COMPOSITION FOR PROMOTING PRODUCTION OF STEM CELL-DERIVED EXOSOMES AND INCREASING STEMNESS
20220195384 · 2022-06-23
Assignee
Inventors
Cpc classification
C12N2501/385
CHEMISTRY; METALLURGY
C12N5/0606
CHEMISTRY; METALLURGY
A61P29/00
HUMAN NECESSITIES
A61K35/51
HUMAN NECESSITIES
A61P9/10
HUMAN NECESSITIES
C12N5/0668
CHEMISTRY; METALLURGY
C12N2501/405
CHEMISTRY; METALLURGY
A61K35/30
HUMAN NECESSITIES
C12N2501/335
CHEMISTRY; METALLURGY
C12N5/0696
CHEMISTRY; METALLURGY
A61P1/00
HUMAN NECESSITIES
C12N5/0663
CHEMISTRY; METALLURGY
A61K35/545
HUMAN NECESSITIES
A61K35/28
HUMAN NECESSITIES
International classification
Abstract
The present disclosure relates to a composition for promoting the production of stem cell-derived exosomes or increasing the stemness of stem cells. When the composition of the present disclosure is used in culturing stem cells, the stemness of stem cells and the yield of stem cell-derived exosomes are increased, and thus good-quality stem cells and stem cell-derived exosomes can be produced more efficiently, and accordingly, can be advantageously used in related research and development and commercialization.
Claims
1.-9. (canceled)
10. A method for producing stem cell-derived exosomes, the method comprising a step of: culturing stem cells in a cell culture medium containing at least one selected from the group consisting of exendin-4, phorbol 12-myristate 13-acetate (PMA), interferon-γ, tetrandrine, hyaluronic acid, substance P, resveratrol, and lanifibranor.
11. The method of claim 10, further comprising the steps of: washing the cultured stem cells, followed by additional culturing in a cell culture medium; and isolating exosomes.
12. The method of claim 10, wherein the cell culture medium contains fetal bovine serum free of exosomes.
13. A method for increasing stemness of stem cells, the method comprising a step of: culturing stem cells in a cell culture medium containing at least one selected from the group consisting of exendin-4, phorbol 12-myristate 13-acetate (PMA), interferon-γ, tetrandrine, hyaluronic acid, substance P, resveratrol, and lanifibranor.
14. The method of claim 10, wherein the stem cell is an embryonic stem cell, an adult stem cell, or an induced pluripotent stem cell (iPSC).
15. The method of claim 14, wherein the adult stem cell is an adult stem cell of human or animal tissue origin, a mesenchymal stromal cell of human or animal tissue origin, or an induced pluripotent stem cell of human or animal tissue origin.
16. The method of claim 15, wherein the human or animal tissue is selected from the group consisting of umbilical cord, umbilical cord blood, lipid, muscle, nerve, skin, amnion, and placenta.
17. The method of claim 15, wherein the adult stem cell of human or animal tissue origin is selected from the group consisting of a hematopoietic stem cell, a mammary stem cell, an intestinal stem cell, a vascular endothelial progenitor cell, a neural stem cell, an olfactory neural stem cell, and a testicular stem cell.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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BEST MODE FOR CARRYING OUT THE INVENTION
[0277] A composition for promoting production of stem cell-derived exosomes, the composition comprising at least one selected from the group consisting of exendin-4, phorbol 12-myristate 13-acetate (PMA), interferon-γ, tetrandrine, hyaluronic acid, substance P, resveratrol, and lanifibranor.
DETAILED DESCRIPTION
[0278] A better understanding of the present disclosure will be obtained from the following Examples which are set forth to illustrate the present disclosure and are not to be construed as limiting the present disclosure.
EXAMPLE 1
Isolation of Stem Cell-Derived Exosome According to Treatment with Exendin-4
[0279] Exendin-4 Pretreatment
[0280] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 20 nM of exendin-4, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0281] Additional Culturing
[0282] After completion of culturing, the mesenchymal stem cells pretreated with exendin-4 were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0283] Exosome Isolation
[0284] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 2
Isolation of Stem Cell-Derived Exosome According to Treatment with PMA
[0285] PMA Pretreatment
[0286] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 10 nM of PMA, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0287] Additional Culturing
[0288] After completion of culturing, the mesenchymal stem cells pretreated with PMA were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0289] Exosome Isolation
[0290] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 3
Isolation of Stem Cell-Derived Exosome According to Treatment with Interferon-γ
[0291] Interferon-γ Pretreatment
[0292] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 10 ng/mL interferon-γ, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0293] Additional Culturing
[0294] After completion of culturing, the mesenchymal stem cells pretreated with interferon-γ were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0295] Exosome Isolation
[0296] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 4
Isolation of Stem Cell-Derived Exosome According to Treatment with Tetrandrine
[0297] Tetrandrine Pretreatment
[0298] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 10 μM of tetrandrine, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0299] Additional Culturing
[0300] After completion of culturing, the mesenchymal stem cells pretreated with tetrandrine were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0301] Exosome Isolation
[0302] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 5
Isolation of Stem Cell-Derived Exosome According to Treatment with Hyaluronic Acid
[0303] Hyaluronic Acid Pretreatment
[0304] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 10 μg/mL hyaluronic acid, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0305] Additional Culturing
[0306] After completion of culturing, the mesenchymal stem cells pretreated with hyaluronic acid were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0307] Exosome Isolation
[0308] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 6
Isolation of Stem Cell-Derived Exosome According to Treatment with Substance P
[0309] Substance P Pretreatment
[0310] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 30 nM of substance P, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0311] Additional Culturing
[0312] After completion of culturing, the mesenchymal stem cells pretreated with substance P were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0313] Exosome Isolation
[0314] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 7
Isolation of Stem Cell-Derived Exosome According to Treatment with Resveratrol
[0315] Resveratrol Pretreatment
[0316] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 10 nM of resveratrol, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0317] Additional Culturing
[0318] After completion of culturing, the mesenchymal stem cells pretreated with resveratrol were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0319] Exosome Isolation
[0320] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXAMPLE 8
Stem Cell-Derived Exosome According to Treatment with Lanifibranor
[0321] Lanifibranor Pretreatment
[0322] In a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065); 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] containing 10 μM of lanifibranor, primary umbilical cord-derived mesenchymal stem cells (ATCC No. PCS-500-010; Lot No. 63822428) were cultured for one week.
[0323] Additional Culturing
[0324] After completion of culturing, the mesenchymal stem cells pretreated with lanifibranor were washed and additionally cultured for 72 hours in a culture medium supplemented with 10% fetal bovine serum (FBS) free of exosomes. The reason of employing exosome-free FBS was to prevent FBS-derived exosomes other than those secreted from the cells from being incorporated because generally used FBS contained a large amount of exosomes derived from bovine serum.
[0325] Exosome Isolation
[0326] After 72 hours of culturing, the culture medium in which the pretreated mesenchymal stem cells were cultured was taken and centrifuged at 300×g for 10 min to remove residual cells and cell debris. The supernatant was filtered through a 0.22 μm filter, followed by centrifugation at 10,000×g and 4° C. for 70 min in a high-speed centrifuge. The supernatant thus obtained was subjected again to ultracentrifugation at 100,000×g and 4° C. for 90 min. The exosomes obtained as a pellet were diluted in PBS (phosphate buffered saline) before use in the following experiments.
EXPERIMENTAL EXAMPLE 1
Functional Reinforcement of Stem Cells by Treatment with Exendin-4
[0327] 1-1. Increase of Stem Cell Proliferation by Treatment with Exendin-4
[0328] Mesenchymal stem cells and the exendin-4-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0329] As can be seen in
[0330] 1-2. Increase of Stem Cell Stemness by Treatment with Exendin-4
[0331] Mesenchymal stem cells and the exendin-4-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0332] As can be seen in
EXPERIMENTAL EXAMPLE 2
Increase of Exosome Productivity by Treatment with Exendin-4
[0333] 2-1. Increase in Number of Exosomes by Treatment with Exendin-4
[0334] The exosomes isolated in the Example were counted through the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 1 and
TABLE-US-00001 TABLE 1 Yield (per 10.sup.6 cells) MSC-Exo Exe4-MSC-Exo No. of exosome particles (10.sup.9) 2.30 13.45 (MSC: non-treated mesenchymal stem cells, Exe4-MSC: Exendin-4-treated mesenchymal stem cells)
[0335] As can be seen in Table 1 and
[0336] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0337] 2-2. Increase in Content of Exosomal Protein by Treatment with Exendin-4
[0338] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 2 and
TABLE-US-00002 TABLE 2 Yield (per 10.sup.6 cells) MSC-Exo Exe4-MSC-Exo Exosomal protein (μg) 2.00 10.69 (MSC: non-treated mesenchymal stem cells, Exe4-MSC: Exendin-4-treated mesenchymal stem cells)
[0339] As can be seen in Table 2 and
[0340] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0341] 2-3. Increase in Content of Exosomal RNA by Treatment with Exendin-4
[0342] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 3 and
TABLE-US-00003 TABLE 3 Yield (per 10.sup.6 cells) MSC-Exo Exe4-MSC-Exo Exosomal RNA (ng) 31.00 174.34 (MSC: non-treated mesenchymal stem cells, Exe4-MSC: Exendin-4-treated mesenchvmal stem cells)
[0343] As can be seen in Table 3 and
[0344] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENTAL EXAMPLE 3
Functional Reinforcement of Stem Cells by Treatment with PMA
[0345] 3-1. Increase of Stem Cell Proliferation by Treatment with PMA
[0346] Mesenchymal stem cells and the PMA-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0347] As can be seen in
[0348] 3-2. Increase of Stem Cell Stemness by Treatment with PMA
[0349] Mesenchymal stem cells and the PMA-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0350] As can be seen in
EXPERIMENTAL EXAMPLE 4
Increase of Exosome Productivity by Treatment with PMA
[0351] 4-1. Increase in Number of Exosomes by Treatment with PMA
[0352] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 4 and
TABLE-US-00004 TABLE 4 Yield (per 10.sup.6 cells) MSC-Exo PMA MSC-Exo No. of exosome particles (10.sup.9) 2.3 11.90 (MSC: non-treated mesenchymal stem cells, PMA MSC: PMA-treated mesenchymal stem cells)
[0353] As can be seen in Table 4 and
[0354] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0355] 4-2. Increase in Content of Exosomal Protein by Treatment with PMA
[0356] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 5 and
TABLE-US-00005 TABLE 5 Yield (per 10.sup.6 cells) MSC-Exo PMA MSC-Exo Exosomal protein (ug) 2.0 10.0 (MSC: non-treated mesenchymal stem cells, PMA: PMA-treated mesenchymal stem cells)
[0357] As can be seen in Table 5 and
[0358] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0359] 4-3. Increase in Content of Exosomal RNA by Treatment with PMA
[0360] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 6 and
TABLE-US-00006 TABLE 6 Yield (per 10.sup.6 cells) MSC-Exo PMA MSC-Exo Exosomal RNA (ng) 31.0 125.0 (MSC: non-treated mesenchymal stem cells, PMA-MSC: PMA-treated mesenchymal stem cells)
[0361] As can be seen in Table 6 and
[0362] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENT EXAMPLE 5
Functional Reinforcement of Stem Cells by Treatment with Interferon-γ
[0363] 5-1. Increase of Stem Cell Proliferation by Treatment with Interferon-γ
[0364] Mesenchymal stem cells and the interferon-γ-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0365] As can be seen in
[0366] 5-2. Increase of Stem Cell Stemness by Treatment with Interferon-γ
[0367] Mesenchymal stem cells and the interferon-γ-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0368] As can be seen in
EXPERIMENTAL EXAMPLE 6
Increase of Exosome Productivity by Treatment with Interferon-γ
[0369] 6-1. Increase in Number of Exosomes by Treatment with Interferon-γ
[0370] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 7 and
TABLE-US-00007 TABLE 7 Yield (per 10.sup.6 cells) MSC-Exo IFNγ MSC-Exo No. of exosome particles (10.sup.9) 2.3 15.4 (MSC: non-treated mesenchymal stem cells, IFNγ MSC: IFNγ-treated mesenchymal stem cells)
[0371] As can be seen in Table 7 and
[0372] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0373] 6-2. Increase in Content of Exosomal Protein by Treatment with Interferon-γ
[0374] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 8 and
TABLE-US-00008 TABLE 8 Yield (per 10.sup.6 cells) MSC-Exo IFNγ MSC-Exo Exosomal protein (ug) 2.0 12.4 (MSC: non-treated mesenchymal stem cells, IFNγ MSC: IFNγ-treated mesenchymal stem cells)
[0375] As can be seen in Table 8 and
[0376] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0377] 6-3. Increase in Content of Exosomal RNA by Treatment with Interferon-γ
[0378] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 9 and
TABLE-US-00009 TABLE 9 Yield (per 10.sup.6 cells) MSC-Exo IFNγ MSC-Exo Exosomal RNA (ng) 31.0 155.0 (MSC: non-treated mesenchymal stem cells, IFNγ MSC: IFNγ-treated mesenchymal stem cells)
[0379] As can be seen in Table 9 and
[0380] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENT EXAMPLE 7
Functional Reinforcement of Stem Cells by Treatment with Tetrandrine
[0381] 7-1. Increase of Stem Cell Proliferation by Treatment with Tetrandrine
[0382] Mesenchymal stem cells and the tetrandrine-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0383] As can be seen in
[0384] 7-2. Increase of Stem Cell Stemness by Treatment with Tetrandrine
[0385] Mesenchymal stem cells and the tetrandrine-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0386] As can be seen in
EXPERIMENTAL EXAMPLE 8
Increase of Exosome Productivity by Treatment with Tetrandrine
[0387] 8-1. Increase in Number of Exosomes by Treatment with Tetrandrine
[0388] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 10 and
TABLE-US-00010 TABLE 10 Yield (per 10.sup.6 cells) MSC-Exo Tet MSC-Exo No. of exosome particles (10.sup.9) 2.3 9.84 (MSC: non-treated mesenchymal stem cells, Tet MSC: tetrandrine-treated mesenchymal stem cells)
[0389] As can be seen in Table 10 and
[0390] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0391] 8-2. Increase in Content of Exosomal Protein by Treatment with Tetrandrine
[0392] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 11 and
TABLE-US-00011 TABLE 11 Yield (per 10.sup.6 cells) MSC-Exo Tet MSC-Exo Exosomal protein (ug) 2.0 9.42 (MSC: non-treated mesenchymal stem cells, Tet MSC: Tetrandrine-treated mesenchymal stem cells)
[0393] As can be seen in Table 11 and
[0394] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0395] 8-3. Increase in Content of Exosomal RNA by Treatment with Tetrandrine
[0396] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 12 and
TABLE-US-00012 TABLE 12 Yield (per 10.sup.6 cells) MSC-Exo Tet MSC-Exo Exosomal RNA (ng) 31.0 136.0 (MSC: non-treated mesenchymal stem cells, Tet MSC: Tetrandrine-treated mesenchymal stem cells)
[0397] As can be seen in Table 12 and
[0398] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENT EXAMPLE 9
Functional Reinforcement of Stem Cells by Treatment with Hyaluronic acid
[0399] 9-1. Increase of Stem Cell Proliferation by Treatment with Hyaluronic Acid
[0400] Mesenchymal stem cells and the hyaluronic acid-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0401] As can be seen in
[0402] 9-2. Increase of Stem Cell Stemness by Treatment with Hyaluronic Acid
[0403] Mesenchymal stem cells and the hyaluronic acid-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0404] As can be seen in
EXPERIMENTAL EXAMPLE 10
Increase of Exosome Productivity by Treatment with Hyaluronic Acid
[0405] 10-1. Increase in Number of Exosomes by Treatment with Hyaluronic Acid
[0406] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 13 and
TABLE-US-00013 TABLE 13 Yield (per 10.sup.6 cells) MSC-Exo HA MSC-Exo No. of exosome particles (10.sup.9) 2.3 11.93 (MSC: non-treated mesenchymal stem cells, HA MSC: Hyaluronic acid-treated mesenchymal stem cells)
[0407] As can be seen in Table 13 and
[0408] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0409] 10-2. Increase in Content of Exosomal Protein by Treatment with Hyaluronic Acid
[0410] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 14 and
TABLE-US-00014 TABLE 14 Yield (per 10.sup.6 cells) MSC-Exo HA MSC-Exo Exosomal protein (ug) 2.0 10.7 (MSC: non-treated mesenchymal stem cells, HA MSC: Hyaluronic acid-treated mesenchymal stem cells)
[0411] As can be seen in Table 14 and
[0412] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0413] 10-3. Increase in Content of Exosomal RNA by Treatment with Hyaluronic Acid
[0414] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 15 and
TABLE-US-00015 TABLE 15 Yield (per 10.sup.6 cells) MSC-Exo HA MSC-Exo Exosomal RNA (ng) 31.0 165.0 (MSC: non-treated mesenchymal stem cells, HA MSC: Hyaluronic acid-treated mesenchymal stem cells)
[0415] As can be seen in Table 15 and
[0416] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENT EXAMPLE 11
Functional Reinforcement of Stem Cells by Treatment with Substance P
[0417] 11-1. Increase of Stem Cell Proliferation by Treatment with Substance P
[0418] Mesenchymal stem cells and the substance P-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0419] As can be seen in
[0420] 11-2. Increase of Stem Cell Stemness by Treatment with Substance P
[0421] Mesenchymal stem cells and the substance P-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0422] As can be seen in
EXPERIMENTAL EXAMPLE 12
Increase of Exosome Productivity by Treatment with Substance P
[0423] 12-1. Increase in Number of Exosomes by Treatment with Substance P
[0424] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 16 and
TABLE-US-00016 TABLE 16 Yield (per 10.sup.6 cells) MSC-Exo SubsP MSC-Exo No. of exosome particles (10.sup.9) 2.3 9.93 (MSC: non-treated mesenchymal stem cells, SubsP MSC: Substance P-treated mesenchymal stem cells)
[0425] As can be seen in Table 16 and
[0426] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0427] 12-2. Increase in Content of Exosomal Protein by Treatment with Substance P
[0428] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 17 and
TABLE-US-00017 TABLE 17 Yield (per 10.sup.6 cells) MSC-Exo SubsP MSC-Exo Exosomal protein (ug) 2.0 10.37 (MSC: non-treated mesenchymal stem cells, SubsP MSC: Substance P-treated mesenchymal stem cells)
[0429] As can be seen in Table 17 and
[0430] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0431] 12-3. Increase in Content of Exosomal RNA by Treatment with Substance P
[0432] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 18 and
TABLE-US-00018 TABLE 18 Yield (per 10.sup.6 cells) MSC-Exo SubsP MSC-Exo Exosomal RNA (ng) 31.0 150.84 (MSC: non-treated mesenchymal stem cells, SubsP MSC: Substance P-treated mesenchymal stem cells)
[0433] As can be seen in Table 18 and
[0434] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENT EXAMPLE 13
Functional Reinforcement of Stem Cells by Treatment with Resveratrol
[0435] 13-1. Increase of Stem Cell Proliferation by Treatment with Resveratrol
[0436] Mesenchymal stem cells and the resveratrol-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0437] As can be seen in
[0438] 13-2. Increase of Stem Cell Stemness by Treatment with Resveratrol
[0439] Mesenchymal stem cells and the resveratrol-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0440] As can be seen in
EXPERIMENTAL EXAMPLE 14
Increase of Exosome Productivity by Treatment with Resveratrol
[0441] 14-1. Increase in Number of Exosomes by Treatment with Resveratrol
[0442] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 19 and
TABLE-US-00019 TABLE 19 Yield (per 10.sup.6 cells) MSC-Exo Resv MSC-Exo No. of exosome particles (10.sup.9) 2.3 11.9 (MSC: non-treated mesenchymal stem cells, Resv MSC: Resveratrol-treated mesenchymal stem cells)
[0443] As can be seen in Table 19 and
[0444] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0445] 14-2. Increase in Content of Exosomal Protein by Treatment with Resveratrol
[0446] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 20 and
TABLE-US-00020 TABLE 20 Yield (per 10.sup.6 cells) MSC-Exo Resv MSC-Exo Exosomal protein (ug) 2.0 10.78 (MSC: non-treated mesenchymal stem cells, Resv MSC: Resveratrol-treated mesenchymal stem cells)
[0447] As can be seen in Table 20 and
[0448] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0449] 14-3. Increase in Content of Exosomal RNA by Treatment with Resveratrol
[0450] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 21 and
TABLE-US-00021 TABLE 21 Yield (per 10.sup.6 cells) MSC-Exo Resv MSC-Exo Exosomal RNA (ng) 31.0 145.15 (MSC: non-treated mesenchymal stem cells, Resv MSC: Resveratrol-treated mesenchymal stem cells)
[0451] As can be seen in Table 21 and
[0452] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
EXPERIMENT EXAMPLE 15
Functional Reinforcement of Stem Cells by Treatment with Lanifibranor
[0453] 15-1. Increase of Stem Cell Proliferation by Treatment with Lanifibranor
[0454] Mesenchymal stem cells and the lanifibranor-pretreated mesenchymal stem cells of the Example were each seeded at a density of 3,000 cells/well into 96-well plates containing 100 μL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per well and cultured for 24 hours in a CO.sub.2 incubator. Then, CCK solution (10 μL/well) was added, and incubation was continued for 4 hours in the CO.sub.2 incubator, followed by reading absorbance at a wavelength of 450 nm (420-480 nm) to determine proliferation.
[0455] As can be seen in
[0456] 15-2. Increase of Stem Cell Stemness by Treatment with Lanifibranor
[0457] Mesenchymal stem cells and the lanifibranor-pretreated mesenchymal stem cells of the Example were each seeded at a density of 1,000 cells/dish into 100-mm cell culture dishes containing 100 mL of a culture medium [high glucose DMEM (Gibco, Cat no. 11995-065), 10% fetal bovine serum (HyClone), 1% MEM non-essential amino acids solution (100×) (Gibco, Cat no. 11140-050)] per dish and cultured for 21 days. The cell-attached dishes were washed twice with PBS, followed by fixation with 95% methanol at room temperature for about 2 min. The fixed cells were washed three times with PBS and stained with 0.5% crystal violet [(sigma, C-3886, USA) 5 g, methanol 100 ml] for 5 min. After water washing and drying at room temperature, colonies, each composed of 50 or more cells, were counted for comparison.
[0458] As can be seen in
EXPERIMENTAL EXAMPLE 16
Increase of Exosome Productivity by Treatment with Lanifibranor
[0459] 16-1. Increase in Number of Exosomes by Treatment with Lanifibranor
[0460] The exosomes isolated in the Example were counted the nanoparticle tracking assay (NanoSight NS300, Malvern). For comparison, exosomes were counted in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 22 and
TABLE-US-00022 TABLE 22 Yield (per 10.sup.6 cells) MSC-Exo Lani-MSC-Exo No. of exosome particles (10.sup.9) 2.30 11.4 (MSC: non-treated mesenchymal stem cells, Lani-MSC-Exo: Lanifibranor-treated mesenchymal stem cells)
[0461] As can be seen in Table 22 and
[0462] From the results, it is understood that treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure increases the quantity of exosomes produced by the mesenchymal stem cells.
[0463] 16-2. Increase in Content of Exosomal Protein by Treatment with Lanifibranor
[0464] Proteins were isolated from the exosomes isolated in the Examples, using an exosome protein isolation kit (total exosome RNA and protein isolation kit, Invitrogen), and quantitatively measured by reading absorbance at 595 nm through Bradford analysis. For comparison, proteins were isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 23 and
TABLE-US-00023 TABLE 23 Yield (per 10.sup.6 cells) MSC-Exo Lani-MSC-Exo Exosomal protein (ug) 2.0 9.2 (MSC: non-treated mesenchymal stem cells, Lani-MSC-Exo: Lanifibranor-treated mesenchymal stem cells)
[0465] As can be seen in Table 23 and
[0466] From the results, it is understood that the content of exosomal proteins as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
[0467] 16-3. Increase in Content of Exosomal RNA by Treatment with Lanifibranor
[0468] Using an RNA isolation kit (total exosome RNA and protein isolation kit, Invitrogen), total exosomal RNA was isolated from the exosomes isolated in the Example, followed by measuring RNA concentrations with the aid of a NanoDrop spectrometer. For comparison, total exosomal RNA was isolated from exosomes and quantitated in the same manner with the exception that the cells were not pretreated with any pretreatment substance. The results are given in Table 24 and
TABLE-US-00024 TABLE 24 Yield (per 10.sup.6 cells) MSC-Exo Lani-MSC-Exo Exosomal RNA (ng) 27.00 110.34 (MSC: non-treated mesenchymal stem cells, Lani-MSC-Exo: Lanifibranor-treated mesenchymal stem cells)
[0469] As can be seen in Table 24 and
[0470] From the results, it is understood that the content of exosomal RNA as well as the number of exosomes are increased by treatment of mesenchymal stem cells with the stem cell pretreatment substance according to the present disclosure.
CONCLUSION
[0471] When treated with the stem cell pretreatment substance (exendin-4, phorbol 12-myristate 13-acetate (PMA), interferon-γ, tetrandrine, hyaluronic acid, substance P, resveratrol, or lanifibranor) according to the present disclosure, mesenchymal stem cells were found to exhibit increased stemness. It was also found that the mesenchymal stem cells treated with the stem cell pretreatment substance according to the present disclosure increased in the productivity of exosomal protein and the content of exosomal RNA as well as producing an increased quantity of exosomes.
[0472] Therefore, the stem cell pretreatment substance (exendin-4, phorbol 12-myristate 13-acetate (PMA), interferon-γ, tetrandrine, hyaluronic acid, substance P, resveratrol, or lanifibranor) according to the present disclosure promotes the production of stem cell-derived exosomes and, as such, is expected to be used in the production of highly functional stem cells and the mass production of exosomes.
INDUSTRIAL APPLICABILITY
[0473] The present disclosure relates to a composition for promoting the production of stem cell-derived exosomes and increasing the stemness of stem cells.