WO2005105169A1 - 脂肪組織由来幹細胞から骨髄を形成するための組成物およびその方法 - Google Patents
脂肪組織由来幹細胞から骨髄を形成するための組成物およびその方法 Download PDFInfo
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- WO2005105169A1 WO2005105169A1 PCT/JP2005/008555 JP2005008555W WO2005105169A1 WO 2005105169 A1 WO2005105169 A1 WO 2005105169A1 JP 2005008555 W JP2005008555 W JP 2005008555W WO 2005105169 A1 WO2005105169 A1 WO 2005105169A1
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- adipose tissue
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
- A61L27/3843—Connective tissue
- A61L27/3847—Bones
Definitions
- the present invention relates to compositions and methods for forming bone marrow from adipose tissue-derived stem cells. Furthermore, the present invention relates to a method for producing hematopoietic stem cells and mesenchymal stem cells in the bone marrow.
- Bone marrow is the tissue that resides in the cavity formed within bone tissue (the bone marrow cavity) and is a major hematopoietic tissue in vertebrates, especially higher amphibians and higher animals.
- the bone marrow there are precursors of all blood-based cells such as red blood cells, granulocytes, monocytes-macrophages, megakaryocytes-platelets, mast cells, and lymphocytes. All of these progenitor cells are derived from pluripotent hematopoietic stem cells. That is, a variety of blood cells can be differentiated from a single hematopoietic stem cell.
- Hematopoiesis in bone marrow and cord blood as a treatment for disorders associated with hematopoietic dysfunction in the bone marrow eg, leukemia, aplastic anemia, innate immunity deficiency in children and some congenital metabolic disorders
- Stem cell transplantation or bone marrow transplantation has been performed.
- HLA Human Leukocyte Antigen
- bone marrow transplantation imposes a burden on the patient's own bone marrow due to the use of chemotherapy or radiation therapy to suppress hematopoietic dysfunction, and the physical and mental distress of the patient is great. Furthermore, the physical and mental distress of the donor from whom bone marrow is collected is also great.
- hematopoietic stem cells obtained from a donor are directly transplanted into the blood vessels of the patient.Therefore, there are few cells that are enriched in the bone marrow by blood flow and are captured by other organs in the patient. There are many cases where it is difficult to obtain an effective therapeutic effect.
- stem cells have been found throughout the body, and it has been found that stem cells also exist in adipose tissue. Adipose tissue can be easily collected under local anesthesia. In the field of cosmetic surgery, it has been discarded after being collected by liposuction or lipectomy. On the other hand, bone marrow-derived stem cells, which have been conventionally studied, require general anesthesia to be harvested, and the burden on the patient is extremely large. Therefore, attempts have been made to create various tissues using the fat-derived stem cells, and the occurrence of fat, bone, muscle, and cartilage has been confirmed (see Patent Document 2). ).
- adipose tissue-derived stem cells unlike bone marrow-derived stem cells, are also involved in revascularization (see Non-Patent Document 1).
- adipose tissue-derived stem cells have overwhelmingly excellent proliferation ability over bone marrow-derived stem cells, and can be easily cultured in a short period of time.
- Patent Document 1 JP-A-8-336584
- Patent Document 2 JP 2002-537849 A
- Patent Document 3 JP 2003-523767 A Non-Patent Document 1 Circul at ion. 2004; 109: 656-663. Disclosure of the Invention
- the present invention provides a composition capable of forming bone marrow from adipose tissue-derived stem cells and a method for forming bone marrow from adipose tissue-derived stem cells.
- the burden on the donor is low, and it provides a means for surely resolving bone marrow disorders.
- the present invention provides a means for recovering from injury using the patient's own cells without performing an HLA-compatible test.
- the present inventors have conducted intensive studies to achieve the above object, and as a result, by collecting stem cells from adipose tissue, dispersing them on a support carrier to be a bone marrow support tissue, and subcutaneously transplanting them into animals, It has been found that new bone marrow can be produced.
- the present invention has been accomplished based on these findings.
- the present invention relates to the following embodiments:
- a bone marrow-forming composition comprising adipose tissue-derived stem cells collected from a vertebrate;
- a bone marrow-forming composition comprising cells differentiated from vertebrate adipose tissue-derived stem cells into osteocytes;
- the bone marrow-forming composition according to the above item 4 wherein the support carrier is made of a biocompatible material and has a three-dimensional network porous structure;
- composition capable of forming bone marrow according to any one of to 6; 8.
- adipose tissue-derived stem cells obtained from a vertebrate adipose tissue to produce a bone marrow-forming composition or bone marrow.
- Adipose tissue-derived stem cells refers to pluripotent stem cells present in adipose tissue of mammals. The cells are considered developmentally to be mesenchymal stem cells derived from the mesoderm. Adipose tissue can be collected by a known method from a vertebrate, preferably a mammal, for example, a rodent (rat, mouse, etc.), particularly preferably a human. The collected animal can be of any type, as long as the stem cells in the adipose tissue are alive.
- adipose tissue may be collected from any part of the animal, for example, from general adipose tissue. Such collection may be performed by any known method, and may be performed by a so-called liposuction or lipectomy.
- cell-containing material containing adipose tissue-derived stem cells refers to a state in which adipose tissue-derived stem cells derived from the collected adipose tissue are contained while maintaining viability and pluripotent differentiation ability .
- Adipose tissue collected from an animal individual is often obtained as a tissue mass, and includes tissue fragments, cells, cell fragments, and the like derived from other tissues such as nerve tissue, vascular tissue, and blood cell. Therefore, when used in the present invention, it is possible to completely or partially dissociate the cells of the tissue mass and completely or partially cut the adhesion between the stem cells present in the adipose tissue and the surrounding tissues or adjacent cells. preferable.
- Such dissociation of cells or cleavage of adhesion can be performed by methods known to those skilled in the art. Examples include mechanical cutting, sonication, mechanical stirring, treatment with heat energy, treatment with an enzyme or a drug capable of breaking cell-cell adhesion, and the like.
- the enzyme include collagenase, trypsin, dispase, hyaluronase, DNAase, and the like.
- the enzyme include ethylenediaminetetraacetic acid (EDTA). ) And the like. Also physiological It is preferable to remove tissue debris and the like from the cell-containing substance obtained by treating the fatty tissue pieces by washing with a buffer, centrifugation, filtration and the like.
- These processes can be appropriately selected by those skilled in the art from all known techniques, and in some cases, can be performed in combination. These treatments are performed under conditions capable of maintaining the survival of adipose tissue-derived stem cells, and are performed in a physiological buffer or a cell culture medium.
- the enzyme treatment is preferably carried out at around 37 ° C. due to its properties.
- the cell-containing substance dissociated from the adipose tissue obtained above may be cultured under appropriate culture conditions. During such culture, the medium may be changed appropriately.
- the culturing time may be any time during which the cell damage due to harvesting and processing is expected to recover to some extent.
- Adipocyte-derived stem cells have the ability to self-replicate and can proliferate under appropriate culture conditions. Therefore, the number of cells can be increased by culturing for 3 days or more, or for one to several weeks, and the culturing period may be adjusted according to the required number of cells.
- a part of the aforesaid cell-containing stem cells must be replaced with adipose tissue-derived stem cells.
- the cells can be cultured in a medium capable of inducing differentiation into any cells that can be differentiated from stem cells (eg, bone cells, chondrocytes, skeletal muscle cells, adipocytes, etc.). Such a medium and culture conditions can be appropriately selected by those skilled in the art.
- stem cells eg, bone cells, chondrocytes, skeletal muscle cells, adipocytes, etc.
- Such a medium and culture conditions can be appropriately selected by those skilled in the art.
- morphological changes of cells are observed. Usually, a morphological change can be observed in about 2 to 4 weeks.
- the adipose tissue mass collected from the animal and the cell content after the above treatment and culture are encompassed in the term “cell content including adipose tissue-derived stem cells” in the present specification.
- the above-described treatment and the like are merely examples, and the cell-containing material containing adipose tissue-derived stem cells contained in an adipose tissue mass collected from an animal while maintaining its viability and pluripotent differentiation ability is known in the art. It may be prepared by any method, may have any composition, and can be prepared by a person skilled in the art by an appropriate method as appropriate.
- the term “support carrier” refers to a material that enables three-dimensional growth. Means a three-dimensional structure that serves as a scaffold for cells.
- the support carrier may be any structure as long as the cells can be held in the structure, but it is preferable that the support carrier can smoothly attach and grow the cells.
- the material When implanting in a living body, it is preferable that the material has biocompatibility.
- composition capable of forming bone marrow refers to a composition capable of forming bone marrow, which is a site for hematopoiesis, when cultured in vitro or transplanted into a living body.
- it is obtained by contacting a cell-containing substance containing adipose tissue-derived stem cells with the above-mentioned support carrier. The contact may be performed in a cell culture medium or a physiological buffer.
- the adipose tissue-derived stem cells When the cell material containing adipose tissue-derived stem cells is brought into contact with the support carrier, the adipose tissue-derived stem cells adhere to the support carrier surface, and survive on the support carrier surface.An environment in which self-renewal and / or differentiation can occur is established. .
- the composition of the present invention is transplanted into an animal, so that the adipose tissue-derived stem cells contained in the transplanted composition are transplanted at the transplant site. It can differentiate into osteoblasts and hematopoietic cells, which are tissues surrounding the bone marrow, and form bone marrow. Alternatively, it would be possible to form bone marrow from the composition in vitro.
- Transplant site May be at any site, but is preferably subcutaneous.
- Transplantation of the bone marrow-forming composition of the present invention is most preferably autotransplantation into an animal from which adipose tissue has been collected, but any of allogeneic, allogeneic, and xenotransplantation is applicable.
- Those skilled in the art can appropriately select donors and recipients at the time of transplantation, and can select and implement appropriate transplantation methods and postoperative management.
- contacting a cell-containing substance with a supporting carrier means that the adipose tissue-derived stem cells contained in the cell-containing substance can enter or adhere to the supporting carrier structure.
- Contact in an unfavorable environment in an environment capable of maintaining cell survival and differentiation ability, that is, 0 ° (: ⁇ 50 ° C., preferably 4 ° C. to 40 ° C., and most preferably about 25 ° C. to 37 ° C.)
- the contact is carried out in a physiological buffer or medium at a temperature of the contact time may be any length as long as the time is sufficient for the stem cells to penetrate or adhere to the support structure.
- the cells can be cultured under ordinary culture conditions for several hours to several weeks, for example, for about 24 hours to 10 days, to complete the cell adhesion of the stem cells to the support carrier, or the like.
- the medium may be cultured in a bone differentiation induction medium
- a bone differentiation induction medium Various types of bone differentiation induction medium are known, and any medium capable of inducing differentiation of stem cells into bone cells may be used.
- a cell culture medium eg, DMEM containing 10% fetal serum
- Culture in methasone (DEX) and about 50 such as those obtained by adding M Asukorubin acid and 10mM of / 3 Dariserorin acid and the like.
- a cell culture medium eg, DMEM containing 10% fetal serum
- Culture in methasone (DEX) and about 50 such as those obtained by adding M Asukorubin acid and 10mM of / 3 Dariserorin acid and the like.
- During bone differentiation-inducing medium preferably about 3 days to 10 days.
- vertebrate includes any vertebrate, but particularly refers to a mammal. Further, in a preferred embodiment, the animal is a rodent (such as a heron, guinea pig, rat or mouse) or human, most preferably a human.
- rodent such as a heron, guinea pig, rat or mouse
- human most preferably a human.
- FIG. 1 is a photograph showing a microscope image of adipose tissue-derived cells (cultured in vitro) including adipose tissue-derived stem cells collected from adipose tissue. It turns out that it has a fibroblast-like morphology.
- the scale bar is 50 ⁇ m.
- FIG. 2 is a photograph showing the fluorescence microscope image of FIG. GFP fluoresces green because all cells are derived from mice with GFP introduced.
- the scale bar is
- FIG. 3 is a photograph showing a microscopic image of the cells of FIG. 1 cultured in a bone differentiation induction medium. It changes from a fibroblast-like morphology in Figure 1 to a circle and a square.
- the scale bar is 50 m.
- FIG. 4 is a photograph showing the fluorescence microscope image of FIG. Since GFP can be confirmed, it is probable that the same gun as in Fig. 1 changed.
- the scale bar is FIG. 5 is a photograph showing a microscope image stained with von Kossa to confirm cells differentiated into bone. It can be seen that the lime of the bone matrix is stained black.
- the scale is 50 m.
- FIG. 6 is a photograph showing a microscope image obtained by staining with Al-force phosphatase to confirm cells differentiated into bone. It can be confirmed that the cells having alkaline phosphatase are stained red.
- the scale par is 50 m.
- FIG. 7 is an X-ray photograph of the cells of FIG. 3 seeded on hydroxyapatite having small pores and transplanted subcutaneously into immunoreactive mice.
- Figure 8 is a photograph showing a one-month-old graft implanted subcutaneously in a mouse. It can be confirmed that blood vessels have invaded the hydroxyapatite.
- the scale bar is 1 mm.
- FIG. 9 is a fluorescence micrograph of FIG. Cells having GFP can be confirmed, and it can be confirmed that the cells are alive.
- the scale is one dragon.
- FIG. 10 is a photograph showing an HE-stained image of a paraffin section. Adipocytes and blood cells characteristic of bone marrow are observed in the pores of hydroxyapatite 5 In the living body, a structure like cancellous bone having bone marrow was confirmed.
- arrow 1 indicates bone regenerated along hydroxyapatite
- arrow 2 indicates bone marrow-like tissue of various blood cells proliferating and surrounded by the regenerated bone.
- HA indicates particles of hydroxyapatite.
- the scale bar is FIG. 11 is a photograph showing an immunostaining image with an anti-GFP antibody. In the area in contact with hydroxyapatite, formation of cells and bone, which are considered to be osteocytes, was observed. These are GFP-positive, that is, it is considered to be cells derived from Dona. In the figure, GFP-positive donor-derived cells are indicated by arrows. HA indicates hydroxyapatite.
- FIG. 12 is a photograph showing an enlarged view of the pores of FIG. Weak blood cells of all strains can be seen.
- the scale is 20 m.
- FIG. 13 is a diagram showing the results of analyzing the sample of FIG. 12 by flow cytometry.
- the X-axis shows cell size, and the Y-axis shows cell density.
- the cells on the top of the graph show low density, and those on the right show large cells. That is, in the regenerated bone marrow, the cell fractions of M0; monocytes, LY; lymphocytes, Gr; and granulocytes can be confirmed.
- the adipose tissue-derived stem cells of the present invention can be obtained from adipose tissue by any method. All of these methods require the isolation of adipose tissue from the source animal. As long as the adipose tissue-derived stem cells maintain their differentiation ability and survive, the source animal can be of any type. Also, as long as a sufficient amount of adipose tissue can be obtained, the species of the source animal and the sampling site are not limited. When the source animal is human, it can be collected in the same manner as in liposuction and lipectomy performed in the field of cosmetic surgery.
- the collected adipose tissue has blood vessels, nerves, and other tissues attached to it. Therefore, they are removed by visual inspection with the naked eye, and washed with a physiologically compatible washing solution such as a phosphate buffer. Then, agitate several times with a washing solution and settle to remove components such as damaged tissue, blood, and red blood cells. This is repeated until the supernatant is relatively clear.
- a physiologically compatible washing solution such as a phosphate buffer.
- the tissue is still large to some extent, so transfer to a standard cell culture medium, shred, add culture medium, and then remove the culture medium by centrifugation to minimize damage to the cells themselves. .
- enzymes such as collagenase ⁇ dispase and trypsin, which have the activity of cleaving cell-cell adhesion, under conditions where the enzyme activity can be exerted, the effect on cell viability is minimized.
- the tissue mass is completely degraded by vigorously stirring the fat mass with weak intercellular bonds under standing conditions. The above conditions are known to those skilled in the art.
- any method that does not cause fatal damage to cells such as mechanical stirring, sonic energy treatment, and heat energy treatment, can be used.
- the fat mass is degraded by the enzymatic method, remove the contaminants other than cells by adding the cell culture medium again and removing the medium by centrifugation to deactivate the enzyme activity. I do.
- a cell culture medium is further added to the precipitate, and cell-cell adhesion can be further broken by pipetting with a pipette or the like.
- the cell-containing material containing adipose tissue-derived stem cells obtained in this manner is a stromal cell population containing stem cells.In some cases, even if this is cultured for about one week while replacing the medium with a cell culture medium, Good.
- the mesenchymal stem cells are contained in the cell-containing material containing adipose tissue-derived stem cells prepared in step 2, culture them for about 3 weeks in a medium that induces mesenchymal cell differentiation.
- the medium for inducing differentiation contains stem cells derived from adipose tissue such as bone, cartilage, or fat. Any medium may be used as long as it is a medium for inducing differentiation into cells that can be transformed. Various media are known depending on the cell type. Cells differentiated by each differentiation-inducing medium can be morphologically or biochemically confirmed by any method.
- the cell-containing material containing adipose tissue-derived stem cells may be further subcultured one to several times in a cell culture medium, and then seeded as described above.
- This support carrier is a type II serving as a scaffold for stem cells, and is not limited as long as it can be used as a substitute for a support tissue. However, it is preferable that the cells do not flow out and allow the cells to adhere and grow smoothly.
- the support carrier does not deteriorate in water and medium, has properties that can withstand high-pressure sterilization, and has chemical resistance to weak acids, alkaline acids, and many organic solvents. Those that are stable in nature are preferred. Further, it preferably has a microporous three-dimensional network porous structure. Naturally-derived polymer materials such as polyvinyl formal resin and porous glass, ceramic, hydroxyapatite, chitosan, cellulose, water-soluble collagen and dextran, which have a porous structure are preferred, and among these, hydroxyapa is particularly preferred. Tites are particularly preferred.
- the support carrier seeded with the above-mentioned cell-containing material can be used immediately for bone marrow formation.However, it is cultured for several hours to about a week to adhere the stem cells to the support carrier and adjust the growth environment of the stem cells. You may. Further, the support carrier seeded with the above-mentioned cell-containing material can be cultured in a bone differentiation-inducing medium for about one week to allow bone differentiation to progress to some extent, and then used for bone marrow formation.
- bone marrow In order to form bone marrow from the bone marrow-forming composition prepared above, it may be transplanted into an animal.
- the place for transplantation is not particularly limited, but is preferably subcutaneous, intraperitoneal, or under the renal capsule or subfascia.
- Known transplantation method Use any of the above surgical techniques.
- Such transplantation may be any of autologous, syngeneic, allogeneic, allogeneic and xenotransplantation.
- normal postoperative management is performed. Approximately three weeks after transplantation, hematopoietic function and bone marrow tissue can be observed.
- New bone marrow is formed by transplantation into individuals with diseases associated with impaired hematopoietic dysfunction of the bone marrow (eg, leukemia, aplastic anemia, congenital immunodeficiency in children and some congenital metabolic diseases). As a result, it becomes a new hematopoietic organ and can suppress the progress of the above-mentioned disease and / or treat it.
- the above-mentioned disease can be prevented by transplanting the bone marrow-forming composition to an individual who has a high risk of developing the disease but has not yet developed the disease.
- the above bone marrow-forming composition may be maintained in vitro and promoted differentiation to form bone marrow in vitro.
- the bone marrow-forming composition of the present invention in which adipose tissue-derived stem cells of a mouse were seeded on hydroxyapatite was subcutaneously transplanted into a nude mouse or a GFP-negative immunoreactive syngeneic mouse. The formation of bone marrow was confirmed. Bone marrow formation was confirmed in all mice (10 or more) transplanted according to the method described in the examples.
- GFP transgenic mice provided by Professor Okabe of Osaka University
- GFP protein was introduced and expressed in all cells of the whole body as a mouse from which adipose tissue was collected.
- the mouse was anesthetized with Nembutal (0.1 g / l OOg), and from both lateral diameters The fat mass was collected and washed well with phosphate buffered saline (pH 7.4). Furthermore, after completely removing blood vessels and nerves that can be visually observed from the fat mass, a Dulbecco's modified Eagle's medium (Gibco BRL) containing 10% fetal serum and 1% antibacterial / antimycotic agent was used. ) (Hereinafter referred to as control medium) and transferred to a 100-band 2 culture dish and minced.
- Nembutal 0.1 g / l OOg
- FIG. 1 shows the results, which show that the cells have a fibroblast-like morphology.
- Fig. 2 shows the result of observing the same sample with a fluorescence microscope. Since the cultured cells are cells derived from GFP transgenic mice, it can be seen that the cells emit green fluorescence.
- FIG. 5 shows the results of von Kossa staining
- Fig. 6 shows the results of alkaline phosphatase staining.
- the von Kossa stain stains black for calcium deposits, the main component of the bone matrix, and the alkaline phosphatase stain suggests that the alkaline phosphatase-containing bone cells stained red.
- the cell contents including adipose tissue-derived stem cells collected in the above 1, 37 ° C, 5% C0 2 conditions, and subcultured three times in control medium, so that the cells to 1 X 10 5 cells prepared, high Dorokishiapatai preparative having fine pores (CELLYA D (TM), manufactured by PENTAX Corporation) were seeded into, placed in a culture dish meets bone differentiation-inducing medium, 1 week at 37 ° C, 5% C0 2 Cultured.
- CELLYA D TM
- FIG. 7 shows the X-ray image at this time.
- Eight hydroxypatites were removed 4 to 16 weeks after transplantation.
- Figure 8 shows the hydroxyapatite removed 16 weeks later.
- green fluorescence was observed (FIG. 9), confirming that cells derived from GFP transgenic mice had survived.
- the transplant was similarly established in both nude mice and immunoreactive mice, confirming that immune rejection did not occur when cells from other individuals were used. Fix this with 4% paraformaldehyde,
- a paraffin section was prepared, and a tissue image was observed under a microscope.
- bone marrow morphology was confirmed in the pores of hydroxyapatite (Fig. 10), and in the part in contact with the hydroxyapatite, bone-like tissue was formed. It was confirmed by immunoimmune staining with an anti-GFP antibody that the cells constituting them had GFP.
- Fig. Shown in 11. Cells having GFP are stained brown. In other words, this means that bone was formed by cells of the mouse from which adipose tissue-derived stem cells were collected, and bone marrow was formed therein.
- bone marrow was formed from fat-derived stem cells, and blood and undifferentiated blood cells were also present in the bone marrow, confirming that a hematopoietic system using fat-derived stem cells could be constructed. was done.
- FIG. 12 is an enlarged view of the bone marrow forming part in 4 of Example 1 and FIG. 10. Weak blood cells of all strains can be confirmed.
- Table 1 shows the results of counting blood fractions in stomata where many young blood cells are observed.
- the calculated granulocyte Z erythroid ratio (M / E ratio) was 1.23%. This is a value that is not different from that of a general mouse bone marrow fraction, and strongly suggests that bone marrow tissue has been regenerated.
- Fig. 10 shows the results of analysis of these cells by pharmacocytometry.
- the X-axis shows cell size, and the Y-axis shows cell density.
- the regenerated bone marrow contained a monocyte, granulocyte, and lymphocyte cell fraction. From the above results, bone marrow was formed from adipose-derived stem cells, and in the regenerated bone marrow, many immature blood cells were confirmed, indicating that bone marrow having the function of producing blood was regenerated. Indicated. Industrial applicability
- a new bone marrow can be formed from stem cells derived not from bone marrow but from adipose tissue, so that bone marrow transplantation can be performed with less burden on the donor. Furthermore, the possibility of bone marrow transplantation using the patient's own cells and the ability to restore hematopoietic function without searching for a donor has been opened. Therefore, according to the present invention, diseases associated with hematopoietic dysfunction of bone marrow (for example, leukemia, reproductive anemia, innate immunity deficiency in children and some congenital metachronous diseases) And a new and effective method of treatment or prevention, and a composition for treatment or prevention. All publications cited herein are incorporated by reference in their entirety. It will be readily understood by those skilled in the art that various modifications and alterations of the present invention are possible without departing from the technical concept and the scope of the invention described in the appended claims. The present invention is intended to cover such modifications and variations.
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RU2461621C1 (ru) * | 2011-08-25 | 2012-09-20 | Учреждение Российской академии наук Государственный Научный Центр РФ Институт медико-биологических проблем РАН | Способ стимуляции формирования фиброзно-хрящевого регенерата костной мозоли у млекопитающих |
CN111500533A (zh) * | 2019-01-31 | 2020-08-07 | 华东理工大学 | 干细胞发生器及其构建方法 |
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CN111500533A (zh) * | 2019-01-31 | 2020-08-07 | 华东理工大学 | 干细胞发生器及其构建方法 |
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