WO2012053719A1 - Method for inducing differentiation of mesenchymal stem cells to nerve cells using sonic waves - Google Patents

Abstract

The present invention relates to a method for differentiation of mesenchymal stem cells. More specifically, the invention relates to a method for differentiating mesenchymal stem cells to nerve cells by treating the mesenchymal stem cells with low-frequency sonic waves. The differentiation method of the present invention can induce differentiation even with low-cost mediums rather than induced neural differentiation mediums which are expensive due to addition of growth factors, and the nerve cells differentiated according to the present invention may be useful for treatment of neurological brain diseases.

Description

Induction method of neuronal differentiation of mesenchymal stem cells using sound waves

The present invention relates to a method for differentiating mesenchymal stem cells. More specifically, the present invention relates to a method for differentiating mesenchymal stem cells into neurons by treating mesenchymal stem cells with sound waves of a specific frequency.

As neurons have emerged as candidates for the treatment of neurological diseases such as Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage, and spinal cord injury, research on neurons has been actively conducted recently, and numerous papers and patents have been published. It is true. However, many studies have been conducted on how to induce differentiation of mesenchymal stem cells into neural cells, which are relatively easy to obtain due to difficulty in obtaining cells. According to Lauren's review ( Plast. Reonstr. Surg . 116: 1453, 2005), one of six reports that adipose-derived mesenchymal stem cells were differentiated into neurons in vitro by chemical methods was functionally electrophysiological. Only one was reported. According to Arshak ( Stem Cells and Development , 17: 1123-30, 2008), bone marrow-derived mesenchymal stem cells were induced to differentiate into neurons by chemical differentiation method. Differentiated neurolabeling factors were identified by lot method (B3T, GFAP, MAP-2, NeuN) and ELISA (neural growth factor (NGF), brain-derived neuronal cell factor (BDNF)), but they showed electrophysiological characteristics. Did. Studies on the use of mesenchymal stem cells in neurotherapy by mixed cultures with neurons or neural progenitor cells (Croft AP, Exp. Neurol. , 216 (2): 329-41 (2009)) It is practically impossible to obtain enough human neurons or neural precursor cells to use. Another study aims to induce neuronal gene overexpression using lentivirus to increase the differentiation rate into neurons (Watson, DJ ,, Journal of Neurotrauma , 21: 1723-36. (2004)) (Hofstetter , C., Nature Neuroscience , 8: 346-53. (2005)) are in progress, but are difficult to apply to cell therapy because they are not safe for viruses.

According to Kuh et al. ( Acta Neurochir 147: 985-992, 2005), human cord blood cells were transplanted into spinal cord injured rats and observed at 8 weeks. The experimental results showed similar trends in the recovery scores), and when the cells were transplanted with brain-derived neurotrophic factor (BDNF), they showed similar results 5 weeks after transplantation. It can be seen that transplantation has limitations. Rooney, etc. (Tissue Engineering Part A, Mar. 31 , 2009) is when the respective implant by introducing a glial cell line-derived neurotrophic factor (GDNF) gene in bone marrow-derived mesenchymal stem cells isolated from fluorescence rat glial cell line derived neurotrophic factor gene Survival was observed until the 6th week, but when only mesenchymal stem cells were transplanted, the transplanted cells were not observed after 2 weeks. Therefore, transplantation of mesenchymal stem cells alone is insufficient for the treatment of spinal cord injury. .

Conventionally known neurotherapy technology using wave is a device for applying low frequency energy of less than about 10 Hz to brain tissue. It is a device that induces magnetic field by electric flow by applying electric stimulation directly after implanting electrode in the brain of patient. (Published patent: US20060205993). Zheng developed a technique (JP 2008-543388) to improve brain function by combining high frequency or multiple frequency components to give magnetic stimulation to the central nervous system. Riken manufactures nerve cells by electropulsing embryonic stem cells. Technology (US200740065941) has been developed. Gliner et al. Developed a technique for producing neurons by treating the cells with electric pulses (US20050075679). The above techniques have been applied to implant electrodes by directly implanting electrodes, which is accompanied by pain in the patient, and in the case of embryonic stem cells, the possibility of tumor formation is limited, and thus it is limited to be applied to clinical practice.

Therefore, there is an urgent need for an efficient method for differentiating mesenchymal stem cells into neural progenitor cells.

The present inventors have recognized the above problems and necessities, and have intensively tried to induce differentiation of mesenchymal stem cells, which are relatively easy to obtain, into neurons in order to obtain neural or neural stem cells that are difficult to obtain. The present invention was completed by confirming that can differentiate stem cells.

Accordingly, an object of the present invention is to provide a method for differentiating mesenchymal stem cells into neurons.

Another object of the present invention to provide a composition for treating neurological diseases.

In order to solve the above problems, the present invention provides a method for differentiating mesenchymal stem cells into neurons by treating sound waves to mesenchymal stem cells.

In addition, the sound waves are treated at a frequency of 1 to 500 Hz at an intensity of 0.1 to 5 V, and particularly preferably at 30 to 300 Hz and 0.5 to 3.0 V.

The mesenchymal stem cells of the present invention may be derived from bone marrow, adipose derived, umbilical cord blood or umbilical cord.

The present invention also provides a composition for treating neurological diseases comprising neurons differentiated by the above method.

The neurological disease may include Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage or spinal cord injury.

Stem cell differentiation method and differentiation apparatus using sound waves according to the present invention induces adult stem cells to differentiate into neurons using low frequency waves, thereby making it easy to obtain neurons or neural stem cells, which are difficult to acquire cells. Although it is possible to induce neuronal differentiation media, it is possible to induce neuronal differentiation in low-cost general medium conditions, not expensive neuronal induction media, by adding growth factors. It can be useful for the treatment of brain neurological diseases.

Figure 1 shows the implementation of the sound wave generator for adult stem cell differentiation according to an embodiment of the present invention.

Figure 2 shows the results of observing the morphological changes of umbilical cord-derived mesenchymal stem cells after exposure to sound waves under a light microscope (A is a control, B is 10Hz, C is 20Hz, D is 30Hz, E is 40Hz treatment). One case).

Figure 3 is a figure measuring the number of cells of umbilical cord-derived mesenchymal stem cells after exposure to sound waves (10 ~ 40 Hz).

Figure 4 is an experiment of the conditions for the neural differentiation of umbilical cord-derived mesenchymal stem cells, the expression of nestin, a mesenchymal stem cell marker at the mRNA level (A is RT-PCR, B is real-time PCR).

Figure 5 shows the results of neuronal mRNA expression after induction of neural differentiation of umbilical cord mesenchymal stem cells. It shows the result of confirming the expression of neuronal factors (MAP2, NeuroD1, Neurofilament) expressed after sonication in vitro (A is RT-PCR, B is real-time PCR).

Figure 6 confirms the expression of neuron-associated protein (tau) that is expressed after sonication in vitro.

Figure 7 shows the results of observing the morphological changes of bone marrow-derived and adipose-derived mesenchymal stem cells with light microscopy after exposure to sound waves (A is a control group, B is 10Hz, C is 20Hz, D is 30Hz, E At 40 Hz).

8 is a diagram showing the number of cells of bone marrow-derived and adipose-derived mesenchymal stem cells after exposure to sound waves (10-40 Hz).

Figure 9 shows the results of neuronal mRNA expression after neuronal differentiation of bone marrow-derived and adipose-derived mesenchymal stem cells. The results of confirming the expression of neuronal factors (MAP2, NeuroD1, Neurofilament) expressed after sonication in vitro (A is bone marrow-derived, B is fat-derived mesenchymal stem cells).

10 is a result of mRNA analysis of the expression of neuronal markers of umbilical cord-derived stem cells in 30 ~ 500 Hz sonic stimulation of 1 Volt (V) intensity,

11 shows the results of morphological changes of umbilical cord-derived stem cells at 30-500 Hz sonic stimulation of 1 Volt (V) intensity.

12 is a result of analyzing the expression of neural markers of umbilical cord-derived stem cells by mRNA at 30 ~ 300 Hz sonic stimulation of 2.5 Volt (V) intensity,

13 is a result of observing the morphological changes of umbilical cord-derived stem cells in 30 ~ 300 Hz sonic stimulation of 2.5 Volt (V) intensity,

Figure 14 shows the results of mRNA analysis of the expression of neuronal markers of bone marrow-derived stem cells in 30 ~ 500 Hz sonic stimulation of 1 Volt (V) intensity.

The present invention relates to a method of differentiating mesenchymal stem cells into neurons by treating sound waves to mesenchymal stem cells.

As used herein, the term "sound wave" refers to oscillation of the eardrum in the form of a longitudinal wave by partially changing the pressure in a medium (air) in which the vibration of the object is uniform. In the present invention, it is possible to induce differentiation into neurons by treating the mesenchymal stem cells with vibrations having a specific sonic band frequency in the frequency region of less than 20 kHz.

Herein, messenchymal stem cells may be derived from embryos or adult tissues, and may be derived from bone marrow, adipose derived or umbilical cord.

Stem cells are undifferentiated cells, which can divide and self-renewal for a long time, and can differentiate into various cell types given a certain condition. Stem cells are divided into embryonic stem cells and adult stem cells according to the origin of the tissue. Potential has limited disadvantages than embryonic stem cells, but many therapeutics are studied for adult stem cells without ethical problems and no side effects. have.

Specifically, in the present invention, adult stem cells are used, which may use stem cells commercially available or stem cells separated from biological tissues.

The present invention relates to a method for inducing differentiation of adult stem cells into neurons by using sound waves of a specific frequency, thereby securing a neural differentiation technology of adult stem cells using sound waves in vitro.

In one embodiment of the present invention (Example 1), the sound wave (1.0V, 10 ~ 40Hz) was irradiated for 5 days using the sound wave generator in FIG. 1, and as shown in FIG. Compared to the sonic treatment group (B-E), mesenchymal stem cells were changed to neuronal-like morphology at 30 to 40 Hz, and when the number of cells was measured in FIG. 3, cell proliferation was further progressed at 30 to 40 Hz. It wasn't. In addition, the expression of Nestin was reduced only by the effect of sound waves in a medium without growth factors (FIG. 4). It could be confirmed at the mRNA level (FIG. 5). The effect of this sound wave was also observed at the protein level, the expression of the neuron-related protein tau protein was increased in the sample treated with sound waves (Fig. 6).

In addition, in one embodiment of the present invention, when the sound waves of 1.0 V at 10-40 Hz were exposed to bone marrow-derived and adipose-derived mesenchymal stem cells for 5 days, cells changed to a neuro-like form were observed, and the proliferation of the cells was significantly reduced. It was confirmed. In addition, as a result of confirming the expression of neuronal markers at the mRNA level, it was confirmed that the expression of MAP-2, NeuroD1, NF-L significantly increased at 30 or 40Hz (Fig. 9).

As a result of observing differentiation of neurons by expanding the frequency range of sound waves, it was confirmed that differentiation into neurons was promoted in the sonic stimulation in the range of 30 to 200 Hz of 1V intensity.

In addition, as a result of observing neural differentiation using sound waves of 2.5V intensity, it was confirmed that umbilical cord-derived stem cells were differentiated into neurons at 30 to 300 Hz at not only 1V but also at 2.5V intensity.

Based on the experimental results, the sound wave frequency of the present invention is preferably treated at a frequency of 1 to 500 Hz, and more preferably, at a frequency of 30 to 300 Hz. Most preferably, it is treated with 30 to 70 HZ. In addition, the intensity of the sound wave is preferably treated at an intensity of 0.1 to 5V, more preferably at an intensity of 0.5 to 3V, and most preferably at an intensity of 0.5 to 1.5V.

Mesenchymal stem cells of the present invention can be cultured in 37 ℃, 5% carbon dioxide environment.

Mesenchymal stem cell differentiation apparatus of the present invention may be preferably in the form illustrated in FIG. In addition, even if it is not shown in Figure 1, it may further include a technique well known to those skilled in the art of the present invention, which is included in the scope of the technical idea of the present invention.

The method of differentiation into neurons of the present invention has the advantage of inducing neuronal differentiation even in growth medium conditions.

The present invention also relates to a composition for treating neurological diseases comprising neurons differentiated by the above method.

Since the composition of the present invention is based on neurons differentiated from mesenchymal stem cells, it is nontoxic and safe.

The neurological disease treatment composition may include a pharmaceutical composition well known to those skilled in the art in addition to the neuron of the present invention, and may be modified in various dosage forms, which is a technical idea of the present invention. Obviously included in the category of.

The composition may be formulated and administered in a unit dosage form suitable for administration in the body of a patient according to conventional methods in the pharmaceutical field, and the preparation may be effective to develop alveoli by one or several administrations. Dosage. Formulations suitable for this purpose are preferably parenteral preparations such as injections such as ampoules for injection. The injection ampoule may be mixed with the injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution.

In the pharmaceutical preparations, in addition to the active ingredient, one or more pharmaceutically acceptable inert carriers such as preservatives, analgesic agents, solubilizers or stabilizers in the case of injections, etc. It may further include a base, excipients, lubricants or preservatives.

The composition or pharmaceutical formulation of the present invention thus prepared may be administered in the form of a mixture with or in combination with other stem cells used for transplantation and other uses, using administration methods commonly used in the art. Preferably, but not limited to, engraftment or implantation directly into the lung disease site of the patient in need of treatment or implantation or injection directly into the respiratory tract. In addition, the administration may be both non-surgical administration using a catheter and surgical administration methods such as infusion or transplantation after thoracic incision, but non-surgical administration using a catheter is more preferable.

Neurological diseases of the present invention refers to all of the neurological diseases including Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage, spinal cord injury, etc. The differentiated neurons or neural stem cells according to the present invention is the function of nerve cells in neurological diseases It can function as a therapeutic agent for neurological diseases by restoring.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are provided to illustrate the present invention more easily, and the content of the present invention is not limited by the examples.

Reference Example 1 Isolation of Mesenchymal Stem Cells

Wash the human umbilical cord (umbilical cord) discharged at birth three times with phosphate buffer solution, remove the perivascular smooth muscle area and epithelial layer, and cut the remaining Watton jelly layer into about 3 mm × 3 mm The tissue was allowed to adhere well to the bottom of the vessel by standing in an incubator at 37 ° C. for about 4 hours. Incubate the culture vessel with DMEM (Dulbecco's Modificaion of Eagle's Medium) medium containing 10% fetal bovine serum (FBS) for 1 week, and cells start to flow out of the watton jelly in the umbilical cord. When abnormal growth was performed, subculture was used as a cell source.

Example 1 Confirmation of Differentiation Induction Capacity of Umbilical Cord-derived Mesenchymal Stem Cells to Neurons Using 1 Volt Intensity Sound Wave (10-40 Hz)

The present invention relates to a method for inducing differentiation of mesenchymal stem cells into neurons using sound waves, and the present method has an advantage of inducing neuronal differentiation even under growth medium conditions. Lonza cells were used for the bone marrow-derived mesenchymal stem cells used in the experiment, and invitrogen cells were used for the adipose derived mesenchymal stem cells. Culture medium was incubated for 5 days using NH medium (Mylteni). The primary cultured cord-derived stem cells were passaged three times using NH medium (Mylteni) and inoculated with 8 × 10 ⁴ cells in 100 mm dishes. The mesenchymal stem cells used in the experiment were all incubated at 37 ° C. and 5% carbon dioxide.

The umbilical cord-derived mesenchymal stem cells were exposed to sound waves of 1.0 V at 10 to 40 Hz for 5 days, and the results were observed. As can be seen in FIG. 2, the cells were changed into neuron-like morphology, and the proliferation of the cells was significantly reduced (FIG. 3). As a result of confirming the expression of neuronal markers at the mRNA level, it was confirmed that the expression of MAP-2, NeuroD1, NF-L was significantly increased at 30-40 Hz (FIG. 5), and the expression was very weak at 10-20 Hz. As a result of observing the expression of the neuron-related protein tau at the protein level, the expression of the tau protein was increased (FIG. 6). This expression was different depending on the frequency, and it was judged to differentiate into neurons at specific frequency and intensity.

<Example 2> Confirmation of the ability to induce differentiation of bone marrow-derived and adipose-derived mesenchymal stem cells into neurons using 1 Volt intensity sound waves (10-40 Hz)

As the growth medium, the umbilical cord-derived mesenchymal stem cells were cultured for 5 days using NH medium (Mylteni). The primary cultured cord-derived stem cells were passaged three times using NH medium (Mylteni) and then inoculated with 8 × 10 ⁴ cells in a 100 mm dish. The mesenchymal stem cells used in the experiment were all incubated at 37 ° C. and 5% carbon dioxide.

10 ~ 40Hz, 1.0V sound waves were exposed to the bone marrow and adipose-derived mesenchymal stem cells for 5 days, and the results were observed. As confirmed in FIG. 7, cells changed to a neuron-like form were observed, and it was confirmed that proliferation of the cells was significantly reduced (FIG. 8). As a result of confirming the expression of neuronal marker at the mRNA level, MAP-2, NeuroD1, NF-L expression was increased significantly, especially at 30 or 40Hz (Fig. 9), the expression was very weak at 10 ~ 20Hz. This expression was different depending on the frequency, and it was judged to differentiate into neurons at specific frequency and intensity.

<Example 3> Confirmation of the neural differentiation of umbilical cord-derived stem cells using 1 Volt intensity sound waves (30Hz ~ 500Hz)

The primary cultured cord-derived stem cells were passaged five times using NH medium (Mylteni) and inoculated with 1 × 10 ⁵ cells in a 100 mm dish. The culture group gave no sonic stimulation to the cells, and the rest gave sonic stimulation of 30, 100, 200, 300, and 500 Hz (1.0 V intensity), respectively, and were cultured in NH medium for 3 days at 37 ° C. and 5% carbon dioxide. .

As a result of confirming the expression of neuronal markers at the mRNA level, MAP-2, NeuroD1, and Tau expression were increased in comparison with the political culture group at 30-200 Hz. In particular, NF-L expression was significantly increased at 200 Hz. (FIG. 10). At this time, as a result of observing the cells under an optical microscope, it was observed that neurites were formed (arrows) at 30-200 Hz compared to the stationary culture group and changed into a nerve-like form (FIG. 11).

That is, the cord-derived stem cells were able to confirm that the differentiation of nerve cells in the sonic stimulation in the range of 30 to 200 Hz of 1V intensity.

Example 4 Confirmation of Induction of Neuronal Differentiation of Umbilical Cord-Derived Stem Cells Using 2.5 Volt Intensity Sound Wave (30 ~ 500Hz)

The primary cultured cord-derived stem cells were passaged six times using NH medium (Mylteni) and then inoculated with 1 × 10 ⁵ cells in a 100 mm dish. The culture group gave no sonic stimulation to the cells, and the rest gave sonic stimulation of 30, 100, 200, 300 and 500 Hz (2.5 V intensity), respectively, and were cultured in NH medium for 3 days at 37 ° C. and 5% carbon dioxide. .

As a result of confirming the expression of neuronal markers at the mRNA level, NeuroD1 expression was increased in comparison with the culture culture group at 30-200 Hz, and DCX expression was increased in comparison with the culture culture group at 100 Hz and 300 Hz. In particular, as the expression of Nestin decreases in all sonic stimulation group, it was confirmed that differentiation into neurons is progressing (FIG. 12). At this time, as a result of observing the cells under an optical microscope, neurites were formed (arrows) at 30 to 300 Hz compared to the stationary culture group and changed into a nerve-like form (FIG. 13).

In other words, the cord-derived stem cells were found to promote the differentiation of sonic stimulation into nerve cells at the intensity of 2.5V as well as 1V.

<Example 5> Confirmation of the neural differentiation of bone marrow-derived stem cells using 1 Volt intensity sound waves (30 ~ 500Hz)

Primary cultured bone marrow-derived stem cells were passaged six times using low glucose DMEM / 10% FBS medium (Gibco) and inoculated with 1 × 10 ⁵ cells in 100 mm dishes. The culture group did not give sonic stimulation to the cells, and the rest gave sonic stimulation of 30, 100, 200, 300, and 500 Hz (1 V intensity), respectively, and for 3 days in low glucose DMEM / 10% FBS medium, 37 ℃, Incubated in a 5% carbon dioxide environment.

The expression of neuronal markers at the mRNA level was increased, especially at 100 Hz, compared with the expression of neuroD1 and NF-L. As the expression of Nestin decreases compared with the culture culture group, it can be seen that differentiation into neurons is progressing (FIG. 14).

That is, the bone marrow-derived stem cells were able to confirm that the differentiation into neurons is promoted even in sonic stimulation of 1V, 100Hz intensity.

Stem cell differentiation method and differentiation apparatus using sound waves according to the present invention induces adult stem cells to differentiate into neurons using low frequency waves, thereby making it easy to obtain neurons or neural stem cells, which are difficult to acquire cells. As it can induce neuronal differentiation in low-cost general medium condition, not expensive neuronal induction medium by adding growth factor, it is useful for the treatment of cerebral neurological diseases such as Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage and spinal cord injury. Can be.

Claims (8)

A method of treating sound waves on mesenchymal stem cells to differentiate mesenchymal stem cells into neurons. The method of claim 1, wherein the sound wave is processed at a frequency of 1 to 500 Hz. The method of claim 1, wherein the sound wave is processed at a frequency of 30 to 300Hz. The method of claim 1, wherein the sound wave is treated with an intensity of 0.1 to 5V. The method of claim 1, wherein the sound wave is treated with an intensity of 0.5 to 3V. The method of any one of claims 1 to 5, wherein the mesenchymal stem cells are derived from bone marrow, adipose derived, umbilical cord blood, or umbilical cord. A composition for treating neurological diseases comprising neurons differentiated by the method according to any one of claims 1 to 5. 8. The composition of claim 7, wherein the neurological disease is Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage or spinal cord injury.

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