtained from adult marrow tissue (8, 9, 10).

The gonad and adrenal gland are the primary steroidogenic organs in mammals. In the gonad, male Leydig cells or female granulosa and theca cells are responsible for the production of androgens and estrogens. The adrenal cortex produces glucocorticoids and mineralocorticoids, although some androgens are also produced in many species, except rodents. These steroidogenic organs develop from the common adrenogenital primodium, which originates from the intermediate mesoderm (11). Fetal-type steroidogenic cells appear when the adrenogenital primodium differentiates into the adrenal cortex and the gonads of the two sexes. These are replaced by adult-type steroidogenic cells during the period between birth and puberty (12, 13), but these processes are poorly understood.

One approach to resolving the complexities of organogenesis is to use stem cells as a model system for differentiation. In this study, the differentiation of MSCs into steroidogenic cells was examined in vivo and in vitro by several methods. A number of studies have reported that the injection of MSCs into some tissues leads to the differentiation of the injected cells into tissue-specific cells, probably due to the microenvironment near the injection sites. To determine whether MSCs are able to differentiate into steroidogenic cells, we injected a purified population of rat MSCs into the prepubertal rat testis and examined the fate of these cells by immunohistochemistry. In addition, the spontaneous differentiation of MSCs to specific cells can be monitored by the expression of specific genes in the differentiated cells. One such experimental approach, known as a promoter-sorting method, is to use fluorescence-activated cell sorting (FACS) to select green fluorescence protein (GFP)-positive MSCs in which the expression of GFP is under the control of the promoter of a gene that is expressed in a cell type-specific fashion. In this study, to demonstrate the emergence of steroidogenic cells from isolated MSCs in vitro, a GFP expression vector driven by the CYP11A promoter (CYP11A is a gene encoding the cholesterol side-chain cleavage enzyme, an essential enzyme for steroidogenesis) was integrated into the MSCs, and GFP-positive MSCs were then separated by fluorocytometry. Finally, to achieve the efficient differentiation of the isolated MSCs in vitro, the orphan nuclear receptor, steroidogenic factor (SF)-1 was ectopically expressed in MSCs. MSCs successfully differentiated into steroidogenic cells using any of these procedures. These results indicate that MSCs represent a useful source of stem cells for producing steroidogenic cells that may provide basis for their use in cell and gene therapy.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
GFP transgenic rats [SD TgN(act-EGFP)OsbCZ-004] were kindly provided by Dr. M. Okabe (Osaka University, Osaka, Japan). Sprague Dawley rats were purchased from Sankyo (Shizuoka, Japan). At all times, the animals were treated according to National Institutes of Health guidelines. The donor animals used in this study were generally 4–5 wk old, and the recipient animals were 3 wk old.

Histology and immunofluorescence analysis
Immunohisto- and cytochemical staining with antirat P450 side-chain cleaving enzyme (P450scc) (C-16; Santa Cruz Biotechnology, Santa Cruz, CA), antimouse 3ß-hydroxysteroid dehydrogenase I (3ß-HSD I) (kindly provided by Dr. A. Payne, Stanford University Medical Center, Stanford, CA), antipig cytochrome P450 17 {alpha}-hydroxylase (P450c17) (kindly provide by Dr. D. Hales, University of Illinois at Chicago, Chicago, IL) or anti-GFP (Medical & Biological Laboratories Co., Ltd.) were performed on 10-µm frozen sections or cultured cells on glass slides using standard protocols. Appropriate Cy3- or fluorescein isothiocyanate-conjugated secondary antibodies (Sigma, St. Louis, MO) were used for detection.

Cell culture, stable transfection, and hormone assay
MSCs from GFP transgenic rats were collected and cultured as described by Pochampally et al. (14). Mouse (KUM9) (15) or human (hMSC-hTERT-E6/E7) (16) bone marrow-derived MSCs were maintained in Iscova’s MEM or DMEM with 10% fetal calf serum. Plasmid DNA was transfected using the LipofectAmine PLUS reagent (Invitrogen, Carlsbad, CA) or calcium phosphate coprecipitation. Cells were used for the experiments after 10–12 passages, and steroid hormone production was sustained for at least 4 months. The levels of each steroid hormone in the media were measured by RIA.

Transplantation
Bone marrow cells from TgN(ActbEGFP) transgenic rats (1 x 106) were injected into the testes of 3-wk-old SD rats. Two to three weeks after transplantation, testes were removed to examine histochemically survival and differentiation of transplanted cells.

Plasmid construction
A 2.3-kb fragment of the human CYP11A (P450scc gene) promoter that functions specifically in steroidogenic organs (17) was obtained by PCR using pSCC2300-LacZ (kindly provided by Dr. B. C. Chung, Institute of Molecular Biology, Taipei, Taiwan) as a template and integrated into a promoter-less pEGFP-1 vector (CLONTECH, Palo Alto, CA). The EcoRI-StuI restriction fragment, containing the CYP11A promoter-GFP, was then excised and inserted into EcoRI and SwaI site of pPUR (CLONTECH). The expression vector for rat SF-1 cDNA containing the entire coding region was generated by RT-PCR and subcloned into pIRES-puro2 vector (CLONTECH).

FACS analysis and cell purification
Cells were harvested by treatment with 0.25% trypsin/EDTA, after which they were neutralized with DMEM with 10% fetal calf serum, washed twice with PBS, and filtered through a 35-mm pore size nylon screen. FACS analysis was performed on a flow cytometer with a 488-nm argon laser and GFP-positive cells were isolated.

RT-PCR and real-time PCR
Total RNA from the cultured cells was extracted using the Trizol reagent (Invitrogen). RT-PCR was performed as described previously (18). The reaction mixture was subjected to electrophoresis in a 1.5% agarose gel, and the resulting bands were visualized by staining with ethidium bromide. Real-time PCR was performed as described by Rutledge and Cote (19). Reagents for real-time PCR were purchased from Applied Biosystems (Warrington, UK), except for SYBER green PCR master mix (QIAGEN, Valencia, CA). Reactions were carried out and fluorescence was detected on a GeneAmp 7700 system (Applied Biosystems). The primers used are shown in Table 1Go.


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  		TABLE 1. Primers for RT-PCR and real-time PCR

 
Western blot analysis
The extraction of protein from the cultured cells and subsequent quantification was performed as described previously (20). Equal amounts of protein (50 µg) were resolved by 12.5% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blot analyses of SF-1, steroidogenic acute regulatory protein (StAR), P450scc, 3ß-HSD I, P450c17, and ß-tubulin were carried out with antisera directed against SF-1 (Ad4BP, kindly provided by Dr. K. Morohashi, National Institute of Basic Biology, Okazaki, Japan), StAR (kindly provided by Dr. W. Miller, University of California, San Francisco, CA) (21), P450scc (kindly provided by Dr. B. C. Chung) (22), 3ß-HSD I (kindly provided by Dr. A. Payne), P450c17 (kindly provided by Dr. D. Hales) (23), and ß-tubulin (D-10, Santa Cruz). ECL Western blot reagents (Amersham Pharmacia Biotech, Piscataway, NJ) were used for detection.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Transplantation of rat bone marrow mesenchymal stem cells
In the prepubertal testis, fetal-type Leydig cells are replaced by adult-type Leydig cells, which originate from mesenchymal precursor cells that are present in the testicular interstitium (12). To determine whether MSCs can be engrafted into the testis and converted into steroidogenic cells we took 1 x 106 bone marrow cells from TgN(ActbEGFP) transgenic rats that had been maintained in culture (Fig. 1AGo) and injected them into the testes of 3-wk-old SD rats. As shown in Fig. 1CGo, donor engraftment was confirmed (100%) at various periods after transplantation (1–4 wk). A histochemical examination revealed that the GFP-positive cells present in the testes were located in the interstitium and were not observed within the seminiferous tubules (Fig. 1DGo). An immunohistochemical study showed that most of the GFP-positive cells in the interstitium were also positive for three Leydig cell markers, P450scc (Fig. 1EGo), 3ß-HSD I, and P450c17 (data not shown). These results indicate that donor derived-plastic adhered marrow cells had in fact differentiated into steroidogenic Leydig-like cells in vivo.


Figure 1
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  		FIG. 1. Transplantation of GFP-positive MSCs into the testis. A, Fluorescence view of MSCs from a green rat 3 d after the first passage. Fluorescence microscopic view of testis before (B) or 3 wk after (C) MSC transplantation. Double staining of frozen sections from the testis 5 wk after MSC transplantation with anti-GFP (D) and anti-P450scc (E) antibodies. F, Merged fluorescent image of D and E. ST, Seminiferous tubule.

 
Gene promoter sorting
Although these data suggest that the injected stem cells differentiated into Leydig cells, the apparent stem cell plasticity may also be explained by possible cell-nuclear fusion between donor and recipient cells, as has been recently suggested (24). Therefore, we next performed in vitro experiments to determine whether purified murine MSCs (mMSCs), KUM9 (15), have the capacity to differentiate into steroidogenic cells. To detect a cell population committed to the steroidogenic lineage, we first introduced a human CYP11A1 promoter/GFP gene construct into the mMSCs. This was accomplished by using a 2.3-kb fragment of the promoter region of the human CYP11A1 (a gene that encodes cytochrome P450scc, cholesterol side-chain cleavage enzyme), which has been shown to selectively drive transgene expression to adrenal and gonadal steroidogenic cells (17). In some of the transformed cell lines, GFP fluorescence was detected, as shown in Fig. 2Go, B and C, but the number of GFP-expressing cells was very low. Thus, GFP-positive cells were enriched by sorting with flow cytometry (Fig. 2EGo, 1–5% of total cells). As shown in Fig. 2Go, F and G, enriched GFP-positive cells were also positive for P450scc, indicating that a very small but distinct portion of the mMSCs had spontaneously differentiated into cells that produce the steroid hormone-synthesizing enzyme. Further analysis of the differentiated cells revealed the expression of several genes that are specific to testicular Leydig cells, as shown in Fig. 2HGo. These include a nuclear orphan receptor SF-1, 3ß-HSD types I and VI, and LH receptor (Fig. 2HGo, lane SCC+). LH receptor and 3ß-HSD VI are known to be typical markers for androgen producing cells, such as Leydig cells (25). These observations further support the in vivo findings that rodent MSCs have the capacity to differentiate into Leydig-like cells in the testis.


Figure 2
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  		FIG. 2. Spontaneous differentiation of KUM9 into steroidogenic cells. A, Schematic representation of the SCC-reporter gene (SCC-GFP). The SCC-GFP reporter plasmid contains the 2300-bp upstream sequence of the human CYP11A1 gene and the puromycin-N-acetyltransferase gene (PURO-pA) driven by the Simian virus 40 early promoter (SV40 e.p.). Phase-contrast (B) and fluorescent (C) images of mMSCs transfected with SCC-GFP and selected by puromycin are shown. Flow cytometric analysis of enhanced GFP (EGFP) expression in KUM9 transfected with control-GFP (D) or SCC-GFP (E) are shown. KUM9-derived cells expressing GFP (F) under the control of the human CYP11A1 promoter were immunocytochemically stained with anti-P450scc antibody (G). H, SCC-GFP-positive (SCC+) and negative (SCC–) populations were sorted and analyzed for various marker genes by RT-PCR.

 
Stable transfection of SF-1 into mouse MSCs
It is noteworthy that SF-1 expression was induced in the GFP-positive cells (Fig. 2HGo). SF-1, also known as Ad4BP, regulates the cell-specific expression of a variety of proteins that are involved in steroidogenesis, in addition to its roles in reproduction and gonadal differentiation (26). Therefore, we next examined the effects of the stable transfection of SF-1 to mMSCs. Various cell lines that stably express SF-1 were isolated. As shown in Fig. 3CGo, SF-1-induced morphological changes in the cells, such as the accumulation of numerous lipid droplets. However, the transformed cells did not express steroidogenic enzyme genes or produce any steroid hormones (Fig. 3DGo and Table 2Go). Therefore, we next added cAMP to the cultures because cAMP is known to induce steroidogenesis in a number of steroidogenic cell lines. Treatment of confluent cultures with cAMP was found to induce both P450scc mRNA (Fig. 3DGo) and protein (Fig. 3HGo) in the transformed cell lines, SF7 and SF9, whereas no induction was observed in untransfected (KUM9) or vector-transfected (pIRES) mMSCs (Fig. 3DGo). Treatment of the cells for a period of 7 d further induced the expression of other steroidogenic enzyme genes, as shown in Fig. 3DGo. Several cell lines showed similar expression patterns (two of which are shown in Fig. 3DGo).


Figure 3
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  		FIG. 3. Differentiation of KUM9 into steroidogenic cells. Phase-contrast images of mMSCs untransfected (A) or stably transfected with the control (B) or the SF-1-expression (C) vectors are shown. An immunoblot analysis was performed with an antibody against SF-1 (insets). D, RT-PCR analysis of each clones cultured with or without 8-bromoadenosine-cAMP (8br-cAMP) for 7 d (C: without 8br-cAMP; A: with 8br-cAMP). Data were compared with those from mouse adrenal and testicular tissues. Fluorescent images of 4',6'-diamino-2-phenylindole staining (E and G) and a P450scc immunostaining (F and H) of KUM9 transfected with the SF-1-expressing vector and then cultured with (G and H) or without 8br-cAMP (E and F).

 

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  		TABLE 2. Production of steroid hormones by MSCs stably expressing SF-1 (SF9-KUM9 or SF4-hMSC) in the presence (+) or absence (–) of 8br-cAMP (ng/ml)

 
3ß-HSD types I and VI were induced 3 d after cAMP treatment (Fig. 4Go). In the testis, the formation of testosterone is dependent on 3ß-HSD activity, and isoform types I and VI have been shown to be expressed in the adult mouse testis (27). P450c17 and 17ß-hydroxysteroid dehydrogenase III (17ß-HSD III) were induced 5 d after the treatment (Fig. 4Go). It is interesting to note that the order of induction of the enzymes is similar to the sequential order for the steroid hormone synthetic pathway. 3ß-HSD enzymes are essential for the production of progesterone, and P450c17 and 17ß-HSD III are both required for the production of testosterone in testicular Leydig cells. Consistent with the expression pattern of the steroidogenic enzymes, testosterone was the major sex steroid hormone produced in the transformed cell line, SF9, when treated with cAMP for 7 d (Table 2Go). Two adrenal-specific steroid hormones, glucocorticoids and mineralocorticoids, were not detected in these cells. These results clearly demonstrate that the stable expression of SF-1 and the addition of cAMP induced the differentiation of mMSCs into steroidogenic cells and that these cells have properties that are similar to those of testicular Leydig cells.


Figure 4
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  		FIG. 4. Time-dependent induction of steroidogenic enzymes by cAMP. KUM9 cells stably transfected with SF-1-expression (SF9) or control (pIRES) vector were cultured and treated with 8-bromoadenosine-cAMP for the indicated times. A, P450scc, 3ß-HSD I, 3ß-HSD VI, P450c17, and 17ß-HSD III mRNA levels were analyzed by RT-PCR and real-time PCR. Real-time PCR data are the mean values of at least triplicate assays. The 7-d value was arbitrarily taken as 1.0. B, Immunoblot analyses were performed with antibodies against StAR, P450scc, 3ß-HSD I, P450c17, and ß-tubulin using the same lysates. The data were compared with that from MA-10 cells treated with cAMP (4 h).

 
Stable transfection of SF-1 into human MSCs
We next examined the issue of whether the same approach could also be used to induce the differentiation of human MSCs (hMSCs) into steroidogenic cells. Similar to the results obtained with mMSCs, hMSCs (hMSC-TERT-E6/E7) expressed no steroidogenic enzymes or StAR before transfection with SF-1 even after cAMP treatment (Fig. 5Go). After SF-1 transfection, all the transformed cell lines became positive for StAR gene expression, and the expression levels were further increased by cAMP treatment. Most of the steroidogenic enzymes, P450scc, 3ß-HSD II, P450c17, cytochrome P450 steroid 21-hydroxylase (P450c21), cytochrome P450 aromatase (P450arom), and cytochrome P450 steroid 11 ß-hydroxylase, were also substantially induced by cAMP stimulation. A significant difference between mMSCs and hMSCs was the strong expression of the P450c21 gene in the case of hMSCs. This caused a difference in the kinds of steroids produced by mMSCs and hMSCs. As listed in Table 2Go, glucocorticoids were the major steroids produced by the transformed hMSCs, hSF4, whereas testosterone was the major product from the transformed mMSCs, mSF9. The hSF4 cells mainly produced cortisol, the major glucocorticoid produced by the human adrenal gland. These results clearly demonstrate that the stable expression of SF-1 and subsequent cAMP treatment induced the differentiation of hMSCs into steroidogenic cells. In addition, the cortisol-producing cells also expressed ACTH receptors and can respond to ACTH for the quick production of cortisol at nanomolar levels (data not shown).


Figure 5
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  		FIG. 5. Induction of steroidogenic enzymes in hMSCs. hMSCs were stably transfected with the control (pIRES) or SF-1-expression (SF3, -4) vector. RT-PCR analysis of each clone was cultured with or without 8-bromoadenosine-cAMP (8br-cAMP) for 7 d (C: without 8br-cAMP; A: with 8br-cAMP). The data were compared with that from human testis and NCI-H295R, a human adrenocortical tumor cell line, treated with cAMP (24 h).

 
Human MSCs also expressed P450arom as in the case of the human adrenocortical carcinoma NCI-H295R cell line (Fig. 5Go), whereas normal adrenal cells do not express it (28). However, hSF3 or -4 did not express IGF-II, an adrenocortical tumor marker. It has recently been shown that P450arom is expressed in human bone marrow stroma cells under certain conditions (29). Thus, it is probable that the expression of P450arom in hMSCs was not the result of a malignant phenotype or the differentiation of the cells by SF-1 and cAMP treatment.

Stable transfection of SF-1 into cells other than MSCs
We next examined the effects of transfection of SF-1 into several cell lines other than MSCs, i.e. a human cell line HEK293, murine embryonic stem cells, and murine cell lines F9 and NIH3T3. None of the transfected cell lines autonomously produced steroid hormones, although some were induced to express the P450scc and 3ß-HSD genes (Fig. 6Go).


Figure 6
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  		FIG. 6. Stable transfection of SF-1 and cAMP treatment for F9 (A) and HEK293 cells (B). RT-PCR analysis of steroidogenesis-related genes in each stable cell line transfected with SF-1 or pIRES (control) cultured with or without 8-bromoadenosine-cAMP (8br-cAMP) for 7 d (C: without 8br-cAMP; A: with 8br-cAMP).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The findings presented herein demonstrate that rodent MSCs have the potential to differentiate into steroidogenic cells with characteristics that are very similar to testicular Leydig cells. It has been postulated that mesenchymal progenitors of Leydig cells are present in the testicular interstitium (12). Immature Leydig cells are gradually replaced by mature Leydig cells that are thought to differentiate from these mesenchymal progenitors during the prepubertal period. In fact, the injection of MSCs into the testis during this critical period caused the differentiation of MSCs into steroidogenic cells that were indistinguishable from Leydig cells. Concerning the in vivo experiments, the possibility of cell fusion between donor MSCs and recipient testicular Leydig cells or their progenitor cells cannot be excluded. However, it should be emphasized that very small but distinct portions of mMSCs underwent spontaneous differentiation into Leydig-like cells in vitro. Lo et al. (30) demonstrated, by means of a cell transplantation assay, the presence of stem cells or progenitors for Leydig cells. Therefore, our data strongly suggest that bone marrow-derived MSCs share common properties with testicular MSCs or Leydig cell progenitors. Conversely, testicular MSCs or Leydig cell progenitors might also have pluripotent characteristics, similar to bone marrow-derived MSCs, as has been reported for some other MSCs (4, 31).

In addition, transfection of cultured mMSCs with SF-1 followed by cAMP stimulation resulted in their differentiation into Leydig cells. The same procedure also led to the successful induction of hMSCs into steroidogenic cells. In this case, however, most of the cell lines expressing SF-1 largely produced glucocorticoids rather than testosterone. This was mainly due to the strong induction of P450c21 gene expression in the hMSCs. To investigate the issue of whether hMSCs are able to differentiate into Leydig cells, we also injected hMSCs to the testis of nude mice or rats (data not shown). Unfortunately, the human cells did not survive for more than several weeks in the rodent testis.

Because the established cell lines need much longer times than general steroidogenic cells to produce steroid hormones by cAMP stimulation in this study, we speculate that cAMP treatment of this study is necessary for the induction of the cellular differentiation rather than direct stimulation of gene transcription of steroidogenic enzymes.

In hMSCs, the stable expression of SF-1 and cAMP treatment induced the expression of the StAR gene, which is essential for the transfer of cholesterol from the outer to the inner membrane of mitochondria in which the conversion of cholesterol to steroid hormones begins (21). The same treatment failed to induce StAR gene expression in several cell lines (other than MSCs) including embryonic stem (ES) cells and therefore failed to induce any steroid hormones. The expression of the P450scc or 3ß-HSD gene was induced at low levels in some of them, however (Fig. 6Go). It has been reported that the stable transfection of SF-1 into ES cells results in morphological changes and the induction of P450scc enzyme expression, (32). No autonomous production of steroid hormones was observed, however, probably because of the deficiency of cholesterol storage and mobilization and the lack of StAR protein expression (32). Therefore, our present observations suggest that MSCs, but not ES cells, are excellent precursors of steroidogenic cells. In contrast to human cells, StAR was constitutively expressed in KUM9 as well as the freshly isolated rat MSCs (our unpublished data). Therefore, we speculate that StAR gene expression is not always under the control of SF-1, and the pattern of expression may be different between species, even in the same tissues. In addition to the steroidogenesis, the movement of cholesterol to the inner mitochondrial membrane is also important for its metabolism, because one of the rate-determining steps, the 27-hydroxylation of cholesterol, is catalyzed by sterol 27-hydroxylase, which is located in the inner mitochondrial membrane (33, 34). Cholesterol metabolites, such as oxysterols have been proposed to be potential regulators of genes in cholesterol homeostasis (33). We found that sterol 27-hydroxylase mRNA was detectable in rat and mouse MSCs (data not shown), suggesting that it is involved in cholesterol metabolism. Therefore, it is assumed that the StAR protein in KUM9 is present to promote the cholesterol metabolism, despite the fact that steroidogenesis does not take place. In support of this hypothesis, ectopic expression of the StAR protein increases the metabolism of cholesterol in rat primary hepatocytes (34).

Gondo et al. (35) recently reported that the adenovirus-mediated forced expression of SF-1 transforms primary long-term cultured murine bone marrow cells into ACTH-responsive steroidogenic cells. In contrast to our observation obtained from murine MSCs, their steroidogenic cells produce both gonadal and adrenal steroids. There are two possible explanations for their results: 1) their cells were a mixed adrenal/gonadal phenotype or 2) were a mixture of adrenal or gonadal phenotypic cells. The latter seems to be more likely because our study clearly demonstrated the differentiation of adult stem cells derived from both murine and human into gonadal or adrenal steroidogenic cells. Therefore, with respect to the difference between mouse and human cells, we assume that the mouse MSCs used in our study were already committed to the gonadal lineage, whereas the hMSCs were already committed to the adrenal lineage. In support of this hypothesis, it has frequently been reported that MSCs are heterogeneous populations that have a different differentiation potential (1, 2, 10). In a future study, the same treatment of various mouse or human MSCs need to be carried out, followed by observations of whether both adrenal and gonadal phenotypes are obtained. This might also provide a tool for revealing the pathway leading to the differentiation of the cells into adrenal or gonadal steroidogenic cells.

In summary, we demonstrate here that MSCs have the capacity to differentiate into steroidogenic cells, both in vivo and in vitro. MSCs represent not only a powerful tool for studies of the differentiation of the steroidogenic lineage but may also offer a possible clinical stem cell resource for diseases of steroidogenic organs.


   Acknowledgments
 
We are grateful to Drs. K. Morohashi, W. Miller, B. C. Chung, A. Payne, and D. Hales for providing plasmids and antisera. We also thank Drs. M. Ascoli and J. Toguchida for the generous gifts of MA10 and hMSCs and Ms. Y. Inoue, T. Satake, and K. Matsuura for technical assistance.


   Footnotes
 
This work was supported in part by a grant from the Smoking Research Foundation and the 21st Century Center of Excellence Program (Medical Science).

All authors (T.Y., T.M., K.Y., H.K., T.S., M.Y., T.K., Z.S., A.U., K.M.) have nothing to declare.

First Published Online May 25, 2006

Abbreviations: ES, Embryonic stem; FACS, fluorescence activated cell sorting; GFP, green fluorescence protein; hMSC, human MSC; 3ß-HSD I, 3ß-hydroxysteroid dehydrogenase I; 17ß-HSD III, 17ß-hydroxysteroid dehydrogenase III; mMSC, murine MSC; MSC, mesenchymal stem cell; P450arom, cytochrome P450 aromatase; P450c17, cytochrome P450 17{alpha}-hydroxylase; P450c21, cytochrome P450 steroid 21-hydroxylase; P450scc, P450 side-chain cleaving enzyme; SF, steroidogenic factor; StAR, steroidogenic acute regulatory protein.

Received February 8, 2006.

Accepted for publication May 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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