JP4929447B2 - Method for preparing chondrocytes from mesenchymal stem cells - Google Patents

Description

  The present invention relates to a method for preparing chondrocytes from mesenchymal cells by inducing differentiation of mesenchymal stem cells, which are undifferentiated cells, into chondrocytes.

  Mesenchymal stem cells (MSCs) are undifferentiated cells present in the bone marrow and have the ability to self-proliferate. In addition, bone marrow cells, chondrocytes, adipocytes, skeletal muscles, cardiac muscles, ligaments, tendons, etc. It is a multipotent cell that can differentiate into various cells of the mesenchymal embryo system. The use of MSC for so-called regenerative medicine such as organ regeneration and cell transplantation based on this multi-differentiation is expected.

  In order for MSCs with multipotency to differentiate into specific cells or tissues, it is considered necessary to have specific signals or factors (differentiation induction factors) that induce or determine such differentiation. Yes. Therefore, in order to differentiate MSCs into specific cells or tissues in vitro, various differentiation inducers having a function of specifically determining differentiation into such cells or tissues are used.

  Examples of differentiation inducers include polypeptide differentiation inducers such as bFGF, TGFβ, and insulin (hereinafter referred to as peptide differentiation inducers), phospholipids such as β-glycerophosphate, fluorinated steroids such as dexamethasone, indomethacin Low molecular weight organic compound differentiation inducers such as arylacetic acid non-steroids such as selenium compounds such as sodium selenite, etc. (hereinafter referred to as non-peptidic differentiation inducers) are known. Either of them has been added alone or in combination to a medium for culturing mesenchymal stem cells (Non-patent Document 1).

  For example, it is known that MSC is induced to differentiate into adipocytes by adding dexamethasone, insulin and indomethacin to the medium, and MSC is induced to differentiate into osteoblasts by adding dexamethasone and chryserophosphate to the medium. (Non-Patent Document 2).

  The induction of differentiation of MSC into chondrocytes has extremely important significance such as the possibility of regenerative medicine for diseases such as osteoarthritis. Therefore, peptide differentiation inducers that induce differentiation of MSCs into chondrocytes Alternatively, non-peptide differentiation inducing factors have been actively searched. So far, successful induction of differentiation of MSCs into chondrocytes by adding TGF-β1, IGF-1, FGF-2 or / and BMP-6, which are peptide differentiation inducers, to the medium Examples have also been reported (Non-Patent Document 3, Non-Patent Document 4, Non-Patent Document 5). However, the method using such a factor has a problem that the rate of differentiation into chondrocytes is not necessarily sufficient.

  A major factor that makes it difficult to differentiate MSCs into chondrocytes is that chondrocytes (tissues) are cells / tissues that are desired to be three-dimensional. It is known that mature chondrocytes change into fibroblast-like morphology and eventually become monolayered when division proceeds and the number of passages increases. Therefore, even when differentiation induction from undifferentiated and multipotent cells such as MSC into chondrocytes is confirmed, the results show that chondrocytes differentiate into fibroblast-like cells before they become three-dimensional. Has been.

Edited by Norio Nakatsuji et al., Stem Cell / Clone Research Protocol, 2001, Yodosha, Tokyo Pittenger et al., Science, 1999, 284, 143-147. Fukumoto et al., Osteroarthritis and Cartilage, 2003, 11, 55-64 Chuma et al., Osteroarthritis and Cartilage, 2004, 12: 834-842 Indrawattana et al., Biochem. Biophys. Res. Commn., 2004, 320, 914-919

  As described above, among the techniques for inducing differentiation of MSCs into specific cells, induction of differentiation into chondrocytes is considered as one of the most difficult techniques. Therefore, specifying methods or conditions for efficiently inducing differentiation of mesenchymal cells into chondrocytes, including the identification of differentiation-inducing factors that determine the differentiation of MSCs into chondrocytes, This is a very important issue in application.

  The present inventor has surprisingly found that MSC placed in an environment in which there is no substance known as a peptidic differentiation inducing factor or a compound known as a non-peptidic differentiation inducing factor is efficient. Is one of the conditions that determine the differentiation of MSC into chondrocytes by in vitro culture using a medium that does not contain peptidic differentiation-inducing factor or non-peptidic differentiation-inducing factor. As a result, the present invention has been completed.

  The present invention comprises the following aspects.

1) A method for preparing chondrocytes from mesenchymal stem cells, comprising culturing in a medium not containing a peptide differentiation inducing factor.

2) The preparation method according to 1), wherein the culture is performed for 3 days or more.

3) The preparation method according to 1), which is a medium not containing a non-peptide differentiation inducing factor.

4) The preparation method according to 3), wherein the non-peptidic differentiation inducing factor is a phospholipid, a steroid compound, an arylacetic acid nonsteroid compound, or a selenium compound.

5) The preparation method according to any one of 1) to 4), wherein the medium is a minimal medium.

  The present invention relates to epidemiological extrinsic factors such as conventionally used peptide differentiation inducers, non-peptide components or inducers that are suggested to possibly promote differentiation induction of specific cells, etc. The use of is not necessary. Therefore, when the induced chondrocytes are administered by a living transplantation technique or the like, the possibility of such factor contamination can be eliminated. For example, in the case of TGF-β, these factors may act as growth-promoting factors depending on the cells that act, but in arthritis such as rheumatoid arthritis, TGF-β is involved in other cytokines and destroys the extracellular matrix. It also has the opposite effect of acting as a growth inhibitory factor, such as causing arthritis. For this reason, many peptidic factors have not been approved as pharmaceuticals, and contamination of these factors should be avoided as much as possible when transplanting differentiated cells. Therefore, the method of the present invention which does not use these factors from the beginning is extremely advantageous in that there is no contamination of such factors.

  In addition, most of such factors, particularly peptidic differentiation-inducing factors, are expensive, and the present invention that does not require their use also leads to a reduction in the cost of preparing chondrocytes from MSCs.

  The method of the present invention is a method for preparing chondrocytes from MSCs by culturing MSCs in vitro in the absence of peptidic differentiation inducing factors and further non-peptidic differentiation inducing factors.

  The medium that can be used in this method is not particularly limited except that it does not contain peptidic differentiation inducing factors or non-peptidic differentiation inducing factors. However, in terms of eliminating contamination of these factors, it is fundamental. Use of a medium (Minimum Essential Medium, MEM) is preferred. Specifically, when the total weight of the medium is 100%, about 0 to 25% by weight of serum, particularly fetal bovine serum, about 0.1 to 1% by weight of inorganic salt, 0.01 to 0.1% by weight It is preferable to use a medium containing about 15 amino acids, about 0.1 to 1% by weight of sugar source, and vitamins as trace components.

  Moreover, as long as it is components other than a peptide differentiation induction factor and a non-peptide differentiation induction factor, components suitable for cell culture, for example, cell adhesion factors such as laminin and collagen may be appropriately added to the medium.

  In addition, since MSCs are adherent cells, it is preferable to use a medium and a solid phase substrate that holds the cells. The solid phase substrate is not particularly limited as long as it has a stable physical form to which the MSC can adhere, and the material of the substrate, the surface form of the substrate, the presence or absence of surface coating, and the like.

  For example, as the solid phase substrate, a petri dish, a plate, or other solid phase substrate formed from glass, polystyrene, a metal plate, or the like can be used. Further, the surface of the solid phase substrate may be coated with a cell adhesion factor such as laminin, collagen, fibronectin or the like.

  In addition, as for the culture conditions such as culture temperature and time, there is no particular limitation except that the culture time is preferably 3 days or more, more preferably 10 days or more, and particularly preferably 20 days or more. As will be described in Examples, the method of the present invention can provide chondrocytes from the day after the culture start date of MSC. In addition, the culture may be performed on one solid phase substrate from the start to the end of the culture of undifferentiated mesenchymal stem cells, and the chondrocytes induced to differentiate after the start of the culture are taken out and transferred to another medium for selection. Alternatively, the culture may be performed.

  The culture temperature may be about 35 ° C. to 39 ° C., and the culture may be performed in an atmosphere of 2.5 to 7% carbon dioxide gas.

  It is known that MSCs cultured under the above conditions are differentiated into chondrocytes, based on the method of Pittenger et al. (Non-patent Document 2), that cells in culture are specifically expressed in chondrocytes. It can be monitored using expression of marker genes such as collagen type 2, immunostaining, or toluidine blue staining.

  The chondrocytes prepared according to the present invention are useful for the treatment of osteoarthritis caused by the reduction of cartilage tissue often seen in the elderly, and tendon and ligament tears / damage often found in athletes. Since the regenerative capacity of chondrocytes is inferior to that of other cells and tissues, there is a possibility that they can greatly contribute to various tissue regeneration medicines in the future.

  Hereinafter, specific examples of the present invention will be described in more detail by way of examples, but the present invention is not limited to such examples.

1) Culture method
MSCs derived from rat femur bone marrow purchased by cell line (Dainippon Pharmaceutical Co., Ltd.) were prepared in a minimal medium (Minimam Essential Medium, MEM /) containing 10% fetal bovine serum (Thermo) and 1% streptmycin penicillin (GIBCO invitrogen). GIBCO invitrogen) was added to 10 ⁴ cells / ml, and the culture was performed in an atmosphere of 37 ° C. and 5% CO ₂ , and the culture was continued while changing the medium every 3 to 5 days.

2) Confirmation of differentiated chondrocytes The presence of chondrocytes in cell culture was confirmed by the following method.

2-1) Confirmation of marker gene expression by RT-PCR After culturing, the cultured cells are collected with TRIzol (invitrogen) at regular intervals, mixed with 20% chloroform, and centrifuged at 15000 rpm for 15 minutes at 4 ° C. Thereafter, the supernatant was collected. An equal amount of isopropanol was mixed and centrifuged at 15000 rpm for 10 minutes at 4 ° C. to precipitate the RNA. It was washed with 75% ethanol, 75% ethanol was dried and then dissolved in diethyl pyrocarbonate water.

  From the RNA recovered as described above, cDNA was prepared using RT-PCR kit (takara high Fidelity RNA PCR kit), and PCR was performed using this as a template. Using specific primers for the following four genes known as chondrocyte marker genes, the expression of the genes was confirmed by PCR (qiagen hotstart master mix kit).

Collagen type 2
Forward primer (Fw): GAAGGATGGCTGCACGAAACAC
Reverse primer (Rv): AGTGCAGATCCTAGAGTGACTGC
Bone sialoprotein (BSP)
Fw: TCCAGCTACCCAAGAAGGCTG
Rv: TGGTGCCATAACTGGTCAGCTC
Agrecan
Fw: TCGAATCACTTGCACAGACCCCAACA
Rv: ATGCACGGTCAGCATGGCTG
Collagen type 10
Fw: GCCCTATTGGACCACCAGGTCC
Rv: GAGCACCTACCGCTGGGTAAGC

  By confirming the presence and amount of the fragment amplified by the PCR reaction by agarose gel electrophoresis, the presence or absence of expression of each marker gene and the expression were quantified.

2-2) Toluidine blue staining After the start of culture, 10% formalin solution was added to the cultured cells on the dish at regular intervals to fix the cells. It collect | recovered to the dish of a 9 cm diameter, and 10% formalin solution was added and fixed. 6-9 ml of 0.05% toluidine blue solution (pH 7.0) is added to fixed cells on the dish, and the cells are stained by rotary shaking, and the ratio of stained cells to unstained cells is measured with an optical microscope. Measured below. Toluidine blue is a reagent that specifically stains glycoproteins such as aggrecan, and the stained cells can be judged to be cells that contain more glycoproteins inside, that is, cells that have differentiated into chondrocytes. .

3) Induction of differentiation of MSC into chondrocytes When the culture of mesenchymal stem cells was started under the conditions shown in 1), expression of the BSP gene expressed in the early stage of chondrocytes was observed from the next day. The expression level of the gene reached a peak around the 6th day of culture and then decreased once, but then rose again around the 24th day of culture. The expression of collagen type 2 was confirmed on the sixth day from the start of the culture, and the expression level increased with the number of culture days. The expression of aggrecan was confirmed on the 28th day from the start of the culture, but the subsequent expression level was almost constant. This indicates that chondrocytes appear immediately after the start of the mesenchymal stem cell culture.

  Further, in the visual observation by the method 2-2), a monolayer of chondrocytes was confirmed at the edge of the proliferating cell colony about one week after the start of the culture. After 10 days, if a sufficient amount of cells for laminating the first layer of chondrocytes is laid on the dish surface, even if there is some space on the dish surface, it will not become confluent and the stacking will be confirmed. It was done. Furthermore, after 2 weeks, the cell mass of the chondrocytes can be confirmed, and the cell mass grows with the incubation time, and the chondrocytes in the cell mass grow to a size of about 100-150 μm and are three-dimensional. Chondrocytes taking various forms were confirmed. The differentiated chondrocytes continued from the 4th week after the start of the culture and continued to grow gradually, and grew more three-dimensionally. Further, the chondrocytes fused with other chondrocytes with the incubation time, and the stacked chondrocytes grew not only in the height direction but also in a plane.

  Toluidine blue-stained chondrocytes accounted for about 70 to 80% (visually) of the whole dish after 25 to 30 days of culture. When the culture time was extended to 40 days or more, the three-dimensionalization of chondrocytes further progressed, and the expression of toluidine blue also increased due to higher expression of glycoprotein, and the chondrocytes became about 90% (visually). .

FIG. 1 shows a phase contrast micrograph (left: enlarged view, right: low magnification) of cultured cells after culturing for 24 days according to the example. FIG. 2 shows the results of analyzing the product of the RT-PCR reaction by agarose gel electrophoresis. FIG. 3 shows photographs of toluidine blue staining results of cultured cells after culturing for 6 days (left) and 30 days (right) according to the examples.

Claims (3)

Mesenchymal stem cells derived from bone marrow were adhered to a solid substrate, including serum, and characterized by culturing in MEM medium without bFGF, TGF [beta and insulin not derived from the serum, mesenchymal bone marrow-derived A method for preparing chondrocytes from stem cells.   The preparation method of Claim 1 which culture | cultivates for 3 days or more. The preparation method according to claim 1, wherein the solid phase substrate is formed of glass, polystyrene, or a metal plate.