JP2006122040A - New steroidogenic cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel steroidogenic cell that can be used for treatment of abnormal steroid hormone secretion such as steroid hormone deficiency by transplantation or the like.
The present invention provides a steroidogenic cell characterized by differentiation from a bone marrow cell or a pluripotent stem cell by introducing a factor involved in steroid hormone synthesis. Also provided are methods for producing and using the steroidogenic cells, pharmaceutical compositions using hormones secreted from the cells, and methods for treating various diseases using the cells.
[Selection figure] None

Description

  The present invention relates to the production of steroid-producing cells, and more specifically to a novel steroid-producing cell produced thereby and a production method thereof. The invention also relates to the use of the cells so produced.

  In general, steroid hormones are known as “steroids” used in bronchial asthma and rheumatism in addition to external and internal use for atopic dermatitis. This steroid agent has a strong immunosuppressive action and is used for the treatment of a wide range of diseases, but its use must be carefully performed from the viewpoint of side effects.

  On the other hand, steroid hormones produced in vivo are hormones that play a very important role in maintaining the living body. If the production of steroid hormones in the body is insufficient, the regulation and maintenance of glucose, the regulation of Na · K balance, the promotion of protein synthesis, the control of inflammatory and immune responses, sexual development, reproductive function and the like are reduced. When this steroid hormone is missing or decreased for various reasons, a pathological condition called “steroid hormone deficiency” is caused.

  One such steroid hormone deficiency is adrenocortical dysfunction. This adrenal cortex dysfunction is caused by a lack of adrenal steroid hormones, and specific symptoms include generalized fatigue due to lack of secretion of the stress-responsive hormone glucocorticoid cortisol. Therefore, hypotension, hypoglycemia, hyponatremia, hyperkemia, pigmentation, etc. may be seen as medical findings. In the worst case, acute adrenal insufficiency may result in death due to an absolute shortage of adrenal steroid hormones or a sudden increase in the amount required due to stress such as infection. Adrenocortical dysfunction often results in poor secretion of aldosterone, a mineralocorticoid hormone, which further promotes hyponatremia and hyperkemia.

  Other steroid hormone deficiencies include gonadal dysfunction. Gonadal dysfunction is a condition in which the secretion of gonadal steroid hormones from the gonadal (testes in men and ovaries in women) decreases due to various etiologies such as genetic factors, tumors, inflammation, surgery, and radiation. Point to. Basically it is not a life-threatening problem, but when it occurs in young people in their 10s and 20s, secondary sexual characteristics may be impaired and there may be complex problems due to the lack of “masculinity / femininity” . At the same time, it can be a problem affecting future fertility. In addition to such effects on the reproductive system, there is also a problem that osteoporosis and arteriosclerosis are likely to occur due to a long-term absence of gonadal hormones.

  Steroid hormone replacement therapy has been established as a current treatment method for these steroid hormone deficiencies, and provides many benefits as described in Non-Patent Document 1, for example. However, especially in cases of adrenal cortex dysfunction, it is necessary to continue supplementation throughout the lifetime. Generally, glucocorticoids are replenished, but mineral corticoids are also added if that alone does not correct the electrolyte dysfunction. Depending on the patient, it is necessary to take a fixed dose by itself, which is difficult to cope with when the steroid requirement increases rapidly due to infection, etc., and if the steroid excess continues, various side effects may occur.

  Accordingly, new therapies such as gene therapy and steroidogenic cell transplantation are eagerly desired, but a definitive treatment method has not yet been developed.

Laureti, S .; Falonni, A .; , And Santeusanio, F.A. "Improvement of treatment of primary adryn insufficiency by administration of cortisone acetate in three days indoles." J Endocrinol Investments. 2003 (11): 1071-1075

  The present invention has been made in view of such problems, and an object of the present invention is to provide a novel steroid-producing cell that can be used for treatment of abnormal steroid hormone secretion such as steroid hormone deficiency. Provides a steroidogenic cell capable of secreting physiologically diverse steroid hormones, its production method, use, pharmaceutical composition using the hormone secreted from the cell, and treatment methods for various diseases using the cell For that purpose.

  Recently, many studies have suggested that bone marrow cell transplantation may contribute to regeneration of hematopoietic and mesenchymal cell groups in various organs. In other words, bone marrow cells are thought to contain pluripotent progenitor cells that differentiate into various organs. Bone marrow stem cells are known to grow into hematopoietic and mesenchymal cells, but prior to the present invention, they were not known to differentiate into steroidogenic cells. However, the present inventors have focused on the fact that bone marrow cells can be differentiated into muscles and fats that are derived from the mesenchymal system as well as the adrenal cortex and gonadal, and obtained new knowledge that steroid-producing cells are differentiated from bone marrow cells. As a result of repeated sincerity studies and experiments based on this, steroid-producing cells having several properties described below have been successfully generated.

  That is, according to the first main aspect of the present invention, there is provided a steroidogenic cell characterized in that a factor involved in steroid hormone synthesis is introduced into bone marrow cells and differentiated from the bone marrow cells.

  According to such a configuration, a novel steroid hormone-producing cell that can be used for treatment of abnormal steroid hormone secretion such as steroid hormone deficiency can be obtained by transplantation or the like.

  Here, the bone marrow cells include, but are not limited to, bone marrow stem cells, bone marrow mesenchymal stem cells, hematopoietic stem cells, and pluripotent progenitor cells.

  According to a second main aspect of the present invention, there is provided a steroidogenic cell characterized by introducing a factor involved in steroid hormone synthesis into a pluripotent stem cell and causing the pluripotent stem cell to differentiate. Provided. Here, the pluripotent stem cells are preferably somatic pluripotent stem cells including mesenchymal stem cells and hematopoietic stem cells.

  The pluripotent stem cells are preferably derived from bone marrow cells. However, the pluripotent stem cells of the present invention are not limited to those derived from bone marrow, but may be those derived from a tissue in which pluripotent stem cells are present.

  The factor involved in steroid synthesis is not limited to this, but is a transcriptional regulator of steroid hormone synthase. It is preferable that the transcription factor of this steroid hormone synthase is Steroidogenic factor 1 (SF-1).

  According to one embodiment of the present invention, the steroid hormone produced in the steroidogenic cells is pregnenolone, progesterone, deoxycorticosterone, corticosterone, 18 hydroxy. Corticosterone (18-hydroxycorticosterone), Aldosterone (aldosterone), 17α-hydroxypregnenolone (17α-hydroxypregnerone), 17α-hydroxyprogesterone (17α-hydroxyprogesterone), 11 deoxycortisol (or-deoxycortisol) sol), DHEA (dehydroepiandrosterone), androstenedione (androstenedione), estrone (estrone), androstenediol (androstenediol), testosterone (testosterone), is one or more of the steroid hormone consisting of estradiol (Estradiol).

  The steroid-producing cells secrete steroid hormones over a predetermined period. The predetermined period is preferably at least 2 weeks. As a result, the frequency of treatment can be reduced and the burden on the patient can be reduced.

  Furthermore, according to one embodiment of the invention, the steroidogenic cells are adrenocorticotropic hormone (ACTH) responsive. Also, ACTH responsiveness here is dose dependent. Thus, for example, when the steroidogenic bone marrow cells obtained in the present invention are transplanted to a patient with adrenal insufficiency, ACTH is secreted if the corticosteroid in the living body is insufficient, and the corticosteroid is obtained from the cell. If secreted and, conversely, in excess, ACTH is suppressed, and as a result, corticosteroid secretion can also be suppressed.

  According to one embodiment of the present invention, the steroid-producing cells produce gonadal steroid hormones by culturing in a retinoic acid-containing medium or a medium containing retinoic acid and hCG (human chorionic stimulating hormone). .

  According to a third main aspect of the present invention, (a) a step of preparing bone marrow cells, and (b) introducing a factor involved in steroid hormone synthesis in the bone marrow cells, There is provided a method for producing a steroidogenic cell, comprising the step of differentiating into a cell. This makes it possible to produce steroidogenic cells capable of secreting various steroid hormones effective for treatment.

  According to one embodiment of the present invention, the method further comprises culturing in a retinoic acid-containing medium, or a retinoic acid and hCG (human chorionic stimulating hormone) -containing medium, and induces differentiation into gonadal steroid-producing cells. This is a method for producing steroidogenic cells.

  According to a fourth main aspect of the present invention, there is provided a pharmaceutical composition comprising physiologically diverse steroid hormones secreted from the steroidogenic cells and a pharmaceutically acceptable carrier. According to the pharmaceutical composition thus obtained, it becomes possible to effectively treat steroid hormone secretion abnormalities such as steroid hormone deficiency and various self-exemptions.

  Further features and remarkable effects of the present invention will become apparent to those skilled in the art from the description of the embodiments of the present invention described below.

  Hereinafter, embodiments of the present invention will be described in detail.

  As described above, according to the present invention, a steroid-producing cell obtained by introducing a factor involved in a steroid synthase into bone marrow cells or pluripotent stem cells and differentiating the cells is provided.

Bone marrow cells and pluripotent stem cells Recently, many studies have suggested that bone marrow cell transplantation may contribute to the regeneration of hematopoietic and mesenchymal cell groups in various organs. In other words, bone marrow cells are thought to contain pluripotent progenitor cells that differentiate into various organs. Bone marrow stem cells are known to grow into hematopoietic and mesenchymal cells, but prior to the present invention, they were not known to differentiate into steroidogenic cells. However, the present inventors focused on the fact that bone marrow cells can be differentiated into muscles and fats derived from the mesenchymal system as well as the adrenal cortex / gonads, and obtained new findings that differentiate steroidogenic cells from bone marrow cells, As a result of conducting sincerity studies and experiments based on this, steroid-producing cells having several desirable properties described below were successfully produced.

  Here, the bone marrow cells and pluripotent stem cells are not limited to mammals such as humans and mice, as long as they can differentiate into steroidogenic cells. The cell collection method may be a known method.

Introduction of transcriptional regulatory factor of steroid synthase To differentiate bone marrow cells and pluripotent stem cells into steroidogenic cells, it is considered necessary to synthesize steroid hormones by introducing factors involved in steroid synthesis.

  Here, SF-1, which is a tissue-specific transcription factor of steroid hormone synthase, also known as adrena 4 binding protein (Ad4BP), has been reported to belong to the nuclear receptor superfamily and to be involved in many steroidogenic genes ( 1). SF-1 is believed to be particularly important for steroidogenesis and steroidogenic tissue growth, as a complete lack of adrenal glands and gonads was observed in this SF-1 knockout mouse.

  On the other hand, when SF-1 was continuously expressed in embryonic stem cells, the embryonic stem cells acquired steroid-producing ability, and P450scc was induced in a cAMP- and retinoic acid-dependent manner to produce progesterone. There are also reports. However, this ability to produce steroids is limited to the level of progesterone secretion, and addition of 20α-hydroxycholesterol, which is a foreign substrate and passes through the outer mitochondrial membrane, requires no physiological secretion. However, these results strongly suggest that SF-1 is key to steroidogenic cell differentiation.

  In order to verify whether steroidogenic cells are generated by introduction of SF-1 into bone marrow cells, the present inventors have created an adenovirus having bovine SF-1 (Adx-bSF-1) and derived from a mouse. It was revealed that a significant amount of various steroids were produced when Adx-bSF-1 was infected with long-term cultured bone marrow cells.

  However, in the present invention, the transcriptional regulatory factor is not limited to Steroidogenic factor 1 (SF-1), and may be any factor necessary for synthesizing steroid hormones in the cells.

  In the present invention, as a method for introducing and forcibly expressing a target gene in bone marrow cells, according to one embodiment of the present invention, for example, adenovirus vector, adeno-associated virus vector, retrovirus vector, lentivirus vector, HIV vector It is performed using a vector such as However, it can be carried out not only by a method using such a virus-derived vector but also by a known technique such as electroporation and a direct protein introduction method.

  Furthermore, the method for differentiating the bone marrow cells and the like into steroid-producing cells is considered to be not only the introduction of endogenous factors involved in steroid hormone synthesis but also the method of allowing exogenous factors to act. That is, the bone marrow cells can be differentiated into target steroid-producing cells by culturing them in a culture medium and medium containing exogenous factors.

Novel steroidogenic cells, their function and their use According to the novel steroidogenic cells of the present invention, the following pregnenolone, progesterone, deoxycorticosterone, corticosterone, 18 Hydroxycorticosterone (18-hydroxycorticosterone), aldosterone (aldosterone), 17α-hydroxypregnenolone (17α-hydroxypregnone), 17α-hydroxyprogesterone (17α-hydroxyprogesterone), 11 deoxycortisol (or 11 deoxycortisol) sol), DHEA (dehydroepiandrosterone), androstenedione (androstenedione), estrone (estrone), androstenediol (androstenediol), testosterone (testosterone), at least one or more is produced steroid hormones consisting of estradiol (Estradiol).

  According to the analysis of the present inventors, the production of steroid hormones by the steroidogenic cells according to the present invention is due to the increase in the amount of steroid hormone synthase in the bone marrow cells by introducing the synthase transcription regulatory factor. I think that the. That is, in the steroidogenic cells according to the present invention, it was found from an experiment using RT-PCR that the mRNA expression of the steroid hormone synthase is enhanced by forcibly expressing the transcription regulatory factor. Furthermore, an immunostaining experiment using an anti-cytochrome P450 scc antibody showed that the expression of the steroid hormone synthase is enhanced at the protein level.

  Further, the steroid-producing bone marrow cells obtained in the present invention are considered to require stable production of steroid hormones (at least 2 weeks, preferably 3 weeks or more) for a certain period or longer. Hormone production continued for at least 112 days. According to the analysis by RT-PCR, this long-term sustained steroidogenic ability is not due to the induction of endogenous mouse SF-1, but is due to the introduced adenovirus-derived bovine SF-1. It was confirmed. That is, even if the expression level of bovine SF-1 by adenovirus is low, it is sufficient for long-term and diverse steroid production of the bone marrow cells, or the SF-1 is necessary for initiation of steroid production. It may not be important for maintenance.

  The results of flow cytometry suggested that the steroidogenic cells originated from pluripotent and immature hematopoietic / mesenchymal stem cells.

  Furthermore, it was found that the novel steroid-producing cells can control steroid secretion in response to adrenocorticotropic hormone (ACTH). This ACTH responsiveness is also dose dependent. Also in this respect, the steroidogenic cells obtained by the present invention are considered beneficial. For example, when the steroidogenic bone marrow cells obtained in the present invention are transplanted for a patient with adrenal insufficiency, ACTH is secreted if the steroids of the living adrenal cortex are insufficient, and corticosteroids are secreted from the cells, Conversely, if it is excessive, ACTH is suppressed, and as a result, corticosteroid secretion can also be suppressed.

  The present inventors further examined how the novel steroidogenic cells are affected by the culture conditions. As a result of the experiment, it was clarified that the novel steroid-producing cells can produce gonadal steroid hormones by culturing them in a retinoic acid-containing medium or a retinoic acid and hCG (human chorionic stimulating hormone) -containing medium. In the prior art, retinoic acid was known to induce adrenal hormone (corticosterone) production, but it was not known prior to the present invention to induce gonadal hormone production.

  In addition, when the steroid-producing cells obtained in the present invention are transplanted, it is presumed that the steroid can be secreted at a high local concentration in the living body in vivo. That is, steroids are secreted as much as necessary at a necessary site, and steroid excess does not become systemic. Therefore, it is considered that no serious side effects are caused.

  Based on the above findings, by using the steroidogenic bone marrow cells obtained by the present invention using means such as autologous cell transplantation, steroid cell transplantation to steroid hormone secretion abnormalities such as steroid hormone deficiency, allergy / It is thought to provide new treatment methods such as autoimmune disease treatment and prevention of rejection of transplanted organs. That is, according to the present invention, there is provided a method for treating steroid hormone secretion abnormalities such as steroid hormone deficiency, autoimmune diseases, rejection of transplanted organs, etc. by transplanting the novel steroid-producing cells to a target site in the living body. Is done.

  Moreover, based on the said knowledge, the pharmaceutical composition characterized by having the steroid hormone secreted from the novel steroid-producing cell obtained above and a pharmaceutically acceptable carrier is provided. The pharmaceutical composition may be used as a “steroidal agent” for treating bronchial asthma, rheumatism, etc., in addition to external and internal use for atopic dermatitis. It can also be used for the treatment of steroid deficiency, allergies, autoimmune diseases and the like.

  Furthermore, it is speculated that the multipotency of bone marrow cells obtained in the present invention into adrenal or gonadal steroid-producing cells can be an important model for elucidating the tissue, site and cell-specific mechanism of adrenal / gonadal cell differentiation. Is done.

  In addition, as described above, the steroidogenic cells obtained in one embodiment of the present invention are presumed to be differentiated from pluripotent stem cells such as bone marrow stem cells, mesenchymal stem cells, and hematopoietic stem cells in bone marrow cells. Is done. As is known, mesenchymal stem cells are also present in fat and muscle. Therefore, it is considered that mesenchymal stem cells and hematopoietic stem cells can be differentiated into the steroidogenic cells of the present invention even if they are not derived from bone marrow cells. When it can be collected from other than the bone marrow, there are advantages that a wide choice and high convenience and safety can be expected.

  Next, an example of the production | generation of the novel steroid producing cell based on this invention is demonstrated in a following example.

  In order to verify whether steroidogenic cells are generated by introducing SF-1 into bone marrow cells, the present inventors first created an adenovirus having bovine SF-1 (Adx-bSF-1), Long-term cultured bone marrow cells were infected with Adx-bSF-1. Thus, it was clarified that a significant amount of various steroids were produced from the bone marrow cells, and steroidogenic cells were produced. Hereinafter, this verification process will be described in detail.

Production of Adenovirus Vector Containing SF-1 A recombinant (recombinant) vector derived from a human 5-adenovirus vector was produced using the Adenovirus Expression Vector Kit (Takara, Osaka, Japan).

  Bovine SF-1 / Ad4BP cDNA (distributed by Prof. Kenichiro Morohashi, National Institute for Basic Biology, Okazaki National Collaborative Research Organization) was digested with BamHI and EcoRI and inserted into the SwaI site of the recombinant cosmid vector pAxCAwt (Takara) having the CAG promoter. . The recombinant bovine SF-1 adenovirus vector (Adx-bSF-1) was prepared according to the manufacturing method based on the report of Miyake et al. (See Proc. Natl. Acad. Sci. USA 93: 132, 1996).

  As a negative control, an adenovirus vector into which only the β-galactosidase gene was transferred was constructed. This vector was named Adx-LacZ.

Collection of mouse bone marrow cells and long-term culture A 3-month-old male B6-GFP (green fluorescence protein) mouse (C57BL / 6Tg14 (act-EGFP) osbY01) was donated by Yamada (Kyoto University). In another experiment, 4-month-old male 129SVJ mice were also used. The bone marrow cells were cultured using conventional techniques with some adjustments. Briefly, fresh complete bone marrow cells were collected by flushing the bone marrow cells from the mice into the culture medium. Culture medium A consists of 2 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin, 0.0125 μg / ml amphotericin (Sigma-aldrich, Irvine, UK), 10-7 M hydrocortisone (Nikkenkayaku, Japan) and 20% donor horse serum Lot 6603F and 7307F, ICN Biochemicals, Aurora, Ohio). The collected cells were seeded in a 75 cm 2 tissue culture flask (Nalge Nunc, Rochester, NY) and incubated with culture medium A at 37 ° C., 5% CO 2. Adherent cells were cultured for several weeks, treated with trypsin, and stored at −80 ° C. in a cell bank until use. The stored BMCs were cultured in culture medium A for 120-180 days (passage number 12-18) with the goal of growing a relatively purified cell population as needed in the experiment. From this cell population, 5 × 10 5 BMCs were again seeded in a 60 mm Petri dish (Nunc) together with the culture medium A. When the BMCs grew sub-confluent, the cells were infected with adenovirus at about 10 plaque forming units / cell. As a control for all experiments, the BMCs were infected with a recombinant adenovirus expressing β-galactosidase (Adx-LacZ).

Measurement of steroid content in the culture solution secreted from the BMCs The following hormones secreted in the culture solution: progesterone (P4), deoxycorticosterone (DOC), corticosterone (B), 17α-hydroxyprogesterone The contents of (17α-OHP4), 11-deoxycortisol (S), dehydroepiandrosterone (DHEA), Δ4-androstenedione (Δ4-A), and testosterone (T) were measured using commercially available RIA kit (Diagnostic Products). Corp., LA) using SRL Co. Ltd .. (Tokyo, Japan) and joint research (each specific RIA system was developed by SRL). The amount of P4 and DOC secreted into the culture medium was also confirmed in the presence / absence of synthetic 1-24ACTH (Shionomi Co., Osaka, Japan). The detection limits of P4, DOC, B, 17α-OHP4, S, DHEA, Δ4-A, and T are 0.1 ng / ml, 0.02 ng / ml, 20.0 ng / ml, 0.1 ng / ml, respectively. 0.04 ng / ml, 0.2 ng / ml, 0.1 ng / ml, and 0.05 ng / ml or less.

Immunocytochemistry
Immunocytochemistry studies of BMCs from 129VJ mice were performed with anti-rabbit cytochrome P450scc antibody (RDI, NJ) using Zenon rabbit IgG labeling kit (Molecular probes, Inc., OR) or with preimmune serum as a control. . The cells were seeded on a collagen type I membrane (Asahi technoglass, Tokyo, Japan) in a 35 mm dish and fixed with 4% paraformaldehyde at 4 ° C. for 1 hour. After that, the manufacturer's instructions were followed. Fluorescence was observed using a fluorescence microscope (BX-51; Olympus, Tokyo, Japan).

Real-time PCR quantitative StAR, ACTH receptor (ACTH-R), and quantitative analysis of various steroidogenic enzymes including P450scc, P450c17, P450C11, P450C21, P450ald, 3β-HSD, and 17β-HSD type 3, LightCycler (Poche Diagnostics) GmbH, Mannheim, Germany). Total RNA was isolated from the cultured BMCs and Y-1 cells using RNasy mini kit (Qiagen) and from mouse testis and adrenal gland using Isogen (Wako Pure Chemical Industries, Osaka, Japan). First-strand complementary DNA was synthesized using 5 μg of total RNA as a template, and PCR was performed on the LightCycler according to the manufacturer's instructions. Sense / antisense primers used were reported by Mukai et al. (Conditionally imprinted and adrenocorative cell lines and undifferentiated states 69-81). PCR conditions are available upon request. LightCycler Software Ver. The threshold was measured when the fluorescence intensity determined in 3.5 was in the geometric phase of amplification. The product was subjected to a 2% agarose gel. The nucleotide sequence of each PCR product was confirmed by direct sequencing using appropriate primers. The relative expression levels of the mRNA were calibrated against their β-actin and its ratio to mouse adrenal cortex Y-1 cells, or mouse testis or adrenal gland.

Flow cytometry The basic protocol of flow cytometry followed a known method. Briefly, 3X105 BMCs were transformed into PE (phycoerythrin) binding anti-mouse c-kit, CD11b, CD34, CD44, CD45, and Sca-1 monoclonal antibodies (BD Biosciences, Japan), or isotype-compatible PE binding rat IgG (BD Biosciences). ) And 4 ° C for 30 minutes. The cells were analyzed with a FACScan flow cytometer (Becton Dickinson).

Statistical 1-factor (1 factor) ANOVA (ANOVA) was used for statistical evaluation. P <0.05 was considered statistically significant.

Infection of long-term cultured bone marrow cells with Adx-bSF-1 (1) Measurement of steroid content in culture medium secreted from BMCs The present inventors generated steroidogenic cells by introducing SF-1 into bone marrow cells. In order to verify whether this is the case, an adenovirus (Adx-bSF-1) having bovine SF-1 was prepared using the method described above. As a result of experiments, the present inventors have revealed that various steroids are produced when long-term cultured bone marrow cells are infected with Adx-bSF-1.

  Long-term (123 days) cultured bone marrow cells of male GFP mice were infected with Adx-bSF-1 or Adx-LacZ as a control and cultured for 7 days. Thereafter, the amount of steroid in the culture medium cultured for 4 days was measured. The result is shown in FIG. 1 (a) to (h) show progesterone (P4), deoxycorticosterone (DOC), corticosterone (B), and 17α-hydroxyprogesterone (17α) in the culture solution of long-term cultured BMCs from GFP mice, respectively. -OHP4), 11-deoxycortisol (S), dehydroepiandrosterone (DHEA), [Delta] 4-androstenedione ([Delta] 4-A), and testosterone (T) basal secretion levels are shown. The values in the figure are shown as mean ± SD (n = 3). S and L indicate BMCs transfected with Adx-bSF-1 and BMCs transfected with Adx-LacZ, respectively. As shown in FIG. 1, Adx-bSF-1-infected bone marrow cells contain significant amounts of progesterone (P4), deoxycorticosterone (DOC), corticosterone (B), 17α-hydroxyprogesterone (17α-OH P4 ), 11-deoxycortisol (S), dehydroepiandrosterone (DHEA), Δ4-androstenedione (Δ4-A), and testosterone (T), but not Adx-LacZ-infected cells. In Adx-LacZ infected control cell cultures, no steroids except S were detected. The trace amount of S detected in the control culture is probably a cross-reaction (9.5%) of S antibody to hydrocortisone and no steroid precursor is produced.

Effect of Adx-bSF-1 expression on bone marrow cells on steroid hormone synthase (1) Measurement by RT-PCR (effect on mRNA expression)
Next, Real-time reverse transcriptase-polymerase chain reaction (RT-PCR) of the cells used in the above experiment was performed. The result is shown in FIG. 2A to 2F are graphs showing the results of real-time PCR of StAR, P450scc, 3β-HSD, P450c11, P450c17, 17β-HSD type 3 and ACTH-R, respectively. Furthermore, the actual specific PCR band in an agarose gel amplified over 40 cycles and stained with ethidium bromide is shown below the figure. Relative mRNA expression levels were calibrated with β-actin. S and L indicate BMCs transfected with Adx-bSF-1 and BMCs transfected with Adx-LacZ, respectively. No significant PCR products of StAR, P450scc, 3β-HSD, P450c11, and 17β-HSD type 3 were obtained from the target cells infected with Adx-LacZ. Values were expressed as mean ± SD (n = 3). * Indicates P <0.05. From these graphs, steroid hormone synthesizing enzyme, stereogenic accurate regulatory protein (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), P450c11, P450c17, and 17β-HSD type Axb 1 was observed in bone marrow cells on day 11 of infection. On the other hand, it was not observed in Adx-LacZ-infected cells. P450ald mRNA could not be verified. Adrenocorticotropic hormone receptor (ACTH-R) was also expressed in Adx-LacZ infected cells, but the expression level was very low (2/1000 of adrenal expression), but more in Adx-bSF-1 infected cells. Declined.

  That is, the relative ratio to the expression of control Y-1 cells is expressed for StAR, P450scc, and 3β-HSD; for mouse adrenal gland, it is expressed for P450c11 and ACTH-R; Is expressed in the case of P450c17 and 17β-HSD type 3. No significant PCR products of StAR, P450scc, 3β-HSD, P450c11, and 17β-HSD type 3 were obtained from the target cells infected with Adx-LacZ. From the above results, it was found that expression of bSF-1 in the bone marrow cells enhances steroid hormone synthase mRNA expression.

  Similar results were observed in 129VJ mice, suggesting little difference by pedigree (data not shown).

(2) Observation by immunocytochemistry (effect on protein expression)
The results of immunostaining of 129VJ mouse bone marrow cells using the antibodies described above are shown in FIG. FIG. 3 revealed the expression of P450scc, which is a steroid hormone synthase. Green fluorescent cells are positive for P450scc. As a negative control, preimmune serum and cells did not react (data not shown). That is, it was found that the expression level of steroid hormone synthase protein is enhanced by expressing bSF-1 in the bone marrow cells.

  In the present invention, steroidogenic cells simultaneously produce a mixture of adrenal and gonadal steroids, that is, DOC, B, DHEA, Δ4-A, and T. Since P450c17, which is a steroid hormone synthase, is expressed in the human adrenal gland but not in the mouse adrenal gland, the marked expression of P450c17 in the bone marrow cells in the present invention is that the mixed production of steroids exceeds the species. It is suggested that it occurs.

  From these results, the bone marrow cells have pluripotency in differentiation into steroid-producing cells, and there is a common origin of steroid-producing tissues; that is, the bone marrow cell-derived steroid-producing cells obtained in the present invention are stem cells. One possibility was suggested. Therefore, this assumption was examined using flow cytometry.

Examination of whether steroidogenic cells differentiated from bone marrow cells are (hematopoietic / mesenchymal) stem cells (1) Analysis of bone marrow cell surface marker by flow cytometry The bone marrow cell surface marker is analyzed using flow cytometry The result of analysis is shown in FIG. The flow cytometry experiment was performed prior to adenovirus infection using the same BMCs used in the experiment of FIG. 4A to 4F show cell surface markers c-kit, CD11b, CD34, CD44, CD45, and Sca-1, respectively. From the experimental results, CD45 specific for hematopoietic cells and CD34 specific for hematopoietic progenitor cells were negative. Also, CD11b, a marker for monocytes and macrophages, was negative. The marker for mouse mesenchymal stem cells has not been fully clarified and is controversial, but the potential marker CD44 was negative in the bone marrow cells. On the other hand, hematopoietic and mesenchymal stem cell / progenitor cell markers c-kit and Sca-1 were positive.

  Although the bone marrow cells obtained in the present invention are heterogeneous, steroidogenic cells were probably pluripotent and originated from immature stem cells. In addition, long-term cultured bone marrow cells differentiated into osteoblasts stained with alkaline phosphatase by treatment with 0.05 mM ascorbic acid, 10 mM β-glycerophosphate, and 0.1 μM dexamethasone (data not shown). This proves that long-term cultured bone marrow cells have mesenchymal properties.

Since the expression of ACTH-R was confirmed in confirming ACTH reactivity of long-term cultured bone marrow cells, ACTH reactivity of bone marrow cells was examined by measuring P4 and DOC. Long-term (100 days) cultured bone marrow cells of male GFP mice were infected with Adx-bSF-1 or Adx-LacZ and stimulated with 2.4 nM to 2.4 μM ACTH every 3-4 days after infection. Steroids were measured. After infection with Adx-bSF-1 or Adx-LacZ (Day 0), cells were treated with 2.4 nM to 2.4 μM ACTH on days 0, 4, 7, 11, 14, 18, 21, 25, 28 It was done. Prior to the addition of ACTH, the culture medium was collected and the steroid concentration was measured. FIG. 5 is a graph showing the results. FIGS. 5A and 5B show the effect of ACTH on the secretion of progesterone (P4) and DOC from cultured BMCs collected from GFP mice, respectively. Values in the figure are shown as mean ± SD (n = 3); a, b, c, d, and e indicate ACTH of 0, 2.4, 24, 240 nM and 2.4 μM, respectively. . * Indicates P <0.05 and ** indicates P <0.01 with respect to the control (in the absence of ACTH). From the experimental results, ACTH produced these steroids in a dose-dependent manner in Adx-bSF-1-infected bone marrow cells. However, it was not generated in Adx-LacZ infected cells (data not shown).

  These results indicate that introduction of bSF-1 into long-term cultured bone marrow cells differentiates into steroid-producing cells that secrete various steroids in the ACTH-stimulated state as well as the basal state.

  In addition, mRNA induction of steroidogenic enzymes P450scc, 3β-HSD, P450c21, P450c11 and 17β-HSD by 2.4 μM ACTH was also confirmed by real-time PCR. However, no induction of ACTH-R mRNA was confirmed (FIG. 6). Cells used in the experiment were treated with 2.4 μM ACTH on days 0, 4 and 7 after infection with Adx-bSF-1 (Day 0) and cultured for 4 days. On day 11, the total RNA of the cells was extracted and real-time PCR was performed. 6 (a) to 6 (f) respectively show P450scc, 3β-HSD, P450c21, P450c11, and 17β-HSD type 3 in the presence of 2.4 μM ACTH (lower right oblique line) and absence (white column). FIG. 5 shows the expression of mRNA of ACTH-R. The values in the figure are shown as mean values ± SD (n = 3). Relative mRNA expression levels were calibrated with β-actin. The relative ratio to the expression of control Y-1 cells is expressed for P450scc and 3β-HSD; for mouse adrenal glands it is expressed for P450c21, P450c11, and ACTH-R; for mouse testis It is expressed in the case of 17β-HSD type 3. * Indicates P <0.05 and ** indicates P <0.01 with respect to the control (in the absence of ACTH).

Measurement of long-term steroidogenic ability from bone marrow cells The duration of bone marrow cell steroid production by Adx-bSF-1 infection was examined. Long-term (180 days) cultured bone marrow cells were infected with Adx-bSF-1 or Adx-LacZ, and P4 and DOC in the culture were measured every 3-4 days. The results are shown in FIG. 7. FIGS. 7 (a) and 7 (b) show the basal secreted amounts of progesterone (P4) and deoxycorticosterone (DOC) in the culture medium of long-term cultured BMCs from GFP mice, respectively. Indicates the time course (time course). The value in the figure shows the average value of the duplicate (duplicate) petri dish. The black column shows the steroid secreted from cells infected with Adx-bSF-1. Secretion of P4 and DOC was not detectable in cultures from BMCs infected with Adx-LacZ (data not shown). From FIG. 7, it was clear that the production of P4 and DOC lasted for at least 112 days. Considering the half-life of adenovirus (2-3 weeks), this long-term steroid production is beyond expectations.

  Moreover, RT-PCR of bSF-1 was performed using RNA extracted from the cells obtained on day 0, 14, 21, and 49 in FIG. Electrophoresis was performed using a 1.5% agarose gel and ethidium bromide was used for staining. FIG. 8 shows the results. Y-1, V, (−), S and L are Y-1 cells (negative control), Adx-bSF-1 (positive control), BMCs before infection, Adx, respectively. -Shows BMCs transfected with bSF-1 and BMCs transfected with Adx-LacZ. In the experiment of expression of bSF-1 derived from Adx-bSF-1, the expression of endogenous mouse SF-1 could not be confirmed, and only adenovirus-derived bovine SF-1 was detected (FIG. 8). This result suggested that long-lasting steroidogenesis was not derived from endogenous mouse SF-1, but was caused by adenovirus-derived bovine SF-1.

Induction of differentiation into gonadal hormone-producing cells The present inventors further examined how the steroid-producing cells of the present invention are affected by the culture conditions. In the same manner as described above, long-term cultured bone marrow cells (LTBMCs) are infected with a bovine SF-1 expression virus or a control virus, and retinoic acid (ATRA), retinoic acid and hCG (human chorionic stimulating hormone) are infected with the virus. And the hormone of the culture medium added at the time of culture medium exchange (day 0, 4, 7, 11, 14) and culture | cultivated to day 14-18 was measured. The results are shown in FIGS. 9A and 9B (when ATRA is added to the medium) and FIGS. 10A and 10B (when ATRA and hCG are added to the medium). The experimental results revealed that production of corticosterone (adrenal hormone) and testosterone (gonad hormone) was promoted. Particularly, since the production of testosterone was remarkably promoted (from FIG. 9B), it was suggested that the steroid-producing cells of the present invention were induced to differentiate into gonadal hormone-producing cells by culturing in a retinoic acid-containing medium. It was.

  Although it is known that retinoic acid stimulates adrenal cells and promotes the production of adrenal hormones, it was first demonstrated by the present invention to induce the production of gonadal hormones.

  While certain preferred embodiments and examples of the invention have been described and illustrated above, the invention is not limited to these embodiments or examples, and does not change the gist of the invention Various modifications can be made within the range.

1 (a) to (h) show progesterone (P4), deoxycorticosterone (DOC), corticosterone (B), and 17α-hydroxyprogesterone (17α) in the culture solution of long-term cultured BMCs from GFP mice, respectively. The graph which shows the basal secretion amount of -OHP4), 11-deoxycortisol (S), dehydroepiandrosterone (DHEA), (DELTA) 4-androstenedione ((DELTA) 4-A), and testosterone (T). 2A to 2F are graphs showing the results of real-time PCR of StAR, P450scc, 3β-HSD, P450c11, P450c17, 17β-HSD type 3 and ACTH-R, respectively. FIG. 3 shows an immunostained image of BMCs collected from 129VJ mice using an anti-cytochrome P450scc antibody. FIGS. 4A to 4F show the results of flow cytometry analysis of c-kit, CD11b, CD34, CD44, CD45, and Sca-1 expression, which are surface markers in cultured BMCs, respectively. 5 (a) and 5 (b) are graphs showing the effect of ACTH on the secretion of progesterone (P4) and DOC from cultured BMCs collected from GFP mice, respectively. 6 (a) to 6 (f) respectively show P450scc, 3β-HSD, P450c21, P450c11, and 17β-HSD type 3 in the presence of 2.4 μM ACTH (lower right oblique line) and absence (white column). , And the graph which shows real-time PCR of ACTH-R. FIGS. 7 (a) and 7 (b) are graphs showing the time course (time course) of the basal secretion amount of progesterone (P4) and deoxycorticosterone (DOC) in the culture medium of long-term cultured BMCs from GFP mice, respectively. FIG. 8 is a diagram showing the expression of AdSF-bSF-1-derived bSF-1 in an agarose gel. FIG. 9 shows corticosterone production (a) and testosterone production (b) when Adx-bSF-1-infected BMCs collected from 129VJ mice were cultured in a medium containing retinoic acid (concentration 0-10 ⁻⁴ M). ). FIG. 10 shows the amount of corticosterone produced when Adx-bSF-1-infected BMCs collected from 129VJ mice were cultured under various culture conditions (including cortisol, retinoic acid, and hCG (human chorionic gonadotropin)). The figure which shows a) and testosterone production amount (b).

Claims (20)

  A steroidogenic cell characterized by being differentiated from the bone marrow cell by introducing a factor involved in steroid hormone synthesis into the bone marrow cell.   A steroidogenic cell differentiated from the pluripotent stem cell by introducing a factor involved in steroid hormone synthesis into the pluripotent stem cell. In the steroidogenic cell according to claim 2,
A steroidogenic cell, wherein the pluripotent stem cell is derived from a bone marrow cell. In the steroidogenic cell according to claim 2,
The pluripotent stem cells are somatic pluripotent stem cells including mesenchymal stem cells and hematopoietic stem cells. In the steroidogenic cell according to claim 1 or 2,
A steroid-producing cell, wherein the factor involved in steroid hormone synthesis is a transcriptional regulator of steroid hormone synthase. In the steroidogenic cell according to claim 5,
A steroid-producing cell, wherein the transcriptional regulatory factor of the steroid hormone synthase is Steroidogenic factor 1 (SF-1). In the steroidogenic cell according to claim 1 or 2,
The steroid producing cell is characterized in that it secretes a steroid hormone for a predetermined period or more. In the steroidogenic cell according to claim 7,
The steroid-producing cell, wherein the predetermined period is at least 2 weeks. In the steroidogenic cell according to claim 1 or 2,
The steroid-producing cell is a corticotrophic hormone (ACTH) -responsive steroid-producing cell. In the steroidogenic cell according to claim 9,
A steroidogenic cell, wherein the ACTH reactivity is ACTH dose-dependent. In the steroidogenic cell according to claim 1 or 2,
The steroidogenic cells are pregnenolone, progesterone, deoxycorticosterone, corticosterone, 18-hydroxycorticosterone, 18-hydroxycorsterosterone (17α-hydroxypreglone), 17α hydroxyprogesterone (17α-hydroxyprogesterone), 11 deoxycortisol (11-deoxycortisol), cortisol (cortisol), DHEA (dehydroepiandrosterone) Steroid production characterized in that it produces at least one steroid hormone consisting of, androstenedione, estrone, androstenediol, testosterone, estradiol cell. In the steroidogenic cell according to claim 1 or 2,
The steroid-producing cells are those that produce gonadal steroid hormones by culturing in a retinoic acid-containing medium. In the steroidogenic cell according to claim 1 or 2,
The steroid-producing cell is a steroid-producing cell that produces a gonadal steroid hormone by culturing in a medium containing retinoic acid and hCG (human chorionic stimulating hormone). (A) preparing bone marrow cells;
(B) introducing a factor involved in steroid hormone synthesis into the bone marrow cells, and causing the bone marrow cells to differentiate into steroid-producing cells. The method for producing a steroidogenic cell according to claim 14,
A method for producing a steroid-producing cell, further comprising the step of culturing the bone marrow cell in a retinoic acid-containing medium, wherein the bone marrow cell is induced to differentiate into a gonadal steroid-producing cell. The method for producing a steroidogenic cell according to claim 14,
A method for producing a steroid-producing cell, further comprising the step of culturing the bone marrow cell in a medium containing retinoic acid and hCG (human chorionic stimulating hormone), and induction of differentiation into a gonadal steroid-producing cell.   A pharmaceutical composition comprising the steroid hormone secreted from the steroidogenic cell according to claim 1 or 2 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 17,
The said pharmaceutical composition is for treating steroid hormone secretion abnormality, The pharmaceutical composition characterized by the above-mentioned. The pharmaceutical composition according to claim 17,
The said pharmaceutical composition is for treating an autoimmune disease, The pharmaceutical composition characterized by the above-mentioned. The pharmaceutical composition according to claim 17,
The said pharmaceutical composition is used as an immunosuppressant at the time of organ transplantation, The pharmaceutical composition characterized by the above-mentioned.

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