JP2005176659A - Method for producing neuron from stem cell and culture medium for culturing stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing neurons from stem cells. <P>SOLUTION: This method for producing the neurons from the stem cells comprises culturing the neurons in a culture medium and then culturing the stem cells in the consequently obtained mixture. The culture medium is a culture medium for culturing the stem cells prepared by culturing the neurons in a basic culture medium. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for generating neurons from stem cells and a medium for culturing stem cells.

  Many clinically common chronic neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis result from neuronal degeneration and death in the central nervous system (CNS). Neurons cannot regenerate rapidly, unlike epithelial cells and hepatocytes. There are indeed stem cells in the CNS that can replicate and differentiate under normal conditions or when neurons are damaged, but their replication is very slow and the rate of differentiation of neurons is very low. Most stem cells in the CNS differentiate into glial cells and therefore cannot rescue neurodegenerative diseases.

  Neuron transplantation has been taken up for the purpose of treating neurodegenerative diseases. Researchers have attempted to generate neurons from various stem cells both in vitro and in vivo. Stem cells are thought to have certain characteristics including self-renewal, pluripotency, proliferation, longevity and differentiation. The fetus, cord blood and bone marrow are common sources of human stem cells for clinical use. In fetuses, stem cells that can be used for treatment of pathological changes in the central nervous system were mainly blastocyst embryonic stem cells and neural stem cells (Non-patent Document 1). Researchers have transplanted embryonic stem cells (Non-patent document 2) or neural stem cells (Non-patent document 3) to repair spinal cord injury, and these cells differentiated mainly into astrocytes and poor cells. It becomes an oligodendrocyte, revealing that very few cells become neuronal cells. In addition to difficult recovery, low survival after transplantation, and very low rate of change to neurons, fetal tissue transplantation is also hampered by discussions from humanitarian, legal, and ethical perspectives. It is rare.

  Repairing damaged nerve cells by direct transplantation of cord blood hematopoietic stem cells has not been studied. However, the in vitro model converts umbilical cord blood hematopoietic stem cells cultured in 0.001% β-mercaptoethanol (BME) into neural progenitors, and then 10% of these cells in addition to neuronal cells and It has been shown that it can be differentiated into glial cells (Non-Patent Documents 4 and 5). Because the conversion rate of cord blood stem cells to neuronal cells is relatively low, the study and application of cord blood has been mainly focused on blood diseases.

  Bone marrow contains hematopoietic stem cells that provide a continuous source of progenitors of red blood cells, platelets, monocytes, granulocytes, and lymphocytes. In addition, the bone marrow contains cells that meet the criteria for non-hematopoietic tissue stem cells. It has been reported that hematopoietic stem cells obtained from adult bone marrow can differentiate into both microglia and macroglia in the mouse brain (Non-patent Document 6). Non-hematopoietic stem cells obtained from bone marrow stroma are called bone marrow stromal cells (BMSC) or bone marrow mesenchymal cells. BMSCs can differentiate in vitro to become osteogenic, chondrogenic, adipogenic, myogenic and other strains (7, 8). Recently, BMSCs (9, 10) have been reported to be able to differentiate into neurons in vivo.

  All of the above methods for generating neurons from pluripotent cells have one common problem. That is, most pluripotent stem cells differentiate to become glial cells (astrocytes, oligodendrocytes, etc.) instead of neurons. Only for in vitro culture of BMSCs, it has been observed that BME has a significant effect on neuronal differentiation, which causes about 80% of the cultured BMSCs to differentiate into neurons (11). However, BME is toxic to proteins and is therefore not suitable for use in the human body (12). Therefore, there remains a need for effective methods for converting stem cells into neurons.

R.L.Rietze et al., 2001, Nature 412: 736-739 J.W.McDonald et al., 1999, Nature Medicine 5: 1410-1412 Q.L.Cao et al., 2001, Experimental Neurology 167: 48-58 J. R. Sanchez-Ramos et al., 2001, Experimental Neurology 171: 109-115 Y. Ha et al., 2001, Neuroreport 12 (16): 3523-3527 M. A. Eglitis et al., 1997, Proc. Natl. Acad. Sci. 94: 4080-4085 M.F.Pittenger et al., 1999, Science 284: 143-147 B. E. Petersen et al., 1999, Science 284: 1168-1170 S. A. Azizi et al., 1998, Proc. Natl. Acad. Sci. 95: 3908-3913 G. C. Kopen et al., 1999, Proc. Natl. Acad. Sci. 96: 10711-10716 D. Woodbury et al., 2000, Journal of Neuroscience Research 61: 364-370 K. White et al., 1973, Journal of Pharmaceutical Sciences 62: 237-241

The present invention provides a method of generating neurons from stem cells, comprising culturing the neurons in a medium and culturing the stem cells in the resulting mixture.
The present invention further provides a medium for culturing stem cells, which is prepared by culturing neurons in a basal medium.

Surprisingly, stem cells cultured in media prepared by culturing neurons in basal culture media for 6-9 days can differentiate to a high percentage (87%) of functional post-mitotic neurons. I found it.
Without wishing to be limited to a particular theory, it is believed that some substance is released from the cultured neurons, which provides the optimal conditions for stem cells to differentiate and become neurons.
The term “stem cell” as used in the present invention means any undifferentiated pluripotent or polymorphic latent mammalian cell, for example embryonic stem (ES) cell, embryonic reproductive (EG) cell, embryo Include, but are not limited to, sex tumor (EC) cells, bone marrow stromal cells (BMSC), bone marrow hematopoietic cells, umbilical cord blood cells and umbilical mesenchymal cells. It has been reported that all the stem cells except umbilical mesenchymal cells can differentiate into neurons and / or glial cells. However, with the culture method of the present invention, even umbilical mesenchymal cells can be differentiated into functional neurons at a high rate.

Accordingly, any mammalian stem cell is suitable for use in the methods of the present invention. Preferably, mesenchymal cells such as BMSC or umbilical mesenchymal cells are used.
Neurons used to prepare the media of the present invention can be obtained from both the peripheral and central nervous systems of mammals. Preferably, the neurons are from the CNS, more preferably from the brain, and most preferably from the hippocampus.
Neurons can be cultured in any conventional medium to obtain the medium of the present invention. Suitable media for use as the basal media of the present invention include, but are not limited to, Dulbecco's modified eagle media supplemented with fetal bovine serum (FBS-DMEM).
In the basal medium used in the present invention, other additives generally used for cell culture can be added.

In the present invention, neurons are cultured in a basal medium by a usual method for 3 to 6 days, preferably 5 days. The resulting mixture can be used directly for culturing stem cells.
In the present invention, the stem cells are cultured in the medium of the present invention in the usual manner for 6 to 12 days, and then mature neurons are harvested. Preferably, neurons are harvested on the ninth day of culture.
After removing the cultured neurons, the media of the invention can be aliquoted and stored for further use.

The following analysis is performed to characterize the neurons prepared by the method of the present invention.
NF immunocytochemistry The neurofilament (NF) is the major cytoskeleton in neurons, which regulates the size, appearance and diameter of neurons' dendrites. Only late developmental or mature neurons express NF. NF immunostaining is used to recognize late-stage neurons.
NeuN immunocytochemistry Neuron-specific nuclear protein (NeuN) is a specific protein present in the nucleus of neurons in the central nervous system of vertebrates. NeuN binds to DNA in vitro. NeuN is known to appear earlier than NF during neuron development. Thus, NeuN immunostaining is used to identify neurons in the early stages of transformation.

Western blotting of GluR5, GluR6, GluR7 and KA2 Glutamate is the major excitatory neurotransmitter in the mammalian CNS. Most neurons have glutamate receptors to receive excitatory signals. There are two main groups of glutamate receptors: ionotropy and metabotropy. Ionotropic type receptors can be further divided by their different selective agents: NMDA (N-methyl-D-aspartate) receptor, AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) ) Receptors and KA (kainate) receptors. The KA receptor is formed from the subunits GluR5, GluR6, GluR7, KA1 and KA2. Thus, Western blotting of GluR5, GluR6, GluR7 and KA2 is utilized to confirm the ability of neurons to synthesize functional proteins.

Ca ²⁺ binding protein immunocytochemistry Ca ²⁺ plays an important role in the signal transduction pathway of cells. However, most Ca ²⁺ does not act alone in cells, but acts by being bound by Ca ²⁺ binding proteins. Ca ²⁺ binding protein buffers the concentration of Ca ^{2+ in} the cell and further regulates the activity of Ca ²⁺ . There are many Ca ²⁺ binding proteins in the CNS, the most widely distributed being parvalbumin (PV), calbindin-D28K (CB) and calretinin. Both PV and CB belong to the EF hand type Ca ²⁺ binding protein family. During development of the embryonic nervous system, PV and CB are thought to be Ca ²⁺ binding proteins that are specifically expressed by neurons. Thus, PV and CB immunostaining is utilized to confirm the ability of neurons to synthesize functional proteins.

BrdU and DAPI double label DAPI (4 ', 6-diamidino-2-phenylindole dihydrochloride) is a fluorescent dye that can freely cross cells and nuclear membranes and bind to DNA in the nucleus, usually the number of cells Is used to quantify. In addition, during replication, the cells need to synthesize an additional set of chromosomes and incorporate an added BrdU (bromodeoxyuridine), an analog of thymidine, into the synthesized chromosome. Thus, observation of BrdU incorporation is used to confirm cell replication. BrdU and DAPI double labeling can confirm neuronal replication.

BrdU and NF double labeling As noted above, NF immunostaining is used to confirm neurons and BrdU labeling is used to confirm cell replication. NF and BrdU double labeling can confirm neuronal replication ability.
GAD immunocytochemistry The synthesis of GABA, one of the neurotransmitters, from glutamate is catalyzed by the enzyme glutamate decarboxylase (GAD). Thus, immunostaining for GAD is used to confirm the ability of neurons to synthesize the neurotransmitter GABA.
Neurons generated from the methods of the present invention may be used in the treatment of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, and amyotrophic lateral sclerosis or neuronal death induced from stroke and trauma. it can.
The invention is further illustrated by the following examples, which should not be construed as a limitation on the scope of the invention.

Preparation of Human Umbilical Mesenchymal Cells Human umbilical cords were obtained from Tsai Obstetric Clinic (72, Sec. 1, Shi Pai Rd., Beitou, Taipei), a clinic that handles labor. The umbilical cord was collected by aseptic operation and stored at 4 ° C. for 24 hours or less in HBSS (Biochrom L201-10).
The umbilical cord was first immersed in 75% ethanol for 30 seconds for sanitization. The sanitized umbilical cord is placed in a sterile laminar flow in Ca ²⁺ / Mg ²⁺ buffer (CMF, Gibco 14185-052) and cut longitudinally with an autoclaved tool from which vascular and mesenchymal tissue (hospital) Wharton's jelly) was removed. The mesenchymal tissue was diced into approximately 0.5 cm ³ cubes and centrifuged at 250 × g for 5 minutes. The supernatant was removed, an appropriate amount of serum-free DMEM (Gibco12100-046) was added, and the precipitate (mesenchymal tissue) was washed twice by centrifuging at 250 × g for 5 minutes. Mesenchymal tissue was treated with collagenase at 37 ° C. for 14-18 hours, washed, and then treated with 2.5% trypsin (Gibco15090-046) with stirring at 37 ° C. for 30 minutes. FBS (Hyclone SH30071.03) was added to the mesenchymal tissue to inactivate trypsin activity. At this point, the mesenchymal tissue became mesenchymal cells.
Mesenchymal cells were dispersed by treatment with 10% FBS-DMEM and counted. Mesenchymal cells can now be used directly in culture.

Preparation of neuron-cultured FBS-DMEM Neurons used for the preparation of neuron-cultured FBS-DMEM were obtained from the brain of the 7-day-old Sprag Dory rat (The Animal Center of National Yang-Ming University, Taipei, Taiwan, ROC). did.
A 50 ml centrifuge tube containing 24 ml of 10% FBS-DMEM was placed in a 37 ° C. incubator prior to further manipulation.
i. p. Rats were administered 0.3 ml of 10% chloride hydrate by injection. After 3-4 minutes, the whole body of a general anesthetized rat was disinfected with 75% ethanol. During sterile laminar flow, rat brains were removed and placed in CMF. The brain was centrifuged at 900 rpm for 5 minutes. The supernatant was removed and 10% FBS-DMEM was added to the precipitate (brain tissue). Brain tissue was pipetted 15 times and dispersed in single cells. The cell dispersion was added to the 50 ml centrifuge tube prepared as described above and mixed well with FBS-DMEM inside. The mixture was added into wells coated with poly-L-lysine in a 24-well plate and cultured in a 37 ° C. incubator. The next day, Arac was added to the culture to a final concentration of 2 μM.
On the fifth day of culture, the medium was removed and used to culture umbilical mesenchymal cells.

Conversion of Umbilical Mesenchymal Cells to Neurons Umbilical mesenchymal cells obtained in Example 1 were cultured in the neuron culture FBS-DMEM or FBS-DMEM prepared in Example 2 (control) in a 37 ° C. incubator. In culture. Cultured cells were harvested on days 3, 6, 9, and 12.
As shown in FIG. 1 (A), the morphology of umbilical mesenchymal cell cultures in FBS-DMEM for 6 days (control) showed a spindle shape and was closely bound to each other for proliferation. Umbilical mesenchymal cells cultured in neuronal culture FBS-DMEM for 6 days showed a neuronal phenotype including cell body regression, process processing, and cell clusters (FIG. 1B).

Analysis The cells collected in Example 3 are subjected to the following analysis, and the results are described below:
(I) NF immunostaining Cells growing on a cover glass for 6 days were washed twice with 0.1 M phosphate buffer (0.1 M PBS, pH 7.4) for 5 minutes; fixer (0.1 M PBS) Fixed for 20 minutes; washed 3 times for 5 minutes with 0.1 M PBS; then with blocking solution (0.05% Triton X-100, 5% normal goat serum and 3% bovine serum albumin) Treatment for 30 minutes prevented specific antibody-antigen binding.
The treated cells were then reacted with the first antibody (rabbit anti-neurofilament 200 polyclonal IgG, 1: 250 dilution, Chemicon AB1982) at 4 ° C. for at least 12-18 hours; washed 3 times for 5 minutes with 0.1 M PBS. Reacted with a second antibody (biotin-conjugated goat anti-rabbit IgG, 1: 1000 dilution, Sigma b-8895) for 1 hour at room temperature; washed again 3 times for 5 minutes with 0.1M PBS; avidin-biotinylated horseradish Reaction with peroxidase complex (ABC KIT, Vectorlabs PK-4000) at room temperature for 1 hour; wash 3 times with 0.1M PBS for 5 minutes; then 3,3′-diaminobenzidine (5 mg DAB, 10 ml of 50 mM Tris buffer) Color was developed with 3.5 μl of 30% H ₂ O ₂ ).

The resulting samples were dehydrated with ethanol with increasing concentrations of 50%, 70%, 80%, 90%, 95% and 100%, and xylene (5 minutes each). The cover glass was sealed on the slide with a par mount and observed under an optical microscope (Olympus BH-2).
As shown in FIG. 2 (A), umbilical mesenchymal cells cultured for 6 days in neuronal culture FBS-DMEM expressed NF, suggesting that they were converted to neurons.
At various times after treatment with neuronal culture FBS-DMEM, the percentage of umbilical mesenchymal cells expressing NF was measured. As shown in FIG. 3, 59.4% of umbilical mesenchymal cells express NF on the third day after treatment, and the ratio of cells expressing NF reaches a maximum of 87.4% on the sixth day after treatment. did. This rate did not increase or decrease over time (n = 3, one-way ANOVA, followed by LSD test; p <0.01).

(II) NeuN immunostaining:
Cells growing for 6 days on coverslips are washed twice for 5 minutes with 0.1 M phosphate buffer (0.1 M PBS, pH 7.4); 20 times with fixer (4% paraformaldehyde in 0.1 M PBS). Fixed for 5 minutes; washed 3 times for 5 minutes with 0.1 M PBS; then treated with blocking solution (0.05% Triton X-100, 5% normal goat serum and 3% bovine serum albumin) for 30 minutes to give non-specificity Sex antibody-antigen binding was prevented.
The treated cells are reacted with the first antibody (mouse anti-neuronal nuclear monoclonal IgG, 1: 100 dilution, Chemicon MAB377) at 4 ° C. for at least 36 to 42 hours; washed 3 times for 5 minutes with 0.1 M PBS; Reaction with a second antibody (biotin-conjugated goat anti-mouse IgG, 1: 250 dilution, Chemicon AP124B) for 1 hour at room temperature; wash 3 times with 0.1M PBS for 5 minutes; avidin-biotinylated horseradish peroxidase complex (ABC KIT, Vectorlabs PK-4000) for 1 hour at room temperature; washed 3 times with 0.1 M PBS for 5 minutes; 3,3′-diaminobenzidine (5 mg DAB, 3.5 μl in 10 ml of 50 mM Tris buffer) The color was developed with 30% H ₂ O ₂ ).
The resulting samples were dehydrated with ethanol with increasing concentrations of 50%, 70%, 80%, 90%, 95% and 100%, and xylene (5 minutes each). The cover glass was sealed on the slide with a par mount and observed under an optical microscope (Olympus BH-2).
As shown in FIG. 2 (B), umbilical mesenchymal cells cultured for 6 days in neuronal culture FBS-DMEM expressed NF, suggesting that they were converted to neurons.

(III) Western blotting of GluR5, GluR6, GluR7 and KA2 (a) Cell membrane preparation Cell membranes were prepared from the brains of umbilical mesenchymal cells and 7-day-old rats treated with neuronal culture FBS-DMEM at different times. The cultured cells were washed twice with 0.1 M phosphate buffer for 3 minutes. Tris-HCl buffer (50 mM, pH 7.4) was added to the cells at 4 ° C., and the cells were scraped from the culture dish with a scraper. The scraped cells were placed in a 15 ml Teflon glass homogenizer, ground 20 times to homogenize, and then centrifuged at 30000 g for 30 minutes at 4 ° C. The resulting pellet, which was mostly cell membrane, was ground again, dissolved in an appropriate volume of Tris-HCl buffer and stored in a −20 ° C. refrigerator for later use.
(B) Determination of protein concentration For use in Western blotting analysis, the concentration of protein in each sample obtained above was determined by measuring the absorbance at 595 nm. The protein concentration was measured based on the Bradford method (MM. Bradford, 1976, Analytical Biochemistry 72: 248-254.).

(C) Electrophoresis Proteins in the sample were separated by 20% SDS-PAGE. The separated proteins in the gel were then transferred onto PVDF paper (NEW, NEF1002) for Western blotting analysis.
(D) Western blotting PVDF paper containing protein was washed twice for 30 minutes with TBS solution (0.9% NaCl in 50 mM Tris-HCl, pH 7.4). Blocking was performed for 60 minutes using 5% skim milk powder dissolved in TBS buffer. The PVDF paper was then immersed in blocking solution (0.05% Triton-X100, 5% normal goat serum and 3% BSA) for 60 minutes, washed with TBS buffer, and the first antibody (rabbit anti-KA2 polyclonal IgG 1: 1800 dilution, UPSTATE06-315; goat anti-GluR5 polyclonal IgG, 1:30 dilution, Santa Cruz sc-7617; goat anti-GluR6 polyclonal IgG, 1:30 dilution, Santa Cruz sc-7618: goat anti-GluR7 polyclonal IgG, 1:30 dilution, Santa Cruz sc-7620; and rabbit anti-glutamate decarboxylase polyclonal IgG, 1: 1500 dilution, Chemicon AB108) at 4 ° C for 12-18 hours.

After the reaction was completed, the PVDF paper was washed twice with TTBS (Tween-20 in 0.05% TBS) for 30 minutes, soaked in blocking solution for 60 minutes, then the second antibody (for GluR5 / 6/7) Was reacted with biotin-anti-goat / sheep IgG, 1: 200 dilution, Sigma B-3148; and for KAR2 / GAD with biotin-anti-rabbit IgG, 1: 250 dilution, Chemicon AP187B) for 1 hour at room temperature. The reacted PVDF paper was washed twice with TTBS for 30 minutes, reacted with avidin-biotinylated-horseradish peroxidase complex (ABC KIT, Vectorlabs PK-4000) for 1 hour at room temperature, and again washed twice with TTBS for 30 minutes. Finally, color was developed with DAB (5 mg DAB, 3.5 μl 30% H ₂ O ₂ , pH 7.4 in 10 ml Tris-HCl).
As shown in FIG. 4, the kainate receptor subunit was not expressed in umbilical mesenchymal cells prior to treatment of neuronal culture FBS-DMEM. On day 6 of treatment with neuronal culture FBS-DMEM, kainate receptor subunits including GluR6, GluR7 and KA2 were expressed in small amounts, and GluR5, GluR6, GluR7 and KA2 were significantly expressed on day 12.

(IV) Parvalbumin (PV) and Calbindin-D28K (CB) immunostaining:
Cells growing for 6 days on coverslips were washed twice for 5 minutes with 0.1 M phosphate buffer (0.1 M PBS, pH 7.4); fixer (2% paraformaldehyde and 7 in 0.1 M PBS) Fixed with 5% pyric acid) for 20 minutes; washed 3 times with 0.1 M PBS for 5 minutes; 30 with blocking solution (0.05% Triton X-100, 5% normal goat serum and 3% bovine serum albumin) Treatment for minutes prevented nonspecific antibody-antigen binding.
Treated cells were then treated with primary antibody (goat anti-parvalbumin polyclonal IgG, 1:40 dilution, Santa Cruz sc-7449; and goat anti-calbindin polyclonal IgG, 1:40 dilution, Santa Cruz sc-7691) and 4 React for at least 12-18 hours at ° C; wash 3 times for 5 minutes with 0.1 M PBS; 1 second at room temperature with a second antibody (biotin-conjugated mouse anti-goat / sheep monoclonal IgG, 1: 250 dilution, Sigma B3148) React with time; again wash 3 times with 0.1M PBS for 5 minutes; react with avidin-biotinylated-horseradish peroxidase complex (ABC KIT, Vectorlabs PK-4000) for 1 hour at room temperature; 5 with 0.1M PBS Wash 3 times for 3 minutes, 3,3'-diaminobe And developed with their own half (5mg DAB, _30% of 50 mM Tris buffer 3.5μl of 10ml H _{2 O} 2).

The resulting samples were dehydrated with ethanol with increasing concentrations of 50%, 70%, 80%, 90%, 95% and 100%, and xylene (5 minutes each). The cover glass was sealed on the slide with a par mount and observed under an optical microscope (Olympus BH-2).
As shown in FIG. 5, umbilical mesenchymal cells cultured for 9 days in neuronal culture FBS-DMEM expressed both parvalbumin (FIG. 5A) and calbindin-D28K (FIG. 5B), indicating that two This suggests that the Ca ²⁺ binding protein was converted to a neuron capable of synthesis.

(V) GAD immunostaining and Western blotting:
Cells growing for 6 days on coverslips were washed twice for 5 minutes with 0.1 M phosphate buffer (0.1 M PBS, pH 7.4); fixer (2% paraformaldehyde in 0.1 M PBS and 7. Fix with 20% 5% pyric acid); wash 3 times with 0.1M PBS for 5 minutes; 30 minutes with blocking solution (0.05% Triton X-100, 5% normal goat serum and 3% bovine serum albumin) Treatment to prevent non-specific antibody-antigen binding.
The treated cells are then reacted with the first antibody (rabbit anti-glutamate decarboxylase polyclonal IgG, 1: 1500 dilution, Chemicon AB108) at 4 ° C. for at least 12-18 hours; washed 3 times for 5 minutes with 0.1M PBS Reacted with a second antibody (biotin-conjugated goat anti-rabbit IgG, 1: 300 diluted, Sigma b-8895) for 1 hour at room temperature; washed again with 0.1M PBS for 5 minutes; avidin-biotinylated Reaction with horseradish peroxidase complex (ABC KIT, Vectorlabs PK-4000) at room temperature for 1 hour; wash 3 times with 0.1 M PBS for 5 minutes; 3,3′-diaminobenzidine (5 mg DAB, 10 μl of 50 mM Tris Color was developed with 3.5 μl 30% H ₂ O ₂ ) in buffer.

The resulting samples were dehydrated with ethanol with increasing concentrations of 50%, 70%, 80%, 90%, 95% and 100%, and xylene (5 minutes each). The cover glass was sealed on the slide with a par mount and observed under an optical microscope (Olympus BH-2).
As shown in FIG. 6 (A), umbilical mesenchymal cells cultured for 6 days in neuronal culture FBS-DMEM expressed GAD, which was converted into neurons that could synthesize the neurotransmitter GABA. I suggest that. As shown in FIG. 6 (B), the ability of umbilical mesenchymal cells to express GAD was induced on the 6th day after treatment, and the GAD expression level was significantly increased on the 12th day.
The results of (III), (IV) and (V) demonstrate that the neurons induced by the method of the invention are functional and can synthesize proteins and neurotransmitters characteristic of neurons.

(VI) DAPI staining After treatment, cells were washed twice with 0.1 M phosphate buffer for 5 minutes each and then stained with 50 μg / ml DAPI for 30 minutes. The cells were then washed thoroughly with Tris buffer (Tris-HCl 50 mM pH = 7.3), mounted with mounting medium (Tris: glycerol = 1: 1), and the cells were observed and counted under a fluorescence microscope.
FIG. 7A is a photomicrograph showing cell density. DAPI labeling (blue) was performed to determine the number of cells on days 0, 3, 6, and 9 after treatment. FIG. 7 (B) shows the cell density of umbilical mesenchymal cells after various times of treatment in neuronal culture FBS-DMEM. At various post time points, DAPI staining was performed to measure changes in cell density. Results showed that the cell density on day 9 after treatment was significantly higher than day 3 (n = 3, one-way ANOVA followed by LSD test; P <0.01).
(VII) BrdU and DAPI double staining Cells growing on coverslips yielded a final concentration of 50 μM BrdU (Sigma B-5002, prepared as a 50 mM stock solution), which was further grown for 24 hours. Cells were washed twice for 5 minutes with 0.1 M phosphate buffer (0.1 M PBS, pH 7.4); fixer (70% ethanol in 50 mM glycine buffer, pH 2.0) at −20 ° C. for 30 Fixed for 1 minute; washed 3 times for 5 minutes with 0.1 M PBS; treated with blocking solution (0.05% Triton X-100, 5% normal goat serum and 3% bovine serum albumin) for 30 minutes to produce specific antibody -Prevented antigen binding.

Treated cells were then treated with primary antibody (mouse anti-BrdU monoclonal IgG, Chemicon MAB3222, 0.66 mM CaCl ₂ in 66 mM Tris buffer and 1: 100 dilution with 1 mM β-mercaptoethanol) at 4 ° C. for at least 36-42 hours. Reaction; wash 3 times for 5 minutes with 0.1 M PBS; 1 hour at room temperature with second antibody (fluorescein-conjugated goat anti-mouse IgG, 1:50 dilution, Chemicon AP124F) and 1 mg / ml DAPI (Sigma D9542) Reacted; washed again 3 times for 5 minutes with 0.1 M PBS. A cover glass was mounted on the slide with a mounting medium (Vector H-1000), sealed with nail polish, and observed under a fluorescence microscope (Olympus BX50). Count the total number of cells within the unit area and the number of BrdU-labeled cells.

(VIII) BrdU and NF double staining Cells growing on coverslips yielded a final concentration of 50 μM BrdU (Sigma B-5002, prepared as a 50 mM stock solution), which was further grown for 24 hours. Cells were washed twice for 5 minutes with 0.1 M phosphate buffer (0.1 M PBS, pH 7.4); fixer (70% ethanol in 50 mM glycine buffer, pH 2.0) at −20 ° C. for 30 Fixed for 1 minute; washed 3 times for 5 minutes with 0.1 M PBS; treated with blocking solution (0.05% Triton X-100, 5% normal goat serum and 3% bovine serum albumin) for 30 minutes to produce specific antibody -Prevented antigen binding.

Treated cells were then treated with the first antibody (mouse anti-BrdU monoclonal IgG, Chemicon MAB3222, 0.66 mM CaCl ₂ and 1 mM β-mercaptoethanol in 66 mM Tris buffer) at 4 ° C. for at least 12-18 hours. React; react with another first antibody (rabbit anti-neurofilament polyclonal IgG, 1: 250 dilution, Chemicon AB1982) at 4 ° C. for at least 12-18 hours; wash 3 times with 0.1 M PBS for 5 minutes; React with a second antibody (fluorescein-conjugated goat anti-mouse IgG, 1:50 dilution, Chemicon AP124F; and rhodamine-conjugated goat anti-rabbit IgG, 1:50 dilution, Chemicon AP132R) for 1 hour at room temperature; again with 0.1 M PBS 5 It was washed three times between. A cover glass was mounted on the slide with a mounting medium (Vector H-1000), sealed with nail polish, and observed under a fluorescence microscope (Olympus BX50).

FIG. 8 shows the results of proliferation analysis of umbilical mesenchymal cells treated with neuron culture 10% FBS-DMEM for 3 days (AF). Most of the cells labeled with DAPI (blue in A) were BrdU positive (green in B). (C) is a combination of the results of (C) and (D). On the third day after treatment, these cells had already differentiated into NF expressing cells (red in D) but were still labeled with BrdU (green in E). (F) is a combination of the results of (D) and (E). Cells treated on day 9 after neuronal culture 10% FBS-DMEM lost their proliferative capacity (G-L). All cells labeled with DAPI (blue in G) were BrdU negative (green in H). (I) is a combination of the results of (G) and (H). On day 9 after treatment, these cells that differentiated into NF-expressing cells (red in J) were hardly labeled with BrdU (green in K). (L) is a combination of the results of (J) and (K). In summary, most of the cells were converted to neurons and stopped growing on day 9, suggesting that day 9 neurons entered the post-mitotic stage.
The results of (VII) and (VIII) indicate that umbilical mesenchymal cells cultured with neuronal culture FBS-DMEM can proliferate, differentiate and become neurons.

  From the foregoing, those skilled in the art can readily understand the essential features of the present invention, and various changes and modifications can be made to the present invention without departing from the spirit and scope thereof. Can be applied.

(A) Morphology of umbilical mesenchymal cells cultured in neuron culture medium for 6 days in FBS-DMEM (control) and (B). (A) NF and (B) NeuN immunostaining of umbilical mesenchymal cells cultured in neuronal culture medium for 6 days. The percentage of umbilical mesenchymal cells expressing NF at various times after treatment with neuron culture medium is shown. The result of the western blotting about GluR5, GluR6, GluR7, and KA2 of the umbilical mesenchymal cell cultured in the neuron culture medium is shown. Immunostaining for (A) parvalbumin (PV) and (B) calbindin-D28K (CB) of umbilical mesenchymal cells cultured in neuronal culture medium for 9 days is shown. (A) Immunostaining and (B) Western blotting for GAD of umbilical mesenchymal cells cultured in neuronal culture medium for 6 days. FIG. 7 (A) shows the altered cell density of umbilical mesenchymal cells induced by neuronal culture medium at 0, 3, 6, and 9 days after treatment (scale bar: 100 μm); FIG. 7 (B) Shows the quantitative cell density of umbilical mesenchymal cells treated with neuronal culture medium using DAPI staining. Shown are DAPI + BrdU double staining and NF + BrdU double staining of umbilical mesenchymal cells cultured for 3 days (3D) and 9 days (9D) in neuronal culture medium.

Claims (19)

A method for preparing neurons from stem cells, comprising culturing neurons in a medium and then culturing the stem cells in the resulting mixture. The method of claim 1 wherein the neurons are obtained from the brain. The method of claim 2 wherein the neurons are obtained from the hippocampus. The method according to claim 1, wherein the neurons are cultured in a medium for 3 to 6 days before adding the stem cells. The method of claim 4, wherein the neurons are cultured for 5 days. Stem cells from the group consisting of embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic tumor (EC) cells, bone marrow stromal cells (BMSC), bone marrow hematopoietic cells, cord blood cells and umbilical mesenchymal cells The method according to claim 1, which is selected. The method according to claim 6, wherein the stem cells are bone marrow stromal cells (BMSC) or umbilical mesenchymal cells. The method according to claim 1, wherein the medium used for culturing neurons is FBS-DMEM. The method according to claim 1, wherein the stem cells are cultured in the mixture for 6 to 12 days. 2. The method of claim 1, further comprising the step of collecting neurons from the stem cell culture mixture. 11. The method of claim 10, wherein the neurons are collected during a period from day 6 to day 12 of culture. 11. The method of claim 10, wherein the neurons are harvested during a period from day 6 to day 9 of culture. A medium for culturing stem cells, which is prepared by culturing neurons in a basal medium. The method according to claim 13, wherein the basal medium is FBS-DMEM. The medium of claim 13, wherein the neurons have been removed from the basal medium. 14. The medium of claim 13, wherein the neurons are obtained from the brain. 17. A medium according to claim 16, wherein the neurons are obtained from the hippocampus. The medium according to claim 13, wherein the neurons are cultured in a basal medium for 3 to 6 days. The medium according to claim 18, wherein the neurons are cultured in a basal medium for 5 days.

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