JP5888670B2 - Culture substrate for inducing osteoblast differentiation, method for inducing osteoblast differentiation, and method for producing osteoblast - Google Patents

Description

  The present invention relates to a culture substrate for inducing osteoblast differentiation, an osteoblast differentiation inducing method, an osteoblast production method, and an osteoblast obtained by the production method. According to the present invention, osteoblasts can be induced from mesenchymal stem cells.

  Osteoblasts are cells that form bone in bone tissue and produce and secrete proteins and the like to create a bone matrix. Specifically, osteoblasts secrete matrix proteins such as collagen in vivo and produce matrix vesicles called Matrix Vesicles there. Then, calcium phosphate is deposited around the matrix vesicles to complete the bone matrix, and the osteoblasts eventually become bone cells in the matrix.

  This osteoblast is differentiated from mesenchymal stem cells, and Non-Patent Document 1 discloses that differentiation can be induced by allowing dexamethasone, β-glycerophosphate, and ascorbic acid to act on mesenchymal stem cells. (Non-Patent Document 1). Patent Document 1 discloses that by using a compound having a Rho kinase inhibitory activity such as (+)-trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane and an osteoinductive factor in combination, It discloses that mesenchymal cells are induced to differentiate into osteoblasts. Furthermore, Patent Document 2 discloses that a calcium antagonist induces differentiation of mesenchymal cells into osteoblasts (Patent Document 2).

  However, the osteoblast differentiation-inducing activity, particularly the early differentiation-inducing activity by the above-mentioned compounds has not been sufficient. Further, dexamethasone and (+)-trans-4- (1-aminoethyl) -1- (4-pyridylcarbamoyl) cyclohexane are not originally present in the living body, and these are not present in the obtained osteoblasts. The effects of the synthesized compounds have not been fully investigated. Furthermore, when differentiating mesenchymal stem cells into osteoblasts, culture is performed using about 10% bovine fetal serum, etc., and pathogens of zoonotic diseases such as prions are present in bovine serum. The possibility of doing was also considered.

International Publication No. 2001/017562 International Publication No. 2006/123699 JP 2006-257014 A

“Science”, 1999, 284, p. 143-147

  An object of the present invention is to provide a method for inducing differentiation of osteoblasts more efficiently and powerfully than the conventional method, and in particular, to provide a method capable of inducing initial differentiation. Another object of the present invention is to provide an osteoblast differentiation induction method or an osteoblast production method capable of differentiating mesenchymal stem cells into osteoblasts in the presence or absence of serum at a low concentration. Furthermore, it is providing the osteoblast manufactured by the said manufacturing method.

As a result of earnest research on an osteoblast differentiation induction method or an osteoblast production method capable of efficiently and powerfully differentiating mesenchymal stem cells into osteoblasts, the present inventors have surprisingly found that fish It was found that mesenchymal stem cells can be differentiated into osteoblasts by contacting the cell culture substrate coated with the derived collagen with mesenchymal stem cells.
The present invention is based on these findings.

That is, the present invention
[1] A culture substrate for inducing osteoblast differentiation, having a contact surface with mesenchymal stem cells coated with fish-derived collagen,
[2] The culture substrate for osteoblast differentiation induction according to [1], wherein the fish-derived collagen is collagen derived from tilapia scales,
[3] The mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells, auricles Peritoneal membrane-derived mesenchymal stem cell, peripheral blood-derived mesenchymal stem cell, umbilical cord blood-derived mesenchymal stem cell, ligament-derived mesenchymal stem cell, tendon-derived mesenchymal stem cell, ES cell-derived mesenchymal stem cell, or iPS cell-derived The culture substrate for inducing osteoblast differentiation according to [1] or [2], which is a mesenchymal stem cell,
[4] The culture substrate for inducing osteoblast differentiation according to any one of [1] to [3], wherein the mesenchymal stem cells are derived from human,
[5] The bone according to any one of [1] to [4], which has a contact surface with a mesenchymal stem cell coated with at least one second collagen other than the coated first fish-derived collagen. Culture substrate for blast differentiation induction,
[6] The second collagen is a collagen selected from the group consisting of mammal-derived collagen, avian-derived collagen, reptile-derived collagen, amphibian-derived collagen, fish-derived collagen, invertebrate-derived collagen, and combinations thereof. The culture substrate for inducing osteoblast differentiation according to any one of [1] to [5],
[7] An osteoblast differentiation induction kit comprising the osteoblast differentiation induction culture substrate according to any one of [1] to [6],
[8] The osteoblast differentiation inducing kit according to [7], further comprising an osteoblast differentiation inducing factor,
[9] A method for inducing osteoblast differentiation comprising a fish-derived collagen contact step in which a cell culture substrate coated with fish-derived collagen is contacted with a mesenchymal stem cell,
[10] The mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells, auricles Peritoneal membrane-derived mesenchymal stem cell, peripheral blood-derived mesenchymal stem cell, umbilical cord blood-derived mesenchymal stem cell, ligament-derived mesenchymal stem cell, tendon-derived mesenchymal stem cell, ES cell-derived mesenchymal stem cell, or iPS cell-derived The osteoblast differentiation induction method according to [9], which is a mesenchymal stem cell,
[11] The osteoblast differentiation inducing method according to [9] or [10], wherein the mesenchymal stem cells are derived from humans.
[12] The method further includes a second collagen contact step in which a cell culture substrate coated with at least one second collagen other than the coated first fish-derived collagen is contacted with a mesenchymal stem cell. ] The osteoblast differentiation inducing method according to any one of [11] to [11],
[13] The second collagen is a collagen selected from the group consisting of mammal-derived collagen, avian-derived collagen, reptile-derived collagen, amphibian-derived collagen, fish-derived collagen, invertebrate-derived collagen, and combinations thereof. [9] The method for inducing differentiation of osteoblasts according to any one of [9] to [12],
[14] The osteoblast differentiation inducing method according to any one of [9] to [13], further comprising an osteoblast differentiation inducing factor contact step of contacting the mesenchymal stem cell with an osteoblast differentiation inducing factor,
[15] The osteoblast differentiation induction method according to any one of [9] to [14], wherein the first fish-derived collagen is tilapia scale-derived collagen,
[16] The osteoblast differentiation inducing method according to any one of [9] to [15], wherein the contact step is performed in the presence of 5% or less of serum or in the absence of serum,
[17] An osteoblast production method comprising a fish-derived collagen culturing step of culturing mesenchymal stem cells with a cell culture substrate coated with fish-derived collagen,
[18] The mesenchymal stem cells are bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells, auricles Peritoneal membrane-derived mesenchymal stem cell, peripheral blood-derived mesenchymal stem cell, umbilical cord blood-derived mesenchymal stem cell, ligament-derived mesenchymal stem cell, tendon-derived mesenchymal stem cell, ES cell-derived mesenchymal stem cell, or iPS cell-derived The osteoblast production method according to [17], which is a mesenchymal stem cell,
[19] The osteoblast production method according to [17] or [18], wherein the mesenchymal stem cell is derived from a human.
[20] The cell culture substrate has a contact surface with mesenchymal stem cells coated with at least one second collagen other than the coated first fish-derived collagen. [17] to [19] An osteoblast production method according to any one of
[21] The second collagen is a collagen selected from the group consisting of mammal-derived collagen, avian-derived collagen, reptile-derived collagen, amphibian-derived collagen, fish-derived collagen, invertebrate-derived collagen, and combinations thereof. [17] to [20], the osteoblast production method according to any one of
[22] The osteoblast production method according to any one of [17] to [21], wherein the culture medium of the mesenchymal stem cells is a medium containing 5% or less serum or a serum-free medium,
[23] The method for producing osteoblasts according to any one of [17] to [20], wherein the culture medium for mesenchymal stem cells is a medium containing an osteoblast differentiation factor.
[24] The osteoblast production method according to any one of [17] to [23], wherein the fish-derived collagen is collagen derived from tilapia scales,
[25] Osteoblasts obtained by the osteoblast production method according to any one of [17] to [24],
[26] Use of fish-derived collagen as a cell culture substrate for induction of osteoblast differentiation,
[27] Use of fish-derived collagen as a cell culture substrate for the production of osteoblasts,
About.

  According to the osteoblast differentiation inducing method or the osteoblast production method of the present invention, mesenchymal stem cells can be efficiently and strongly differentiated into osteoblasts. In particular, it is possible to strongly induce early differentiation induction of osteoblasts. In addition, mesenchymal stem cells can be differentiated into osteoblasts in the presence or absence of serum at a low concentration.

It is the graph which showed the activity of alkaline phosphatase when a human bone marrow origin mesenchymal stem cell is cultured on a fish origin collagen coated cell culture plate (contact). As controls, pig-derived collagen-coated cell culture plates and uncoated cell culture plates were used. When human bone marrow-derived mesenchymal stem cells are cultured for 3 days (A) or 7 days (B) in fish-derived collagen-coated cell culture plates in the presence (Dex +) or absence (Dex −) of dexamethasone It is the graph which showed the activity of alkaline phosphatase. As controls, pig-derived collagen-coated cell culture plates and uncoated cell culture plates were used. “Dex + 3%” means dexamethasone 10 nM and FBS 3%, “Dex-3%” means dexamethasone 0 nM and FBS 3%, “Dex + 15%” means dexamethasone 10 nM and FBS 15%, “Dex-15%” means dexamethasone. 0 nM and FBS 15% are shown. Graph showing an increase in the amount of DNA of cells when human bone marrow-derived mesenchymal stem cells were cultured in fish-derived collagen-coated cell culture plates in the presence (Dex +) or absence (Dex-) of dexamethasone. It is. Human bone marrow-derived mesenchymal stem cells were cultured in the presence of 3% or 15% fetal bovine serum (FBS). As controls, pig-derived collagen-coated cell culture plates and uncoated cell culture plates were used. It is the photograph which observed the surface (A) of the well coat | covered with the tilapia scale origin collagen, or the surface (B) of the well coat | covered with the pig origin collagen with the phase-contrast microscope. It is the graph which showed the expression of BMP2 (A) or SPP1 (B) when a human bone marrow origin mesenchymal stem cell is cultured (contacted) with a fish origin collagen coated cell culture plate. The expression of each mRNA was corrected by the expression level of GAPDH. As controls, pig-derived collagen-coated cell culture plates and uncoated cell culture plates were used. BMP2 (A) or human bone marrow-derived mesenchymal stem cells when cultured (contacted) for 3 days in fish-derived collagen-coated cell culture plates in the presence (Dex +) or absence (Dex −) of dexamethasone It is the graph which showed the expression of SPP1 (B). The expression of each mRNA was corrected by the expression level of GAPDH. “Dex + 3%” means dexamethasone 10 nM and FBS 3%, “Dex-3%” means dexamethasone 0 nM and FBS 3%, “Dex + 15%” means dexamethasone 10 nM and FBS 15%, “Dex-15%” means dexamethasone. 0 nM and FBS 15% are shown. As controls, pig-derived collagen-coated cell culture plates and uncoated cell culture plates were used. Graph (A) showing the activity of alkaline phosphatase when human bone marrow-derived mesenchymal stem cells were cultured (contacted) on a fish-derived collagen-coated cell culture plate and human bone marrow-derived mesenchymal stem cells in the presence of dexamethasone ( It is the graph (B) which showed the activity of the alkaline phosphatase when it culture | cultivates for 3 days by the fish origin collagen-coated cell culture plate in the absence (Dex +). As controls, pig-derived collagen-coated cell culture plates (coated at a concentration of 0.3 wt%) and uncoated cell culture plates were used. “Dex +” indicates dexamethasone 10 nM and FBS 3%, and “Dex−” indicates dexamethasone 0 nM and FBS 3%. In the presence of 10 nM dexamethasone, a graph (Day 0) showing the expression of BMP2 (A) or SPP1 (B) when human bone marrow-derived mesenchymal stem cells were cultured (contacted) on a fish-derived collagen-coated cell culture plate. FIG. 3 is a graph showing the expression of BMP2 (A) or SPP1 (B) when cultured (contacted) on a fish-derived collagen-coated cell culture plate for 3 days (Day 3) or 7 days (Day 7). The expression of each mRNA was corrected by the expression level of GAPDH. As controls, pig-derived collagen-coated cell culture plates (coated at a concentration of 0.3 wt%) and uncoated cell culture plates were used.

[1] Culture substrate for inducing osteoblast differentiation The culture substrate for inducing osteoblast differentiation of the present invention has a contact surface with mesenchymal stem cells coated with fish-derived collagen.
(Fish-derived collagen)
The fish-derived collagen used for the culture substrate for inducing osteoblast differentiation of the present invention is not particularly limited as long as it is type I collagen. For example, the types of fish include tilapia, gonzui, rabeo rojita, katra, carp, thunderfish, piraluk, thailand, flounder, shark, and salmon. Fish that live in high rivers, lakes, or seas are preferred. Specific examples of such fish include fish of the genus Oreochromis, with tilapia being particularly preferred. Collagen with a relatively high denaturation temperature can be obtained from fish of the genus Oreochromis. For example, Nile tilapia (Oreochromis niloticus), which is cultivated for food in Japan and China, is easily available, and a large amount of collagen can be obtained. it can.

  The part of the fish from which fish-derived collagen is obtained is not limited. For example, scales, skin, bones, cartilage, fins, organs and the like can be mentioned, but scales are preferred. This is because the scale has few lipids that cause fishy odor. Moreover, collagen derived from fish scales is more likely to be fibrillated than other collagens, and the fiber formation rate is significantly faster.

  Fish type I collagen has a “triple helix structure” made up of three polypeptide chains having a molecular weight of about 100,000, and has a molecular weight of about 300,000. It is shaped like one hard bar with a length of 300 nm and a diameter of 1.5 nm. It is the amino acid sequence of the polypeptide chain that forms the unique “triple helix structure” of fish type I collagen. The polypeptide chain is made up of a series of units “GXY” in which three amino acids are arranged. G often represents glycine, X is proline, and Y is often hydroxyproline. Hydroxyproline is not contained in ordinary proteins and is an amino acid unique to collagen, but it is believed that the triple helix structure is stabilized by hydrogen bonding between the hydroxyl group of hydroxyproline and hydrated water.

  When the temperature rises, the “triple helical structure” composed of three polypeptides is dissolved, and the three polypeptides are separated into gelatin. The change from collagen to gelatin is called denaturation. Once denaturation occurs, it is difficult to return to the “triple helical structure” even if the temperature is lowered again. The denaturation temperature of collagen is usually slightly higher than the inhabitant temperature of the organism from which the collagen is derived. Therefore, the denaturation temperature of fish scales inhabiting fish is not so high.

The fish-derived collagen used in the present invention is not limited by its denaturation temperature, but fish-derived collagen having a high denaturation temperature is preferred, specifically 20 ° C or higher, more preferably 25 ° C or higher, and 28 More preferably, it is 30 ° C. or higher. In the osteoblast differentiation inducing method of the present invention, the temperature at which fish-derived collagen and mesenchymal stem cells are brought into contact is often around 37 ° C., and gelatin in which the “triple helix structure” is destroyed is mesenchymal. This is because it is considered that differentiation induction of stem cells into osteoblasts does not occur.
However, even with fish-derived collagen having a denaturation temperature of less than 20 ° C., contact with mesenchymal stem cells at less than 20 ° C. allows the mesenchymal stem cells to become osteoblasts without causing collagen denaturation. Can be differentiated.

  The denaturation temperature of collagen used in the present invention is increased stepwise by using a CD spectrometer (Jasco model 725 spectrometer) according to the method described in International Journal of Biological Macromolecules, 32, 199 (2003). Seek by letting That is, the collagen aqueous solution is diluted with dilute hydrochloric acid having a pH of 3, adjusted to 0.03 wt%, and placed in a quartz cell having an optical path length of 1 mm. The cell temperature is increased at 1 ° C./min, and the optical rotation at 221 nm is measured every 0.2 ° C. When the optical rotation at each temperature is plotted against temperature, a denaturation curve is obtained in which the value of optical rotation changes rapidly from the value of the collagen helix to the value of the random coil. The temperature that gives an intermediate value of the optical rotation values, that is, the temperature at which the helix (%) becomes 50% is defined as the denaturation temperature.

  The method for obtaining fish-derived collagen is not particularly limited as long as the triple helix structure of collagen is not destroyed. For example, it can be obtained by the obtaining method disclosed in Patent Document 3. Below, the manufacturing method of the collagen derived from a scale is demonstrated as a typical example.

  First, remove unnecessary items such as fish skin and sea bream from the collected scales with water. If necessary, adhere to the scale surface using alcohols such as methanol, ethanol and isopropanol, hydrophilic organic solvents such as acetone, surfactants, salts such as sodium chloride, alkaline solutions such as sodium hydroxide, etc. Substances that cause odor, such as proteins and lipids other than collagen, may be removed from the scales.

  Furthermore, in the decalcification treatment solution, the mixture is gently stirred using a stirring blade for 1 to 48 hours to remove the inorganic component (calcium phosphate) contained in the scale. The treatment solution only needs to be able to dissolve inorganic components, and may be an inorganic acid such as hydrochloric acid or phosphoric acid, an organic acid such as acetic acid or citric acid, or an aqueous solution such as ethylenediaminetetraacetic acid. Acetic acid is preferably used. The amount used is not particularly limited, and the scale after decalcification may be washed with water.

  The scales from which contaminants were removed in this manner were solubilized by gradual stirring using an agitating blade or the like in an acidic solution to which protease was added for 2 to 72 hours, thereby breaking the cross-links between collagen molecules. Collagen is extracted. The operation after this extraction step should be performed at a denaturation temperature or lower, preferably at a denaturation temperature of −5 ° C. or lower in order to prevent collagen denaturation.

  As the type of protease, pepsin, proctase, papain and the like having high activity in an acidic solution are preferably used. The pH of the solution may be in a range where the protease activity is high, and is generally about 2 to 5. The amount of protease used is not particularly limited, but is usually about 1 to 15% with respect to the dry weight of fish scales, and if the concentration is determined so that the scale can be uniformly stirred. good. The acid to be used is not particularly limited, but is preferably selected from those having high safety for living organisms such as hydrochloric acid, acetic acid, citric acid and malic acid, and hydrochloric acid and acetic acid are particularly preferably used. By such a method, collagen (atelocollagen) in which non-helical regions (telopeptides) present at both ends of the collagen molecule are decomposed can be extracted.

  Collagen solubilized in this way is separated from unscaled fish scale residue by centrifugation, filtration or the like. The fish scale residue can be extracted again by solubilizing collagen by treating it in an acidic solution to which a protease is added. Therefore, the yield may be increased by repeating it about 2 to 4 times.

  The collagen solution thus obtained contains a protein other than protease, collagen, gelatin (denatured collagen) and the like, and thus is purified as necessary. As for the purification method, collagen is precipitated by adding a salt such as sodium chloride to the solubilized collagen solution and increasing the salt concentration. For example, sodium chloride is added to a solubilized collagen aqueous solution so as to have a final concentration of 1 M, and the mixture is allowed to stand for about 5 minutes to 24 hours, whereby collagen is precipitated.

  Precipitation can also be achieved by adding sodium hydroxide or the like to bring the pH to neutral or higher. For example, a sodium hydroxide solution is added until pH becomes about 7-9, and collagen is deposited by leaving still for about 5 minutes to 24 hours. Such collagen precipitation can be easily confirmed by the cloudiness of the solution.

  The precipitated collagen is collected by a general solid-liquid separation method such as centrifugation or filtration, and this is gently dissolved in an acidic solution and redissolved. For example, it may be gently stirred for about 1 to 48 hours in a hydrochloric acid solution having a pH of about 2 to 4. In this way, collagen can be purified, and the purity can be further increased by repetition. The salts used in the purification step can be removed by desalting against pure water using a dialysis membrane or the like.

  Since coexistence of bacteria and the like becomes a serious problem when cells are cultured, the obtained collagen solution is preferably used after being sterilized by a method that does not destroy the structure of collagen such as filtration sterilization.

(Cell culture substrate)
The cell culture substrate that coats the fish-derived collagen can be used without limitation as long as it is a substrate that is usually used for cell culture. As a material, a substrate of glass, a porous body (for example, polycarbonate), or plastic (for example, polystyrene or polypropylene) can be used, and as a shape, a flask, a petri dish, a multi-culture plate (for example, a 6-well multi-plate) A substrate in the form of a culture plate, a 12-well multi-culture plate, or the like) or a bead can be used. However, since the collagen solution is hydrophilic, a hydrophilic material substrate or a substrate having a hydrophilized surface is preferable.

(Contact surface with mesenchymal stem cells)
The contact surface with the mesenchymal stem cells is a portion of the cell culture substrate that is coated with fish-derived collagen. Examples thereof include a cell culture surface of a flask, a cell culture surface of a petri dish, a bottom surface of a well of a multiculture plate, a bead surface, or a porous body surface. These parts are coated with fish-derived collagen and used as a contact surface with mesenchymal stem cells.

(Coating of cell culture substrate with collagen)
The cell culture substrate can be coated with fish collagen according to a conventional method.
The pH of the solution for dissolving collagen is preferably 2 to 5 which is lower than the isoelectric point of collagen. The acid source of the solution is not particularly limited, but is preferably selected from those that are highly safe for living organisms, such as hydrochloric acid, acetic acid, citric acid, or malic acid, and more preferably hydrochloric acid or acetic acid that vaporizes during drying. If the pH is too low, the structure of the collagen may be destroyed. If the pH is too high, the solubility may be reduced, resulting in thickening or gelation.

  Although the collagen concentration of the coating solution to be used is not particularly limited, it is generally 0.01 to 1% by weight, more preferably 0.03 to 0.3% by weight. When the concentration is low, the amount of collagen that can be coated on the substrate with a single coating decreases. Higher concentrations can increase the amount of collagen that covers the substrate, but only increase the thickness. The effect of the present invention is exerted mainly on the surface layer portion, so even if the thickness of the coated collagen increases, an effect commensurate with the amount of collagen cannot be expected.

  As a coating method, a collagen solution adjusted to an appropriate concentration using an acidic solution is poured onto a cell culture substrate such as a dish so that it does not become irritated. Remove. By drying this, the fibrillated collagen is uniformly coated on the substrate. This is a phenomenon peculiar to fish scale-derived collagen, and cannot be achieved by conventionally used collagen derived from mammals.

Since contamination with bacteria becomes a serious problem during cell culture, it is desirable to perform the collagen coating operation in a sterile environment such as a clean room or a clean bench using a clean sterile substrate. As needed, you may add the sterilization process which does not destroy the structure of collagen, such as ethylene oxide and UV irradiation.
Speaking of the temperature during coating and drying, it is carried out at a modification temperature or lower, preferably at a modification temperature of -5 ° C or lower. When the treatment is performed at a temperature higher than the denaturation temperature, the fibrosis function may be lost due to the denaturation of the collagen before the formation of the bonds between the base materials and the collagen fibers, and the characteristics as collagen may not be exhibited.
Although the drying time varies depending on the humidity and the like, it is dried in about 5 minutes to 2 hours under ventilation such as in a clean bench. Insufficient drying may result in inadequate collagen fibrillation and adhesion to the substrate, and when the cell culture solution is added, the effect of the present invention is fully exerted by re-dissolution or peeling of the coating. May not. If it is in a sufficiently dry and fibrotic state, it can exert its effect even after being stored for several months. Storage is preferably at a temperature lower than the denaturation temperature, preferably refrigerated storage.

  The culture substrate for inducing osteoblast differentiation of the present invention may have a contact surface with a mesenchymal stem cell coated with at least one second collagen other than the coated first fish-derived collagen. . That is, the culture substrate for inducing osteoblast differentiation according to the present invention can be a composite material coated with collagen of different origin.

(Second collagen)
The second collagen is not particularly limited as long as it is different from the coated first fish-derived collagen. For example, fish-derived collagen other than the first fish-derived collagen can be used. It is. Specific examples of the second collagen include mammal-derived collagen (eg, bovine-derived collagen, pig-derived collagen, sheep-derived collagen, goat-derived collagen, or monkey-derived collagen), avian-derived collagen (eg, chicken-derived collagen, goose Derived collagen, duck-derived collagen, or ostrich-derived collagen), reptile-derived collagen (eg, crocodile-derived collagen), amphibian-derived collagen (eg, frog-derived collagen), fish-derived collagen (eg, tilapia-derived collagen, Thai-derived collagen, flounder Derived collagen, shark-derived collagen, or salmon-derived collagen), invertebrate-derived collagen (eg, jellyfish-derived collagen), and combinations thereof. As a combination of the second collagen, mammal-derived collagen and avian-derived collagen may be combined, or mammal-derived collagens having different origins, for example, bovine-derived collagen and pig-derived collagen may be combined. The second collagen can be produced according to a conventionally known method.

(Coating of second collagen on cell culture substrate)
Similar to the first fish-derived collagen, the second collagen can be coated on the contact surface of the cell culture substrate with the mesenchymal cells. The first fish-derived collagen and the second collagen may be mixed and coated on the contact surface, or may be separately coated on the contact surface in order. In addition, the first fish-derived collagen and the second collagen may be coated on the same contact surface or different contact surfaces.
For example, when the first fish-derived collagen and the second collagen are coated on different contact surfaces, the bottom surface of the well of the multiculture plate is divided into two or more, and the first fish-derived collagen and the second collagen are separated. Each may be coated. Alternatively, the bottom of the well of the multiculture plate may be coated with the first fish-derived collagen, the surface of the bead may be coated with the second collagen, and the multiculture plate and beads may be combined and used as a cell culture substrate. Good.

(Mesenchymal stem cells)
The mesenchymal stem cells to be brought into contact with the cell culture substrate coated with fish-derived collagen are not limited as long as they can be induced to differentiate into medulloblasts. For example, bone marrow mesenchymal stem cells, fat Tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth embryo-derived mesenchymal stem cells, auricular perichondrium-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord Examples include blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cell-derived mesenchymal stem cells, or iPS cell-derived mesenchymal stem cells. The animal species from which the mesenchymal stem cells are derived is not particularly limited, and examples of mammals include humans, monkeys, dogs, cats, pigs, sheep, goats, cows, horses, rabbits, guinea pigs, rats, and mice. Can be mentioned. Birds can include chickens, quails, ducks, geese, ostriches and guinea fowls. Reptiles can include crocodiles, turtles and lizards. Amphibians include frogs and newts. Fish can include tilapia, Thailand, flounder, shark, and salmon. In addition, examples of invertebrates include crabs, shellfish, jellyfish, and shrimps.
The mesenchymal stem cells are cells capable of differentiating into cells belonging to the mesenchymal system such as osteoblasts, adipocytes, myocytes, or chondrocytes, and can constitute bone, blood vessels, or myocardium It will be.

  The mesenchymal stem cell may be a cell line, can be prepared according to a conventional method, and a commercially available cell can also be used. In addition, mesenchymal stem cells (eg, bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells) , Auricular perichondrium-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cells-derived mesenchymal stem cells, or iPS cell-derived mesenchymal stem cells) can be induced to differentiate into osteoblasts by the osteoblast differentiation inducing method of the present invention. In this case, the cells collected from the living body are preferably prepared by removing connective tissue and the like according to a conventional method. Furthermore, it is preferable to use primary cultured cells as cells collected from a living body. Although it is possible to use after passage, the number of passages is preferably small, preferably 10 times or less, more preferably 5 times or less, and most preferably 2 times or less.

(Osteoblast)
Osteoblasts that are induced to differentiate by the method of the present invention are cells that form bone in bone tissue, the cytoplasm is basophile, and has alkaline phosphatase activity. Osteoblasts may also have androgen and estrogen receptors, androgen reduces osteoblast activity, and estrogen stimulates osteoblasts. Differentiation induction from mesenchymal stem cells into osteoblasts can be confirmed by measuring the activity of alkaline phosphatase. The measurement of alkaline phosphatase can be performed according to a conventional method as shown in Examples described later.

[2] Osteoblast differentiation induction kit and osteoblast production apparatus The osteoblast differentiation induction kit of the present invention includes a culture substrate for inducing osteoblast differentiation. The culture substrate for inducing osteoblast differentiation has a contact surface with mesenchymal stem cells coated with fish-derived collagen. Specifically, the culture substrate for inducing osteoblast differentiation of the present invention can be included. Further, using the fish-derived collagen according to the osteoblast differentiation induction method [1] and a cell culture substrate, according to the method described in “Coating of collagen on a cell culture substrate” and a known method, It is possible to produce a culture substrate for inducing osteoblast differentiation.
The contact surface with the mesenchymal cells is coated with fish-derived collagen, specifically, a flask or petri dish cell culture surface, a bottom surface of a multiculture plate well, a bead surface, or a porous surface It is the surface of the body and can be contacted by mesenchymal cells.

Moreover, the osteoblast differentiation inducing kit of the present invention may contain an osteoblast differentiation inducing factor. Examples of osteoblast differentiation inducers include dexamethasone, immunosuppressive agents such as FK-506 or cyclosporine, bone morphogenetic proteins (BMP: Bone Morphogenic Proteins) such as BMP2, BMP4, BMP5, BMP6, BMP7 or BMP9, bones such as TGFβ Mention may be made of forming liquid factors. The osteoblast differentiation inducing kit of the present invention can contain one or more selected from these osteoblast differentiation inducing factors.
In addition, the osteoblast differentiation induction kit of the present invention may include an instruction manual specifying that it is for induction of osteoblast differentiation from mesenchymal stem cells. The statement that it is for inducing differentiation of osteoblasts from mesenchymal stem cells may be attached to the container of the kit.

The osteoblast production apparatus of the present invention includes a culture substrate for inducing osteoblast differentiation. The culture substrate for inducing osteoblast differentiation has a contact surface with mesenchymal stem cells coated with fish-derived collagen and is the same as that contained in the osteoblast differentiation inducing kit of the present invention. Can be used.
Moreover, the osteoblast production apparatus of the present invention may include a CO ₂ incubator for cell culture. Osteoblasts from mesenchymal stem cells can be produced using a culture substrate for inducing osteoblast differentiation in a CO ₂ incubator.

[3] Osteoblast differentiation inducing method The osteoblast differentiation inducing method of the present invention comprises a step of contacting a cell culture substrate coated with fish-derived collagen and a mesenchymal stem cell (hereinafter referred to as a fish collagen contact step). In some cases).
In the osteoblast differentiation inducing method of the present invention, the fish-derived collagen described in the culture substrate for osteoblast differentiation induction and the cell culture substrate can be used, and the fish-derived collagen is used as the cell culture substrate. The coating can be performed according to the description in the above-mentioned “Coating of collagen on cell culture substrate” and known methods. However, it is not necessary to use a cell culture substrate coated with fish-derived collagen that is specifically limited to the use for inducing osteoblast differentiation.
Further, the mesenchymal stem cells used in the osteoblast differentiation induction method can also use the mesenchymal stem cells described in the culture substrate for osteoblast differentiation induction. It is as follows.

(contact)
In the osteoblast differentiation induction method, the medium used for contacting the cell culture substrate and mesenchymal stem cells is not limited as long as it is a medium that can maintain mesenchymal stem cells, but is usually used for culturing mesenchymal stem cells. What is necessary is just to use the culture medium to be used. For example, a known medium such as a MEM medium, an α-MEM medium, or a DMEM medium can be appropriately selected and used according to the cells to be cultured.
The contact time is not limited as long as differentiation induction of mesenchymal stem cells into osteoblasts occurs, but the lower limit is substantially 10 minutes or more, preferably 1 hour or more, more preferably 1 More than a day. The upper limit of the contact time is not limited, but is 30 days or less, preferably 15 days or less, and more preferably 7 days or less.
The contact temperature can be appropriately determined according to the optimal culture temperature for mesenchymal stem cells. For example, in the case of mammalian mesenchymal stem cells, it is 30 ° C to 40 ° C, preferably 35 ° C to 37 ° C. However, it is also possible to contact at a temperature higher or lower than the culture temperature. For example, when fish-derived collagen with a low denaturation temperature is used, the cell culture substrate is contacted with the mesenchymal stem cells at a contact temperature lower than the culture temperature, and the mesenchyme is raised to the optimal culture temperature. It is also possible to culture stem cell.

Usually, osteoblast differentiation is induced in the presence of about 10 to 15% serum. In the osteoblast differentiation inducing method of the present invention, differentiation can be induced in the presence of about 10 to 15% serum, but bone can be obtained even in the presence of low-concentration serum or in the absence of serum. It is possible to induce blast differentiation. In order to eliminate the influence of various factors in serum when osteoblast differentiation is induced from mesenchymal stem cells, it is preferable to contact in the presence of a low concentration of serum. For example, the presence of 10% or less of serum is preferable, the presence of 5% or less of serum is more preferable, the presence of 3% or less of serum is more preferable, and the absence of serum is most preferable.
The serum that can be used in the induction of osteoblast differentiation is not particularly limited as long as it is mammalian serum, but serum from humans, cows, horses, sheep, goats, and the like can be used. However, when the mesenchymal stem cells are separated from the living body, it is most preferable to use the serum of the individual from which the mesenchymal stem cells are collected. By using the serum of the individual itself, it is possible to prevent contact with pathogens of zoonotic diseases that may be contained in the serum of cattle and the like.

(Osteoblast differentiation factor)
The osteoblast differentiation inducing method of the present invention can include a step of bringing the mesenchymal stem cells into contact with an osteoblast differentiation inducing factor (hereinafter sometimes referred to as an osteoblast differentiation inducing factor contact step). . The osteoblast differentiation inducing factor contact step may be performed simultaneously with the fish collagen contact step, or may be performed before or after the fish collagen contact step. The osteoblast differentiation inducer contact step can be performed by adding an osteoblast differentiation inducer to a medium containing mesenchymal stem cells.
The osteoblast differentiation-inducing factor is not limited as long as it can induce differentiation of mesenchymal stem cells into osteoblasts. For example, an immunosuppressant such as dexamethasone, FK-506 or cyclosporine, BMP2, BMP4, BMP5 , BMP6, BMP7, BMP9 and other bone morphogenetic proteins (BMP: Bone Morphogenic Proteins) and TGFβ and other osteogenic fluid factors. By adding one or more selected from these osteoblast differentiation inducing factors to the medium, the osteoblast differentiation inducing factor contact step can be performed.
The concentration of the osteoblast differentiation inducing factor can be appropriately determined according to the type of osteoblast differentiation inducing factor used. For example, when dexamethasone is used, it can be used at a concentration of 1 to 100 nM, and 10 nM is particularly preferable.

In the osteoblast differentiation inducing method of the present invention, a second collagen contact step of contacting a cell culture substrate coated with at least one second collagen other than the first fish-derived collagen with a mesenchymal stem cell is performed. Further, it may be included.
As the second collagen used in the second collagen contact step, the second collagen described in the osteoblast differentiation induction culture substrate of the present invention can be used. The coating of the second collagen on the cell culture substrate can also be performed according to the method described in the culture substrate for inducing osteoblast differentiation of the present invention.

The fish collagen contact step, the second collagen contact step, and the osteoblast differentiation inducer contact step in the osteoblast differentiation inducing method of the present invention may be performed simultaneously or separately. When performing each process separately, three processes may be performed separately, two processes may be performed simultaneously, and another process may be performed separately.
The order of performing each step is not particularly limited, but the fish collagen contact step strongly induces the initial differentiation of osteoblasts, so when performing the fish collagen contact step and the second collagen contact step separately, It is preferred to perform the fish collagen contact step and then the 2 collagen contact step. This is because the osteoblasts differentiated by the fish collagen contact step are further induced to differentiate by the second collagen contact step.

  The culture substrate for inducing osteoblast differentiation of the present invention can be used in the above-described method for inducing osteoblast differentiation of the present invention.

[4] Osteoblast production method and osteoblast The osteoblast production method of the present invention includes a step of culturing mesenchymal stem cells with a cell culture substrate coated with fish-derived collagen. Is.
Osteoblasts can be produced using the osteoblast differentiation induction method. That is, in the osteoblast production method of the present invention, the fish-derived collagen described in the culture substrate for osteoblast differentiation induction and the cell culture substrate can be used, and the fish-derived collagen is used as the cell culture substrate. Can be coated according to the description in “Coating of cell culture substrate with collagen”. However, it is not necessary to use a cell culture substrate coated with fish-derived collagen that is specifically limited to the use for inducing osteoblast differentiation.
The mesenchymal stem cells described in the culture substrate for inducing osteoblast differentiation can also be used as the mesenchymal stem cells used in the osteoblast differentiation inducing method.

(culture)
The medium in the osteoblast production method of the present invention is a mesenchymal stem cell to be used, for example, bone marrow mesenchymal stem cell, adipose tissue-derived mesenchymal stem cell, synovial tissue-derived mesenchymal stem cell, dental pulp-derived mesenchymal stem cell, tooth Embryonic-derived mesenchymal stem cells, auricular perichondrium-derived mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cells A mesenchymal stem cell or an iPS cell-derived mesenchymal stem cell can be appropriately selected. For example, in the case of bone marrow mesenchymal stem cells, MEM medium, α-MEM medium, DMEM medium, or the like can be used. Moreover, you may add antibiotics etc. to the said culture medium.

The culture time is not limited as long as differentiation induction of mesenchymal stem cells into osteoblasts occurs, but it is 1 to 30 days, preferably 1 to 14 days, more preferably 3 to 10 days. Day.
The culture temperature can be appropriately determined according to the optimal culture temperature for mesenchymal stem cells, but in the case of mammalian mesenchymal stem cells, it is preferably 30 ° C to 40 ° C, more preferably 35 ° C to 37 ° C. However, when fish-derived collagen having a low denaturation temperature is used, it can be cultured at a temperature lower than the optimum temperature for culturing mesenchymal stem cells. The culture is preferably performed in the presence of 3 to 10% CO _{2, and} more preferably in the presence of 5% CO ₂ .

Usually, mesenchymal stem cells are cultured in the presence of about 10 to 15% serum to produce osteoblasts. In the osteoblast production method of the present invention, it is possible to produce osteoblasts in the presence of about 10 to 15% serum, but even in the presence of a low concentration of serum or in the absence of serum. It is possible to produce osteoblasts. In order to eliminate the influence of various factors in serum when producing osteoblasts from mesenchymal stem cells, it is preferable to produce them in the presence of a low concentration of serum. For example, the presence of 10% or less of serum is preferable, the presence of 5% or less of serum is more preferable, the presence of 3% or less of serum is more preferable, and the absence of serum is most preferable.
The serum that can be used in the production of osteoblasts is not particularly limited as long as it is mammalian serum, but serum from humans, cows, horses, sheep, goats, and the like can be used. However, when the mesenchymal stem cells are separated from the living body, it is most preferable to use the serum of the individual from which the mesenchymal stem cells are collected. By using the serum of the individual itself, it is possible to prevent contact with pathogens of zoonotic diseases that may be contained in the serum of cattle and the like.

(Osteoblast differentiation factor)
In the osteoblast production method of the present invention, an osteoblast differentiation inducing factor can be included in the culture medium. The osteoblast differentiation inducing factor may be added from the start of the culture or may be added during the culture.
The osteoblast differentiation-inducing factor is not limited as long as it can induce differentiation of mesenchymal stem cells into osteoblasts. For example, an immunosuppressant such as dexamethasone, FK-506 or cyclosporine, BMP2, BMP4, BMP5 , BMP6, BMP7, BMP9 and other bone morphogenetic proteins (BMP: Bone Morphogenic Proteins) and TGFβ and other osteogenic fluid factors. By adding one or more selected from these osteoblast differentiation inducing factors to the medium, the osteoblast differentiation inducing factor contact step can be performed.
The concentration of the osteoblast differentiation inducing factor can be appropriately determined according to the type of osteoblast differentiation inducing factor used. For example, when dexamethasone is used, it can be used at a concentration of 1 to 100 nM, and 10 nM is particularly preferable.

In the osteoblast production method of the present invention, the cell culture substrate has a contact surface with a mesenchymal stem cell coated with at least one second collagen other than the coated first fish-derived collagen. Also good. That is, in the osteoblast production method of the present invention, culture using a cell culture substrate coated with the second collagen may be further performed.
As the second collagen, the second collagen described in the culture substrate for inducing osteoblast differentiation of the present invention can be used. The coating of the second collagen on the cell culture substrate can also be performed according to the method described in the culture substrate for inducing osteoblast differentiation of the present invention.

  The culture using the cell culture substrate coated with the second collagen may be performed simultaneously with the culture using the cell culture substrate coated with the first fish-derived collagen, or may be performed separately. When the cell culture substrate in which the first fish-derived collagen and the second collagen are coated on the same contact surface is used, each culture is performed simultaneously. On the other hand, when the first fish-derived collagen and the second collagen are coated on different contact surfaces, the respective cultures can be performed simultaneously or separately. When culturing at the same time, for example, cell culture in which the first fish-derived collagen is coated on the bottom of the well of the multiculture plate, the second collagen is coated on the surface of the bead, and the multiculture plate and beads are combined. A substrate may be used. Furthermore, each culture may be performed in the presence of an osteoblast differentiation inducing factor or in the absence of an osteoblast differentiation inducing factor.

  The culture substrate for inducing osteoblast differentiation of the present invention can be used in the above-described method for producing osteoblasts of the present invention.

(Osteoblast)
The osteoblast of the present invention is an osteoblast that can be obtained by the production method of the present invention, and can perform bone formation in bone tissue. The osteoblasts are differentiated from mesenchymal stem cells into osteoprogenitor cells and further differentiated into osteoblasts. The cytoplasm of osteoblasts is basophil and has alkaline phosphatase activity. Furthermore, the osteoblasts of the present invention may have androgen and estrogen receptors. Androgens reduce osteoblast activity and estrogens stimulate osteoblasts.

[5] Use of fish-derived collagen The use of fish-derived collagen of the present invention is intended to induce osteoblast differentiation or produce osteoblasts. Specifically, it is used for a cell culture substrate, and more specifically, a cell culture substrate is coated. As the fish-derived collagen, the fish-derived collagen described in the osteoblast differentiation inducing method [1] can be used. The cell culture substrate is coated with fish-derived collagen according to the method described in “Coating of cell culture substrate with collagen” in the osteoblast differentiation induction method [1] and a known method. Is possible.
Furthermore, fish-derived collagen can be used for the production of the osteoblast differentiation induction kit of the present invention. The present specification also discloses the use of the osteoblast differentiation inducing kit of the present invention.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention.

<Measurement of alkaline phosphatase activity>
Measurement of alkaline phosphatase in osteoblasts obtained in the examples was performed as follows.
The cells were washed with PBS (−) (pH 7.4), collected with a scraper, suspended in 200 μL of 100 mM Tris-HCl (pH 7.5), 5 mM MgCl ₂ , 0.2% Triton X-100, and ultrasonicated. It was crushed. After crushing, the supernatant was collected by centrifugation at 6,000 g for 10 minutes. 20 μL of supernatant (appropriately diluted with 100 mM Tris-HCl (pH 7.5), 5 mM MgCl ₂ ) was used, and alkaline phosphatase activity was measured using LabAssay ALP (Wako).

<< Production Example 1: Production of fish-derived collagen >>
A method for producing collagen from tilapia scales is described below.
The tilapia scales were thoroughly washed with water and further washed with a 10% sodium chloride solution to remove impurities such as wrinkles, and then dried at room temperature. The water content was 18.5%.
1 kg of this tilapia scale was dispersed in 9 kg of a hydrochloric acid solution of pH 2 and gently stirred at 25 ° C. for 2 hours with the pH kept at 2 while adding a 1M hydrochloric acid solution to elute the inorganic components contained in the scale. . This was raised to a colander and washed thoroughly with water, and then a hydrochloric acid solution having a pH of 2 was added so that the total weight was 4 kg.

  To this, 24 g of pepsin (Wako Pure Chemicals 1: 10000) was added and gently stirred at 25 ° C. for 24 hours using a stirring blade to elute collagen from the scales. This was raised to a monkey and separated from the scale residue, and the supernatant was further collected by centrifugation (10000 G, 60 min) to separate it from the fine scale residue. To this, 0.5 g of pepsin was added and held at 25 ° C. for 24 hours.

  The obtained solution (2.4 kg) was filtered through a 0.45 μm membrane filter, and then sodium chloride was added to the final concentration of 1.0 M and gently stirred. After salting out by standing at 25 ° C. for 30 min, the supernatant was removed by centrifugation (10000 G, 20 min), and the collagen precipitate was recovered. 400 mL of pH 2 hydrochloric acid solution was added to the precipitate and dissolved by gently stirring at 5 ° C. for 24 hours. This salting-out purification process was repeated three times to obtain a collagen hydrochloric acid aqueous solution.

<< Production Example 2: Production of Cell Culture Substrate Coated with Fish Scale Collagen >>
The collagen solution derived from tilapia scale obtained in Production Example 1 was diluted to 0.3 wt% using a 0.01 M acetic acid solution having a pH of 3. On an ice plate, 500 μL of the collagen solution was spread thinly on a polystyrene 12-well cell culture plate (manufactured by FALCON) and allowed to stand at 25 ° C. for 60 minutes. Excess collagen solution was removed and dried in a clean bench at 25 ° C. for 60 minutes.
FIG. 4A shows a photograph of the surface of a well coated with tilapia scale-derived collagen, observed with a phase contrast microscope. Collagen fibers having a thickness of about 50 to 200 nm were bonded to the substrate and uniformly coated.

<< Comparative Production Example 1: Production of cell culture substrate coated with porcine-derived collagen >>
Manufactured except that instead of tilapia scale collagen solution (0.3 wt%), porcine collagen (Nitta Gelatin, Cellmatrix Type I-C) solution (0.03 wt%) was used. The operation of Example 2 was repeated to create a well coated with porcine collagen.
FIG. 4B shows a photograph of the surface of a well coated with porcine collagen, observed with a phase contrast microscope. No collagen fibers were seen and some aggregates were seen.

<< Example 1 and Comparative Example 1: Differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts >>
In this example, differentiation from human bone marrow-derived mesenchymal stem cells into osteoblasts was performed using the cell culture plate coated with fish-derived collagen obtained in Production Example 1.
(1) Example 1
Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza, Lot. No. 6F3974) were seeded at 1.0 × 10 ⁴ cells / well on a cell culture plate coated with fish-derived collagen. As the medium, α-MEM supplemented with 15% FBS was used and cultured at 37 ° C. in a 5% CO ₂ atmosphere for 20 hours. Cells were harvested and alkaline phosphatase activity was measured. The results are shown in FIG.
The collected cells were quantified using a Quant-iT ^™ Pico Green ^R ds DNA assay kit (Invitrogen), and the activity of alkaline phosphatase was corrected based on the measured DNA amount.

(2) Comparative Example 1
As a comparative example, the bone marrow-derived mesenchymal stem cells are differentiated into osteoblasts using the cell culture plate coated with porcine-derived collagen obtained in Comparative Production Example 1 or the cell culture plate not coated with collagen. Tried.
The operation of Example 1 except that instead of the cell culture plate coated with fish scale-derived collagen, a cell culture plate coated with pig-derived collagen or a cell culture plate not coated with collagen was used. Was repeated. The results are shown in FIG.

  As shown in FIG. 1, human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates had very low alkaline phosphatase activity. On the other hand, human bone marrow-derived mesenchymal stem cells cultured with a plate coated with tilapia scale-derived collagen have high alkaline phosphatase activity, and contact with a plate coated with tilapia scale-derived collagen leads to human bone marrow. Mesenchymal stem cells differentiated into osteoblasts.

<< Example 2 and Comparative Example 2: Differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts >>
In this example, differentiation from human bone marrow-derived mesenchymal stem cells into osteoblasts was performed in the presence of 3% or 15% FBS using a cell culture plate coated with fish-derived collagen.
(1) Example 2
Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza, Lot. No. 6F3974) were seeded on a plate at 1.0 × 10 ⁴ cells / well. Pre-culture was performed for 20 hours at 37 ° C. in a 5% CO ₂ atmosphere using 15% FBS-added α-MEM.
The medium was removed and replaced with α-MEM medium containing 3% FBS or 15% FBS. The cells were cultured for 7 days under an atmosphere of 37 ° C. and 5% CO ₂ . Cells were collected after 3 days and 7 days, and the activity of alkaline phosphatase was measured. The results are shown in FIGS. 2A and B (Dex-3% and Dex-15%).
Note that the collected cells ^was quantified the amount of DNA using the ^{Quant-iT TM Pico Green R ds} DNA assay kit (invitrogen , Inc.) were corrected alkaline phosphatase activity by measuring the amount of DNA. FIG. 3A (Dex-3% and Dex-15%) shows the proliferation of cells on the 3rd, 7th, and 10th days from the start of culture, based on the amount of DNA.

(2) Comparative Example 2
As a comparative example, the bone marrow-derived mesenchymal stem cells are differentiated into osteoblasts using the cell culture plate coated with porcine-derived collagen obtained in Comparative Production Example 1 or the cell culture plate not coated with collagen. Tried.
The procedure of Example 2 was used except that a cell culture plate coated with pig-derived collagen or a cell culture plate not coated with collagen was used instead of the cell culture plate coated with fish scale-derived collagen. Was repeated. The results are shown in FIGS. 2A and B (Dex-3% and Dex-15%).
Note that the collected cells ^was quantified the amount of DNA using the ^{Quant-iT TM Pico Green R ds} DNA assay kit (invitrogen , Inc.) were corrected alkaline phosphatase activity by measuring the amount of DNA. 3B and C (Dex-3% and Dex-15%) show the proliferation of cells on the 3rd, 7th, and 10th days from the start of culture according to the amount of DNA.

As shown in FIG. 2A, human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates showed alkaline phosphatase activity 3 days after induction of osteoblast differentiation at both serum concentrations of 3% and 15%. Was very low. On the other hand, human bone marrow-derived mesenchymal stem cells cultured on a plate coated with collagen derived from tilapia scales have high alkaline phosphatase activity 3 days after induction of osteoblast differentiation at both serum concentrations of 3% and 15%. The human bone marrow-derived mesenchymal stem cells differentiated into osteoblasts by contacting with a plate coated with collagen derived from human scales.
As shown in FIG. 2B, human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates have particularly high alkaline phosphatase activity 7 days after induction of osteoblast differentiation at a serum concentration of 3%. It was low. On the other hand, human bone marrow-derived mesenchymal stem cells cultured on a plate coated with collagen derived from tilapia scales have particularly high alkaline phosphatase activity 7 days after osteoblast differentiation induction at a serum concentration of 3%. The human bone marrow-derived mesenchymal stem cells differentiated into osteoblasts by contact with the collagen-coated plate.

<< Example 3 and Comparative Example 3: Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells into Osteoblasts >>
In this example, differentiation from human bone marrow-derived mesenchymal stem cells into osteoblasts was performed using a cell culture plate coated with fish-derived collagen and an osteoblast differentiation inducing factor (dexamethasone).
(1) Example 3
Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza, Lot. No. 6F3974) were seeded on a plate at 1.0 × 10 ⁴ cells / well. Using α-MEM containing 15% FBS, the cells were cultured for 20 hours at 37 ° C. in a 5% CO ₂ atmosphere.
The medium was removed and replaced with α-MEM medium containing 3% FBS or 15% FBS and 10 nM dexamethasone, 50 μg / mL L-ascorbic acid, 10 mM β-glycerophosphate. The cells were cultured for 7 days under an atmosphere of 37 ° C. and 5% CO ₂ . Cells were collected after 3 days and 7 days, and the activity of alkaline phosphatase was measured. The results are shown in FIGS. 2A and B (Dex + 3% and Dex + 15%).
Note that the collected cells ^was quantified the amount of DNA using the ^{Quant-iT TM Pico Green R ds} DNA assay kit (invitrogen , Inc.) were corrected alkaline phosphatase activity by measuring the amount of DNA. In addition, FIG. 3A (Dex + 3% and Dex + 15%) shows the proliferation of cells on the 3rd, 7th, and 10th days from the start of culture according to the amount of DNA.

(2) Comparative Example 3
As a comparative example, using a cell culture plate coated with porcine collagen obtained in Comparative Production Example 1 or a cell culture plate not coated with collagen, bone marrow-derived mesenchymal stem cells were transformed into osteoblasts. Tried differentiation.
The procedure of Example 3 was used except that a cell culture plate coated with pig-derived collagen or a cell culture plate not coated with collagen was used instead of the cell culture plate coated with fish scale-derived collagen. Was repeated. The results are shown in FIGS.
Note that the collected cells ^was quantified the amount of DNA using the ^{Quant-iT TM Pico Green R ds} DNA assay kit (invitrogen , Inc.) were corrected alkaline phosphatase activity by measuring the amount of DNA. Also, in FIGS. 3B and C (Dex + 3% and Dex + 15%), the proliferation of cells on the 3rd, 7th, and 10th days from the start of culture was shown by the amount of DNA.

As shown in FIG. 2A, human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates showed alkaline phosphatase activity 3 days after induction of osteoblast differentiation at both serum concentrations of 3% and 15%. Was very low. On the other hand, human bone marrow-derived mesenchymal stem cells cultured on a plate coated with tilapia scale-derived collagen had high alkaline phosphatase activity 3 days after induction of osteoblast differentiation at both serum concentrations of 3% and 15%.
In addition, as shown in FIG. 2B, human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates are alkaline phosphatase 7 days after induction of osteoblast differentiation at both serum concentrations of 3% and 15%. The activity of was very low. On the other hand, human bone marrow-derived mesenchymal stem cells cultured on a plate coated with tilapia scale-derived collagen had high alkaline phosphatase activity 7 days after induction of osteoblast differentiation at both serum concentrations of 3% and 15%.

(Results of Examples 2 and 3)
As shown in FIG. 2A, in the presence of 3% FBS or 15% FBS (Example 2), human bone marrow-derived mesenchymal stem cells 3 days after the start of culture by contact with a fish-derived collagen-coated cell culture plate. Alkaline phosphatase activity was elevated, confirming that human bone marrow-derived mesenchymal stem cells differentiated into osteoblasts. In particular, the increase in the activity of alkaline phosphatase in the presence of 3% FBS was large. However, when contacted with a porcine-derived collagen-coated plate or an uncoated plate, the activity of alkaline phosphatase in human bone marrow-derived mesenchymal stem cells is 1/6 of Example 2 even in the presence of 3% FBS or 15% FBS. It was as follows, and differentiation into osteoblasts could not be confirmed.
Further, in Example 3 to which dexamethasone, which is an osteoblast differentiation inducing factor, was added, alkaline phosphatase of human bone marrow-derived mesenchymal stem cells was contacted with the fish-derived collagen-coated cell culture plate 3 days and 7 days after the start of culture. Activity increased (FIGS. 2A and B). As in the case where dexamethasone was not added, the increase in alkaline phosphatase activity in the presence of 3% FBS was large. However, even when dexamethasone is added, the activity of alkaline phosphatase of human bone marrow-derived mesenchymal stem cells is small in contact with porcine-derived collagen-coated plates or non-coated plates, and even after 3 days, 1 of Example 3 The activity of alkaline phosphatase was only slightly increased after 7 days.
That is, according to the method of the present invention, it was found that human bone marrow-derived mesenchymal stem cells are strongly induced to differentiate into osteoblasts at the initial stage of contact with the fish-derived collagen-coated cell culture plate. No differentiation into osteoblasts was observed in cell culture plates coated with porcine collagen. Furthermore, differentiation of mesenchymal stem cells into osteoblasts by contact with a fish-derived collagen-coated cell culture plate was stronger than that of dexamethasone, which is a typical osteoblast differentiation inducing factor.
As shown in FIG. 3, there is no significant difference in cell proliferation (DNA amount) when using any of the fish-derived collagen-coated cell culture plate, pig-derived collagen-coated plate, or uncoated plate. It was. Therefore, it is considered that differentiation of human bone marrow-derived mesenchymal stem cells by contact with a fish-derived collagen-coated cell culture plate occurs regardless of cell proliferation.

<< Example 4 and Comparative Example 4: Expression of bone differentiation marker gene >>
In this example, the differentiation of human bone marrow-derived mesenchymal stem cells in Example 1 into osteoblasts was confirmed by detection of the bone differentiation marker gene mRNA.
(1) Example 4
Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza, Lot. No. 6F3974) were seeded at 1.0 × 10 ⁴ cells / well on the plate obtained in Production Example 2. As the medium, α-MEM containing 15% FBS was used and precultured at 37 ° C. in a 5% CO ₂ atmosphere for 20 hours. Total RNA was recovered from the cells using RNeasy Micro Kit (QIAGEN) according to the attached protocol. Using Transcriptor First Strand cDNA Synthesis Kit (Roche), cDNA was synthesized from the obtained total RNA. Using the synthesized cDNA as a template, the expression of bone morohogenetic protein 2 (BMP2) and Osteopontin (SPP1) was measured by real-time PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO). The primers used are as follows. In addition, Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) whose expression does not change during osteoblast differentiation was measured as a control. The results are shown in FIGS.

BMP2
Forward primer: 5'-TCA AGC CAA ACA CAA ACA GC -3 '
Reverse primer: 5'- ACG TCT GAA CAA TGG CAT GA -3 '
The length of the amplified PCR product is 200 bp.
Osteopontin (SPP1)
Forward primer: 5'- TTC GCA GAC CTG ACA TCC AGT ACC C -3 '
Reverse primer: 5'- GGG GAT GGC CTT GTA TGC ACC ATT C -3 '
The length of the amplified PCR product is 98 bp.
GAPDH
Forward primer: 5'-CAT GGA GAA GGC TGG GGC TCA TTT G -3 '
Reverse primer: 5'- GTT CAC ACC CAT GAC GAA CAT GGG G -3 '
The length of the amplified PCR product is 97 bp.

(2) Comparative Example 4
As comparative examples, the expression of BMP2 and SPP1 was confirmed using the cell culture plate coated with porcine collagen obtained in Comparative Production Example 1 or the cell culture plate not coated with collagen.
The operation of Example 4 except that a cell culture plate coated with pig-derived collagen or a cell culture plate not coated with collagen was used instead of the cell culture plate coated with fish scale-derived collagen. Was repeated. The results are shown in FIGS.

  As shown in FIG. 5, gene expression of BMP2 and SPP1 in hMSCs cultured on tilapia collagen-coated plates was extremely high at 20 hours of culture (contact) compared to porcine collagen-coated plates and non-coated plates. That is, human bone marrow-derived mesenchymal stem cells differentiated into osteoblasts at the beginning of contact with a plate coated with collagen derived from tilapia scales.

<< Example 5 and Comparative Example 5: Expression of a bone differentiation marker gene >>
In this example, the differentiation of human bone marrow-derived mesenchymal stem cells in Examples 2 and 3 into osteoblasts was confirmed by detection of mRNA of a bone differentiation marker gene.
Human bone marrow-derived mesenchymal stem cells (hMSC; Lonza, Lot. No. 6F3974) were seeded at 1.0 × 10 ⁴ cells / well on the plate obtained in Production Example 2. As the medium, α-MEM containing 15% FBS was used and precultured at 37 ° C. in a 5% CO ₂ atmosphere for 20 hours. The medium was removed, bone differentiation-inducing factor (10 nM dexamethasone), 50 μg / mL L-ascorbic acid and 10 mM β-glycerophosphate added, and bone differentiation-inducing factor-free 3% FBS or α containing 15% FBS -Replaced with MEM. Cells were collected on the third day, and total RNA was collected from the cells using RNeasy Micro Kit (QIAGEN) according to the attached protocol. Using Transcriptor First Strand cDNA Synthesis Kit (Roche), cDNA was synthesized from the obtained total RNA. Using the synthesized cDNA as a template, the expression of bone morohogenetic protein 2 (BMP2) and Osteopontin (SPP1) was measured by real-time PCR using THUNDERBIRD SYBR qPCR Mix (TOYOBO). The primer described in Example 4 was used as the primer. The results are shown in FIGS. 6A and B.

(2) Comparative Example 5
As comparative examples, the expression of BMP2 and SPP1 was confirmed using the cell culture plate coated with porcine collagen obtained in Comparative Production Example 1 or the cell culture plate not coated with collagen.
The operation of Example 4 except that a cell culture plate coated with pig-derived collagen or a cell culture plate not coated with collagen was used instead of the cell culture plate coated with fish scale-derived collagen. Was repeated. The results are shown in FIGS. 6A and B.

  As shown in FIG. 6, the expression of BMP2 and SPP1 mRNA decreased compared to the initial stage of culture (contact) with a plate coated with collagen derived from tilapia scales. However, compared to human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates, human bone marrow-derived mesenchymal stem cells cultured on plates coated with tilapia scale-derived collagen are BMP2 and The expression of SPP1 mRNA was high. In particular, when cultured in the presence of 3% FBS, the expression of MP2 and SPP1 mRNA was high.

  Since BMP2 and Osteopontin are genes that are highly expressed in the early stage of differentiation into osteoblasts, it is a bone differentiation marker gene that hMSCs differentiate into osteoblasts at the initial stage of contact with the fish-derived collagen-coated culture plate. It was also shown from the expression of.

<< Production Example 3: Production of Cell Culture Substrate Coated with Fish Scale Collagen >>
In this production example, collagen from tilapia was diluted with hydrochloric acid to produce a culture substrate for inducing osteoblast differentiation.
500 μL of tilapia scale-derived collagen solution (0.3 wt% / 0.01 M HCl) was thinly spread on a polystyrene 12-well cell culture plate (manufactured by FALCON) and allowed to stand at 25 ° C. for 60 minutes. Excess collagen solution was removed and dried in a clean bench at 25 ° C. for 60 minutes.

<< Comparative Production Example 2: Production of Cell Culture Substrate Coated with Pig-derived Collagen >>
In this comparative production example, pig-derived collagen was diluted with hydrochloric acid and coated at a concentration of 0.3 wt% to produce a cell culture substrate.
Manufactured except that instead of tilapia scale collagen solution (0.3 wt%), porcine collagen (Nitta Gelatin, Cellmatrix Type I-C) solution (0.3 wt%) was used. The operation of Example 3 was repeated to produce wells coated with porcine collagen.

<< Example 6 and Comparative Example 6: Differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts >>
In this example, differentiation from human bone marrow-derived mesenchymal stem cells into osteoblasts was performed using the cell culture plate coated with fish-derived collagen obtained in Production Example 3.
(1) Example 6
The plate obtained in Production Example 3 was used in place of the plate obtained in Production Example 2, and D-MEM (high-glucose) added with 10% FBS was used instead of α-MEM containing 15% FBS. Except for, the operation of Example 1 was repeated. The results are shown in FIG. 7A.

(2) Comparative Example 6
As a comparative example, bone marrow-derived mesenchymal stem cells are differentiated into osteoblasts using the cell culture plate coated with porcine-derived collagen obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen. Tried.
The procedure of Example 6 was repeated except that the plate obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen was used in place of the plate obtained in Production Example 3. The results are shown in FIG. 7A.

  As shown in FIG. 7A, human bone marrow-derived mesenchymal stem cells cultured on porcine-derived collagen-coated plates or uncoated plates had very low alkaline phosphatase activity. On the other hand, human bone marrow-derived mesenchymal stem cells cultured with a plate coated with tilapia scale-derived collagen have high alkaline phosphatase activity, and contact with a plate coated with tilapia scale-derived collagen leads to human bone marrow. Mesenchymal stem cells differentiated into osteoblasts.

<< Example 7 and Comparative Example 7: Differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts >>
In this example, differentiation from human bone marrow-derived mesenchymal stem cells into osteoblasts was performed in the presence of 3% FBS using a cell culture plate coated with fish-derived collagen.
(1) Example 7
The plate obtained in Production Example 3 was used in place of the plate obtained in Production Example 2, and D-MEM (high-glucose) added with 10% FBS was used instead of α-MEM containing 15% FBS. The procedure of Example 2 was repeated except that D-MEM (high-glucose) medium containing 3% FBS was used instead of α-MEM medium containing 3% or 15% FBS. Cells were collected after 3 days. The results are shown in FIG. 7B (Dex (−)).

(2) Comparative Example 7
As a comparative example, bone marrow-derived mesenchymal stem cells are differentiated into osteoblasts using the cell culture plate coated with porcine-derived collagen obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen. Tried.
The procedure of Example 7 was repeated except that the plate obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen was used in place of the plate obtained in Production Example 3. The results are shown in FIG. 7B (Dex (−)).

<< Example 8 and Comparative Example 8: Differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts >>
In this example, differentiation from human bone marrow-derived mesenchymal stem cells into osteoblasts was performed using a cell culture plate coated with fish-derived collagen and an osteoblast differentiation inducing factor (dexamethasone).
(1) Example 8
The plate obtained in Production Example 3 was used in place of the plate obtained in Production Example 2, and D-MEM (high-glucose) added with 10% FBS was used instead of α-MEM containing 15% FBS. The procedure of Example 3 was repeated except that a D-MEM (high-glucose) medium containing 3% FBS was used instead of the α-MEM medium containing 3% or 15% FBS. Cells were collected after 3 days. The results are shown in FIG. 7B (Dex (+)).

(2) Comparative Example 8
As a comparative example, bone marrow-derived mesenchymal stem cells to osteoblasts were obtained using the cell culture plate coated with porcine collagen obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen. Tried differentiation.
The procedure of Example 8 was repeated except that the plate obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen was used in place of the plate obtained in Production Example 3. The results are shown in FIG. 7B (Dex (+)).

(Results of Examples 7 and 8)
As shown in FIG. 7B, the activity of alkaline phosphatase of human bone marrow-derived mesenchymal stem cells 3 days after the start of culture by contact with a fish-derived collagen-coated cell culture plate in the presence of 3% FBS (Example). It was confirmed that human bone marrow-derived mesenchymal stem cells differentiated into osteoblasts. However, the contact with swine-derived collagen-coated plates is less than 1/2 of Example 7, and the contact with uncoated plates is that the activity of alkaline phosphatase of human bone marrow-derived mesenchymal stem cells is less than 1/5 of Example 7. there were.
Furthermore, also in Example 8 to which dexamethasone, an osteoblast differentiation inducing factor, was added, alkaline phosphatase activity of human bone marrow-derived mesenchymal stem cells was brought into contact with the fish-derived collagen-coated cell culture plate 3 days after the start of culture. Increased (FIG. 7B). In contact with the porcine-derived collagen-coated plate or non-coated plate, the increase in the activity of alkaline phosphatase of human bone marrow-derived mesenchymal stem cells was small, and about 3 times less than or about 1/5 of Example 8 after 3 days Met.
That is, according to the method of the present invention, it was found that human bone marrow-derived mesenchymal stem cells are strongly induced to differentiate into osteoblasts at the initial stage of contact with the fish-derived collagen-coated cell culture plate. No differentiation into osteoblasts was observed in cell culture plates coated with porcine collagen. Furthermore, differentiation of mesenchymal stem cells into osteoblasts by contact with a fish-derived collagen-coated cell culture plate was stronger than that of dexamethasone, which is a typical osteoblast differentiation inducing factor.

<< Example 9 and Comparative Example 9: Expression of a bone differentiation marker gene >>
In this example, the differentiation of human bone marrow-derived mesenchymal stem cells into osteoblasts in Example 6 was confirmed by detection of mRNA of bone differentiation marker genes (BMP2 and SPP1).
(1) Example 9
The plate obtained in Production Example 3 was used instead of the plate obtained in Production Example 2, and D-MEM (high-glucose) containing 10% FBS was used instead of α-MEM containing 15% FBS. Except for, the operation of Example 4 was repeated. The results are shown in FIGS. 8A and B (Day 0).

(2) Comparative Example 9
The procedure of Example 9 was repeated except that the plate obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen was used in place of the plate obtained in Production Example 3. The results are shown in FIGS. 8A and B (Day 0).

  As shown in FIG. 8, gene expression of BMP2 and SPP1 in hMSCs cultured on tilapia collagen-coated plates was extremely high at 20 hours of culture (contact) compared to porcine collagen-coated plates and non-coated plates. That is, human bone marrow-derived mesenchymal stem cells differentiated into osteoblasts at the beginning of contact with a plate coated with collagen derived from tilapia scales.

<< Example 10 and Comparative Example 10: Expression of bone differentiation marker gene >>
In this example, the differentiation of human bone marrow-derived mesenchymal stem cells in Examples 7 and 8 into osteoblasts was confirmed by detection of mRNA of the bone differentiation marker gene.
(1) Example 10
The plate obtained in Production Example 3 was used instead of the plate obtained in Production Example 2, and 3% or 15% FBS-containing α-MEM was used instead of 3% FBS-containing D-MEM (high-glucose). The procedure of Example 5 (addition of 10 nM dexamethasone of Example 3) was repeated except that it was used and the cells were collected on the third and seventh days. The results are shown in FIGS. 8A and 8B (Day 3 and Day 7).

(2) Comparative Example 10
As comparative examples, the expression of BMP2 and SPP1 was confirmed using the cell culture plate coated with porcine collagen obtained in Comparative Production Example 2 or the cell culture plate not coated with collagen.
The procedure of Example 10 was used except that a cell culture plate coated with pig-derived collagen or a cell culture plate not coated with collagen was used instead of the cell culture plate coated with fish scale-derived collagen. Was repeated. The results are shown in FIGS. 8A and 8B (Day 3 and Day 7).

  As shown in FIGS. 8A and 8B, the expression of BMP2 and SPP1 mRNA decreased compared to the initial stage of culture (contact) with a plate coated with collagen derived from tilapia scales.

  Since BMP2 and SPP1 are genes that are highly expressed in the early stage of differentiation into osteoblasts, it is a bone differentiation marker gene that hMSCs differentiate into osteoblasts at the initial stage of contact with the fish-derived collagen-coated culture plate. It was also shown from the expression of.

Osteoblasts can be induced from mesenchymal stem cells using the osteoblast differentiation inducing method of the present invention. In addition, osteoblasts can be produced from mesenchymal stem cells using the osteoblast production method of the present invention.
Furthermore, according to the present invention, an osteoblast differentiation induction kit and an osteoblast production apparatus can be produced.

Claims (22)

Fish-derived collagen coated, has a contact surface with the mesenchymal stem cells, osteoblast differentiation inducing a culture substrate, and osteoblast differentiation inducers including osteoblast differentiation inducing kit. The osteoblast differentiation induction kit according to claim 1, wherein the fish-derived collagen is tilapia scale-derived collagen . The mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells, auricular cartilage membrane Mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cell-derived mesenchymal stem cells, or iPS cell-derived mesenchymal cells The osteoblast differentiation inducing kit according to claim 1 or 2, which is a stem cell . The osteoblast differentiation induction kit according to any one of claims 1 to 3, wherein the mesenchymal stem cell is derived from a human . The osteoblast differentiation according to any one of claims 1 to 4, which has a contact surface with a mesenchymal stem cell coated with at least one second collagen other than the coated first fish-derived collagen. Induction kit . The second collagen is a collagen selected from the group consisting of mammal-derived collagen, avian-derived collagen, reptile-derived collagen, amphibian-derived collagen, fish-derived collagen, invertebrate-derived collagen, and combinations thereof. The osteoblast differentiation induction kit according to any one of 1 to 5 .   An osteoblast differentiation inducing method comprising a fish-derived collagen contact step in which a cell culture substrate coated with fish-derived collagen is contacted with a mesenchymal stem cell. The mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells, auricular cartilage membrane Mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cell-derived mesenchymal stem cells, or iPS cell-derived mesenchymal cells The osteoblast differentiation inducing method according to claim 7 , which is a stem cell. The osteoblast differentiation induction method according to claim 7 or 8 , wherein the mesenchymal stem cells are derived from human. Further comprising the coated first and second collagen at least one cell culture substrate coated non fish-derived collagen, second collagen contacting step of contacting a mesenchymal stem cell according to claim 7-9 The osteoblast differentiation inducing method according to any one of the above. The second collagen is a collagen selected from the group consisting of mammal-derived collagen, avian-derived collagen, reptile-derived collagen, amphibian-derived collagen, fish-derived collagen, invertebrate-derived collagen, and combinations thereof. The osteoblast differentiation inducing method according to any one of 7 to 10 . The osteoblast differentiation inducing method according to any one of claims 7 to 11 , further comprising an osteoblast differentiation inducing factor contact step of contacting the mesenchymal stem cells with an osteoblast differentiation inducing factor. Before SL Fish-derived collagen is a collagen from tilapia scales, osteoblast differentiation inducing method according to any one of claims 7 to 12. The osteoblast differentiation induction method according to any one of claims 7 to 13 , wherein the contacting step is performed in the presence of 5% or less of serum or in the absence of serum.   An osteoblast production method comprising a fish-derived collagen culturing step of culturing mesenchymal stem cells with a cell culture substrate coated with fish-derived collagen. The mesenchymal stem cells are derived from bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, synovial tissue-derived mesenchymal stem cells, dental pulp-derived mesenchymal stem cells, tooth germ-derived mesenchymal stem cells, auricular cartilage membrane Mesenchymal stem cells, peripheral blood-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, ligament-derived mesenchymal stem cells, tendon-derived mesenchymal stem cells, ES cell-derived mesenchymal stem cells, or iPS cell-derived mesenchymal cells The osteoblast production method according to claim 15 , which is a stem cell. The method for producing osteoblasts according to claim 15 or 16 , wherein the mesenchymal stem cells are derived from human. Said cell culture substrate, the coated first fish other than collagen from the second collagen least one has a contact surface with the mesenchymal stem cells coated, any one of claims 15-17 The method for producing osteoblasts according to 1. The second collagen is a collagen selected from the group consisting of mammal-derived collagen, avian-derived collagen, reptile-derived collagen, amphibian-derived collagen, fish-derived collagen, invertebrate-derived collagen, and combinations thereof. The method for producing osteoblasts according to any one of 15 to 18 . In the culturing step, MahakeimikiHoso cells are cultured in medium or serum-free medium containing 5% serum, osteoblasts process according to any one of claims 15-19. In the culturing step, MahakeimikiHoso cells are cultured in a medium containing osteoblast differentiation inducing factor, osteoblast process according to any one of claims 15-20. The osteoblast production method according to any one of claims 15 to 21 , wherein the fish-derived collagen is collagen derived from tilapia scales.