JP2007202435A - Smooth muscle stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide smooth muscle stem cells, especially uterine muscle stem cells, and to provide methods for utilizing the stem cells. <P>SOLUTION: The smooth muscle stem cells, which are derived from mammal smooth muscle, can be obtained as side population cells when smooth muscle cells constituting the uterine muscle of mammal, especially humans, are stained with Hoechst 33342 and analyzed by flow cytometry with the fluorescence intensity of Hoechst Red and Hoechst Blue as indicators. The smooth muscle stem cells have the ability to differentiate into smooth muscle by their transplantation to smooth muscle. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a tissue stem cell derived from smooth muscle, an isolation method and a culture method thereof, and use thereof.

  In recent years, it has become clear that unique living body (tissue) stem cells existing in various organs and tissues are responsible for the maintenance and regeneration of the respective organs and tissues. To date, stem cells derived from various tissues have been reported, and stem cells have been isolated.

  In addition, a method for isolating tissue stem cells has also been reported (see Non-patent Document 1). In this method, a cell population that is not stained with DNA binding dye Hoechst33342 (Hoechst is a registered trademark) is referred to as “side population (SP)”. It has been collected as cells and confirmed that the cell population exhibits high tissue stem cell activity.

  Examples of stem cells isolated so far include skeletal muscle stem cells, cardiac muscle stem cells, liver stem cells, neural stem cells, pancreatic stem cells, epidermal stem cells, adipose tissue stem cells and the like.

  Regenerative medicine is currently attracting a great deal of attention as a medicine that restores and restores the function of human cells and tissues that have been damaged or lost due to diseases or trauma. If it becomes possible to isolate and identify the above tissue stem cells and replicate and proliferate, it is considered that regenerative medicine will be possible.

  As described above, stem cells have been isolated in various tissues and there are increasing reports on stem cells, but there are no reports on smooth muscle stem cells. To date, even attempts to isolate and identify stem cells have been conducted. I have not been told.

Experimental Medicine Special Issue Forefront of stem cell research and its application to regenerative medicine Vol.19, No.15, 2002, p.68-73

  An object of the present invention is to provide smooth muscle stem cells, particularly uterine muscle stem cells, and to provide utilization of those stem cells.

  Uterus muscle, a type of smooth muscle, is a very unique tissue known to show a marked increase and an increase in the number of cells during pregnancy and delivery, and to undergo rapid apoptosis during the postpartum period.

  The present inventors focused on a series of events such as increase in the number of cells and apoptosis from pregnancy to postpartum, and considered the possibility that tissue stem cells exist in uterine smooth muscle, which is the main constituent tissue of myometrium. It was.

Therefore, the present inventors collected normal myometrium, obtained dispersed cells by enzyme treatment, stained with Hoechst33342, and then used side population (SP) from the dispersed cells for myometrial SP (Myometrial SP: MSP). ) Isolated by FACS.
The present inventors have further found that isolated MSP can be cultured alone under hypoxic conditions.

  Next, the cell cycle of uterine myocytes other than MSP and MSP (non-MSP) and the expressed genes and proteins were analyzed to confirm that they had the characteristics of stem cells. Furthermore, we found that MSP can be induced to differentiate into bone and adipocytes in vitro, and that when MSP is transplanted into immunodeficient mice, myometrial tissue can be reconstructed. It was confirmed to be a stem cell.

That is, the present invention is as follows.
[1] Smooth muscle stem cells derived from mammalian smooth muscle.
[2] The smooth muscle stem cells according to [1], which can be obtained as side population cells when mammalian smooth muscle cells are stained with Hoechst33342 and analyzed by flow cytometry using the fluorescence intensity of Hoechst red and Hoechst blue as an index.
[3] The smooth muscle stem cell according to [1] or [2], wherein the smooth muscle is a uterine muscle.
[4] The smooth muscle stem cell according to any one of [1] to [3], wherein the mammal is a human.
[5] The smooth muscle stem cell according to any one of [1] to [4], which has the ability to differentiate into smooth muscle by transplanting into smooth muscle.
[6] The smooth muscle stem cell of [5] derived from uterine muscle having the ability to differentiate into uterine muscle by transplanting into the uterine muscle.
[7] Obtain dispersed cells from smooth muscle tissue, and subject the dispersed cells only to staining with Hoechst33342, then subject to flow cytometry, and collect side population cells using fluorescence intensity of Hoechst red and Hoechst blue as indicators. A method of isolating smooth muscle stem cells.
[8] A method for culturing and smoothing smooth muscle stem cells, comprising culturing the smooth muscle stem cells according to any one of [1] to [6] under conditions where the culture oxygen concentration is 2.5% or less.
[9] The method for culturing and proliferating smooth muscle stem cells according to [8], wherein the cultured oxygen concentration is 2%.
[10] A method for inducing differentiation of smooth muscle stem cells according to any one of [1] to [6] into smooth muscle cells in vitro.
[11] A composition for regenerating smooth muscle tissue comprising the smooth muscle stem cells of any one of [1] to [6].

  As shown in the examples, MSP has tissue stem cell characteristics such as 1) differentiation state, 2) pluripotency, and 3) self-organization ability. Therefore, MSP is a myometrial tissue stem cell. We succeeded in isolating tissue stem cells of myometrium for the first time in the world. The isolated MSP is used to analyze the etiology of uterine muscle-derived diseases such as uterine fibroids, cell mechanisms responsible for uterine muscle growth, regression, and functional expression during pregnancy and parturition. It becomes a useful biological resource. MSP cells are also expected to be applied clinically as cell materials in the treatment of other organs.

  The stem cells of the present invention are smooth muscle-derived tissue stem cells isolated from smooth muscle. Examples of the smooth muscle from which the stem cells of the present invention are derived include uterine smooth muscle, visceral smooth muscle and vascular smooth muscle, preferably uterine smooth muscle. These tissue pieces are collected, the cells are dispersed, and stem cells can be isolated from the dispersed cells. For example, a uterine fibroid piece can be used. The animal species from which the smooth muscle is collected is not limited, and mammals such as mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans can be used.

  In the present invention, the “stem cell” refers to a cell having self-renewal ability and pluripotency. Stem cells are usually able to regenerate the tissue when the tissue is damaged. Unlike embryonic stem cells, tissue stem cells exist at specific positions in the tissue and have an undifferentiated intracellular structure. Tissue stem cells have a high nucleus / cytoplasm ratio and poor intracellular organelles. Tissue stem cells generally have pluripotency, have a slow cell cycle, and maintain proliferative ability over the life of the individual. In the present invention, the term “stem cell” refers to a cell population containing at least a certain amount of stem cells, and includes, for example, a cell population containing 90% or more, preferably 95% or more of stem cells.

  Cell dispersion can be performed by chopping tissue pieces and treating them with an enzyme such as collagenase. Since dispersed cells are isolated by flow cytometry (FACS), it is desirable to disperse them to a single cell state. For example, a cell strainer or the like is used, and a specific gravity solution such as Ficoll-Paque (registered trademark) is further added. It is possible to disperse as a single cell by performing density gradient centrifugation using the solution and further performing trypsin treatment or the like. Conventionally, cells collected from tissues have been cultured and used for research, but in the method for isolating stem cells of the present invention, cells are obtained and dispersed from smooth muscle tissue, and the dispersed cells are directly subjected to flow cytometry. To analyze.

  The stem cells of the present invention can be obtained as side population cells (SP cells) when dispersed smooth muscle cells are separated by flow cytometry. For isolation of stem cells of the present invention, tissue cells are stained with Hoechst33342 (Hoechst is a registered trademark). Hoechst33342 is a fluorescent dye incorporated into the AT base sequence of DNA, has strong cell membrane permeability, and can stain cells in a live state. Hoechst33342 emits fluorescence with two wavelengths of around 424/44 nm and around 670 nm, and cells incorporating Hoechst33342 can be separated using flow cytometry with the fluorescence intensity at 424/44 nm and 670 nm as an index. Stem cells excrete Hoechst33342 out of the cell by a pump-like molecule that is an ABC transporter, resulting in lower fluorescence intensity at 424/44 nm and 670 nm than other cell populations. When two-dimensional analysis of the fluorescence intensity of 424/44 nm (Hoechst blue) on the Y-axis side and 670 nm (Hoechst red) on the X-axis side is performed by flow cytometry, normal G0 / G1 phase, S / G2 In addition to the / M phase fraction, there is a prominent cell population with lower fluorescence intensity than the G0 / G1 fraction, and this fraction protrudes slightly laterally in two dimensions compared to other fractions. Is called side population (SP) cells (FIG. 1). SP cells are described in detail in Experimental Medicine, Special Issue, Frontier of Stem Cell Research and Application to Regenerative Medicine Vol.19, No.15, 2002, p.68-73. Many stem cells are contained in SP cells, and the stem cells of the present invention can be isolated and recovered as SP cells. In the present invention, for example, uterine muscle SP cells are referred to as MSP (myometirial side population) cells. SP cells may contain cells other than stem cells. However, as described above, in the present invention, the stem cell refers to a cell aggregate containing at least a certain amount of stem cells, and the stem cell includes SP cells. Isolation of SP cells can be performed, for example, as described in J. Experimental Medicine, 183, 1797-1806 (1996).

  The smooth muscle stem cells of the present invention remain in the G0 phase of the cell cycle. The cell cycle can be determined by staining cells with Pyronin Y and Hoechst33342 and analyzing by flow cytometry.

  The smooth muscle stem cells of the present invention further express stem cell markers ABCG2 and CD34, and can be isolated using flow cytometry using these surface-expressed antigens as indicators.

  In addition, when smooth muscle SP cells are analyzed using flow cytometry by the above method, smooth muscle markers calponin (calponin) and smoothin ( The expression of smoothelin) is significantly low.

  The isolated stem cell of the present invention can be grown by culture. In this case, the medium to be used is not limited, and a known medium (for example, DMEM (Dulbecco's modified Eagle's medium) medium) can be used. In particular, it is desirable to use a known stem cell culture medium. For example, mesenchymal stem cell basal medium (MSCBM)) (Cambrex Bio Science), mesenchymal stem cell growth medium (MSCGM) (Cambrex Bio Science), POWEREDBY10 medium (Med Jyotori Co., Ltd.) ), PLUSOID-M (Med Jotori Co., Ltd.), etc. can be used. To the medium, serum such as fetal bovine serum, antibiotics such as penicillin and streptomycin, and various physiologically active substances may be appropriately added. In addition, when culturing the stem cells of the present invention, the culturing oxygen concentration is carried out under a condition that is lower than 20% during normal culturing. Preferably it is 5% or less, more preferably 2.5% or less, particularly preferably 2%. By culturing by this method, stem cells can be proliferated. The cells may be seeded and cultured in a culture dish for tissue culture.

  Furthermore, the isolated stem cells of the present invention can be induced to differentiate into specific tissue cells in vitro. Since the stem cell of the present invention has pluripotency, it can basically be induced to differentiate into any tissue cell. Differentiation-inducing media for various tissues are commercially available, and differentiation can be induced using these commercially available media. For example, when inducing differentiation into bone cells, a bullet kit osteoblast differentiation medium (Cambrex Bio Science) may be used. When inducing differentiation into adipocytes, a bullet kit adipocyte differentiation medium (Cambrex Bio Science) is used. What is necessary is just to culture | cultivate using a bullet kit chondrocyte differentiation medium (Cambrex Bio Science), when differentiation-inducing to a chondrocyte. The smooth muscle stem cells of the present invention can also be induced to differentiate into smooth muscle tissue cells such as uterine muscle tissue cells. At this time, the oxygen concentration is performed under normal cell culture conditions, that is, about 20%. Cells differentiated into these tissue cells can be further cultured to construct a tissue.

  Whether or not stem cells have been induced to differentiate into tissue cells can be determined by examining the expression of markers specific to each tissue cell. For example, differentiation into bone cells can be examined by the presence or absence of alkaline phosphatase staining positive cells, and differentiation into adipocytes can be examined by the presence or absence of oil red O staining positive cells.

  Furthermore, the present invention includes cell lines in which the smooth muscle stem cells of the present invention are immortalized. Cell immortalization can be performed by treatment with radiation, mutagens, viruses, etc., and also by introducing human telomerase reverse transcriptase gene (hTERT) or SV40T gene into cells. be able to. The cell line has both proliferative ability and pluripotency, and can be used by growing as many times as necessary. The established cell line can be used as a research tool for smooth muscle differentiation and development.

  The smooth muscle stem cells of the present invention can be used for regenerative medicine and the like. For example, by transplanting and administering the smooth muscle stem cells of the present invention to a damaged smooth muscle site by injection using a syringe, the stem cells are differentiated into smooth muscle cells, and the damaged smooth muscle can be regenerated. Further, as described above, the stem cells of the present invention may be induced to differentiate in vitro, a tissue is constructed, and the tissue may be transplanted. In these regenerative medicine, in order to avoid rejection of the transplanted cells or tissues by the recipient, a tissue piece is collected from a patient who is going to receive regenerative medicine, and the smooth muscle stem cell of the present invention is isolated from the tissue piece. It is desirable to use. For example, due to a disease such as uterine sarcoma, a tissue piece is collected from the remaining myometrium of a patient whose part of the uterus has been excised, and myometrial stem cells are isolated from the tissue piece to regenerate the uterus of the patient. It is possible to use it. Further, as described above, the smooth muscle stem cells of the present invention can be differentiated into other tissues. For example, a tissue piece is removed from a smooth muscle such as a uterine muscle from a patient who has damaged a specific tissue other than smooth muscle, and smooth muscle stem cells are isolated and induced to differentiate into damaged tissue. It can be used for regenerative medicine of tissues other than muscles. The present invention also includes a composition for regenerative medicine containing the stem cells of the present invention, that is, a preparation for regenerative medicine.

  Furthermore, a model animal partially having human smooth muscle tissue can be obtained by transplanting human-derived stem cells of the present invention into an immunodeficient animal other than human. For example, by transplanting human-derived smooth muscle stem cells of the present invention into the smooth muscle of an immunodeficient animal, the stem cells of the present invention differentiate into smooth muscle, and a human smooth muscle tissue is partially constructed in the animal body. The fact that human smooth muscle tissue has been constructed in the animal body may be confirmed by measuring proteins specific to human smooth muscle expressed by cells and morphologically having smooth muscle characteristics. For example, when the smooth muscle is a uterine muscle, it may be confirmed that it is vimentin-positive, that is, derived from a human and is α-SMA-positive cell and morphologically uterine smooth muscle. The animal partially having human smooth muscle tissue thus obtained can be used as a model animal having human smooth muscle tissue. For example, when the smooth muscle is a myometrium, it can be used for screening for an agent that contracts the uterus or relaxes the uterus. In addition, when the smooth muscle stem cells of the present invention are transformed into cancer and transplanted, a model animal having human uterine cancer or the like can be produced and used for screening for therapeutic agents. Immunodeficient animals include immunodeficient mice such as scid mice.

  The present invention will be specifically described by the following examples, but the present invention is not limited to these examples.

Example 1 Preparation of Myometrial Side Population (MSP) Cells (1) Preparation of Single Dispersed Uterus Myocytes The uterine fibroids isolated from the human uterus were cut into small pieces of about 2 mm ³ with scissors, 0.2 % (W / v) collagenase (Wako Pure Chemicals, Osaka), DMEM medium containing 0.05% (w / v) DNaseI (GIBCO, CA, USA) (1% antibiotic-antimycotic [GIBCO] In Dulbecco's Modified Eagle Medium (DMEM, Sigma-Aldrich, Missouri, USA) containing 10% fetal calf serum [BioWest, Florida, USA] The cells were enzymatically dispersed by shaking at 16 ° C. for 16 hours. Subsequently, after filtering through a 400 μm pore size polyethylene mesh, the cells were passed through a 40 μm pore size cell strainer (BD Biosciences, Massachusetts, USA) to be dispersed into a single cell state. Subsequently, these dispersed cells were layered on Ficoll-Paque PLUS (Amersham Biosciences, NJ, USA) and subjected to density gradient centrifugation at 780 × g for 15 minutes, and a single cell dispersion was removed from the interface layer. It was collected. This was made into a completely dispersed cell population by enzyme treatment and pipetting with 0.05% (w / v) trypsin-EDTA (Sigma-Aldrich) + 0.05% (w / v) DNaseI solution.

(2) Nuclear staining with Hoechst 33342 The above dispersed single myometrial cell population was isolated from Hank's equilibration buffer containing 2% fetal bovine serum, 10 mM HEPES, and 1% penicillin and streptomycin (no calcium and magnesium, HBSS ⁺ ) Was suspended at a concentration of 2 × 10 ⁶ , Hoechst 33342 (Sigma) was added to a final concentration of 5 μg / ml, and the mixture was reacted at 37 ° C. for 90 minutes. In parallel, reserpine treatment was performed at a concentration of 1 μg / ml. Subsequently, it was centrifuged at 4 ° C., suspended in 2 ml of the above Hanks solution, and stained with Propidium Iodide (PI) for selection of dead cells.

(3) Isolation of myometrial SP cells (MSP)
Hoechst 33342 emits fluorescence with two wavelengths of 424/44 nm (Hoechst blue) and 670 nm (Hoechst red) with 350 nm excitation light, and based on this, the cell population is developed two-dimensionally by flow cytometry. The stained dispersed cells were analyzed using flow cytometry (FACS Vantage SE, Becton Dickinson) and analysis software (Cell-Quest, Becton Dickinson).

FIG. 1 shows the cell distribution diagram. In this way, 1x10 ⁵ cells are collected while developing two-dimensionally, and finally the side population (SP) fraction is collected to form myometrial SP cells (MSP). The cell population was designated as myometrial MP cells (MMP) (FIG. 1A). Further, when treated with reserpine, this SP fraction disappeared (FIG. 1B), and from its characteristic kinetics, it was clearly clarified that this fraction was the SP fraction.

(4) Expression analysis of differentiation marker gene Total RNA was extracted from the above isolated MSP and MMP using Trizol (Invitrogen, CA), and the expression of messenger RNA of several markers characteristic of myometrium was reversed. Analysis was performed by -transcriptase polymerase chain reaction (RT-PCR). FIG. 2 shows the result. Since myometrium is the target tissue / organ of sex steroid hormones, we investigated the expression of two types of estrogen receptors (ER-α and ER-β) and progesterone receptors (PR), which are sex steroid hormone receptors. As compared with MMP, the expression was decreased or hardly detected in MSP. Similarly, calponin and smootherin, which are smooth muscle differentiation markers, were found to be expressed in MMP, but the expression level was clearly lower in MSP. The expression level of GAPDH, an internal standard, was not different between the two. This indicates that MSP is in an immature stage where the degree of differentiation is lower than that of MMP, and MSP has properties that match the characteristics of tissue stem cells.

(5) Cell cycle analysis The collected MSP and MMP were washed twice with HBSS ⁺ and stained with Pyronin Y (PY) and Hoechst 33342. The mixture was reacted with both Hoechst 33342 and reserpine at 37 ° C. for 45 minutes, and PY was added as it was to a final concentration of 1 μg / ml, followed by further reaction for 45 minutes. Washed once with an excess of HBSS ⁺ and subjected to flow cytometry analysis.

  As a result, 96.2% of MSP remained in the stationary phase (G0) of the cell cycle (FIG. 3A), whereas MMP was in the G0 phase at 20.2% and the remaining 80% was in the cell rotation cycle ( G1 phase in G1 → S → G2 / M) (FIG. 3B). This indicates that MSP is a cell population in which the activity is stopped in myometrial cells, and MSP has a property that matches the characteristics of tissue stem cells.

Example 2 Cultivation of myometrial side population (MSP) cells Although culturing of separated MSPs was attempted, mesenchymal stem cell growth medium containing 10% fetal bovine serum and 1% penicillin and streptomycin (not only a normal medium) Even when MSCGM, manufactured by Cambrex Bio Science, Sanko Junyaku Co., Ltd., product number PT-4105) was used, culture was impossible. Further, even when a culture dish coated with various extracellular substrates such as Matrigel and collagen was used, the culture was impossible.

  Therefore, although the same MSCGM as described above was used, the cells were cultured in a multi-gas incubator (Astech, Fukuoka) under a low oxygen concentration of 2% instead of the normal oxygen concentration of 20%.

  As a result, when cultured under a 20% oxygen concentration, the cells did not grow at all even after 2 weeks (2 left in FIG. 4A). At that time, a cell colony was formed (upper right of FIG. 4A), and at 2 weeks, the cells proliferated until they were almost confluent (lower right of FIG. 4A).

Example 3 Differentiation induction assay of myometrial side population (MSP) cells In order to examine whether the above cultured MSP has multipotency, which is one of the stem cell characteristics, MSP is differentiated into adipocytes and bone cells, respectively. The cells were cultured in an inducible medium and examined for their ability to differentiate into different cell lineages.

A prekit osteoblast differentiation medium (manufactured by Cambrex Bio Science, Sanko Junyaku Co., Ltd., product number PT-3002) was used as a differentiation induction medium for bone cells, and a prekit adipocyte differentiation medium ( Cambrex Bio Science, Sanko Junyaku Co., Ltd., product number PT-3004) was used. The cultured MSP and MMP were seeded in a 96-well plate at a concentration of about 5 × 10 ³ / well and cultured under an oxygen concentration of 2% (low oxygen concentration) for about 2-3 weeks under MSCGM medium until confluence. At the start of the following culture for differentiation induction, differentiation induction was performed by returning the oxygen concentration to the usual 20%.

Differentiation into bone Differentiation of the above cultured MSP into bone cells was performed. The bone cell induction medium was changed every 3 to 4 days and cultured for 2 to 3 weeks. Differentiation into bone cells was evaluated by the appearance of alkaline phosphatase staining positive cells. As a result, a large number of alkaline phosphatase staining positive cells (purple) appeared in MSP (FIG. 4B left), but almost no MMP showed (FIG. 4B right).

Differentiation into fat Differentiation of the cultured MSP into adipocytes was performed. First, the cells were cultured in the above-described adipocyte basal medium for 3 days, and then cultured in an adipocyte differentiation induction medium for 1 to 3 days. This was performed as one cycle for 3 cycles. Finally, the cells were cultured in a basal medium for a maximum of one week, washed and fixed, and then stained with oil red O to stain lipid droplets. As a result, oil red O staining positive cells (red) appeared in MSP (FIG. 4C left), but almost no MMP was observed (FIG. 4C right).

Example 4 Transplantation experiment of myometrial side population (MSP) cells into severely immunodeficient mice Severely immunodeficient mice were injected intraperitoneally with 40 μl phosphate buffer (Sigma) containing 10% pentobarbital (Dainippon Pharmaceutical). Anesthesia was performed and the ovaries were removed to remove the effects of endogenous estrogen. At the same time, one tablet of estrogen (E ₂ ) sustained release pellet (Innovative Research of America, Florida, USA) was implanted subcutaneously in order to obtain a constant estrogen environment. About 1 × 10 ⁵ MSPs and MMPs separated as described above were injected into each uterine horn of NOG mice using a 29 gauge needle, and then reared for about 10 weeks.

Fluorescent tissue staining Approximately 10 weeks after the above transplantation, the uterus was removed, embedded in Tissue-Tek OCT compound (Sakura Finetech, California, USA), frozen, and cryostat (Leica Microsystems, Wetzlar, Germany) Was continuously sliced at a thickness of 6 μm. The obtained frozen section was fixed with 4% paraformaldehyde at room temperature for 20 minutes and then washed with a phosphate buffer. The cell membrane was permeabilized with 0.2% Triton X-100-containing phosphate buffer for 10 minutes, and then immersed in a 10% bovine serum albumin solution for 30 minutes for blocking treatment. Subsequently, these treated sections were reacted with an antibody against uterine smooth muscle marker α-smooth muscle actin (αSMA) (clone 1A4, 200-fold diluted, DAKO Cytomation, Denmark) at 4 ° C overnight. The primary antibody was detected using Alexa Fluor 488 (for green fluorescence) labeled secondary antibody (Molecular Probes, Inc., Oregon, USA) (1000-fold dilution, 37 ° C., 1 hour). Subsequently, fluorescent double staining was performed by reacting with a vimentin antibody (clone V9) that reacts only with human cells, directly labeled with Cy3 (Sigma-Aldrich), a red fluorescent dye.

  Furthermore, the sections were counterstained with the nuclear stain DAPI (Sigma) for 5 minutes. Sections after each staining were encapsulated with VECTASHIELD (Vector Laboratories, California, USA), examined with DMIRE2 inverted fluorescence microscope (Leica Microsystems), and images were captured with VB-700 CCD camera (Keyence Corp., Osaka City) It is.

  As a result, as shown in FIG. 5 (upper four sheets), the uterus transplanted with MSP is vimentin positive, that is, derived from human (red), and is also uterine smooth muscle morphologically with αSMA positive cells (green). A tissue (yellow) appeared. On the other hand, in the uterus transplanted with MMP, the human-derived tissue seen in the above MSP was not observed, and human-derived cells were present (red) as shown in FIG. Was diffusely distributed in the interstices of mouse smooth muscle tissue (green) and negative for αSMA (no yellow cells observed).

  The above indicates that MSP has the ability to build uterine smooth muscle, while MMP does not have that ability, and MSP specifically has tissue stem cell characteristics of myometrium.

It is a figure which shows the result of the flow cytometry analysis of the myometrial dispersion cell which gave Hoechst 33342 dyeing | staining. FIG. 1B shows the result of further reserpine treatment on the cells of FIG. 1A. It is a figure which shows the result of the expression analysis by RT-PCR of the sex steroid receptor and smooth muscle differentiation marker in MSP and MMP. In the figure, “total” refers to myometrial cells when dispersed by mechanical / enzymatic treatment (before sorting MSP and MMP). DDW uses distilled water as a template to indicate a negative control. . It is a figure which shows the result of the flow cytometry analysis of MSP (FIG. 3A) and MMP (FIG. 3B) which performed the double dyeing | staining by Hoechst 33342 and Pyronin Y. It is a photograph which shows the phase-contrast microscope image of MSP and MMP cultured under normal oxygen concentration 20% or low oxygen concentration 2%. It is a photograph which shows the alkaline phosphatase dyeing | staining microscope image of MSP (left figure) and MMP (right figure) cultured in the osteocyte induction medium. It is a photograph which shows the oil red O dyeing | staining microscope image of MSP (left figure) and MMP (right figure) cultured on the adipocyte induction | guidance | derivation medium. It is a photograph which shows the fluorescence immunostaining image of the NOG mouse | mouth uterus which transplanted MSP and MMP.

Claims (11)

  Smooth muscle stem cells derived from mammalian smooth muscle.   The smooth muscle stem cells according to claim 1, which can be obtained as side population cells when mammalian smooth muscle cells are stained with Hoechst33342 and analyzed by flow cytometry using fluorescence intensity of Hoechst red and Hoechst blue as an index.   The smooth muscle stem cell according to claim 1 or 2, wherein the smooth muscle is uterine muscle.   The smooth muscle stem cell according to any one of claims 1 to 3, wherein the mammal is a human.   The smooth muscle stem cell according to any one of claims 1 to 4, which has an ability to differentiate into smooth muscle by transplanting into smooth muscle.   6. The smooth muscle stem cell according to claim 5, which is derived from a uterine muscle having an ability to differentiate into a uterine muscle when transplanted into the uterine muscle.   Including obtaining dispersed cells from smooth muscle tissue, subjecting the dispersed cells only to staining with Hoechst33342, and then subjecting the cells to flow cytometry, and collecting side population cells using the fluorescence intensity of Hoechst red and Hoechst blue as indicators. A method for isolating smooth muscle stem cells.   A method for culturing and proliferating a smooth muscle stem cell, comprising culturing the smooth muscle stem cell according to any one of claims 1 to 6 under a condition in which a culture oxygen concentration is 2.5% or less.   The method for culturing and proliferating smooth muscle stem cells according to claim 8, wherein the culture oxygen concentration is 2%.   A method for inducing differentiation of smooth muscle stem cells according to any one of claims 1 to 6 into smooth muscle cells in vitro.   A composition for regenerating smooth muscle tissue, comprising the smooth muscle stem cell according to any one of claims 1 to 6.

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