Method of differentiating adult stem cells into nerve cells by using high-intensity electromagnetic field
10167465 ยท 2019-01-01
Assignee
Inventors
- Young Kwon Seo (Seongdong-gu, KR)
- Jung Keug Park (Seodaemun-gu, KR)
- Hee Hun Yoon (Bucheon-si, KR)
- Hyun Jin Cho (Seocho-gu, KR)
- Hee Jung Park (Seongbuk-gu, KR)
- Yu Mi Kim (Songpa-gu, KR)
- Bo-Young Yoo (Gangdong-gu, KR)
- Sang Eun Cho (Yangju-si, KR)
- Sang Heon Kim (Bupyeong-gu, KR)
Cpc classification
C12N2506/1353
CHEMISTRY; METALLURGY
C12N2529/00
CHEMISTRY; METALLURGY
C12N5/0622
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention provides methods of differentiating mesenchymal stem cells or adult stem cells into nerve cells by treating the mesenchymal stem cells or the adult stem cells with an electromagnetic field having a high intensity of 100 to 1,500 mT and a low frequency of 0.01 to 100 Hz. These methods also provide injecting the mesenchymal stem cells or adult stem cells into a subject prior to treating the mesenchymal stem cells or adult stem cells with an electromagnetic field having a high intensity of 100 to 1,500 mT and a low frequency of 0.01 to 100 Hz.
Claims
1. A method of differentiating mesenchymal stem cells or adult stem cells into nerve cells, comprising: treating the mesenchymal stem cells or the adult stem cells under culturing in DMEM medium with an electromagnetic field having a high intensity of 100 to 1,500 mT and a frequency of 1 to 100 Hz for 5 to 30 min/day over 3 to 15 days wherein said mesenchymal stem cells or adult stem cells differentiate into nerve cells.
2. The method of claim 1, wherein the nerve cells comprise astrocytes or oligodendrocytes.
3. The method of claim 1, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, or an umbilical cord.
4. The method of claim 1, wherein the adult stem cells comprise dental pulp stem cells or neural progenitor cells.
5. A method of differentiating mesenchymal stem cells or adult stem cells into nerve cells, comprising: injecting the mesenchymal stem cells or the adult stem cells into a subject; and treating the mesenchymal stem cells or the adult stem cells with an electromagnetic field having a high intensity of 100 to 1,500 mT and a frequency of 1 to 100 Hz for 5 to 30 min/day over 3 to 15 days wherein said mesenchymal stem cells or adult stem cells differentiate into nerve cells.
6. The method of claim 5, wherein the nerve cells comprise astrocytes or oligodendrocytes.
7. The method of claim 5, wherein the mesenchymal stem cells are derived from bone marrow, adipose tissue, or an umbilical cord.
8. The method of claim 5, wherein the adult stem cells comprise dental pulp stem cells or neural progenitor cells.
Description
DESCRIPTION OF DRAWINGS
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BEST MODE
(25) Hereinafter, the present invention will be described in detail.
(26) The present invention relates to a system for regeneration of damaged nerves, and a method of promoting differentiation of mesenchymal stem cells or adult stem cells into neuron-associated cells. Specifically, the present invention relates to a system for promoting differentiation into neuron-like cells through in vivo physical stimulation or promoting regeneration of damaged neurons with stem cells injected into the body. More specifically, the present invention relates to a system for promoting differentiation of mesenchymal stem cells or adult stem cells into neuron-like cells by exposing the mesenchymal stem cells or adult stem cells to a certain low-frequency/high-intensity electromagnetic field, or a system for promoting recovery of damaged nerve tissue by injecting mesenchymal stem cells or adult stem cells into the body and exposing the mesenchymal stem cells or adult stem cells to a low-frequency/high-intensity electromagnetic field. The present invention relates to a system capable of improving the established differentiation efficiency of an electromagnetic field by applying a high-intensity electromagnetic field to mesenchymal stem cells or adult stem cells for a short time. The electromagnetic field of the present invention can differentiate mesenchymal stem cells or adult stem cells into nerve cells only when the mesenchymal stem cells or the adult stem cells are treated with certain frequencies. Therefore, the present inventors have ensured a technique of inducing in vitro neuronal differentiation of adult stem cells using a high-intensity electromagnetic field according to the present invention.
(27) According to one exemplary embodiment of the present invention, it is confirmed that, when SH-SY5Y neural progenitor cells are treated with a high-intensity electromagnetic field daily for 20 minutes over 5 days, the length of dendrites increases at frequencies of 60 Hz and 75 Hz to improve the differentiation of the neural progenitor cells, as shown in
(28) According to one exemplary embodiment of the present invention, the neuronal differentiation of mesenchymal stem cells is induced for 8 days by treatment with high-intensity/low-frequency electromagnetic waves, and observed under a microscope (
(29) Also, according to one exemplary embodiment of the present invention, human mesenchymal stem cells are injected into a rat stroke model, and treated with a low-intensity electromagnetic field (EMF) and a high-intensity electromagnetic field (h-EMF), and tissue is collected and subjected to Western blotting. As a result, it is confirmed that the expression of the neuron-associated proteins MAP 2 and Neuro D1 increases, indicating that the increased expression of the neuron-associated proteins is caused due to an increase in p-ERK and p-CREB. The immunostaining results show that the expression of MMP in the high-intensity electromagnetic field-treated group is reduced, resulting in decreased inflammation (
(30) Also, in the present invention, after the undifferentiated mesenchymal stem cells or differentiated nerve cells are injected into the body, an affected part may be treated with a high-intensity electromagnetic field daily for 20 minutes to maximize therapeutic efficiency in the damaged nerve tissue.
MODE FOR INVENTION
(31) Hereinafter, the present invention will be described in further detail with reference to examples thereof. However, it should be understood that the following examples provided herein are merely intended to provide a better understanding of the present invention and are not intended to limit the scope of the present invention.
Example 1: Effect of High-Intensity Electromagnetic Field on Differentiation of Nerve Cells Using Nerve Cell Line
Example 1.1: Culturing of Nerve Cell Line
(32) An SH-SY5Y neural progenitor cell line (Catalog No. CRL-2266) was purchased from the American Type Culture Collection (ATCC), and cultured in a DMEM medium supplemented with 5% (v/v) FBS and 5 M retinoic acid. The cell line was seeded in a culture plate, and then cultured in a CO.sub.2 incubator maintained at a constant temperature of 37 C. while the medium was replaced with a fresh one every three days.
Example 1.2: Confirmation of Differentiation of Neural Progenitor Cell Line Using High-Intensity Electromagnetic Field
(33) The cultured neural progenitor cells were seeded in a 12-well plate at a concentration 210.sup.4 cells/well, and cultured in a CO.sub.2 incubator at 37 C. for 5 days while the medium was replaced with a fresh one every three days.
(34) The treatment with the high-intensity electromagnetic field was performed twice daily for 15 minutes over a culture period. Here, the treatment was performed while a culture plate was placed over a high-intensity electromagnetic field. The neural progenitor cells were cultured for 5 days under an electromagnetic field having a frequency of 0 Hz (an untreated group), 60 Hz (at 630 mT) and 75 Hz (at 570 mT), and then cultured in a DMEM medium supplemented with 5% (v/v) FBS and 5 M retinoic acid. After the culturing, the neural progenitor cells were observed under an optical microscope to check a morphological change of the neural progenitor cells. The results are shown in
(35) As shown in
(36) Also,
(37) In addition,
(38) Further,
Example 2: Culturing of Adult Stem Cells Using High-Intensity Electromagnetic Field
Example 2.1: Primary Culturing of Adult Stem Cells
(39) Human adult stem cells whose passage number was 2 were purchased from Lonza (Walkersville, Md.), put into an NH medium, and then centrifuged at 800 rpm for 5 minutes. A supernatant obtained by the centrifugation was discarded, and the remaining cells were again seeded in a 100 mm culture plate containing 10 ml of an NH medium, and then cultured in a CO.sub.2 incubator maintained at a constant temperature of 37 C. while the medium was replaced with a fresh one every three days.
Example 2.2: Confirmation of Differentiation of Adult Stem Cells Cultured Using High-Intensity Electromagnetic Field into Nerve Cells
(40) After the primary culturing, the medium was removed, and the cultured cells were washed with 10 ml of phosphate buffered saline (PBS) one or more times. 1 ml of a solution containing 0.05% (w/v) trypsin and 0.01% (w/v) EDTA was added to the washed cells, and the cells were treated at 37 C. for 5 minutes so that the cells were detached from the bottom of the culture plate to float in the solution. The cell solution was mixed with 15 ml of an NH medium containing 10% (v/v) FBS, and the mixture was then centrifuged at 800 rpm for 5 minutes to discard a supernatant and collect the cells. The collected cells were sub-cultured up to five times in a CO.sub.2 incubator.
(41) The cultured cells were seeded in a DMEM medium-containing 60 mm culture plate at a concentration of 0.2510.sup.5 cells/culture plate, and then cultured in a CO.sub.2 incubator at 37 C. for 8 days while the medium was replaced with a fresh one every three days. In this case, the experimental groups were treated with a high-intensity electromagnetic field.
(42) The treatment with the high-intensity electromagnetic field was performed once daily for 20 minutes over a culture period. When the treatment was performed, the 60 mm culture plate was placed over the high-intensity electromagnetic field, and the cells were then cultured for 7 days under the electromagnetic field having a frequency of 0 Hz (an untreated group), 30 Hz (at 1,120 mT), 45 Hz (at 890 mT), 50 Hz (at 680 mT), 60 Hz (at 630 mT), and 70 Hz (at 570 mT). The cells were cultured in a DMEM medium supplemented with 5% FBS, 10 ng/ml EGF, and 10 M Forskolin. After the culturing, the adult stem cells were observed under an optical microscope to determine morphological changes of the adult stem cells at 0 Hz (an untreated group), 30 Hz (at 1,120 mT), 45 Hz (at 890 mT), 50 Hz (at 680 mT), 60 Hz (at 630 mT), and 70 Hz (at 570 mT). The results are shown in
(43) As shown in
(44) Also, a lactate dehydrogenase (LDH) assay was carried out to evaluate an effect of the high-intensity electromagnetic field on cytotoxicity. The results are shown in
(45) Also, the cells were cultured and collected to analyze expression levels of the nerve cell-associated proteins Tau, MAP 2, and Neuro D1. As shown in
(46) Further, the in vitro viability of the adult stem cells at 60 Hz was analyzed to evaluate in order to perform an animal efficacy assay (
Example 3: Evaluation of Efficiency of High-Intensity Electromagnetic Field Using Small Animal Stroke Model
Example 3.1: Establishment of Small Animal Stroke Model
(47) To establish an animal stroke model, three-week-old SD rats (weighing 45 to 50 g) were used, and 0.1 cc/100 g (50 mg/kg) of Zoletil (250 mg/5 cc; Virbac) and 0.025 to 0.04/100 g (5 to 10 mg/kg) of Rompun 2% (Bayer) were mixed to prepare an anesthetic, and the anesthetic was intraperitoneally administered to anesthetize the rats. A method of establishing the animal stroke model was performed using a photochemical method, as follows. 300 l of a systemic photoactive dye, Rose Bengal (10 mg/ml), was administered, and the rats' skulls were then irradiated with light beams to induce a stroke in the rats.
Example 3.2: Stroke Therapeutic Effect of High-Intensity Electromagnetic Field Using Small Animal Model
(48) To evaluate an effect of the high-intensity electromagnetic field, rats were divided into four experimental groups: a first group (control) in which physiological saline (100 l) was administered to a rat stroke model; a second group in which adult stem cells were administered to a rat stroke model (cell number: 110.sup.6); a third group in which adult stem cells were administered to a rat stroke model and treated with low-intensity electromagnetic field (at 60 Hz and 1 mT for 60 min/day); and a fourth group in which adult stem cells were administered to a rat stroke model and treated with high-intensity electromagnetic field (at 60 Hz and 630 mT for 20 min/day).
(49) A method of injecting the adult stem cells was performed, as follows:
(50) A rat stroke model was established. After 24 hours, the adult stem cells were administered through a tail vein in the rat stroke model. The cells were added to 500 l of physiological saline so that the concentration of the cells was adjusted to be 110.sup.6 cells, and then slowly injected for 2 minutes. An electromagnetic field was placed over the skull of a rat whose brain was positioned in the rat stroke model so that the adult stem cells were treated with the electromagnetic field, and the treatment with the electromagnetic field was carried out daily for 20 minutes over 14 days after 24 hours of the cell administration.
(51) After 14 days, the rat was euthanized, and tissue around the cerebral infarction was taken, and then subjected to Western blotting, hematoxylin & eosin (H&E) staining, and an immunohistochemical assay.
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(53) Also,
(54) Further, to compare effects of induction of differentiation of the adult stem cells according to the treatment with the high-intensity/low-frequency electromagnetic field, the expression levels of MMP 1, Neuro D1, and NF-L were determined through immunohistochemical staining. The results are shown in
Example 4: Evaluation of Cell Differentiation Efficiency According to Various Frequencies of High-Intensity Electromagnetic Field
Example 4.1: Culturing of Mesenchymal Stem Cells Derived from Human Bone Marrow
(55) Human bone marrow-derived mesenchymal stem cells whose passage number was 2 were purchased from Lonza (Walkersville, Md.), put into an NH medium, and then centrifuged at 800 rpm for 5 minutes. A supernatant obtained by the centrifugation was discarded, and the remaining cells were again seeded in a 100 mm culture plate containing 10 ml of an NH medium, and then cultured in a CO.sub.2 incubator maintained at a constant temperature of 37 C. while the medium was replaced with a fresh one every three days.
Example 4.2: Evaluation of Differentiation Ability of Mesenchymal Stem Cells According to Various Frequencies of High-Intensity Electromagnetic Field
(56) After the primary culturing, the medium was removed, and the cultured cells were washed with 10 ml of phosphate buffered saline (PBS) one or more times. 1 ml of a solution containing 0.05% (w/v) trypsin and 0.01% (w/v) EDTA was added to the washed cells, and the cells were treated at 37 C. for 5 minutes so that the cells were detached from the bottom of the culture plate to float in the solution. The cell solution was mixed with 15 ml of an NH medium containing 10% (v/v) FBS, and the mixture was then centrifuged at 800 rpm for 5 minutes to discard a supernatant and collect the cells. The collected cells were sub-cultured up to five times in a CO.sub.2 incubator.
(57) The cultured cells were seeded in a DMEM medium-containing 60 mm culture plate at a concentration of 0.2510.sup.5 cells/culture plate, and then cultured in a CO.sub.2 incubator at 37 C. for 8 days while the medium was replaced with a fresh one every three days. In this case, the experimental groups were treated with a high-intensity electromagnetic field.
(58) The treatment with the high-intensity electromagnetic field was performed once daily for 20 minutes over a culture period. When the treatment was performed, the 60 mm culture plate was placed over the high-intensity electromagnetic field, and the cells were then cultured for 7 days under the electromagnetic field having a frequency of 0 Hz (an untreated group), 50 Hz, 60 Hz, 75 Hz, and 85 Hz. The cells were cultured in a DMEM medium supplemented with 5% FBS, 10 ng/ml EGF, and 10 M Forskolin. After the culturing, a FACS assay was performed to check neuronal differentiation efficiency at 0 Hz (an untreated group), 50 Hz, 60 Hz, 75 Hz, and 85 Hz. The results are shown in
(59) The cultured cells of each experimental group were collected, and subjected to a FACS assay. As a result, it was confirmed that the expression of CD105 and CD73 at 60 Hz and 75 Hz was reduced, as shown in
(60) After the culturing, the cells were collected to determine mRNA expression levels of the nerve cell-associated factors Neuro D1, Map2, Tau, MBP, DCX, and NF-L. As shown in
(61) To compare the expression levels of the neuron-associated proteins, the mesenchymal stem cells were cultured, collected, and then subjected to Western blotting. The results are shown in
(62) The mesenchymal stem cells were cultured under a high-intensity electromagnetic field having a frequency of 50 Hz, 60 Hz, 75 Hz, and 85 Hz, and then observed under an optical microscope to determine a morphological change of the bone marrow-derived cells. The results are shown in
Example 5: Evaluation of Efficiency of High-Intensity Electromagnetic Field Using Mouse Stroke Model
Example 5.1: Evaluation of Effect of High-Intensity Electromagnetic Field on Mouse Stroke Model
(63) To establish an animal stroke model, six-week-old C57BL/6N mice (weighing 20 to 22 g) were used, and 0.1 cc/100 g (50 mg/kg) of Zoletil (250 mg/5 cc; Virbac) and 0.025 to 0.04/100 g (5 to 10 mg/kg) of Rompun 2% (Bayer) were mixed to prepare an anesthetic, and the anesthetic was intraperitoneally administered to anesthetize the rats. A method of establishing the animal stroke model was performed using a photochemical method, as follows. 100 l of a systemic photoactive dye, Rose Bengal (10 mg/ml), was administered, and the mice' skulls were then irradiated with light beams (KL 1,500 LCD (SCHOTT); a wavelength of 532 nm) for 15 minutes to induce a stroke in the mice.
(64) To select mice used for experiments, before a mouse stroke model was established, a Rotarod test (20 rpm) was carried out on all the mice for one week to select the mice whose motor ability was maintained for 80 seconds. After 3 days of stroke occurrence, the Rotarod test was again carried out to select only the mice which fell from a rotarod within 40 seconds, and the selected mice were used in experiments.
(65) To evaluate a nerve regeneration effect of the high-intensity electromagnetic field using a mouse stroke model, the mice were divided into three experimental groups: a first group (negative control) in which physiological saline was administered to a mouse stroke model; a second group in which adult stem cells were administered to a mouse stroke model (cell number: 110.sup.5; control group), and a third group in which adult stem cells were administered to a mouse stroke model and then treated with a high-intensity electromagnetic field (at 60 Hz and 500 mT for 15 min/day).
(66) A method of treating the adult stem cells with the high-intensity electromagnetic field was as follows. A mouse was put into a 50 cc syringe to be fixed therein, and positioned on a high-intensity electromagnetic coil so that the head of the mouse faced inward, and the adult stem cells were then treated with the high-intensity electromagnetic field. The treatment with the high-intensity electromagnetic field was performed for 2 weeks.
Example 5.2: Evaluation of Motor Recovery Ability of Stroke Mouse
(67) A Rotarod test was performed to evaluate motor recovery abilities of the stroke mice prepared in Example 5.1.
(68)
Example 5.3: Histological Examination and Protein Analysis after Treatment of Mouse Stroke Model with High-Intensity Electromagnetic Field
(69) After 2 weeks, the mice prepared in Example 5.1 were euthanized, and tissues around stroke lesions were taken, and then subjected to hematoxylin & eosin staining and an immunohistochemical assay.
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