KR102453246B1 - Method for Preparing Stem Cells Having Improved Secretion of Neurotrophic Factors Using Aspirin or Conditioned Medium of Urine Stem Cells - Google Patents

Abstract

The present invention relates to a method for producing adipose-derived stem cells with improved secretion of neurotrophic factors necessary for neural differentiation using a urine stem cell culture medium and aspirin from normal people of low age, wherein the secretion of neurotrophic factors according to the present invention The improved method for producing stem cells is a method of culturing adipose-derived stem cells from a neurodegenerative disease patient in a medium containing urine stem cell culture medium and aspirin. Therefore, it is very useful clinically and industrially.

Description

{Method for Preparing Stem Cells Having Improved Secretion of Neurotrophic Factors Using Aspirin or Conditioned Medium of Urine Stem Cells}

The present invention relates to a method for producing adipose-derived stem cells with improved neurotrophic factor secretion using a urine stem cell culture medium or aspirin, and more particularly, to a medium containing urine stem cell culture medium or aspirin of a normal person of a young age. To a method for producing adipose stem cells with improved secretion of neurotrophic factors required for neuronal differentiation by culturing adipose-derived stem cells from patients with degenerative neurological diseases.

Human stem cell biology is one of the most modern and attracting attention in the field of neurobiology. Stem cell biology has attracted the attention of neurobiologists as well as the general public, perhaps as a result of research over the past decade, not only during development, but also in the brain of adult mammals, including humans. ) and can continuously divide and multiply (Johansson CB et al., Cell 96: 25-34, 1996; Magavi SS et al., Nature 405: 951-5, 2000). That is, the fact that the regenerative neurogenesis of nerve cells exists in the adult central nervous system (CNS) also causes death of nerve cells that cause various neurodegenerative CNS disorders. and the destruction of the neural network can be fundamentally restored through the supply or replacement of healthy cells. This treatment, so-called regenerative cell replacement, cultivates undifferentiated cells in a special way, differentiates them into specific nerve cells, and then transplants them into the lesion. Alternatively, it can be carried out by directly transplanting undifferentiated cells into the damaged area to induce proliferation and differentiation according to the signaling mechanisms of the nervous tissue itself.

Therefore, as a material for cell therapy of degenerative central nervous diseases, undifferentiated cells that can be differentiated into neurons suitable for the purpose are essential, and for this purpose, neural precursor cells, adult stem cells and embryonic stem cells Research on embryonic stem cells (ESCs) is being intensively conducted.

On the other hand, urine stem cells are a representative non-invasive method for obtaining stem cells from urine, and they do not cause any pain or sequelae compared to other mesenchymal stem cells, so they are safer and easier than conventional invasive methods. In addition, urine stem cells can be harvested multiple times, so the number of stem cells required for culturing is higher than that of existing cell sources, and urine stem cells can be used as autologous cell therapy that does not induce an immune response due to low HLA-DR expression. In this respect, clinical approach is very easy. In addition, the technology for developing pharmaceutical materials using stem cell conditioned mdeium, which has skin regeneration efficacy, anti-inflammatory and wound healing effects, has a high potential for the future and is meaningful in terms of presenting a new bio-industry model with new market development. can be said to be large.

Accordingly, the present inventors have made diligent efforts to develop a safer and more effective therapeutic agent for degenerative neurodegenerative diseases. As a result, when culturing adult adipose-derived stem cells using children's urine stem cell culture medium (CM: conditioned media) or aspirin, nerve differentiation occurs. It was confirmed that the secretion of necessary neurotrophic substances increased, and the present invention was completed.

It is an object of the present invention to prepare adipose stem cells with improved secretion of neurotrophic substances necessary for neurodifferentiation by culturing adipose-derived stem cells of a patient with a neurodegenerative disease using children's urine stem cell culture medium (CM: conditioned media) or aspirin. To provide a method and a therapeutic agent for autologous stem cell degenerative neurological disease using the same.

In order to achieve the above object, the present invention provides a method for producing stem cells with improved secretion of neurotrophic factors, comprising the step of culturing the stem cells in a medium containing a urine stem cell culture solution.

The method for producing stem cells with improved secretion of neurotrophic factors using a urine stem cell culture medium and aspirin according to the present invention can use autologous stem cells with significantly increased therapeutic efficacy as a therapeutic agent for stem cell treatment of patients with degenerative neurological disease. It is very useful both commercially and industrially.

1 shows a culture schedule of adipose stem cells in a patient with degenerative neurological disease in a urine stem cell culture medium and aspirin-added medium.
Figure 2 shows the measurement of BDNF in the culture medium recovered after culturing the adipose stem cells of Parkinson's patients in a medium containing the children's urine stem cell condition culture medium by ELISA.
Figure 3 shows the measurement of GDNF in the culture medium recovered after culturing the adipose stem cells of Parkinson's patients in a medium containing the children's urine stem cell condition culture medium by ELISA.
Figure 4 shows the measurement of IGF-1 by ELISA in the culture medium recovered after culturing adipose stem cells of Parkinson's patients in a medium containing the children's urine stem cell condition culture medium.
5 is a graph showing the measurement of BDNF in the culture medium recovered after culturing the adipose stem cells of a Parkinson's patient in a medium containing aspirin by ELISA.
6 is a graph showing the measurement of GDNF in the culture medium recovered after culturing adipose stem cells of a Parkinson's patient in a medium containing aspirin by ELISA.
7 is a graph illustrating the measurement of IGF-1 by ELISA in a culture medium recovered after culturing adipose stem cells from a Parkinson's patient in a medium containing aspirin.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is those well known and commonly used in the art.

In the present invention, adipose stem cells from a neurodegenerative disease patient are isolated and then cultured in a children's urine stem cell conditioned medium or a medium containing aspirin to improve the secretion of neurotrophic factors such as BDNF, GDNF and IGF-1. Derived stem cells were prepared. The adipose stem cells prepared in this way can be used as a very safe and effective cell therapy for autologous stem cell treatment of patients with neurodegenerative diseases.

Accordingly, the present invention, in one aspect, relates to a method for producing stem cells with improved secretion of neurotrophic factors, comprising culturing the stem cells in a medium containing a urine stem cell culture solution.

In the present invention, it may be characterized in that it further comprises the step of culturing in a culture medium containing urine stem cells and aspirin.

In the present invention, the stem cells are preferably adipose-derived stem cells, and any method that has been used in the past can be used as a method for isolating and culturing stem cells from adipose tissue.

The isolated or cultured stem cells may be stored by methods known in the art until use. In general, stem cells can be stored frozen after cryoprotection treatment. The cryoprotection treatment is known in the art DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol , using a cryoprotectant such as i-inositol, D-lactose or choline chloride.

The culture of the adipose stem cells isolated above can be performed in a cell culture medium known in the art, for example, but not limited to, DMEM, Defined Keratinocyte-SFM, α-MEM, IMDM, F12 and It can be performed in a basal medium selected from the group consisting of DMEM/F12. More preferably, the basal medium, L-ascorbic acid 2-phosphate (vitamin C), fetal bovine serum and a urine stem cell culture medium ( conditioned medium) or aspirin is added, but is not limited thereto.

In the present invention, the urine stem cells are preferably derived from the urine of a normal child of a low age group, and the low age group is more preferably a normal child aged 8 to 19 years old, and most preferably about 10 years old. However, the present invention is not limited thereto.

In the present invention, the medium used for culturing urine stem cells is preferably KCM-N culture medium, more preferably KSFM, DMEM, 5% FBS, 0.2mM ascorbic acid, 10ng/mL bFGF, 10ng/mL EGF, 5 ng/mL selenium, 5 ng/mL insulin. 75ng/mL hydrocortisone, 0.09mM calcium, 2mM N-acetyl-L-cysteine is a medium containing, but is not limited thereto.

The ratio of KSFM to DMEM is preferably 1:1, but is not limited thereto.

As used herein, the term "urine stem cell (USC)" refers to a cell having stem cell characteristics isolated and cultured in urine.

In the present invention, "medium (culture media)" means a culture medium that can support the growth and survival of stem cells in in vitro culture conditions, and includes all conventional media used in the art suitable for culturing stem cells. . In addition, the medium and culture conditions can be selected according to the type of cell.

In the present invention, "passage culture" refers to a method of continuously culturing cells in succession while changing the culture medium after periodically transferring a part of the cells to a new culture vessel in order to continuously culture the cells in a healthy state for a long period of time. do. As the number of cells increases in a culture vessel with a limited space, the growth nutrients are consumed or contaminants accumulate and the cells die naturally after a certain period of time, so it is used as a method to increase the number of healthy cells. Changing the culture vessel) or culturing the cell group by dividing it is called one passage can be performed.

In the present invention, "conditioned medium" or "conditioned culture medium" refers to a medium in which only the culture medium is collected after exchanging the cells with a serum-free medium when the cells reach the logarithmic growth phase, the peak phase of cell division. This refers to the use of unknown growth factors and cytokines extracted from dividing cells in the medium. The conditioned medium includes a solution in which urine or adipose-derived mesenchymal stem cells are removed after culturing urine or adipose-derived mesenchymal stem cells in a serum-free medium, and growth factors derived from urine or adipose-derived mesenchymal stem cells; It may contain substances such as cytokines in abundance.

In the present invention, the neurotrophic factor may be characterized in that BDNF, GDNF or IGF-1.

As used herein, the term "brain-derived neurotrophic factor (BDNF)" protein is the most abundantly distributed neurotrophin in the brain. It is known to promote the growth of neurons, and to regulate the synthesis, metabolism, release and activity of neurotransmitters. In addition, it is known that BDNF protein is decreased in the prefrontal cortex or hippocampus of depressed patients, and can treat depression by increasing its concentration.

The term "Glial cell line-Derived Neurotrophic Factor (GDNF)" is a small protein that potently promotes the survival of many types of neurons, signaling through the GFRα receptor, specifically GFRα1. In addition, "nerve growth factor (nerve growth factor, NGF)" is a neurotrophic factor mainly involved in the regulation of growth, maintenance, proliferation and survival of specific target neurons.

"Insulin-Like Growth Factors (IGF-1)" of the present specification promotes cell growth, division, and survival similar to growth hormone, and increases sensitivity to insulin to activate cell metabolism. IGF-1 secretion is stimulated by growth hormone/somatotropin, which is secreted according to the commands of the hypothalamus and pituitary gland. IGF-1 contributes to aging and promotes the growth of cancer cells.

In the present invention, the degenerative neurological disease is Alzheimer's disease (AD), Parkinson's disease (PD), Lou Gehrig's disease (Amyotrophic Lateral Sclerosis, ALS), cerebral infarction, chronic brain injury (chronic brain injury), ischemic From the group consisting of brain disease, spinal cord injury, Huntington's disease (HD), Rett's disease (RD), stroke, multiple sclerosis, traumatic brain injury and neonatal hypoxic ischemic encephalopathy can be selected.

The present invention prepared a culture medium or conditioned medium containing neurotrophic factors using urine stem cells that can be easily obtained and cultured at a low cost compared to other adult stem cells. In the present invention, adipose stem cells with improved secretion of neurotrophic factors can be obtained by culturing adipose stem cells in a conditioned medium obtained from urine stem cells isolated from urine of a young age group.

[Example]

Hereinafter, the present invention will be described in more detail by way of Examples. These examples are merely for illustrating the present invention in more detail, and thereby it will be apparent to those skilled in the art that the technical scope of the present invention is not limited to these examples.

Example 1: Isolation and culture of adipose-derived stem cells

Adipose-derived stem cells were isolated from Parkinson's patients and normal adults. Adipose tissue separated from abdominal fat by liposuction was washed with PBS, and then the adipose tissue was cut into small pieces and then using DMEM medium supplemented with collagenase type 1 (1mg/ml) at 37°C for 2 hours. tissue was disassembled. The collagenase-treated tissue was washed with PBS and then centrifuged at 1000 rpm for 5 minutes to remove the supernatant, and the pellet was washed with PBS and then centrifuged at 1000 rpm for 5 minutes. After filtering through 100 mesh to remove suspended matter, it was washed with PBS, and cultured in DMEM medium supplemented with 10% FBS, 2mM NAC (N-acetyl-L-cysteine), and 0.2mM ascorbic acid. After overnight, non-adherent cells were washed with PBS, 5% FBS, 2 mM NAC, 0.2 mM ascorbic acid, 0.09 mM calcium, 5 ng/ml rEGF, 5 ng/ml insulin, 10 ng bFGF, 74 ng/ml hydrocortisone and 1 ng/ml. K-SFM (Keratinocyte Serum Free Medium) and DMEM medium containing ml of selenium were subcultured while replacing every 2 days, and cultured for about 4 days at each passage. Adipose-derived stem cells were isolated by culturing until passage 5.

Example 2: Isolation of urine stem cells and preparation of conditioned medium

2-1: Isolation of urine stem cells

More than 200 mL of urine from children (around 10 years of age) and normal adults was collected in a urine collection container, and 40 mL each was dispensed into a 50 mL tube and centrifuged at 1,600 rpm for 5 minutes. After removing the supernatant, the pellet was washed with PBS containing antibiotics, and the PBS of 4-5 tubes was collected and centrifuged again at 1,600 rpm for 5 minutes, and then the supernatant was removed. After the second washing with PBS 20mL, filtered through a 100㎛ cell strainer, centrifuged once more to remove the supernatant. The cell precipitate was suspended in adhesion medium (DMEM/F12: KSFM (1:1), 10% FBS, 2X Antibiotic-Antimycotic), and then seeded in a 12-well plate coated with collagen type I.

The medium was exchanged for the first time after 48 hours, and medium exchange was performed every 48 hours until cells were generated. After urine stem cells are generated, AMSC culture medium (KSFM, DMEM, 5% FBS, 0.2mM ascorbic acid, 10ng/mL bFGF, 10ng/mL EGF, 5ng/mL selenium, 5ng/mL mL insulin, 75 ng/mL hydrocortisone, 0.09 mM calcium, 2 mM N-acetyl-L-cysteine).

2-2: Subculture of urine stem cells and preparation of conditioned medium

Urine stem cells cultured at a confluency of 90% or more were enzymatically treated and subcultured in a 12-well plate 1:2 (passage 1). Cultures were performed while exchanging the medium with RKCM-N medium every 48 hours, and when confluency reached 90%, subcultures were performed at 1:4 (passage 2).

Urine stem cells (passage 2) cultured at 90% confluency or more were enzymatically treated and inoculated into T175cm ² flasks and cultured to 90% or more confluency in RKCM-N medium. The cultured urine stem cell (passage 3) flask was washed twice with PBS, exchanged for serum-free DMEM medium, and cultured for 5 days, and only the culture solution was recovered and filtered with a 0.2 μm syringe filter. The serum-free medium treatment exposes the urine stem cells to a specific extreme environment to release as much of the protein secreted by the cells as possible into the culture medium.

As a control, adipose-derived mesenchymal stem cells (passage 3) were also cultured in the same manner to prepare a conditioned medium.

Example 3: Culture of adipose stem cells using urine stem cell culture medium and confirmation of neurotrophic factor secretion

The third generation adipose-derived stem cells from Parkinson's patients isolated in Example 1 were cultured in AMSC culture medium (KSFM, DMEM, 5% FBS, 0.2 mM ascorbic acid, 10 ng/mL bFGF, 10ng/mL EGF, 5ng/mL selenium, 5ng/mL insulin, 75ng/mL hydrocortisone, 0.09mM calcium, 2mM N-acetyl-L-cysteine) was inoculated, and cultured at 37°C, CO ₂ in an incubator for 2 days. After 2 days, the culture medium was replaced with the same medium containing the children's urine stem cell condition culture medium, and the culture medium was recovered. BDNF, GDNF, and IGF-1 secreted by adipose stem cells from Parkinson's patients were measured in the recovered culture medium. Cytokine analysis using an ELISA kit (abcam) for each factor was performed, absorbance was checked with a Microplate (Microplate reader. EPOCH, BioTek), and the results were analyzed with [myassays, four parameter logistic curve].

Control: Culture medium recovered after culturing adipose stem cells from Parkinson's patients in stem cell culture medium

Experimental group: A culture medium recovered after culturing adipose stem cells from Parkinson's patients in a medium containing children's urine stem cell condition culture medium

As a result, the secretion of BDNF, GDNF, and IGF-1 was increased in the experimental group compared to the control group ( FIGS. 2 to 4 ).

Example 4: Culture of adipose stem cells using aspirin and confirmation of neurotrophic factor secretion

Passage 3 adipose-derived stem cells from Parkinson's patients isolated in Example 1 were cultured in AMSC culture medium (KSFM, DMEM, 5% FBS, 0.2mM ascorbic acid, 10ng/mL bFGF, 10ng/mL EGF, 5ng/mL selenium, 5ng /mL insulin, 75ng/mL hydrocortisone, 0.09mM calcium, 2mM N-acetyl-L-cysteine) was inoculated, and cultured at 37°C, CO ₂ in an incubator for 2 days. After 2 days, the culture medium was replaced with an AMSC culture medium supplemented with 0.5 mM aspirin, and the culture medium was recovered for 48 hours. BDNF, GDNF, and IGF-1 secreted by adipose stem cells from Parkinson's patients were measured in the recovered culture medium. Cytokine analysis using each ELISA kit (abcam) was performed, absorbance was checked with a Microplate (Microplate reader. EPOCH, BioTek), and the results were analyzed with [myassays, four parameter logistic curve].

Control: Culture medium recovered after culturing adipose stem cells from Parkinson's patients in stem cell culture medium

Experimental group: A culture medium recovered after culturing adipose stem cells from Parkinson's patients in a stem cell culture medium containing aspirin

As a result, the secretion of GDNF, BDNF and IGF-1 was increased in the experimental group compared to the control group ( FIGS. 5 to 7 ).

Example 5: Culture of adipose stem cells using urine stem cell culture medium and aspirin and confirmation of neurotrophic factor secretion

The third generation adipose-derived stem cells from Parkinson's patients isolated in Example 1 were cultured in AMSC culture medium (KSFM, DMEM, 5% FBS, 0.2 mM ascorbic acid, 10 ng/mL bFGF, 10ng/mL EGF, 5ng/mL selenium, 5ng/mL insulin, 75ng/mL hydrocortisone, 0.09mM calcium, 2mM N-acetyl-L-cysteine) was inoculated, and cultured at 37°C, CO ₂ in an incubator for 2 days. After 2 days, the culture medium was replaced with a medium supplemented with 0.5 mM aspirin in the same medium containing the children's urine stem cell condition culture medium, and the culture medium was recovered. BDNF, GDNF, and IGF-1 secreted by adipose stem cells from Parkinson's patients were measured in the recovered culture medium. Cytokine analysis using an ELISA kit (abcam) for each factor was performed, absorbance was checked with a Microplate (Microplate reader. EPOCH, BioTek), and the results were analyzed with [myassays, four parameter logistic curve].

As a result, a synergistic effect in which the secretion of BDNF, GDNF, and IGF-1 was significantly increased in the experimental group treated with the urine stem cell conditioned medium and aspirin was confirmed as compared to the urine stem cell conditioned medium alone or aspirin alone.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby It will be obvious. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents. Simple modifications or changes of the present invention can be easily used by those of ordinary skill in the art, and all such modifications or changes can be considered to be included in the scope of the present invention.

Claims (5)

A method for producing stem cells with improved secretion of neurotrophic factors, comprising the step of culturing stem cells in a medium containing the urine-derived urine stem cell culture medium of a normal person of a low age.
The method according to claim 1, further comprising the step of culturing in a medium containing urine stem cell culture medium and aspirin.
delete The method according to claim 1, wherein the stem cells are adipose-derived stem cells.
The method according to claim 1, wherein the neurotrophic factor is BDNF, GDNF, or IGF-1.

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