JPWO2006117925A1 - Pancreatic β-cell regeneration method and apparatus - Google Patents

Abstract

An object of the present invention is to provide a method for regenerating pancreatic β cells for cell transplantation treatment from easily harvested cells such as bone marrow cells. According to the present invention, there is provided a method for regenerating pancreatic β cells by culturing stem cells collected from the stroma in a medium to which a differentiation-inducing factor is added.

Description

  The present invention relates to a technique for highly efficiently regenerating pancreatic β cells, which are insulin-producing cells, by culturing and inducing differentiation of bone marrow stem cells.

  Diabetes has increased rapidly since the latter half of the 20th century, and now the patient population in Japan is estimated to be 13 million including the reserve army, which is becoming a major national problem. Type I diabetes is a disease in which insulin production in the pancreas is inhibited and requires insulin injection. Type II diabetes accounts for over 90% of diabetes, and insulin is produced, but somatic cells exhibit insulin resistance. In type II diabetes, supplementation of insulin is necessary as symptoms progress.

  The main treatment for diabetes is insulin injection to compensate for the decrease in pancreatic insulin production. Recently, oral insulin preparations have been developed, and allografts of beta cells (pancreatic insulin-producing cells) and animals Transplantation of artificial islets in which β-cells are encapsulated in immunoisolation capsules is also being investigated. On the other hand, in recent years, research to regenerate β cells themselves has been actively carried out with the development of regenerative medicine. Pancreas, liver, intestinal tract, ES cell, bone marrow and the like have been studied as cell sources for producing β cells in vitro.

  For β-cell regeneration from pancreatic duct cells, consideration is given to DDS preparations of retinoic acid, use of growth factors such as activin and betacellulin, transcription factor proteins such as pdx-1, and gene transfer such as the pdx-1 gene or NeuroD gene Insulin-expressing cells have been induced to differentiate from pancreatic ductal epithelial cells with a high efficiency of about 70% (regeneration of insulin-prone cells by intra-pancreatic administration of β-cell-related transcription factors and differentiation growth factor-expressing adenovirus vectors) , Yoichi Oikawa et al. The 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)).

  To regenerate β cells from the liver, Fujinomiya et al. Introduced the NeuroD and betacellulin genes into mice using a viral vector, and regenerated pancreatic islets into the liver. The blood glucose level remained normal for over 4 months. Moreover, hepatocytes could be differentiated in vitro (Induction of differentiation into pancreatic endocrine cells using adult rat liver stem-like cells, Atsuko Yamada et al. The 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)) .

  In β-cell regeneration from the intestinal epithelium, Taniguchi et al. Were able to differentiate into insulin-expressing cells by collecting mouse intestinal epithelial cells, adding glucagon-like peptide and culturing them (induction of differentiation of insulin-secreting cells in small intestinal epithelial cells, Hideki Taniguchi 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)).

In the regeneration of β cells from ES cells, Joi et al. Introduced the nhx2.2 gene into mouse ES cells and were able to induce differentiation into insulin-producing cells with high efficiency (differentiation from nkx2.2 gene-transferred ES cells into insulin-producing cells). Trial of induction and treatment, Kei Shiroi et al. The 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)). However, 1 × 10 ⁶ cells were transplanted into the mouse kidney, but no normal blood glucose level was obtained.

  For β-cell regeneration from bone marrow mesenchymal cells (MSC), Nakata et al. Collected bone marrow cells from ICR mouse bone marrow, cultured under mouse pancreatic islet cell culture conditions, and differentiated using growth factors such as bFGF Insulin-expressing cells were able to be induced (attempts to induce differentiation of glucose-responsive insulin-secreting cells from bone marrow cells, Masanori Nakata et al. The 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)). Glucose responsiveness was also observed but incomplete. Also, only a few percent of bone marrow MSCs were able to differentiate.

  For in vitro β-cell regeneration, Izumida et al. Transplanted bone marrow cells collected from the same type of rat femur into hyperglycemic model rats, labeled with PKH-26, from the tail vein, and removed the pancreas 48 days later. Insulin staining confirmed that insulin-producing cells derived from transplanted bone marrow cells existed (examination of differentiation induction of bone marrow cells into insulin-producing cells in a rat diabetes model, 3rd Japan Society for Regenerative Medicine General meeting (March 2004)). However, differentiation induction is not performed.

  In β-cell regeneration from placental cells, Matsumoto et al. Induced insulin-producing cells that formed spheres by suspension culture of mouse placenta-derived cells in serum-free medium supplemented with EGF, bFGF, and Nicotinamide (placental-derived insulin secretion). Possibility of treating cells with diabetes, Shuichiro Matsumoto et al. The 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)). The cells did not grow in vitro.

  Apart from the method of regenerating β cells in vitro as described above and transplanting them to the pancreas with reduced function, the cells are regenerated in vivo by stimulating the stem cells in the body. There is also research to try to make it happen. Yamaguchi et al. Improved insulin secretion by administering all trans retinoic acid DDS preparation to type I diabetic model rats (possibility of induction of differentiation of somatic stem cells into pancreatic β cells by vitamin A active substance and its DDS preparation) , Mariko Kawakami et al. The 3rd Annual Meeting of the Japanese Society for Regenerative Medicine (March 2004)). This is presumed that somatic stem cells have differentiated into β cells (from the 3rd Annual Meeting of the Japanese Society for Regenerative Medicine 2004).

  Most research aimed at the regeneration of pancreatic β-cells is aimed at regeneration based on pancreatic cells and ES cells, but these cell sources are not readily available or have ethical problems, so clinical application It cannot be said that it is suitable for. The most realistic stem cell source is considered to be bone marrow mesenchymal stem cells (MSCs) (including pluripotent stem cells: MAPC here). Recently, it has been clarified that cells having the same differentiation potential are also present in adipose tissue and placenta, and the existence in umbilical cord blood and peripheral blood has also been suggested. Since these cells can be collected non-invasively, it can be said that they are suitable cell sources for clinical application. An object of the present invention is to provide a method for regenerating pancreatic β cells for cell transplantation treatment from easily harvested cells such as bone marrow cells.

  Although β cell induction from bone marrow MSCs has been studied, the number of bone marrow MSCs that can be differentiated is very small, about several percent. The present invention provides a more efficient method for inducing differentiation of β cells from MSCs. The present inventors succeeded in obtaining natural cell lines by subculture of bone marrow cells and colony assay in mice and humans. It has been found that this cell line can be induced to differentiate into insulin-positive cells with a high efficiency of 10% or more by combining appropriate growth factors. The present invention has been completed based on these findings.

  That is, according to the present invention, there is provided a method for regenerating pancreatic β-cells, comprising obtaining insulin-expressing cells by culturing stem cells collected from the stroma in a medium supplemented with a differentiation-inducing factor.

Preferably, insulin-expressing cells are obtained by culturing established stromal stem cells in a medium supplemented with a differentiation-inducing factor.
Preferably, insulin-expressing cells are obtained by culturing stromal stem cells collected from mesenchymal tissue and established in a medium supplemented with a differentiation-inducing factor.
Preferably, the mesenchymal tissue is bone marrow, adipose tissue, peripheral blood, umbilical cord blood, or placenta.

  Preferably, stem cells collected from the stroma are cultured in a medium supplemented with one or more of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide to express insulin-expressing cells To get.

  Preferably, insulin-expressing cells are obtained by culturing stem cells collected from the stroma in a medium to which at least one of Transferrin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, and Progesterone is added.

  Preferably, cells collected from the bone marrow are subcultured to prepare a bone marrow stromal cell line, and this bone marrow stromal cell line is cultured in a medium to which FBS, Ascorbic acid, and dexamethasone are added. Insulin-expressing cells are obtained by changing to a medium containing Transferrin, Insulin, sodium selenite, BSA, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide.

  According to another aspect of the present invention, somatic stem cells, ES cells or dedifferentiated mature cells are any one of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide. There is provided a method for regenerating pancreatic β-cells, comprising obtaining insulin-expressing cells by culturing in a medium supplemented with the above.

  According to yet another aspect of the present invention, a sealed container for culturing somatic stem cells, ES cells or dedifferentiated mature cells, Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, A mechanism for adding one or more of Progesterone and Nicotinamide to the container in a time-controlled order and in a controlled amount, a mechanism for changing the medium at a controlled time, and sterilized air A regenerating apparatus for pancreatic β cells comprising a mechanism for circulating, a mechanism for controlling the temperature of the container, a mechanism for monitoring the culture state of the cells, and a mechanism for filling and discharging the cells from the container Is provided.

  According to yet another aspect of the present invention, somatic stem cells, ES cells or dedifferentiated mature cells are grown and transferredrin, insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide. There is provided a method for treating diabetes, which comprises regenerating pancreatic β cells by culturing in a medium containing any one or more of them, and transplanting the regenerated pancreatic β cells into a diabetic patient.

Hereinafter, embodiments of the present invention will be described in detail.
The method for regenerating pancreatic β cells according to the present invention is a method characterized in that insulin-expressing cells are obtained by culturing stem cells collected from the stroma in a medium supplemented with a differentiation-inducing factor.

  The stroma is a supporting tissue contained in bone marrow or the like, and is generally composed of stromal cells and an extracellular matrix. In the present invention, stem cells present in tissues such as stroma are collected and used. The stem cells to be collected are preferably mesenchymal stem cells. The mesenchyme is a loose connective tissue that fills the space between the germ layers, and is generally derived from the mesoderm, but some is derived from the ectoderm. Examples of the tissue containing mesenchymal stem cells include bone marrow, adipose tissue, peripheral blood, umbilical cord blood, and placenta.

  Stem cells are ectoderm (dental pulp cells (including dental pulp fibroblasts), epithelial cells (tooth), enamel epithelial basement membrane cells, neurons, odontoblasts, cement blasts, etc.), mesoderm (osteoblasts, It has pluripotency that can differentiate into cells such as chondrocytes, bone cells, renal basement membrane cells, blood cells), endoderm (gastrointestinal epithelial cells, gastrointestinal parenchymal cells), or promote their repair. It is an undifferentiated cell. As stem cells, somatic stem cells contained in each tissue / organ and whose direction of differentiation is determined to some extent may be used, or pluripotent stem cells such as embryonic stem cells may be used. . Further, in the present invention, dedifferentiated mature cells may be used as stem cells.

  In the present invention, the stem cells can be collected from any stroma containing the cells, but preferably can be collected from bone marrow, adipose tissue, peripheral blood, umbilical cord blood, placenta, or the like. From the viewpoint that a large amount of cells can be collected and collection is easy, it is preferable to collect from the femur, tibia or pelvis (iliac bone). A method for collecting bone marrow-derived mesenchymal stem cells is known to those skilled in the art, and for example, a normal collection method used in medicine can be used. Specifically, for humans, with informed consent to the patient, a syringe or puncture needle is used from tissues and organs in which stem cells such as human femur, iliac bone, jawbone, and peripheral blood vessels are present. Collect as much bone marrow and peripheral blood as necessary and inoculate them in a culture vessel and use them by separating floating cells and adherent cells, or use techniques such as flow cytometry or density gradient centrifugation. By using it, stem cells can be collected and separated. When collecting mesenchymal stem cells from the bone marrow of mammals other than humans, for example, cut both ends of the bone (femur, tibia), wash the inside of the bone with a medium suitable for culturing mesenchymal stem cells, Mesenchymal stem cells can be obtained from the washed culture.

  In order to perform primary culture and / or subculture of mesenchymal stem cells, the collected and separated cells are used for tissue culture using an appropriate medium (for example, DMEM (Dulbecco's modified Eagle's medium) medium, α-MEM medium, etc.). Cells are seeded in a culture dish for primary culture and subculture. As the serum used for the culture, fetal bovine serum (FBS) can be used.

Cell culture can be performed using normal serum-containing medium or serum-free medium used for animal cell culture under normal animal cell culture conditions (eg, room temperature to 37 ° C; 5% CO ₂ incubator, etc.) Can be done below. Although the form of culture is not particularly limited, for example, it can be performed by static culture.

  In the present invention, differentiation induction culture from stem cells to pancreatic β cells, which are insulin-producing cells, can be performed by culturing primary and subcultured stem cells using a differentiation induction medium and inducing cell differentiation. it can. Here, the differentiation-inducing medium refers to a medium to which a differentiation-inducing factor described below is added.

  The differentiation inducing factor used in the present invention is not particularly limited as long as it is a factor capable of differentiating stem cells into insulin-expressing cells. Specific examples of differentiation-inducing factors include transferrin, insulin, insulin selenite, serum albumin, ascorbic acid, dexamethasone, EGF (epidermal growth factor) ), Progesterone, and nicotinamide. In the present invention, any one or more of the nine types of differentiation inducing factors can be used, and preferably two or more (more preferably three or more) of the nine types of differentiation inducing factors are combined. It is possible to use, and particularly preferably, all nine types of differentiation inducers described above can be used. According to another preferred embodiment, six types of differentiation induction of transferrin, sodium selenite, serum albumin, ascorbic acid, dexamethasone, and progesterone Factors can be used in combination.

  When transferrin is used, the concentration of transferrin in the medium is not particularly limited as long as the effect of the present invention can be achieved, but is generally 5 to 500 μg / ml, preferably 10 to 200 μg / ml. It is.

  When insulin is used, the concentration of insulin in the medium is not particularly limited as long as the effect of the present invention can be achieved, but is generally 0.5 to 50 μg / ml, preferably 1 to 20 μg / ml. is there.

  In the case of using sodium selenite, the concentration of sodium selenite in the medium is not particularly limited as long as the effect of the present invention can be achieved, but is generally 0.5 to 50 ng / ml, preferably 1-20 ng / ml.

  When serum albumin is used, the concentration of serum albumin in the medium is not particularly limited as long as the effect of the present invention can be achieved, but is generally 0.1 to 10 mg / ml, preferably 0.2 to 5 mg / ml.

  When ascorbic acid is used, the concentration of ascorbic acid in the medium is not particularly limited as long as the effects of the present invention can be achieved, but is generally 5 to 500 μg / ml, preferably 10 to 200 μg / ml. is there.

  When dexamethasone is used, the concentration of dexamethasone in the medium is not particularly limited as long as the effect of the present invention can be achieved, but it is generally 1 to 100 nM, preferably 2 to 50 nM.

  When EGF is used, the concentration of EGF in the medium is not particularly limited as long as the effect of the present invention can be achieved, but is generally 0.2 to 20 ng / ml, preferably 0.5 to 10 ng / ml. ml.

  When progesterone is used, the concentration of progesterone in the medium is not particularly limited as long as the effect of the present invention can be achieved, but is generally 2.5 to 250 ng / ml, preferably 5 to 100 ng / ml. is there.

  When nicotinamide is used, the concentration of nicotinamide in the medium is not particularly limited as long as the effects of the present invention can be achieved, but it is generally 1 to 100 mM, preferably 2 to 50 mM.

  Insulin-producing cells can be obtained by culturing stem cells according to the method of the present invention described above and inducing differentiation thereof. As for the presence or absence of insulin expression in differentiation-induced cells, mRNA level expression can be confirmed by RT-PCR or Northern blot, in situ hybridization, etc., and protein level expression can be confirmed by Western blot, immunostaining, etc. . RT-PCR, Northern blotting, and in situ hybridization are known to those skilled in the art using primers or probes that can specifically detect the insulin gene (primers or probes for detecting proinsulin may be used). Can be done by law. In addition, Western blotting and immunostaining can be performed by an ordinary method known to those skilled in the art using an anti-insulin antibody. In addition, by using the presence or absence of expression of the Pdx1 gene (an important gene involved in the development of the pancreas, which is also thought to play a role in the generation and maintenance of functions of insulin-producing cells), pancreatic β cells You can also check the playback.

  The pancreatic β cells regenerated by the method of the present invention can express insulin and can be treated for diabetes by transplantation into a diabetic patient.

  Furthermore, the present invention relates to a sealed container for culturing somatic stem cells, ES cells or dedifferentiated mature cells, and any of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide. A mechanism for adding one or more of them in a time-controlled order and in a controlled amount to the container, a mechanism for changing the medium at a controlled time, a mechanism for circulating sterilized air, The present invention relates to an apparatus for regenerating pancreatic β cells, comprising a mechanism for controlling the temperature of a container, a mechanism for monitoring the culture state of the cells, and a mechanism for filling and discharging the cells from the container.

  FIG. 4 shows an example of a culture apparatus for automatically performing a series of steps for regenerating pancreatic β cells from the collected cells. Somatic stem cells, ES cells or dedifferentiated mature cells are subjected to necessary measures such as selection, dilution or concentration, and then injected from the cell container 1 into the device. The cells are led from the inlet through the pipe 2 to the culture vessel 3. Transferrin, Insulin, Sodium selenite, Serum albumin, Ascorbic acid, Dexamethasone, EGF, Progesterone, Nicotinamide, or a differentiation inducer reservoir group 4 with a combination of them was controlled in a time-controlled order. An amount is added to the culture vessel 3. In addition, the medium is supplied to the culture container 3 from the medium reservoir 5 at a controlled time, and the medium containing the waste is discharged. Supply and discharge of the differentiation-inducing factor and the medium are performed by a pump mechanism 7 controlled by a computer 6. Air and carbon dioxide sterilized by the circulation mechanism 8 are supplied to the culture container. The temperature of the culture vessel is controlled by a temperature sensor and a heating / cooling mechanism 9. The shape and number of cells in the culture vessel and the growth rate are monitored by the optical monitor system 10. The pH and glucose concentration of the medium are monitored by the chemical sensor 11 in the waste liquid channel. The β cells grown to the required number are detached by enzyme treatment or the like and then discharged into the container 12 by the pump system 7.

  The following examples further illustrate the present invention, but the present invention is not limited to the examples.

Example 1
Bone marrow cells collected from mice (C57B1 / 6, male, 6 weeks old) were subcultured to obtain bone marrow stromal cell lines by spontaneous cell lines (2 strains). To obtain a bone marrow stromal cell line, a cylinder method using a cloning ring was performed. From bone marrow stromal cells seeded in a petri dish thinly, arbitrary one cell was surrounded by a cloning ring, and one detached cell was passaged to a 24-well plate. In a 24-well plate, cells that reached confluence from a single cell were subcultured to a 6-well plate with a slightly larger culture area, and cultured until confluence. Furthermore, cells that reached confluence in a 6-well plate were subcultured into a T-75 flask with a larger culture area, and continued to culture, thereby obtaining a single bone marrow stromal cell-derived cell. The cells were cultured for about six months to one year, and it was confirmed that the differentiation ability and proliferation ability were stable before and after culturing by a known method and used as a bone marrow stromal cell line.

  The bone marrow stromal cell line obtained above was subcultured and then cultured for 12 hours in an α-MEM medium supplemented with 10% FBS, 50 μg / ml Ascorbic acid, and 10 nM dexamethasone. After that, the medium was 5% FBS, 50 μg / ml Transferrin, 5 μg / ml Insulin, 5 ng / ml sodium selenite, 1 mg / ml BSA, 50 μg / ml Ascorbic acid, 10 nM dexamethasone, 2 ng / ml EGF, 25 ng / ml Progesterone, 10 mM It changed to the DMEM culture medium which added Nicotinamide.

  Seven to 10 days after the medium change, colonies in which cells were densely formed were formed as shown in FIG. 1, and a large number of granules were formed in the cytoplasm. Gene expression was examined for the following 8 genes. That is, gene expression was examined by RT-PCR using primers having the nucleotide sequences described in SEQ ID NOs: 1 to 10, respectively. RT-PCR was performed as follows.

  Total RNA was collected from cultured cells using an RNA collection kit (TRIzol Reagent: Invitrogen). cDNA was synthesized from 2 μg of total RNA using a cDNA synthesis kit (Superscript First-Strand Synthesis System for RT-PCR: Invitrogen). Next, for each sample tube, 1 μl of cDNA, 11.3 μl of sterilized water, 2 μl of 10 × PCR buffer (Takara Bio), 1.6 μl of dNTP (Takara Bio), 2 μl of sense primer (final concentration 0.5 μM, see below), Antisense primer (final concentration 0.5 μM, see below) 2 μl and Taq polymerase (manufactured by Takara Bio Inc.) were mixed to prepare a sample with a total amount of 20 μl. The adjusted sample was set in a thermal cycler, PCR reaction was performed in 20 to 35 cycles according to the target gene for detection, and finally the target gene was detected by electrophoresis.

1. Proinsulin 1 (gttggtgcac ttcctacccc tg (SEQ ID NO: 1) and gtagagggag cagatgctgg tg (SEQ ID NO: 2), 300 bp)
2. Proinsulin 2 (gtggatgcgc ttcctgcccc tg (SEQ ID NO: 3) and gtagagggag cagatgctgg tg (SEQ ID NO: 4), 300 bp)
3. Glucagon (tcatgacgtt tggcaagtt (SEQ ID NO: 5) and cagaggagaa ccccagatca (SEQ ID NO: 6), 202 bp)
4. Somatostatin (gacctgcgaa ctagactgac (SEQ ID NO: 7) and tttgggggag agggatcag (SEQ ID NO: 8), 294 bp)
5. Pax 4 (gctttgtacc caggacaagg ct (SEQ ID NO: 9) and gaggtgtcac tggaacatct ac (SEQ ID NO: 10), 552 bp)
6. Pax 6 (aaccagagaa gacaggccag (SEQ ID NO: 11) and aggttcactc ccgggaagaa (SEQ ID NO: 12), 420 bp)
7. PDX1 (ggccacacag ctctacaagg (SEQ ID NO: 13) and ttccacttca tgcgacggtt (SEQ ID NO: 14), 582 bp)
8. G3PDH (accacagtcc atgccatcac (SEQ ID NO: 15) and tccaccaccc tgttgctgta (SEQ ID NO: 16), 452 bp)

  The results are shown in FIG. Proinsulin 1, Proinsulin 2 and PDX-1 mRNA expression was clearly observed. Moreover, the result of having investigated the expression of protein by Western Blot is shown in FIG. Western Blot was performed as follows. For the cultured cells, the medium was removed and the protein was recovered using a protein lysing agent (such as Cytobaster Novagen). The sample was suspended and heated with a sonicator and adjusted to a protein concentration of 1 mg / ml, and the sample was electrophoresed using a 10% acrylamide gel. The protein fraction contained in the electrophoresed gel was transferred to a PVDF membrane, and finally the target protein was detected with a primary antibody of anti-insulin and a secondary antibody of peroxidase-labeled anti-primary antibody.

Primary antibody: Goat anti-Insulin antibody (Santa Cruz sc-7838, 1: 200)
Secondary antibody: Horseradish peroxides-conjugated anti-Goat IgG antibody (Pierce 31402, 1: 5000)
It was. As shown in FIG. 3, the expression of insulin was observed.

  As a result of confirming the ratio of differentiated cells with a microscope, the ratio of insulin-positive cells was high in the method of the present invention.

  By inducing differentiation of the bone marrow stromal cell line, expression of pancreatic development master genes pdx-1, Proinsulin 1 and Proinsulin 2 was observed, and further, the expression of insulin protein was observed. It was confirmed that it was differentiated. Due to differentiation induction from the cell line, there is no possibility of cell fusion with the pancreatic cells present in this experiment. Insulin added to the medium may have been detected in cells, but granules were observed in differentiated cells and expression of mRNA for Proinsulin. This contribution is recognized.

  As described above, cell lines derived from bone marrow stromal cells have been described. Since cells in adipose tissue, placenta, umbilical cord blood and peripheral blood also have differentiation potential similar to bone marrow stromal cells, It is expected that the cells can be differentiated into β cells. Furthermore, it is expected that general somatic stem cells, ES cells, or dedifferentiated mature cells can be differentiated into β cells by the same treatment.

  According to the method of the present invention, pancreatic β cells can be regenerated from mesenchymal stem cells that can be easily collected, such as bone marrow stroma. Therefore, the pancreatic β cells regenerated by the method of the present invention are β cells for diabetes. It is effective as a cell source for transplantation therapy. In addition, since a cell line is used in the present invention, a large number of cells can be obtained by repeated passage. It can also be stored and differentiated and transplanted again when necessary. Since the method of the present invention does not use gene transfer or special proteins or cytokines, clinical application is easy.

FIG. 1 shows regenerated β cells. FIG. 2 shows mRNA expression of regenerated β cells. FIG. 3 shows protein expression of regenerated β cells. FIG. 4 shows a differentiation / culture apparatus diagram of β cells. 1 cell container, 2 pipes, 3 culture containers, 4 differentiation inducer reservoir groups, 5 medium reservoir, 6 computer, 7 pump mechanism, 8 circulation mechanism, 9 heating / cooling mechanism, 10 optical monitoring system, 11 chemical sensor, 12 containers

Claims (10)

A method for regenerating pancreatic β cells, comprising obtaining insulin-expressing cells by culturing stem cells collected from stroma in a medium to which a differentiation-inducing factor is added. The method according to claim 1, wherein insulin-expressing cells are obtained by culturing established stromal stem cells in a medium to which a differentiation-inducing factor is added. The method according to claim 1 or 2, wherein insulin-expressing cells are obtained by culturing stromal stem cells collected from mesenchymal tissue and established in a medium supplemented with a differentiation-inducing factor. The method according to any one of claims 1 to 3, wherein the mesenchymal tissue is bone marrow, adipose tissue, peripheral blood, umbilical cord blood, or placenta. Insulin-expressing cells are obtained by culturing stem cells collected from the stroma in a medium supplemented with one or more of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide The method according to claim 1. Insulin-expressing cells are obtained by culturing stem cells collected from the stroma in a medium containing at least one of Transferrin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, and Progesterone. The method in any one of. Cells collected from the bone marrow are subcultured to produce a bone marrow stromal cell line, this bone marrow stromal cell line is cultured in a medium containing FBS, Ascorbic acid, and dexamethasone, and then the medium is FBS, Transferrin, The method according to any one of claims 1 to 6, wherein insulin-expressing cells are obtained by culturing by changing to a medium to which insulin, sodium selenite, BSA, ascorbic acid, dexamethasone, EGF, progesterone, and nicotinamide are added. . By culturing somatic stem cells, ES cells or dedifferentiated mature cells in a medium supplemented with one or more of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide A method for regenerating pancreatic β cells, comprising obtaining insulin-expressing cells. A sealed container for culturing somatic stem cells, ES cells or dedifferentiated mature cells, and one or more of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide A mechanism for adding to the container in a time-controlled order and in a controlled amount, a mechanism for changing the medium at a controlled time, a mechanism for circulating sterilized air, and controlling the temperature of the container An apparatus for regenerating pancreatic β cells, comprising: a mechanism for monitoring the culture state of the cells; and a mechanism for filling and discharging the cells from the container. Somatic stem cells, ES cells or dedifferentiated mature cells are grown and cultured in a medium containing at least one of Transferrin, Insulin, sodium selenite, serum albumin, Ascorbic acid, dexamethasone, EGF, Progesterone, and Nicotinamide A method for treating diabetes comprising regenerating pancreatic β cells by transplanting the regenerated pancreatic β cells to a diabetic patient.