JP2015039307A - Mesenchymal stem cell separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method to separate mesenchymal stem cells easily and efficiently.SOLUTION: A mesenchymal stem cell separation method is characterized by separating mesenchymal stem cells with ossein used as material.

Description

  The present invention relates to a method for separating mesenchymal stem cells and a mesenchymal stem cell obtained by the separation method.

  Mesenchymal stem cells (MSCs) are contained in mesenchymal tissues at a low frequency, and are considered to exist in tissues and organs throughout the body, albeit with a difference in frequency. Conventionally, cells obtained from bone marrow were cultured, and cells showing fibroblast-like morphology attached to a culture dish were identified as MSCs (Non-patent Document 1).

  This cell has attracted attention because it has been reported that MSCs isolated from human bone marrow can be induced to differentiate into bone, cartilage, and adipocytes in vitro (Non-patent Document 2). . Thereafter, it was reported that bone formation was promoted by transplanting mesenchymal stem cells derived from allogeneic bone marrow into patients with osteogenesis imperfecta (Non-patent Document 3). Furthermore, differentiation into nerve cells (Non-Patent Document 4), differentiation into cardiomyocytes (Non-Patent Document 5), differentiation into hepatocytes (Non-Patent Document 6), differentiation into vascular cells (Non-Patent Document 7), Since the differentiation into alveolar epithelial cells (Non-patent Document 8) has been reported one after another, this cell has attracted attention as a source of transplanted cells in regenerative medicine.

  As regenerative medicine using MSC, for example, a treatment for regenerating alveolar bone by transplanting MSC obtained from bone marrow or the like is performed on a patient who is difficult to implant due to insufficient strength. In animal experiments related to this, it has been reported that the use of MSCs obtained from bone marrow together with platelet-rich plasma in the defect site effectively increases the osseous bond of dental implants (Non-patent Document 9). In addition, for MSCs to be transplanted, research is being conducted to determine whether not only bone marrow-derived cells but also fat-derived cells can be used (Non-patent Document 10).

  On the other hand, for example, regenerative medicine of cartilage is known as regenerative medicine that does not use MSC. As a history of autologous chondrocyte transplantation, first in 1994, a method was introduced in which chondrocytes were cultured in a monolayer from their own cartilage pieces and then suspended in a cartilage defect covered with periosteum. (Non-Patent Document 11). This method was approved by the US FDA in 1997, and by the year 2007, more than 20,000 cases were to be implemented worldwide. However, although this method can reduce the risk of an immune reaction and the like by using autologous cartilage, there is a problem of invasion of normal tissue associated with collection of cartilage, and fixation of cells to tissue because cells are used in a floating state. There is also a problem that cartilage formation is incomplete due to the poor nature and the limited ability of chondrocytes to proliferate. In recent years, it has been reported that the engraftment of chondrocytes is improved by the method of transplanting after co-culture of chondrocytes and atelocollagen, but this problem has not yet been solved. Therefore, development of a method using MSC having higher proliferation / regeneration ability is demanded.

  In addition to the above regenerative medicine, MSC is useful for immune control and treatment of graft versus host disease (GVHD) (Non-patent Documents 12 and 13), and support for hematopoietic stem cells (HSC) It is known that it is useful as a cell (Non-Patent Documents 14 to 16).

  Thus, it is known that MSC can be used in various fields such as regenerative medicine, immune control, and support / amplification of HSC. At present, it has been reported that MSC can be separated from bone marrow and umbilical cord blood (Non-patent Document 17). However, in view of the many fields of application of MSC, development of a method for separating MSC more efficiently is required. It has been.

Friedenstein AJ, et al .: Fibroblast precursors in normal and irradiated mouse hematopoietic organs.Exp Hematol 4: 267-274,1976. Pittenger MF, et al .: Multilineage potential of adult human mesenchymal stem cells.Science 284: 143-147,1999. Horwitz EM, et al .: Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implication for cell therapy of bone.Proc Natl Acad Sci USA 99: 8932-8937,2002. Kopen GC, et al .: Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains.Proc Natl Acad Sci USA 96: 10711-10716,1999. Makino S, et al .: Cardiomyocytes can be generated from marrow stromal cells in vitro.J Clin Invest 103: 697-705,1999. Sato Y, et al .: Human mesenchymal stem cells xenografted directly to rat liver are differentiated into human hepatocytes without fusion.Blood 106: 756-763,2005. Copland I, et al .: CD34 expression on murine marrow-derived mesenchymal stromal cells: impact on neovascularization.Exp Hematol 36: 93-103,2008. Kotton DN, et al .: Bone marrow-derived cells as progenitors of lung alveolar epithelium.Development 128: 5181-5188,2001. Yamada Y, et al .: Tissue-engineered injectable bone regeneration for osseointegrated dental implants. Clin Oral Impl Res 15: 589-597,2004. Masanori Sagawa: Regenerative medicine of bone and teeth using somatic stem cells. Asahikawa Medical University Research Forum 12: 2-11,2011. Brittberg M, et al .: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation.New Engl J Med 331: 889-895,1994. Le Blanc K, et al .: HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells.Exp Hematol 31: 890-896,2003. Le Blanc K, et al .: Treatment of severe acute graft-versus-host disease with third party haploidentical mesenchymal stem cells. Lancet 363: 1439-1441,2004. Mendez-Ferrer S, et al .: Mesenchymal and hematopoietic stem cells form a unique bone marrow niche.Nature 466: 829-834,2010. Wang J, et al .: SCID-repopulating cell activity of human cord blood-derived CD34- cells assured by intra-bone marrow injection.Blood 101: 2924-2931,2003. Masuda S, et al .: Cotransplantation with MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation in nonhuman primates.Exp Hematol 37: 1250-1257,2009. Peters R, et al .: Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture.PlosOne 5: e15689. Deans RJ, Moseley AB: Mesenchymal stem cells: Biology and potential clinical uses.Exp Hematol 28: 875-884,2000. Morikawa S, et al .: Prospective identification, isolation, and systemic transplantation of multipotent mesenchymal stem cells in murine bone marrow.J Exp Med 206: 2483-2496,2009. Morikawa S, et al .: Development of mesenchymal stem cells partially originate from the neural crest.BBRC 379: 1114-1119,2009. Quirici N, et al .: Isolation of bone marrow mesenchymal stem cells by anti-nerve growth factor receptor antibodies.Exp Hematol 30: 783-791,2002. Sorrentino A, et al .: Isolation and characterization of CD146 + multipotent mesenchymal stromal cells.Exp Hematol 36: 1035-1046,2008. Battula VL, et al .: Isolation of functionally distinct mesenchymal stem cell subsets using antibodies against CD56, CD271, and mesenchymal stem cell antigen-1.Haematologica 94: 173-184,2009. Gang EJ, et al: SSEA-4 identifies mesenchymal stem cells from bone marrow. Blood 109: 1743-1751,2007.

  An object of this invention is to provide the method of isolate | separating a mesenchymal stem cell simply and efficiently. More specifically, an object of the present invention is to provide a method for easily preparing a large amount of mesenchymal stem cells required for treatment using mesenchymal stem cells and research related to the cells. Furthermore, another object is to provide mesenchymal stem cells that differentiate into a specific tissue (or do not differentiate into a specific tissue).

  As a result of extensive research, the present inventors have more mesenchymal stem cells in bone quality than bone marrow, which is well known to contain stem cells such as mesenchymal stem cells and hematopoietic stem cells. I found out. And it discovered that a large amount of mesenchymal stem cells could be separated easily and efficiently by using bone quality as a material. Furthermore, the present inventors also found that mesenchymal stem cells isolated using specific cell surface markers (SSEA-4 and CD271) as an index have the property of differentiating into specific tissues (or not differentiating into specific tissues). As a result of further research based on these findings, the present invention was completed.

  That is, the present invention includes the following aspects.

Item 1.
A method for isolating mesenchymal stem cells, comprising isolating mesenchymal stem cells using bone quality as a material.

Item 2.
Item 2. The separation method according to Item 1, wherein mesenchymal stem cells are separated using CD271 negative as an index.

Item 3.
Item 2. The separation method according to Item 1, wherein mesenchymal stem cells are separated using SSEA-4 negative and CD271 positive as indices.

Item 4.
Item 4. The method according to any one of Items 1 to 3, wherein the mesenchymal stem cells are separated using at least one selected from the group consisting of a negative blood cell marker, a negative apoptosis marker, and a negative dead cell marker as an index.

Item 5.
Item 5. A mesenchymal stem cell isolated by the separation method according to any one of Items 1 to 4.

Item 6.
Mesenchymal stem cells that are (a) derived from bone quality, (b) negative for hematopoietic cell markers, (c) negative for apoptosis markers and / or dead cell markers, and (d) negative for CD271.

Item 7.
(A) derived from bone quality, (b) negative for hematopoietic cell marker, (c) negative for apoptosis marker and / or dead cell marker, (e) negative for SSEA-4, and (f) positive for CD271 A mesenchymal stem cell.

  According to the present invention, since mesenchymal stem cells can be efficiently separated, a large amount of mesenchymal stem cells can be easily prepared. Further, the bone quality as a material is a portion that remains after removing bone marrow, which is a useful portion, and is usually discarded. Therefore, the separation method of the present invention using bone material as the material is extremely excellent in that the material can be easily prepared in large quantities and at low cost. Also, by separating CD271 negative as an index, mesenchymal stem cells that show differentiation potential to cartilage but not bone differentiation can be obtained, SSEA-4 negative and CD271 positive Separation using a certain index as an index makes it possible to obtain mesenchymal stem cells that show differentiation potential into bone but do not show differentiation ability into cartilage. Such cells are very useful tools for regenerating only cartilage tissue or bone tissue, for example.

The separation result by FACS of Example 1 is shown. The separation result by FACS of Comparative Example 1 is shown. The fractionation result by FACS of Example 2 is shown. The CFU-F assay result of Example 2 is shown. The measurement result of the forward scattered light of Example 2 is shown. The fractionation result by FACS of the comparative example 2 is shown. The cell image which is a result of the mesenchymal stem cell establishment test of the comparative example 2 is shown. The separation efficiency of bone quality-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells in Test Example 1 is shown. The measurement results of forward scattered light of bone quality-derived mesenchymal stem cells and bone quality-derived microstem cells in Test Example 2 are shown. The growth characteristics of bone quality-derived mesenchymal stem cells and bone quality-derived microstem cells in Test Example 3 are shown. 10 shows the bone differentiation ability of bone quality-derived mesenchymal stem cells in Test Example 4. 10 shows the fat differentiation ability of bone quality-derived mesenchymal stem cells of Test Example 4. 10 shows the cartilage differentiation ability of bone quality-derived mesenchymal stem cells in Test Example 4. The bone differentiation ability, fat differentiation ability, and cartilage differentiation ability of the bone marrow-derived mesenchymal stem cells of Test Example 4 are shown.

1． TECHNICAL FIELD The present invention relates to a method for isolating mesenchymal stem cells, which comprises isolating mesenchymal stem cells using bone material as a material.

  Bone quality is a bone tissue part mainly composed of a bone matrix, and is a tissue that is distinguished from bone marrow. In the present invention, the bone quality is not particularly limited as long as it is a portion other than the bone marrow of bone (usually composed of periosteum, bone quality, and bone marrow in the case of bone). Specifically, the bone quality includes, for example, a part composed of periosteum and bone in a hard bone, preferably a bone part (comprised of dense bone and cancellous bone) not including the periosteum in a hard bone, and more preferably bone in a hard bone. The cortex (compact bone part) can be mentioned. Bone quality as a material may be used alone or in combination of two or more.

  The bone-derived organism is not particularly limited as long as it is an organism having bone. Specifically, the bone-derived organisms include, for example, mammals such as humans, mice, rhesus monkeys, rabbits, and rats; birds such as chickens; amphibians such as Xenopus; or fishes such as zebrafish, medaka, and tiger pufferfish. Preferred examples include mammals such as humans, mice, rhesus monkeys, rabbits, and rats, more preferred examples include humans, mice, and rats, and even more preferred examples include humans. Bone-derived organisms may be used alone or in combination of two or more.

  The kind of bone which is a bone material is not particularly limited, and various kinds of bones can be adopted. Specifically, bone types from which bone quality is derived include, for example, long bones such as the humerus, radius, ulna, femur, tibia, and radius; short bones such as the carpal bone or tarsal bone; Flat bones such as the occipital bone, ribs, or sternum; or aerated bones such as the maxilla, preferably long bones such as the humerus, ribs, ulna, femur, tibia, and ribs, more preferably Femur or tibia. The bone material may be used alone or in combination of two or more.

  Bone quality can be obtained by using bone containing bone marrow as a raw material, for example, by cutting the bone and removing the bone marrow present in the bone lumen. Bone marrow can be removed by a known method. For example, a method of washing away the bone lumen using a buffer solution such as PBS as a washing solution can be mentioned. In addition, when bone that does not contain bone marrow is used as a raw material, bone can be used as bone quality as it is. In addition, as a bone which is a raw material, for example, a bone head removed by an operation such as a bone head replacement can be employed.

  The obtained bone quality may be used as a material as it is, but it is preferable to use it after crushing or crushing by a known method. Examples of the crushing or crushing method include a method of crushing on a mortar and a method using various crushers or crushers.

  Separation of mesenchymal stem cells can be performed according to a known method. For example, there is a method in which cells are extracted from bone material as a material, and the cells are separated from the extracted cells using a property known as a property of mesenchymal stem cells as an index.

  Extraction of cells can be performed according to a known method. For example, it is performed by treating bone quality with a known proteolytic enzyme used for cell extraction. By this treatment, proteins such as collagen constituting the bone matrix in the bone quality are decomposed, and cells existing in the bone matrix are extracted.

  The proteolytic enzyme is not particularly limited as long as it is an enzyme having an activity of degrading the protein constituting the bone matrix. Examples of the proteolytic enzyme include collagenase, dispase, trypsin, or pronase, preferably collagenase or dispase, more preferably a mixture of collagenase and dispase. As a mixture of collagenase and dispase, the weight ratio (collagenase weight: dispase weight) is, for example, 1.0: 0.1-10, preferably 1.0: 0.5-4, more preferably 1.0: 1.0-1.5. The mixture which is is mentioned. Proteolytic enzymes may be used alone or in combination of two or more.

  The treatment with a proteolytic enzyme is carried out by reacting bone quality (preferably bone crush) in a known enzyme treatment method, specifically, for example, in a solvent containing a proteolytic enzyme.

  The solvent is not particularly limited as long as the proteolytic enzyme is not inactivated and the cells are not denatured. As the solvent, for example, a buffer solution such as PBS, or a cell culture medium such as α medium can be used.

  The concentration of the proteolytic enzyme is not particularly limited as long as the proteolytic enzyme exhibits activity, and can be appropriately set according to the type of the proteolytic enzyme. The concentration of the proteolytic enzyme is, for example, 0.1 to 10 mg / mL, preferably 1.0 to 6.0 mg / mL, more preferably 2.5 to 4.0 mg / mL.

  The reaction temperature is not particularly limited as long as the proteolytic enzyme is not inactivated and the cells are not denatured, and can be appropriately set according to the type of the proteolytic enzyme. As reaction temperature, 20-50 degreeC, for example, Preferably 30-40 degreeC is mentioned.

  The treatment time is not particularly limited as long as the proteolytic enzyme can exert its activity. Examples of the treatment time include 0.5 to 4 hours, preferably 1 to 2 hours.

  In addition, when using a proteolytic enzyme whose activity is inhibited by calcium ion or magnesium ion (for example, trypsin described below), a chelating agent such as EDTA or EGTA is further added to a concentration of 0.5 to 1.5 mM, for example. It is good to add further to. Further, during the reaction, it is preferable to gently stir so that the bone quality in the reaction solution does not precipitate.

  Separation of cells from the extracted cells using the known properties of mesenchymal stem cells as an index can be performed according to known methods.

  As known properties of mesenchymal stem cells, for example, as described in Non-Patent Document 1 and the like, it is known that when cultured, it adheres to a culture dish and exhibits a fibroblast-like morphology. Therefore, for example, mesenchymal stem cells can be isolated using as an indicator that when cultured in a culture dish, they adhere to the culture dish and show a fibroblast-like morphology.

  As known properties of mesenchymal stem cells, for example, as described in Non-Patent Documents 18 to 24, it is known that a specific cell surface marker is positive or negative. Non-patent document 18 describes mesenchymal stem cells as adhesion molecules (CD44, CD50, CD54, CD58, CD56, CD62L, CD106, CD166, etc.), cytokine receptors (CD71, CDw119, CD120a, b, CD121a, b). , CD123, CD124, CD126, CD127, CD140a, etc.), integrin family (CD29, CD49a, CD49b, CD49c, CD49e, CD49f, CD61, CD104, etc.), and other markers (CD9, CD73, CD90, CD105, CD146, etc.) And CD157) are described as being positive. Non-Patent Documents 19 and 20 describe that CD45 negative, TER119 negative, PDGFRα positive, and Sca-1 positive cells can be isolated from mouse bone marrow cells as mesenchymal stem cells. Non-Patent Document 21 describes that CD271 is useful for efficient separation of mesenchymal stem cells. Non-Patent Document 22 describes that CD146 (MUC18) is useful for efficient separation of mesenchymal stem cells. Non-patent document 23 describes that mesenchymal stem cells can be separated using CD271 positive and CD56 positive, or MSCA-1 positive and CD56 positive as indicators. Non-Patent Document 24 describes that SSEA-4, which has been considered as an ES cell marker, is useful for efficient separation of mesenchymal stem cells. Therefore, mesenchymal stem cells can be isolated using positive or negative of these markers as an index.

  As known properties of mesenchymal stem cells, for example, it is known that blood cell markers are negative. Therefore, it is good also as one parameter | index that a blood cell system cell marker is negative. Examples of blood cell markers include blood cells such as leukocytes such as neutrophils, eosinophils, basophils, lymphocytes, monocytes, or macrophages, red blood cells, platelets, mast cells, or dendritic cells. As long as it is a known marker. Specific examples include B220, CD11b, Ly6G, TcRαβ, TcRγδ, Ter119, CD2, CD3, CD4, CD14, CD19, CD24, CD41, CD56, CD66c, CD235a, or CD45. Preferably, in the case of mice, B220, CD11b, Ly6G, TcRαβ, TcRγδ, and Ter119 are all used as lineage markers, and in the case of humans, CD2, CD3, CD4, CD14, CD19, CD24, CD41, CD56, CD66c, And all of CD235a can be used as Lineage markers. The blood cell marker may be used alone or in combination of two or more.

  Moreover, you may remove in advance the apoptotic cell and the dead cell in the extracted cell by isolate | separating by making it the parameter | index that an apoptosis marker and / or a dead cell marker are negative. Examples of the apoptosis marker include AnnexinV, TUNEL, JC-1, cytochrome C, Rhodamine 123, TMRE / TMRM, and preferably AnnexinV. Examples of the dead cell marker include 7ADD, PI, and Hoechst 33258, and preferably 7ADD. These may be used alone or in combination of two or more.

  As one embodiment of the present invention, it is preferable to use CD271 negative as an index. By using this as an index, mesenchymal stem cells that show differentiation potential into cartilage but do not show differentiation ability into bone can be isolated. Another embodiment of the present invention preferably includes using SSEA-4 negative and CD271 positive as an index. By using this as an index, mesenchymal stem cells that show differentiation potential into bone but do not show differentiation ability into cartilage can be isolated.

  In regenerative medicine, for example, when mesenchymal stem cells are injected to regenerate a deficient cartilage, the risk that not only cartilage but also bone is formed in the deficient part becomes a problem. Therefore, as described above, mesenchymal stem cells that show differentiation ability into cartilage but do not show differentiation ability into bone, or mesenchymal stem cells that show differentiation ability into bone but not differentiation ability into cartilage. Is very useful to solve such problems.

  You may employ | adopt the said parameter | index, combining 1 type (s) or 2 or more types.

  Separation using the marker as an index can be performed according to a known method. For example, it is performed by staining cells, judging positive or negative based on the amount of fluorescence emitted by the stained cells, and sorting out positive or negative cells. Specifically, the series of operations can be performed using, for example, a flow cytometer having a cell sorting function.

  For example, when the marker is a cell endogenous protein, the cell can be stained by immunostaining the cell using a labeled antibody against the marker, and the marker is a cell endogenous factor other than the protein (eg, cell surface). Phosphatidylserine, etc.) can be performed by staining cells with a marker labeled, such as AnnexinV (apoptosis marker). In the case of a label (fluorescent substance) (for example, 7ADD (dead cell marker)) that binds to a cellular endogenous factor (for example, DNA or the like), the cell can be stained using the marker.

  Various commercially available products can be used as the labeled antibody against the marker and the labeled marker. Alternatively, an antibody against a marker or a marker may be bound to cells according to a known method, and then a label may be further bound to the antibody or the marker. As a kind of label | marker, FITC, PE, Pacific Blue, APC, PE-Cy7, PE-Cy5, PerCP, Brilliant Violet 570, or Brilliant Violet421 is mentioned. For the selection of the label, it is preferable to select a label in which the fluorescence excitation wavelength and the peak of the fluorescence wavelength of the label do not overlap as much as possible.

  The positive or negative judgment is made based on the amount of fluorescence emitted by the stained cells according to a known method. This determination can be made according to a known method. For example, the amount of fluorescence emitted by a negative control cell (specifically, for example, a cell stained with an isotype control antibody) is defined as a background fluorescence amount, and a sufficiently high fluorescence amount relative to the background is used as a reference. A cell having a low fluorescence amount is determined as a negative cell, and a cell having a higher fluorescence amount than the reference is determined as a positive cell.

  Sorting of positive or negative cells can be performed according to a known method, for example, by a droplet charging method or a cell capture method.

  By such a method of the present invention, mesenchymal stem cells can be easily and efficiently separated, whereby a large amount of mesenchymal stem cells can be easily prepared. In addition, since the bone material, which is a material, is a part that is normally discarded, the separation method using the bone material as a material is excellent in that the raw material cost is very low and the raw material procurement is easy. The obtained mesenchymal stem cells can be used in various fields such as regenerative medicine such as cartilage and bone, immune control, treatment of graft-versus-host disease, and support / amplification of hematopoietic stem cells (HSC).

2． Bone quality-derived mesenchymal stem cells The present invention relates to mesenchymal stem cells isolated by the method described in "1. Method for separating mesenchymal stem cells" (hereinafter referred to as "the mesenchymal stem cells of the present invention"). . Since the mesenchymal stem cells of the present invention are derived from bone quality, they are different in nature from the conventionally known bone marrow and umbilical cord blood-derived mesenchymal stem cells. Therefore, the mesenchymal stem cell of the present invention is a novel cell different from the conventional mesenchymal stem cell, and is provided as a new research material.

  As a preferred embodiment of the mesenchymal stem cell of the present invention, (a) derived from bone substance, (b) negative for blood cell marker, (c) negative for apoptosis marker and / or dead cell marker, and ( d) Mesenchymal stem cells that are negative for CD271. These cells show the ability to differentiate into cartilage but do not show the ability to differentiate into bone. Therefore, for example, it is useful for regenerative medicine of cartilage tissue.

  Another preferred embodiment of the mesenchymal stem cells of the present invention is (a) derived from bone, (b) negative for hematopoietic cell markers, (c) negative for apoptosis markers and / or dead cell markers, (E) Mesenchymal stem cells that are SSEA-4 negative and (f) CD271 positive. These cells show the ability to differentiate into bone but do not show the ability to differentiate into cartilage. Therefore, it is useful for regenerative medicine of bone tissue, for example.

  EXAMPLES The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

Example 1: Separation of mouse bone quality-derived mesenchymal stem cells Already known as an index for isolating bone marrow-derived mesenchymal stem cells, an index that Lineage and CD45 are negative and that Sca-1 and PDGFRα are positive ( Based on Non-Patent Documents 19 and 20), mesenchymal stem cells were isolated from mouse bone material. Specifically, it was performed as follows.

[Preparation of cell fluid]
A total of 6 tibias were collected from 3 8 week old female C57BL / 6 mice. The epiphysis of the tibia was excised and the bone marrow present in the tibia lumen was washed away with PBS + 2% FCS. The bone part from which the bone marrow was removed was pulverized with a mortar, and the obtained pulverized product was washed with PBS + 2% FCS. The pulverized material remaining after washing was placed in a beaker, and the collagenase-dispase solution was added to such an extent that the pulverized material was sufficiently immersed. The beaker was placed on a shaking plate in a 37 ° C. incubator, and the liquid in the beaker was treated for 1 hour with gentle stirring. The obtained treatment solution was used as a cell solution. The collagenase-dispase solution (20 ml in total) used in the above preparation was prepared by using the collagenase type I (Invitrogen) solution (10 mg / ml) and Dispase (Invitrogen) solution (10 mg / ml) using PBS. Each was prepared separately, sterilized with a millipore filter, mixed with 3 ml of sterilized Collagenase solution and 4 ml of sterilized Dispase solution, and further mixed with 13 ml of α medium containing 20% FCS.

[Immunostaining]
Cells in the cell fluid were labeled with FITC-labeled Lineage markers (B220, CD11b, Ly6G, TcRαβ, TcRγδ, Ter119), PE-labeled CD45 antibody, Pacific Blue-labeled Sca-1 antibody, APC-labeled Immunostaining was performed according to a conventional method using PDGFRα antibody, 7ADD, and FITC-labeled AnnexinV.

[FACS analysis]
By FACS (FACS Aria (BD Bioscience)), cells that were negative for Lineage and CD45 and positive for Sca-1 and PDGFRα were separated as mesenchymal stem cells from the cell solution in which the cells were immunostained. The boundary between positive and negative was set according to a standard method. Fig. 1 shows the results of separation by FACS.

Comparative Example 1: The same procedure as in Example 1 was performed, except that the bone marrow removed in [Preparation of cell fluid] in Example 1 was used as the separated cell fluid of mouse bone marrow-derived mesenchymal stem cells . The results of separation by FACS are shown in FIG.

Example 2: Isolation of mesenchymal stem cells derived from human bone mesenchymal stem cells were isolated from human bone material. Specifically, it was performed as follows.

[Preparation of cell fluid]
Under the approval of the Kansai Medical University Ethics Committee (Kan Medical School No. 1114), provision of bone cortex blocks (1-2 cm square) at the end of the femur removed during femoral artificial head replacement Received. In addition, written consent was obtained from the patient with the artificial bone head replacement. The bone cortex block was washed several times with physiological saline, then dispersed in collagenase-dispase-DNase solution and shaken at 37 ° C. for 1 hour. After the shaking treatment, the bone cortical block became sponge-like and a bone cortical cell suspension was obtained. Thereafter, bone fragments and cell clumps were removed through a 70 μm cell strainer. Furthermore, hemolysis was performed to remove red blood cells, and a bone quality-derived cell fluid was obtained.

[Immunostaining]
Cells in the obtained cell fluid are labeled with FITC-labeled lineage markers (CD2, CD3, CD4, CD14, CD16, CD19, CD24, CD41, CD56, CD66c, CD235a) antibody, CD45 antibody labeled with BV510, PE Immunostaining was performed using a labeled CD271 antibody, APC-labeled SSEA-4 antibody, 7-AAD, and FITC-labeled Annexin V according to a conventional method.

[FACS analysis]
Using FACS, doublet was removed by FSC / SSC gate from the cell fluid in which cells were immunostained. Furthermore, 7-ADD (dead cell marker), AnnexinV (apoptosis marker), Lineage (blood cell marker), and CD45 (blood cell marker) negative cells were collected. This was further divided into four (SSEA-4 negative and CD271 negative fraction (DN (Double Negative)), SSEA-4 positive and CD271 negative fraction (SSEA-4 SP (Single Positive)), SSEA- Subfractionation into 4 negative and CD271 positive fractions (CD271 SP (Single Positive)) and SSEA-4 positive and CD271 positive fractions (DP (Double Positive))). FACS fractionation results are shown in FIG.

[CFU-F assay]
Four fractions of cells obtained by FACS were suspended in a medium (α-MEM containing 10% FCS), the cell suspension was seeded on a 10 cm ² plastic dish, and cultured according to a standard method. Twelve days after the start of the culture, the presence or absence of CFU-F formation as an indicator of the presence or absence of mesenchymal stem cells was observed with an inverted phase contrast microscope. The results of CFU-F assay are shown in FIG.

[Measurement of cell size]
For each of the four fractional cells obtained by FACS, forward scattered light (FSC) indicating the cell size was measured. The measurement result of the forward scattered light is shown in FIG.

[result]
From FIG. 3, the fraction of each fractional cell was 6.9% for the DP fraction, 14.8% for the CD271 SP fraction, 2.5% for the SSEA-4 SP fraction, and 61.5% for the DN fraction. From FIG. 4, it was confirmed that all four fractions formed CFU-F. This showed that all four fractions contained mesenchymal stem cells. From the cell size distribution pattern shown in FIG. 5, the four fractions showed similar peaks in the vicinity of FSC 250-600, but the distribution pattern was different in the vicinity of FSC 800-2000.

Comparative Example 2: Isolation of mesenchymal stem cells derived from human bone marrow Mesenchymal stem cells were isolated using human bone marrow as a material. Specifically, it was performed as follows.

[Preparation of cell fluid]
At the time of the allogeneic allogeneic bone marrow transplantation or the unrelated allogeneic bone marrow transplantation, the cells remaining in the filter bag usually discarded were collected and washed several times with PBS-. The mononuclear cell fraction was recovered from the washed cells by Ficoll-Paque specific gravity centrifugation. A cell solution was obtained by removing lineage positive cells from the collected mononuclear cell fraction with immunomagnetic beads. Regarding the use of a filter bag for allogeneic allogeneic bone marrow transplantation, with the approval of the Kansai Medical University Ethics Committee (Seki Medical Lingen No. 611), written consent from a healthy donor for the bone transplantation Got. In addition, the use of filter bags for unrelated allogeneic bone marrow transplantation was approved by the Data and Sample Management Committee of the Foundation for Bone Marrow Transplantation (December 19, 2006), and healthy people with such bone marrow transplantation. Written consent was obtained from the donor.

[FACS analysis]
As in Example 2, the cell fluid was divided into 4 fractions (SSEA-4 negative and CD271 negative (DN (Double Negative)), SSEA-4 positive and CD271 negative (SSEA-4 SP ( Single Positive)), SSEA-4 negative and CD271 positive fraction (CD271 SP (Single Positive)), and SSEA-4 positive and CD271 positive fraction (DP (Double Positive))) . Fractionation was performed multiple times with different cell fluids. One of the FACS fractionation results is shown in FIG.

[Mesenchymal stem cell establishment test]
Four fractions of cells obtained by FACS were suspended in a medium (α-MEM containing 10% FCS), the cell suspension was seeded on a 10 cm ² plastic dish, and culture was started according to a standard method. After the culture was started, when the cells on the dish became confluent, a part of the cells was subcultured to a new dish. At the 4th passage (about 1 month from the start of culture), the presence or absence of cell proliferation and the cell morphology were observed to evaluate whether or not mesenchymal stem cells could be established. The observation results of cell morphology are shown in FIG.

[result]
As a result of FACS analysis, the ratio of the number of cells in each fraction to the number of cells in the cell solution was 0.1 to 3.1% for the DP fraction, 0.1 to 6.8% for the CD271 SP fraction, and 3.3 to 42.5 for the SSEA-4 SP fraction. %, DN fraction was 50.7-93.4%. In addition, as shown in FIG. 7, mesenchymal stem cells were successfully established in three fractions (DN, CD271 SP, DP) other than the SSEA-4 SP fraction.

Comparative Example 3: Isolation of mouse bone quality-derived microstem cells In [FACS analysis] of Example 1, Lineage (blood cell marker), CD45 (blood cell marker), and PDGFRα (stem cell marker) were negative, and Sca Cells positive for -1 were isolated as microstem cells.

Test example 1: Separation efficiency of bone-derived mesenchymal stem cells Separation efficiency when separating mesenchymal stem cells from bone (Example 1) and when separating mesenchymal stem cells from bone marrow (Comparative Example 1) (Number of mesenchymal stem cells per 1 × 10 ⁶ cells in cell fluid) was compared. The comparison results are shown in FIG. FIG. 8 shows that mesenchymal stem cells can be separated about 1000 times more efficiently when bone material is used than bone marrow.

Test Example 2: Size of bone quality-derived mesenchymal stem cells The size of bone quality was compared between bone quality-derived mesenchymal stem cells (Example 1) and bone quality-derived microstem cells (Comparative Example 3). Specifically, forward scattered light (FSC) indicating the size of the cells was measured, and the measured values were compared. The comparison results are shown in FIG. In FIG. 9, a indicates the distribution of the size of bone quality-derived mesenchymal stem cells, and b indicates the distribution of the size of bone quality-derived microstem cells. From Fig. 9, the peaks of both cells are greatly different (mesenchymal stem cells: around FSC 550, micro-stem cells: around FSC 50), and the distribution pattern is also different. It was suggested.

Test Example 3: Proliferation characteristics of bone quality-derived mesenchymal stem cells Growth characteristics were compared between bone quality-derived mesenchymal stem cells (Example 1) and bone quality-derived microstem cells (Comparative Example 3). Specifically, each cell was cultured in a medium (α-MEM containing 20% FCS) for 2 weeks according to a conventional method. Cells in culture were observed with an inverted phase contrast microscope. The observation results are shown in FIG. Mesenchymal stem cells (MSCs) proliferated and formed clusters. In addition, accumulation of fat droplets was also observed. On the other hand, no division / proliferation was observed in bone quality-derived microstem cells. Thus, both cells have different growth characteristics, suggesting that they are distinct cells that are clearly distinguished.

Test Example 4: Analysis of differentiation potential of bone quality-derived mesenchymal stem cells Differentiation of bone quality-derived mesenchymal stem cells (Example 2) and bone marrow-derived mesenchymal stem cells (Comparative Example 2) into bone, fat, and chondrocytes The ability was analyzed. Specifically, it went as follows.

[Analysis of differentiation potential of bone quality-derived mesenchymal stem cells]
Each of the four fractions of bone quality-derived mesenchymal stem cells (DP, CD271 SP, SSEA-4 SP fraction, and DN fraction) is divided into a bone differentiation induction kit, a fat differentiation induction kit, or a cartilage differentiation induction kit (all Differentiation was induced with R & D, Mesenchymal Stem Cell Functional Identification Kit), and cells on day 21 after induction were fixed with 4% paraformaldehyde (PFA). Regarding the sample in which bone differentiation was induced, a sample fixed with 4% PFA was stained with alkaline phosphatase according to a conventional method, and a stained image was observed (CKX41, Olympus) (FIG. 11). As for the sample in which adipose differentiation was induced, a sample fixed with 4% PFA was stained with Sudan II (Oil Red O staining) according to a conventional method, and a stained image was observed (CKX41, Olympus) (FIG. 12). For the sample induced to differentiate cartilage, a frozen section was prepared from a cell mass fixed with 4% PFA, the section was stained with Alcian Blue according to a standard method, and a stained image was observed (CKX41, Olympus) (FIG. 13). .

[Results of analysis of differentiation potential of bone-derived mesenchymal stem cells]
As shown in FIGS. 11 to 13, the mesenchymal stem cells established from nucleated cells derived from whole bone cells (whole bone) were able to induce differentiation into three cells of bone, fat, and cartilage. Similar to the control, the mesenchymal stem cells of the DP fraction were able to induce differentiation into three cell lines, bone, fat, and cartilage. On the other hand, the mesenchymal stem cells of the DN fraction could induce differentiation into two cell lines, fat and cartilage, but could not induce differentiation into bone. Similarly, the mesenchymal stem cells of the SSEA-4 SP fraction could be induced to differentiate into two cells, fat and cartilage, but could not be induced to differentiate into bone. The CD271 SP fraction mesenchymal stem cells were able to induce differentiation into two cells, bone and fat, but could not induce differentiation into cartilage. The tissues in which mesenchymal stem cells differentiate from each fraction are summarized in Table 1.

From the above, it is suggested that mesenchymal stem cells that differentiate only into specific tissues (or do not differentiate into specific tissues) can be isolated by fractionating bone quality-derived cell fluid using SSEA-4 and CD271 as indices. It was.

[Analysis of differentiation potential of bone marrow-derived mesenchymal stem cells]
Each of the four fractions of bone marrow-derived mesenchymal stem cells (DP, CD271 SP, SSEA-4 SP fraction, and DN fraction) was converted into a bone differentiation induction kit, a fat differentiation induction kit, or a cartilage differentiation induction kit (all R & D, Mesenchymal Stem Cell Functional Identification Kit) was used to induce differentiation, and cells were fixed with 4% PFA on the 21st day after induction. For samples induced to differentiate into bone, 4% PFA-fixed sample was immunostained with anti-osteocalcin antibody (attached to the bone differentiation induction kit) as the primary antibody, and the stained image was fluorescent microscope (BX50, Olympus) Observed with. For samples induced to differentiate into fat, 4% PFA-fixed sample was immunostained with anti-FABP-4 antibody (attached to adipose differentiation induction kit) as the primary antibody, and the stained image was fluorescence microscope (BX50, Olympus) Observed). For the sample in which cartilage differentiation was induced, a frozen section was prepared from a cell mass fixed with 4% PFA, and the section was immunostained using an anti-Aggrecan antibody (attached to the cartilage differentiation induction kit) as a primary antibody, and stained. The image was observed with a fluorescence microscope (BX50, Olympus). An observation image is shown in FIG.

[Results of analysis of differentiation potential of bone marrow-derived mesenchymal stem cells]
As shown in FIG. 14, the mesenchymal stem cells other than the DP fraction were able to induce differentiation into three cells of bone, fat, and cartilage. On the other hand, the mesenchymal stem cells of the DP fraction were able to induce differentiation into three cell lines, bone and cartilage, but could not induce differentiation into fat.

Claims (7)

A method for isolating mesenchymal stem cells, comprising isolating mesenchymal stem cells using bone quality as a material. The separation method according to claim 1, wherein mesenchymal stem cells are separated using CD271 negative as an index. The separation method according to claim 1, wherein mesenchymal stem cells are separated using SSEA-4 negative and CD271 positive as indices. The method according to any one of claims 1 to 3, further comprising isolating mesenchymal stem cells using as an index at least one selected from the group consisting of blood cell marker negative, apoptosis marker negative, and dead cell marker negative. . A mesenchymal stem cell isolated by the separation method according to claim 1. Mesenchymal stem cells that are (a) derived from bone quality, (b) negative for hematopoietic cell markers, (c) negative for apoptosis markers and / or dead cell markers, and (d) negative for CD271. (A) derived from bone quality, (b) negative for hematopoietic cell marker, (c) negative for apoptosis marker and / or dead cell marker, (e) negative for SSEA-4, and (f) positive for CD271 A mesenchymal stem cell.

Images