JP2008278842A - Differentiating and inducing method of adipose tissue-derived stromal cell to nerve cell and differentiated and induced nerve cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To simply and surely differentiate an adipose tissue-derived stromal cell to a nerve cell. <P>SOLUTION: The method for differentiating and inducing an adipose tissue-derived stromal cell comprises a step of collecting the adipose tissue-derived stromal cells from adipose cells and a step of culturing the adipose tissue-derived stromal cell in a culture medium containing dcAMP (dibutyryl cyclic AMP) or forskolin, and differentiates and induces the adipose tissue-derived stromal cell so as to express a marker of immature nerve cell. The method can differentiate and induce nerve cell from adipose tissue-derived stromal cell simply and surely and can obtain the nerve cell for achieving regenerative medical techniques thereby. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for inducing differentiation of stromal cells derived from adipose tissue (adipose tissue stromal cells), the induced cells, and a medium used for the induction of differentiation. In particular, the present invention relates to a method for inducing differentiation of adipose tissue stromal cells so as to express a neuronal phenotype, the induced cells, and a medium used for the differentiation induction.

  In recent years, research on stem cells aimed at tissue regeneration has been actively conducted. The reason is that there is a possibility that the tissue can be recovered by transplanting cells from the outside to the tissue that has already undergone necrosis or degeneration.

  Embryonic stem cells (ES cells) are the first candidates for stem cells that can be used for tissue regeneration. Stem cells used for tissue regeneration need to be able to easily proliferate in an undifferentiated state and be capable of differentiating into a target tissue. ES cells are undifferentiated in vitro. While maintaining the state, it can be proliferated almost infinitely and can be differentiated into cells of various lineages. However, ES cells have an ethical problem because they are collected by destroying embryos. In addition, legal problems have not been solved, and it is not practical to use it for nerve regeneration at this stage. In addition, since ES cells use cells from an individual different from an individual that needs transplantation in principle, there may be a problem of immune rejection (see Non-Patent Document 1).

  Therefore, in recent years, stem cells called somatic stem cells that can be collected from adult tissues have attracted attention. Somatic stem cells are present in the adult brain, skin, bone marrow, fat and the like, and can proliferate in an undifferentiated state in vitro. Moreover, differentiation into the cell of the tissue from which it originates is possible.

  Here, nerve regeneration is one of the most important items in tissue regeneration. Nerve cells have poor self-regenerative ability after the nervous system is constructed by proliferating and distributing in the early stages of development, and once damaged nerve tissue is not repaired. Therefore, there is a demand for a technique for repairing damaged nerve tissue by transplanting nerve cells differentiated from stem cells.

In view of the above-mentioned properties of somatic stem cells, neural stem cells (NSCs) that can be collected from the subcerebral ventricular region are the first conceivable somatic stem cells used for nerve regeneration. There are reports that neural stem cells can proliferate in vitro while forming floating cell masses called neurospheres, and can differentiate into neurons when placed under specific differentiation conditions (see Non-Patent Document 2). However, since neural stem cells cause great damage to the individual cerebrum when collected, it is very difficult to perform nerve regeneration using the same solid cells. Therefore, it is necessary to collect neural stem cells from different individuals, and in the same way as ES cells, not only ethical problems but also immune rejection problems arise.
On the other hand, recently, it has been reported that somatic stem cells can be differentiated into cells of tissues other than the derived tissue (for example, Non-Patent Document 3). In particular, techniques for inducing differentiation of nerve cells from adipose tissue stromal cells, which are one of somatic stem cells, have been reported (for example, Patent Documents 1 and 2).

  Patent Document 1 describes a method of inducing differentiation of nerve cells from adipose tissue stromal cells. In the method of Patent Document 1, adipose tissue stromal cells are cultured in DMEM containing 10% fetal bovine serum and 5% chicken embryo extract, then cultured in DMEM containing 20% fetal bovine serum (FBS), and then BHA. Incubation with serum-free DMEM containing NSE induces the expression of NSE, which is a marker for young neurons, and nestin, which is a marker for neural stem cells. However, in this method, the pretreatment for inducing NSE and nestin is complicated, and there is a problem that a lot difference of a chicken embryo extract that is generally not used in a medium affects its success or failure.

On the other hand, Patent Document 2 describes a method for inducing differentiation of adipose tissue stromal cells into nerve cells. In the method of Patent Document 2, adipose tissue stromal cells are cultured in DMEM containing 20% FBS and then cultured in serum-free DMEM containing 1 mM β-mercaptoethanol, and neurites appear in the cultured cells. Has been. However, it has not been proved that the cell processes obtained by this method are neurites, and even if they begin to differentiate into neurons, what level of differentiation is the level of neurons It is unknown.
Swijinenburg et al., 2005, Circulation 112 (9 suppl): I166-172 Su et al., 2004, Mol Cell Biol. 24: 8018-25 Petersen et al., 1999, Science 284: 1168-1170 Special Table 2003-523767 International Publication No. 03/008592 Pamphlet

  The present invention has been made based on such circumstances, and provides a method for differentiating adipose tissue stromal cells into nerve cells more easily and reliably, the composition of the medium used therefor, and the induced cells. Objective.

  The present invention relates to a method for inducing differentiation of adipose tissue stromal cells, comprising collecting adipose tissue stromal cells from adipose tissue, and the adipose tissue stromal cells containing dcAMP (dibutyryl cyclic AMP) or forskolin (forskolin). It comprises a step of culturing in a medium, and is characterized by inducing differentiation so that adipose tissue stromal cells express a marker of young neurons.

  As another aspect of the present invention, the present invention relates to a method for inducing differentiation of adipose tissue stromal cells, the step of collecting adipose tissue stromal cells from the adipose tissue, and the adipose tissue stromal cells as basic fibroblasts. A first differentiation inducing stage that is cultured in a medium containing cell growth factor and epidermal growth factor to induce differentiation so as to express nestin and NSE, and cells that have been induced to differentiate in the first differentiation inducing stage are treated with retinoic acid, dcAMP And a second differentiation-inducing step for enhancing differentiation of NSE and inducing differentiation to express MAP-2 by culturing in a medium containing Sonic hedgehog and Sonic hedgehog. Is characterized by inducing differentiation to express a neuronal phenotype.

  According to the present invention, by culturing adipose tissue stromal cells in the medium according to the present invention, it is possible to induce differentiation of nerve cells more easily and more reliably from adipose tissue stromal cells. Therefore, it is possible to obtain nerve cells for realizing regenerative medicine.

(First embodiment)
The first embodiment of the present invention will be described in detail below.

  A first embodiment of the present invention is a method for inducing differentiation of adipose tissue stromal cells, comprising collecting adipose tissue stromal cells from adipose tissue, and stromal cells derived from adipose tissue (adipose tissue stromal cells) ) In a test tube in a medium containing dcAMP (dibutyryl cyclic AMP) or forskolin, for example, to induce differentiation so that adipose tissue stromal cells express markers of young neurons It is characterized by that.

  Due to differentiation induction, spindle-shaped adipose tissue stromal cells show a neuron-like morphology extending a plurality of processes from a circular cell body. In addition, differentiation-induced adipose tissue stromal cells express immature neuronal markers. Examples of young neuronal markers include NSE (neuron specific enolase). Adipose tissue stromal cells induced to differentiate strongly express NSE.

  Here, juvenile nerve cells refer to cells such as neural progenitor cells found in the fetal period.

  First, the collection of adipose tissue stromal cells and the culture before differentiation induction that are induced to differentiate in the first embodiment will be described.

  Adipose tissue-derived stromal cells (ATSCs) are somatic stem cells that can be obtained from adipose tissue, and can be collected with minimal damage to the living body. Adipose tissue stromal cells are mesenchymal stem cells present in adipose tissue at various sites in the living body, and easily proliferate in an undifferentiated state in a simple medium containing FBS. In addition, since adipose tissue is present in large amounts in the living body, the number of cells obtained is larger than that of other somatic stem cells.

  Here, the adipose tissue stromal cells according to the first embodiment can be efficiently and reliably collected from the adipose tissue of the dog, and the differentiation induction efficiency according to the first embodiment and the second embodiment described later is high. It is preferable to collect from adipose tissue of dogs. In other words, in the differentiation induction method according to the first embodiment, it is preferable to induce differentiation using canine adipose tissue stromal cells so as to express a marker of young nerve cells.

  In addition, the adipose tissue stromal cells according to the first embodiment are not particularly limited in the collection of adipose tissue, but preferred adipose tissue includes abdominal abdominal adipose tissue present in a large amount in vivo.

  The adipose tissue stromal cells according to the first embodiment can be obtained by, for example, treating peritoneal adipose tissue collected by liposuction with a digestive enzyme such as collagenase and separating the cells. Here, after passing the cell solution containing the separated adipose tissue stromal cells through a nylon mesh, hemolyzing erythrocytes contained in the cell solution using a hemolysis buffer, and then centrifugally washing this with a culture solution, It is possible to obtain adipose tissue stromal cells with high purity.

  The obtained adipose tissue stromal cells can be cultured in DMEM containing 10% FBS to increase the number of cells in an undifferentiated state.

  Subsequently, a process of inducing differentiation of the obtained adipose tissue stromal cells so as to express a marker of young nerve cells will be described.

  In the differentiation induction according to the first embodiment, adipose tissue stromal cells collected from adipose tissue or adipose tissue stromal cells collected and cultured from adipose tissue are cultured in serum-free DMEM containing dcAMP or forskolin. Is done.

  dcAMP is a chemical agent that increases intracellular cAMP concentration. Increasing intracellular cAMP concentration promotes cell survival.

  When differentiation induction is performed using dcAMP, it is preferable to add IBMX (Isobutyl-methylxanthine), which is an inhibitor of phosphodiesterase that degrades cAMP.

  On the other hand, forskolin also increases intracellular cAMP concentration, similar to dcAMP.

  When differentiation is induced using forskolin, it is preferable to add BHA (Butylhydroxyanisole) and valproic acid together in order to increase the certainty of differentiation induction.

  When differentiation is induced by culturing adipose tissue stromal cells in a medium containing dcAMP and IBMX, or a medium containing forskolin, BHA, and valproic acid, both are 50% to 60% of cells and are characteristic of neurons. Cells that have changed into a form that extends multiple protrusions are observed.

  In addition, differentiation-induced adipose tissue stromal cells show a marked increase in NSE expression. That is, adipose tissue stromal cells are differentiated into young neurons by the differentiation induction method of the first embodiment.

(Second Embodiment)
Subsequently, a second embodiment of the present invention will be described.

  A second embodiment of the present invention is a method for inducing differentiation of adipose tissue stromal cells, comprising collecting adipose tissue stromal cells from adipose tissue, and adipose tissue stromal cells as basic fibroblast growth factor ( Incubate in a medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) to induce differentiation so that nestin, a marker of neural stem cells, and NSE are expressed Differentiation induction stage and culture in a medium containing retinoic acid, dcAMP, and Sonic hedgehog to enhance NSE expression and differentiate to express MAP-2, a neuronal marker And a second differentiation-inducing step for inducing differentiation of the adipose tissue stromal cells to express a neuronal phenotype.

  Here, as in the first embodiment, the adipose tissue stromal cells according to the second embodiment can be efficiently and reliably collected from the adipose tissue of the dog, and the efficiency of differentiation induction according to the second embodiment is high. Therefore, it is preferable to collect from the adipose tissue of the dog. In other words, in the differentiation induction method according to the second embodiment, it is preferable to induce differentiation using canine adipose tissue stromal cells so as to express a neuronal phenotype.

  Further, the adipose tissue stromal cells according to the second embodiment are not particularly limited in the adipose tissue to be collected, but preferable adipose tissue includes abdominal adipose tissue that is present in a large amount in vivo. .

  As in the first embodiment, the adipose tissue stromal cells according to the second embodiment are obtained by, for example, treating peritoneal adipose tissue collected by liposuction with a digestive enzyme such as collagenase and separating the cells into cells. ,Obtainable. Here, after passing the cell solution containing the separated adipose tissue stromal cells through a nylon mesh, hemolyzing erythrocytes contained in the cell solution using a hemolysis buffer, and then centrifugally washing this with a culture solution, It is possible to obtain adipose tissue stromal cells with high purity.

  The obtained adipose tissue stromal cells can be subcultured with DMEM containing 10% FBS to increase the number of cells in an undifferentiated state.

  Subsequently, a process of inducing differentiation of the obtained adipose tissue stromal cells so as to express a neuronal phenotype will be described.

  In the first differentiation induction step according to the second embodiment, adipose tissue stromal cells collected from adipose tissue, or adipose tissue stromal cells collected from adipose tissue and cultured, a basic fibroblast growth factor and epithelium It is carried out by culturing in a serum-free Dulbecco modified Eagle medium (DMEM) as a medium containing a cell growth factor, for example, a preferable medium.

  During the first differentiation induction stage, adipose tissue stromal cells form a floating cell mass (sphere) that closely resembles the neurosphere obtained from neural stem cells.

  In addition, the expression of nestin mRNA, a marker of neural stem cells, is not observed in adipose tissue stromal cells before the first differentiation induction stage, whereas in the adipose tissue stromal cells after the first differentiation induction stage, neural stem cells Similar to the neurosphere obtained from nestin, it expresses nestin mRNA.

  Subsequently, a second differentiation induction step is performed on the cells formed in the first differentiation induction step and having formed spheres.

  The second differentiation inducing step according to the second embodiment is performed by culturing the sphere-formed cells in a medium containing retinoic acid, dcAMP, and sonic hedgehog, for example, serum-free DMEM as a preferred medium.

  The second differentiation induction step can be performed, for example, on a PDL / laminin coated cover slip.

  In the second differentiation induction stage, cells that extend the process appear from the cells that have formed cell clusters.

  In addition, the expression of NSE, whose expression was weak after the first differentiation induction stage, is enhanced by the second differentiation induction stage, and the expression of MAP-2, which is a neuronal marker, is confirmed.

  Furthermore, when the adipose tissue stromal cells before differentiation induction are stimulated with glutamic acid, which is a neuroexcitatory drug, there is no significant difference in intracellular calcium concentration before and after stimulation, but the first and second differentiation induction stages. When intracellular cells are stimulated with glutamic acid, an increase in intracellular calcium concentration is observed. That is, adipose tissue stromal cells are differentiated into nerve cells by the differentiation induction method of the second embodiment.

  Next, examples of the present invention will be described.

  The following reagents were used in the implementation.

  Anti-human NSE (neuron specific enolase) monoclonal antibody was purchased from Dako, and anti-porcine NF-68 (Neurofilament 68) monoclonal antibody and anti-bovine MAP-2 (microtubule associated protein 2) monoclonal antibody were purchased from Sigma. Goat serum was purchased from Bethyl, and FITC-labeled anti-mouse IgG antibody was purchased from Santacruz. Fluoro-3 AM (fluo-3 acetoxymethylester), a fluorescent dye, was purchased from Molecular Probe. DMEM (Dulbecco ’s modified Eagle medium) was purchased from Sigma, and fetal bovine serum (FBS), NB (Neurobasal medium-A), and B27 (B27 supplement without vitamin A) were purchased from Gibco. dcAMP (Dybutyril cyclic AMP) was purchased from Sigma, forskolin, IBMX (isoburylmethylxanthine), BHA (butylated hydroxyanisole), and valproic acid from Wako. Basic fibroblast growth factor (bFGF) was purchased from Peprotech and epidermal growth factor (EGF) from Sigma. Retinoic acid was purchased from Sigma and Sonic Hedgehog (SHH) from R & D. Triton-X 100 (TX-100) and Tween-20 were purchased from Sigma. Reagents other than the above were purchased from Wako. The sequences of primers used for detecting various mRNAs by RT-PCR are shown in the nucleotide sequence table.

Example 1
Example 1 shows the induction of differentiation of adipose tissue stromal cells derived from a dog (canine adipose tissue stromal cells) in accordance with the first embodiment.

  As dog adipose tissue stromal cells (ATSCs), stromal cells derived from canine abdominal adipose tissue were collected. Specifically, abdominal adipose tissue of 1 to 2 year old healthy beagle dogs was aseptically collected and used with a collagenase solution (2 mg / ml Collagenase, 4 mg / ml bovine serum albumin (BSA), DMEM containing 20 mM HEPES). Digested for 1 hour at ° C. Next, the obtained solution was passed through a 100 μm nylon mesh, hemolysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) was added, and then washed three times with DMEM containing 10% FBS. Adipose tissue stromal cells were used. The obtained canine adipose tissue stromal cells were cultured in DMEM containing 10% FBS in a φ90 mm petri dish (FIG. 1 top), and those passaged 3 times were used for the experiment. The culture medium was changed once every 5 days.

  Differentiation induction can be done in two ways: a) replacing the medium with DMEM containing 100 μM dcAMP and 125 μM IBMX; All were cultured until the 10th day after the exchange.

  When the medium was changed to an induction medium containing dcAMP, cells changed into a form in which a plurality of protrusions were extended from a small round cell body 3 days after the exchange (Fig. 1, lower left). Ten days after switching to the induction medium, about 60% of the cells showed morphological changes.

  On the other hand, when the medium was replaced with an induction medium containing forskolin, cells extending several protrusions from round cell bodies were also observed 3 days after the replacement (FIG. 1, lower right). However, the cell body was larger than that induced by dcAMP, and about 50% of the cells had changed in morphology 10 days after the medium was changed.

  In cells in which canine adipose tissue stromal cells were differentiated with dcAMP, NSE expression was significantly increased and NF-68 expression was weak. However, no expression of MAP-2 was observed (Figure 2 left). Moreover, when NSE expression after differentiation induction was compared over time, it increased until day 10 after changing to the induction medium (FIG. 3), and then plateau.

  On the other hand, the expression of NSE was increased in cells obtained by inducing differentiation of canine adipose tissue stromal cells with forskolin, but NF-68 expression was not observed. In addition, MAP-2 expression was not observed (Fig. 2, right). In canine adipose tissue stromal cells after differentiation induction with forskolin, strong expression of NSE was observed in cell bodies and processes only in cells with altered morphology (in FIG. 4). Approximately 60% of all cells treated with forskolin were expressed.

  In contrast, NSE expression was observed in about 3% of canine adipose tissue stromal cells before differentiation induction, all of which were weakly expressed (Fig. 4, left).

  In canine adipose tissue stromal cells before differentiation induction, mRNA expression of glial cell transporters GLUT-3 and GLT-1 and nerve cell transporter EAAC1 was already observed (Fig. 5 bottom). Increased expression of GLUT-3, GLT-1, and EAAC1 was observed in cells in which adipose tissue stromal cells were induced to differentiate with dcAMP. In addition, the expression of EAAT4, a neuronal transporter, was not seen as before differentiation induction (FIG. 5, upper panel).

(Example 2)
Example 2 shows the induction of canine adipose tissue stromal cell differentiation according to the second embodiment.

  In the same manner as in Example 1, stromal cells derived from canine adipose tissue were collected as canine adipose tissue stromal cells. That is, abdominal adipose tissue of 1 to 2 year old healthy beagle dogs was aseptically collected and used with a collagenase solution (2 mg / ml Collagenase, 4 mg / ml bovine serum albumin (BSA), DMEM containing 20 mM HEPES). Digested for 1 hour at ° C. Next, the obtained solution was passed through a 100 μm nylon mesh, hemolysis buffer (154 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA) was added, and then washed three times with DMEM containing 10% FBS. Adipose tissue stromal cells were used. The obtained canine adipose tissue stromal cells were cultured in DMEM containing 10% FBS in a φ90 mm petri dish (FIG. 1 top), and those passaged 3 times were used for the experiment. The culture medium was changed once every 5 days.

  In addition, canine neural stem cells (NSCs) were collected for use as a control in Example 2. Specifically, the subcerebral ventricular region of a healthy beagle dog of 1 to 2 years old was aseptically collected and digested with a 0.05% Trypsin solution at 37 ° C. for 1 hour, and then passed through a 25 gauge syringe three times. Then, it was washed 3 times with NB (NB / B27) containing B27, and cultured in NB / B27 containing 20 ng / ml basic fibroblast growth factor and 20 ng / ml epidermal growth factor in a 90 mm petri dish to form a neurosphere I let you. The culture medium was changed 70% of the whole once every 5 days.

  After replacing the adipose tissue stromal cells with serum-free medium containing basic fibroblast growth factor and epidermal growth factor, a neurosphere-like floating cell mass (sphere) is seen from the third day, and the cell mass It grew in the state which formed. Most of the surviving cells formed spheres (Figure 6, upper left). After that, when the obtained sphere was cultured on a cover slip coated with PDL / laminin in a medium containing retinoic acid, dcAMP, and sonic hedgehog, it adhered to the cover slip the next day after replacement, and then from the third day Cells that extended the protrusions were seen (FIG. 6, bottom).

  On the other hand, the neural stem cell used as a control formed a neurosphere in a medium containing basic fibroblast growth factor and epidermal growth factor and proliferated (upper right of FIG. 6). When the medium was replaced with a medium containing retinoic acid, nerve cells attached to the PDL / laminin-coated coverslip and extending the protrusions were observed from the third day after the exchange (FIG. 6, lower right).

  Nestin mRNA expression was not detected in canine adipose tissue stromal cells before differentiation induction. In contrast, the expression of nestin mRNA was detected in the spheres induced to differentiate from canine adipose tissue stromal cells, similar to the neurosphere used as a control (FIG. 7).

  The sphere obtained from the canine adipose tissue stromal cells in the first differentiation induction stage showed weak expression similar to the neurosphere only for NSE. In contrast, cells obtained from spheres in the second differentiation induction stage showed stronger expression of NSE than cells in which neurospheres were induced to differentiate (FIG. 8, upper panel). In addition, when immunoblotting was performed using an anti-MAP-2 monoclonal antibody, both the cells induced to differentiate from the sphere and the cells induced to differentiate from the neurosphere showed a band with a molecular weight (50 KDa) different from the molecular weight detected by brain homogenization (280 Kda). It was seen. Cells with differentiated spheres obtained from canine adipose tissue stromal cells showed stronger protein expression of that molecular weight (lower part of Fig. 8).

  A significant increase in the fluorescence intensity of fluo-3, a calcium ion fluorescence monitor, was detected after glutamate stimulation in cells in which sphere differentiation was induced and in cells in which neurosphere was induced to differentiate (Fig. 9 top). In cells in which sphere differentiation was induced, the fluorescence intensity after the stimulation was enhanced about 2.5 times before the stimulation. On the other hand, in cells in which neurosphere used as a control was induced to differentiate, the fluorescence intensity was enhanced by about 3.5 times that by stimulation with glutamic acid. Both fluorescence intensities decreased immediately after stimulation and reached a plateau 200 seconds after stimulation. In the canine adipose tissue stromal cells before induction, there was no significant difference in fluorescence intensity before and after stimulation (lower part of Fig. 9).

  According to the present invention, there are provided a differentiation induction method, a differentiation-induced nerve cell, and a medium used for the differentiation induction method, which are required for regenerative medicine targeting nerve tissue. According to the differentiation induction method of the present invention, stem cells were obtained from an individual in need of regeneration of neural tissue, differentiated into nerve cells, and regenerated without receiving immunological rejection by returning the cells to the individual. Nervous tissue can be deposited.

It is a figure which shows the form of a dog adipose tissue stromal cell (upper figure) and the canine adipose tissue stromal cell (lower figure) differentiation-induced by dcAMP or forskolin. It is a figure which shows the expression of the neuron marker (NSE, NF-68, or MAP-2) in canine adipose tissue stromal cells and canine adipose tissue stromal cells induced to differentiate by dcAMP or forskolin. It is a figure which shows the time-dependent change of the NSE expression of a dog adipose tissue stromal cell in the process of differentiation induction with dcAMP. It is a figure which shows the expression of NSE in the dog adipose tissue stromal cell differentiation-induced by dcAMP or forskolin. It is a figure which shows the expression of GLUT-3, GLT-1, EAAT4, or EAAC1 mRNA which is a nerve cell transporter or a glial cell transporter in canine adipose tissue stromal cells differentiation-induced by dcAMP. It is a figure which shows the form of the sphere produced | generated from the canine adipose tissue stromal cell, and the Neurosphere produced | generated from the canine neural stem cell. It is a figure which shows the expression of nestin mRNA of Sphere produced | generated from the canine adipose tissue stromal cell. It is a figure which shows the expression of NSE and MAP-2 which are neuron marker proteins of the cell induced differentiation from the neurosphere produced | generated from the canine adipose tissue stromal cell and the canine neural stem cell. It is a figure which shows the calcium image by the glutamate stimulation of the dog adipose tissue stromal cell, the cell induced differentiation from sphere, and the cell induced differentiation from Neurosphere.

Claims (15)

Collecting adipose tissue stromal cells from adipose tissue;
Culturing the adipose tissue stromal cells in a medium containing dcAMP (dibutyryl cyclic AMP) or forskolin (forskolin),
A method of inducing differentiation of adipose tissue stromal cells, wherein the differentiation of the adipose tissue stromal cells is induced so as to express a marker of a young neuronal cell.   The method for inducing differentiation of adipose tissue stromal cells according to claim 1, wherein the medium is serum-free DMEM.   The method for inducing differentiation of adipose tissue stromal cells according to claim 1 or 2, wherein the adipose tissue stromal cells are collected from dog adipose tissue.   The method for inducing differentiation of adipose tissue stromal cells according to any one of claims 1 to 3, wherein the adipose tissue stromal cells are collected from peritoneal adipose tissue.   5. The differentiation induction of adipose tissue stromal cells according to any one of claims 1 to 4, further comprising a step of subculturing the collected adipose tissue stromal cells in a medium containing serum. Method.   A cell characterized in that differentiation is induced from adipose tissue stromal cells by the method for inducing differentiation of adipose tissue stromal cells according to any one of claims 1 to 5. A medium used in the differentiation induction method for adipose tissue stromal cells according to any one of claims 1 to 5,
Serum-free DMEM,
Contains dcAMP (dibutyryl cyclic AMP) or forskolin,
A culture medium characterized by inducing differentiation of cultured adipose tissue stromal cells so as to express a marker of young neurons. Collecting adipose tissue stromal cells from adipose tissue;
Culturing the adipose tissue stromal cells in a medium containing basic fibroblast growth factor and epidermal growth factor to induce differentiation so as to express nestin and NSE; and
The cells induced to differentiate in the first differentiation induction stage are cultured in a medium containing retinoic acid, dcAMP, and Sonic hedgehog so as to enhance NSE expression and to express MAP-2. A second differentiation induction stage for inducing differentiation,
A method of inducing differentiation of adipose tissue stromal cells, wherein the differentiation of the adipose tissue stromal cells is induced to express a neuronal phenotype.   The method for inducing differentiation of adipose tissue stromal cells according to claim 8, wherein the medium for the first differentiation induction stage and the medium for the second differentiation induction stage are serum-free DMEM.   The method for inducing differentiation of adipose tissue stromal cells according to claim 8 or 9, wherein the adipose tissue stromal cells are collected from adipose tissue of a dog.   The method for inducing differentiation of adipose tissue stromal cells according to any one of claims 8 to 10, wherein the adipose tissue stromal cells are collected from peritoneal adipose tissue.   The induction of differentiation of adipose tissue stromal cells according to any one of claims 8 to 11, further comprising a step of subculturing the collected adipose tissue stromal cells in a medium containing serum. Method.   A cell induced to differentiate from adipose tissue stromal cells by the method according to any one of claims 8 to 12. A medium used in the differentiation induction method for adipose tissue stromal cells according to any one of claims 8 to 12,
DMEM,
Basic fibroblast growth factor, and
Epidermal growth factor and
A medium characterized by inducing differentiation of cultured adipose tissue stromal cells to express nestin and NSE. A medium used in the differentiation induction method for adipose tissue stromal cells according to any one of claims 8 to 12,
DMEM,
Retinoic acid,
dcAMP,
Including Sonic Hedgehog,
The cells induced to differentiate by the medium according to claim 12, which are cultured, enhance the expression of NSE,
A medium characterized by inducing differentiation so as to express MAP-2.