Method for promoting stem cell proliferation and proliferation

Abstract

To provide a method for multiplying or proliferating undifferentiated stem cells.SOLUTION: A method includes bringing a population of stem cells in contact with genus Ganoderma immunoregulatory protein having an effective amount of a specific sequence or a recombinant thereof or a fragment thereof having a specific sequence.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 2. Detailed Description of the Invention This application claims the benefit under 35 U.S.C. § 119 of U.S. Provisional Application No. 63 / 087,677, filed October 5, 2020. The entire contents of the prior application are incorporated herein by reference.

[0002] This application contains a Sequence Listing that has been filed electronically in ASCII format and is incorporated by reference in its entirety. The ASCII copy, created on April 20, 2021, is named G4590-10300US_SeqListing.txt and is 3 kilobytes in size.

[0003] FIELD OF THE DISCLOSURE The present disclosure relates to methods for promoting stem cell proliferation. In particular, the present disclosure provides methods for promoting stem cell proliferation using immunomodulatory proteins. [Background technology]

[0004] Pluripotent stem cells are characterized by their ability to self-renew and differentiate. Stem cells are typically characterized by morphology and the presence of characteristic markers. Differentiation of stem cells may result in phenotypic changes in cell morphology. Differentiation of stem cells may also result in the loss of stem cell markers or telomerase activity. Differentiation of stem cells may result in the acquisition of additional markers or morphology characteristic of one or more of the three embryonic germ layers: ectoderm, mesoderm, and endoderm. Currently, culture reagents that eliminate differentiated cells and promote the proliferation of undifferentiated cells are used to propagate undifferentiated pluripotent stem cells.

[0005] However, there is a need in the art to develop methods for promoting the proliferation of undifferentiated stem cells. Summary of the Invention

[0006] The present disclosure has surprisingly found that Ganoderma immunomodulatory proteins or recombinant forms thereof provide advantageous efficacy in growing or propagating undifferentiated stem cells and maintaining stem cells in an undifferentiated state.

[0007] In one aspect, the present disclosure provides a method for growing or propagating undifferentiated stem cells, comprising contacting a population of stem cells with an effective amount of a Ganoderma genus immunomodulatory protein, a recombinant thereof, or a fragment thereof.

[0008] In another aspect, the present disclosure also provides a stem cell culture supplement comprising a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof, in an amount sufficient to grow or propagate undifferentiated stem cells, and a chemically defined stem cell culture medium.

[0009] In one embodiment, the stem cells described herein remain undifferentiated.

[0010] In one embodiment, the Ganoderma immunomodulatory protein or recombinant thereof described herein comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, the recombinant Ganoderma immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 4. In one embodiment, the fragment of the Ganoderma immunomodulatory protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-2.

[0011] The sequences of SEQ ID NOs: 1 to 4 are shown below. TIFF0007785496000001.tif46165TIFF0007785496000002.tif26160

[0012] In one embodiment, the amount of Ganoderma genus immunomodulatory protein or a recombinant or fragment thereof is less than 15 μg / mL, preferably less than 12.5 μg / mL. In some embodiments, the amount of Ganoderma genus immunomodulatory protein or a recombinant or fragment thereof is about 1.0 μg / mL to about 15 μg / mL, about 1.5 μg / mL to about 15 μg / mL, about 2.0 μg / mL to about 15 μg / mL, about 2.5 μg / mL to about 15 μg / mL, about 3.0 μg / mL to about 15 μg / mL, about 3.5 μg / mL to about 15 μg / mL, about 4.0 μg / mL to about 15 μg / mL, about 4.5 μg / mL to about 15 μg / mL, about 5.0 μg / mL to about 15 μg / mL, or about 5.5 μg / mL to about 15 μg / mL. mL, about 6.0μg / mL to about 15μg / mL, about 6.5μg / mL to about 15μg / mL, about 7.0μg / mL to about 15μg / mL, about 7.5μg / mL to about 15μg / mL, about 8.0μg / mL to about 15μg / mL, about 8.5μg / mL to about 15μg / m L, about 9.0μg / mL to about 15μg / mL, about 9.5μg / mL to about 15μg / mL, about 10.0μg / mL to about 15μg / mL, about 10.5μg / mL to about 15μg / mL, about 11.0μg / mL to about 15μg / mL, about 11.5μg / mL to about 15μg / mL, 1.0μg / mL to approximately 12.5μg / mL, approximately 1.5μg / mL to approximately 12.5μg / mL, approximately 2.0μg / mL to approximately 12.5μg / mL, approximately 2.5μg / mL to approximately 12.5μg / mL, approximately 3.0μg / mL to approximately 12.5μg / mL, approximately 3.5μg / mL mL~about 12.5μg / mL, about 4.0μg / mL~about 12.5μg / mL, about 4.5μg / mL~about 12.5μg / mL, about 5.0μg / mL~about 12.5μg / mL, about 5.5μg / mL~about 12.5μg / mL, about 6.0μg / mL~about 12.5μg / mL mL, about 6.5 μg / mL to about 12.5 μg / mL, about 7.0 μg / mL to about 12.5 μg / mL, about 7.5 μg / mL to about 12.5 μg / mL, about 8.0 μg / mL to about 12.5 μg / mL, about 1.5 μg / mL to about 10 μg / mL, about 1.5 μg / mL to about 8.5 μg / mL, about 1.5 μg / mL to about 8.0 μg / mL, about 1.5 μg / mL to about 7.5 μg / mL, about 1.5 μg / mL to about 7.0 μg / mL, about 1.5 μg / mL to about 6.5 μg / mL, and about 1.5 μg / mL to about 6.0 μg / mL.In further embodiments, the amount of Ganoderma immunomodulatory protein or recombinant or fragment thereof is about 1.5 μg / mL, about 1.5625 μg / mL, about 3.0 μg / mL, about 3.125 μg / mL, about 6.25 μg / mL, or about 12.5 μg / mL.

[0013] In one embodiment, the population of stem cells is of human origin.

[0014] In one embodiment, the population of stem cells comprises a population of induced pluripotent stem (iPS) cells, a population of embryonic stem (ES) cells, a population of germ cells, a population of tissue-specific stem cells, or a population of adult stem cells, hi some embodiments, the stem cells are embryonic stem cells, mesenchymal stem cells (MSCs), bone marrow stromal cells, hematopoietic stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, or neural stem cells. [Brief explanation of the drawings]

[0015] [Figure 1-1] Figures 1A and 1B show the effect of GMI on ADSC proliferation. ADSCs (5 x 10 cells) were treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 3 days. After treatment, cells were harvested and the total cell number was analyzed using a hemocytometer (B). Phase-contrast images (A) were taken using an optical microscope. Experiments were performed in triplicate (n = 3). **p<0.01 and ***p<0.001 compared to control cells without GMI treatment. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 2] Figure 2 compares the expression levels of surface markers in ADSCs after GMI treatment. ADSCs (5 × 10 cells) were treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 3 days. After incubation, cells were harvested and stained with specific MSC-positive (CD29, CD146, CD166, CD106, CD90, CD105, CD73, CD44) and -negative (CD34, CD45, CD11b, CD19, HLA-DR) surface marker antibodies. Results were analyzed by flow cytometry (BD Accuri™ C6 Plus). Experiments were performed in triplicate (n = 3). [Figure 3-1] Figures 3A and 3B show the differentiation potential of control ADSCs or GMI-treated ADSCs. ADSCs were treated with different concentrations of GMI (0, 3, and 5 μg / ml) for 3 days. After treatment, control ADSCs (6 × 10 cells) and GMI-treated ADSCs (6 × 10 cells) were cultured in adipogenic differentiation medium for 21 days (A) or osteogenic differentiation medium for 14 days (B). Phase-contrast images of ADSCs were taken at the indicated time points. The upper and middle panels show changes in cell morphology after induction of adipogenic or osteogenic differentiation. The lower panel shows staining results after induction of adipogenic or osteogenic differentiation. The scale bar is 100 mm for osteogenic differentiation and 50 mm for adipogenic differentiation. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4-1] Figures 4A and 4B show the effect of GMI on BMSC proliferation. BMSCs (2 x 10 cells) were treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 3 days. After treatment, cells were harvested and the total cell number was analyzed using a hemocytometer (B). Phase-contrast images (A) were taken using an optical microscope. Experiments were performed in triplicate (n = 3). *p<0.05 and **p<0.01 compared to control cells without GMI treatment. [Figure 4-2] This is a continuation of Figure 4-1. [Figure 5] Figure 5 compares the expression levels of surface markers in BMSCs after GMI treatment. BMSCs (2 × 10 cells) were treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 3 days. After incubation, cells were harvested and stained with specific MSC-positive (CD29, CD146, CD166, CD106, CD90, CD105, CD73, CD44) and -negative (CD34, CD45, CD11b, CD19, HLA-DR) surface marker antibodies. Results were analyzed by flow cytometry (BD Accuri™ C6 Plus). Experiments were performed in triplicate (n = 3). [Figure 6-1]Figures 6A and 6B show the differentiation potential of control BMSCs or GMI-treated BMSCs. BMSCs were treated with different concentrations of GMI (0, 3, and 5 μg / ml) for 3 days. After treatment, control BMSCs (6 × 10 cells) and GMI-treated BMSCs (6 × 10 cells) were cultured in adipogenic differentiation medium for 21 days (A) or osteogenic differentiation medium for 14 days (B). Phase-contrast images of BMSCs were taken at the indicated time points. The upper and middle panels show changes in cell morphology after induction of adipogenic or osteogenic differentiation. The lower panels show staining results after induction of adipogenic or osteogenic differentiation. The scale bar is 100 mm for the osteogenic differentiation photographs and 50 mm for the adipogenic differentiation photographs. [Figure 6-2] This is a continuation of Figure 6-1. DETAILED DESCRIPTION OF THE INVENTION

[0016] Unless otherwise defined, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference.

[0017] In this application, unless otherwise stated, the use of the singular includes the plural, the article "a" or "an" means "at least one," and the use of "or" means "and / or."

[0018] As used herein, "growth" refers to the process of becoming bigger, longer, or more numerous, or an increase in size, number, or volume.

[0019] As used herein, "differentiation" refers to the developmental process by which cells or tissues acquire increasing levels of organization or complexity and more specialized functions.

[0020] As used herein, "effective amount" means an amount sufficient to propagate undifferentiated stem cells and maintain the stem cells in an undifferentiated state.

[0021] As used herein, a "subject" refers to a human or non-human animal.

[0022] As used herein, "pluripotent stem cells" refer to cells that can differentiate into cells of all three embryonic germ layers that form the body's organs, nervous system, skin, muscle, and skeleton, but cannot differentiate into the embryonic components of the trophoblast and placenta.

[0023] As used herein, "stem cell" refers to an undifferentiated cell that has high proliferative potential, the ability to self-renew (to produce more stem cells by cell division), and can generate daughter cells that can ultimately differentiate into two or more distinct cell phenotypes.

[0024] As used herein, "proliferation" refers to the process by which a cell grows and divides to produce daughter cells that have the same phenotype as the cell.

[0025] As used herein, "propagation" refers to the expansion of a population of cells by the successive division of a single cell into identical daughter cells.

[0026] As used herein, "cell culture" or "culture" refers to the maintenance of cells in an artificial (eg, in vitro) environment.

[0027] As used herein, "cell culture medium," "culture medium," or "medium" (all plural media) refers to a nutritional composition that supports the cultivation and / or growth of cells.

[0028] "Chemically defined media" refers to growth media suitable for culturing human or animal cells in vitro or ex vivo, in which all chemical components are known. In certain embodiments, chemically defined media are completely free of animal-derived components and do not contain fetal bovine serum, bovine serum albumin, or human serum albumin, as these products are of bovine or human origin and contain complex mixtures of albumin and lipids.

[0029] Surprisingly, it has been found that culturing stem cells with Ganoderma lucidum immunomodulatory proteins or recombinant or fragments thereof can cause the stem cells to proliferate or multiply but not differentiate. That is, Ganoderma lucidum immunomodulatory proteins or recombinant or fragments thereof can cause undifferentiated stem cells to proliferate or multiply and maintain them in an undifferentiated state for several passages. The resulting undifferentiated stem cells are multipotent or pluripotent.

[0030] Thus, the present disclosure provides a method for growing or propagating undifferentiated stem cells, comprising contacting a population of stem cells with an effective amount of a Ganoderma genus immunomodulatory protein or a recombinant or fragment thereof.

[0031] Particular embodiments of stem cells include embryonic stem cells, mesenchymal stem cells (MSCs), bone marrow stromal cells, hematopoietic stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, and neural stem cells. A particular embodiment of a modified stem cell is an MSC.

[0032] Preparation of Ganoderma lucidum immunomodulatory protein or a recombinant or fragment thereof is described in US 7,601,808. In particular, in US 7,601,808, Ganoderma lucidum immunomodulatory protein is designated as GMI, a recombinant Ganoderma lucidum immunomodulatory protein is designated as reGMI, and fragments of Ganoderma lucidum immunomodulatory protein are designated as SEQ ID NOs: 2 and 3. The Ganoderma lucidum immunomodulatory protein or a recombinant or fragment thereof is present in an amount of less than 15 μg / mL, preferably less than 12.5 μg / mL.

[0033] Ganoderma immunomodulatory proteins or recombinant or fragments thereof can be added to chemically defined stem cell culture media containing essential mineral nutrients, essential salts, essential amino acids, one or more nutritional supplements, and hyaluronic acid. Exemplary commercially available media include classic formulations such as DMEM, DMEM:F12, RPMI, and modifications thereof.

[0034] The present disclosure also provides a stem cell culture supplement comprising a Ganoderma lucidum immunomodulatory protein, a recombinant thereof, or a fragment thereof, in an amount sufficient to grow or propagate undifferentiated stem cells, and a chemically defined cell culture medium.

[0035] Chemically defined cell culture media are known in the art for in vitro culture of stem cells. Ganoderma immunomodulatory proteins, recombinant forms thereof, or fragments thereof can be added to chemically defined cell culture media and chemically defined stem cell culture media for growing or propagating undifferentiated stem cells.

[0036] The culture medium may be used in all cell processing steps, including cryopreservation, thawing, resuspension, conditioning, expansion, or maintenance of cell populations, particularly cell populations comprising hematopoietic stem cells.

[0037] Cells supported in the culture media described herein can be cultured according to experimental conditions determined by the investigator, although it should be understood that optimal plating and culture conditions for a given animal cell type can be determined by one of ordinary skill in the art using only routine experimentation.

[0038] The following examples are offered to illustrate, but not to limit, the claimed invention. [Example]

[0039] Materials and Methods Culture of human bone marrow-derived and adipose tissue-derived mesenchymal stem cells Bone marrow-derived mesenchymal stem cells (BMSCs) extracted from the bone marrow of a 56-year-old donor and tissue-derived mesenchymal stem cells were obtained from ATCC (Manassas, VA, USA). Adipose-derived stem cells (ADSCs) were purchased from Lonza. ADSCs were seeded in 10-cm Petri dishes using mesenchymal stem cell basal medium (American Type Culture Collection, ATCC) containing MSC supplements (2% FBS, 5 ng / ml rhFGF basic, 5 ng / ml rhFGF acidic, and 5 ng / ml EGF) and L-alanyl-L-glutamine (2.4 mM). BMSCs were seeded in 10-cm Petri dishes using a Mesenchymal Stem Cell Growth Kit (ATCC) containing 7% FBS, 15 ng / ml rhIGF-1, 125 pg / ml rhFGF-β, and L-alanyl-L-glutamine (2.4 mM). The culture medium was changed 24 hours after seeding and then every 3 days. Upon reaching confluence, cells were detached with 0.15% trypsin for 4 minutes at 37°C, and the collected cells were passaged at the original cell seeding density every 10 days. Cell morphology was recorded using a phase-contrast microscope.

[0040] MSC proliferation ADSC(5×10 5 cells) and BMSCs (2 × 10 5Cells) were seeded onto 10 cm Petri dishes for 24 h and then treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 72 h. After treatment, cells were washed twice with DPBS and incubated with 1 ml of Accutase at 37°C for 5 min. After incubation, cells were harvested and washed with DPBS. Finally, the total cell number was counted using a hemocytometer.

[0041] Flow cytometry analysis Control and GMI-treated MSCs harvested from different passages were evaluated for cell surface marker expression by flow cytometry. Briefly, cells were harvested, washed with DPBS, and incubated for 30 minutes with a nonspecific blocking solution containing 1% bovine serum albumin. After blocking, cells were washed with DPBS and centrifuged to remove the blocking solution. Cells were then incubated with fluorescently conjugated anti-human antibodies for 45 minutes. Specifically, antibodies against CD29 (557332), CD106 (563525), CD146 (563619), and CD166 (565461) were purchased from BD Pharmingen, and the Human MSC Analysis Kit (562245) was also purchased from BD Pharmingen. An antibody against mouse IgG was used as a negative staining isotype control. Stained MSCs were resuspended in DPBS and further analyzed using flow cytometry (BD Accuri™ C6 Plus).

[0042] Adipogenic and osteogenic differentiation of MSCs To evaluate whether GMI treatment affects the differentiation potential of MSCs, cells were seeded and incubated with different concentrations of GMI in 10 cm Petri dishes for 3 days as previously described. After GMI treatment, cells were harvested and seeded into 24-well plates for adipogenic and osteogenic differentiation assays. Adipogenic and osteogenic differentiation assays were performed using the Mesenchymal Stem Cell Adipogenesis Kit and Mesenchymal Stem Cell Osteogenesis Kit (EMD Millipore Corp., MA, USA), respectively. For adipogenic differentiation, cells were treated with adipogenic induction medium containing 1 mM dexamethasone, 0.5 mM IBMX, 10 μg / ml insulin, and 100 mM indomethacin. For osteogenic differentiation, cells were treated with osteogenic induction medium containing 0.1 mM dexamethasone, 0.2 mM ascorbic acid diphosphate, and 10 mM glycerol diphosphate. When the cells reached 100% confluence, the medium was carefully aspirated from each well and 0.5 ml of adipogenic induction medium or osteogenic induction medium was added. The medium was replaced with fresh induction medium every 2–3 days. BMSCs were able to differentiate into adipocytes or osteocytes 21 or 14 days after induction. MSC differentiation was further analyzed in the following experiments.

[0043] histological staining For the adipogenic differentiation assay, the medium was carefully aspirated from each well, and the adipocytes were then fixed with 4% paraformaldehyde for 30 minutes at room temperature. After fixation, each well was rinsed three times with PBS and then twice with water. A sufficient amount of Oil Red O solution (0.4 ml / well) was added to each well. The plate was then incubated at room temperature for 50 minutes. After incubation, the Oil Red O solution was removed, and the wells were rinsed three times with 1 ml of water. Hematoxylin solution (0.4 ml / well) was used to stain cell nuclei for 5 minutes. Finally, the results were observed microscopically and photographed under a light microscope (magnification, 400x). For the osteogenic differentiation assay, the medium was carefully aspirated from each well, and the adipocytes were fixed by incubating in ice-cold 70% ethanol for 1 hour at room temperature. After fixation, the ethanol was carefully aspirated, and the wells were rinsed twice with water for 5 minutes. The water was aspirated, and enough alizarin red solution was added to cover the wells (0.5 ml / well), followed by incubation at room temperature for 30 minutes. After incubation, the alizarin red solution was removed, and the wells were washed four times with 1 ml of water. Then, 1 ml of water was added to each well to prevent the cells from drying out. Finally, the results were observed under a microscope and photographed under an optical microscope (magnification: 100x).

[0044] statistical analysis Statistical data are shown as mean ± SEM. Comparisons between two groups were performed using Student's t-test, and a p value of less than 0.05 was considered significant.

[0045] [Example 1] Low-dose GMI treatment induces ADSC proliferation GMI manufactured by Mycomagic Biotechnology Co. Ltd. was used in these examples. It is derived from and improved upon Ganoderma microsporum (see U.S. Patent No. 7,601,808, incorporated by reference in its entirety). Human adipose-derived stem cells (ADSC, PT-5006) were purchased from Lonza. Previous studies have shown that GMI inhibits tumor growth and even induces tumor cell death, but there is no evidence demonstrating the effect of GMI on ADSC proliferation. To elucidate the effect of GMI on ADSC proliferation, a cell proliferation assay was performed using a hemocytometer. Adipose-derived stem cells (5 × 10 5 ADSCs (cells) were seeded onto 10-cm Petri dishes using mesenchymal stem cell basal medium (American Type Culture Collection, ATCC) containing MSC nutrient supplements (2% FBS, 5 ng / ml rhFGF basic, 5 ng / ml rhFGF acidic, and 5 ng / ml EGF) and L-alanyl-L-glutamine (2.4 mM). After 24 h of incubation, the cells were treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 72 h. After treatment, the cells were harvested using 1 ml of Accutase solution, and the total cell number was counted using a hemocytometer (Figure 1B). Phase-contrast images (Figure 1A) were taken under an optical microscope (magnification, 100x). Figures 1A and 1B demonstrate that GMI significantly induced the proliferation of ADSCs.

[0046] Surface markers were consistently expressed on ADSCs after GMI treatment Antibodies used for flow cytometry analysis, such as anti-CD29-PE (557332), anti-CD146-AlexaFluor647 (563619), anti-CD166-BB515 (565461), and anti-CD106-Percp-Cy5.5 (563525), were purchased from BD Biosciences. The Human MSC Analysis Kit (562245) was purchased from BD Biosciences. After cell counting, the expression levels of cell surface markers were further analyzed. Control ADSCs and GMI-treated ADSCs were harvested and further stained with anti-CD29, anti-CD106, anti-CD146, anti-CD166, anti-CD90, anti-CD105, anti-CD73, anti-CD44, and a negative marker cocktail antibody for 30 minutes. After staining, cells were washed twice with PBS and resuspended in 1 ml of PBS. Cell surface markers of control and GMI-treated cells were assessed by flow cytometry (BD ACCURI™ C6 Plus). Eight MSC-specific surface markers were assessed: CD29, CD106, CD146, CD166, CD90, CD105, CD73, and CD44, as well as five other markers: CD34, CD11b, CD19, CD45, and HLA-DR. As shown in Figure 2, even after GMI treatment, over 90% of control and GMI-treated ADSCs expressed MSC-positive markers, such as CD29, CD166, CD90, CD105, CD73, and CD44. ADSCs showed lower levels of CD146 and CD106, but the CD146- and CD106-expressing populations were not affected by GMI treatment. Furthermore, negative surface markers of ADSCs, such as CD34, CD11b, CD19, CD45, and HLA-DR, were measured using a negative control antibody cocktail. As shown in Figure 2, after treatment with GMI (5 μg / ml), the population expressing MSC negative markers increased.

[0047] GMI-treated ADSCs maintain their adipogenic and osteogenic differentiation potential As shown in Figure 1, the results demonstrated that GMI can induce ADSC proliferation. To elucidate whether GMI treatment affects the adipogenic and osteogenic differentiation potential of ADSCs, ADSCs were treated with different concentrations of GMI (0, 3, and 5 μg / ml) for 3 days. After GMI treatment, ADSCs were harvested and seeded into 24-well plates for adipogenic and osteogenic differentiation assays. Adipogenic and osteogenic differentiation assays were performed using the Mesenchymal Stem Cell Adipogenesis Kit (SCR020) and Mesenchymal Stem Cell Osteogenesis Kit (SCR028, EMD Millipore Corp., MA, USA), respectively. When the cells reached 100% confluence, the medium was aspirated from each well, and 0.5 ml of adipogenic induction medium or osteogenic induction medium was added to each well. The medium was then replaced with fresh induction medium every 2–3 days. ADSCs were able to differentiate into adipocytes or osteocytes after 21 or 14 days. For the adipogenic differentiation assay (Figure 3A), the medium was aspirated from each well, and the adipocytes were then fixed by incubating them in 4% paraformaldehyde for 30 minutes at room temperature. After fixation, each well was rinsed three times with PBS and then twice with water. The water was removed, and Oil Red O solution (0.4 ml / well) was added to the wells. The wells were then incubated at room temperature for 50 minutes. The Oil Red O solution was removed, and the wells were rinsed three times with 1 ml of water. The resulting cells were stained with hematoxylin solution (0.4 ml / well) for 5 minutes. Finally, the results were observed microscopically and photographed under a light microscope (magnification, 400x). For the osteogenic differentiation assay (Figure 3B), the medium was aspirated from each well, and the bone cells were fixed by incubating them in ice-cold 70% ethanol for 1 hour at room temperature. After fixation, the ethanol was removed, and the bone cells were rinsed twice with water for 5 minutes. The water was aspirated and alizarin red solution was added to cover the wells (0.5 ml / well). The wells were incubated at room temperature for 30 minutes. After incubation, the alizarin red solution was removed and the wells were washed four times with 1 ml of water. To prevent the cells from drying out, 1 ml of water was added to each well. Finally, the results were observed under a microscope and photographed under a light microscope (magnification 0.01). , 100x). As shown in Figure 3A and Figure 3B, adipogenic ADSCs not only changed morphology to polygonal or round, but also produced abundant lipid droplets in the cytoplasm (shown in red). On the other hand, osteogenic ADSCs showed mineral deposition in both control and GMI-treated cells by alizarin staining. These observations indicated that GMI-treated ADSCs maintained their adipogenic and osteogenic differentiation potential.

[0048] [Example 2] Low-dose GMI treatment induces BMSC proliferation Human bone marrow-derived stem cells (BMSCs) were purchased from ATCC. Cell proliferation assays were performed using a hemocytometer. Previous studies have shown that GMI inhibits tumor growth and even induces tumor cell death, but there is no evidence demonstrating the effect of GMI on BMSC proliferation. To investigate the role of GMI on BMSC proliferation, BMSCs (2 × 10 5 BMSCs (BMSCs) were seeded onto 10-cm Petri dishes using a Mesenchymal Stem Cell Growth Kit (ATCC) containing 7% FBS, 15 ng / ml rhIGF-1, 125 pg / ml rhFGF-β, and L-alanyl-L-glutamine (2.4 mM). After 24 hours of incubation, the cells were treated with different concentrations of GMI (0, 1, 3, and 5 μg / ml) for 72 hours. After treatment, the cells were harvested using Accutase and the cell number was counted using a hemocytometer (Figure 4B). Phase-contrast images (Figure 4A) were taken under an optical microscope (magnification, 100x). Figures 4A and 4B demonstrate that GMI significantly induced the proliferation of BMSCs.

[0049] Surface markers were consistently expressed on BMSCs after GMI treatment Antibodies used for flow cytometry analysis, such as anti-CD29-PE (557332), anti-CD146-AlexaFluor647 (563619), anti-CD166-BB515 (565461), and anti-CD106-Percp-Cy5.5 (563525), were purchased from BD Biosciences. The Human MSC Analysis Kit (562245) was also purchased from BD Biosciences. After cell counting, the expression levels of cell surface markers were further analyzed. Control BMSCs and GMI-treated BMSCs were stained for 30 minutes with anti-CD29-PE, anti-CD106-Percp-Cy5.5, anti-CD146-AlexaFluor647, anti-CD166-BB515, anti-CD90-FITC, anti-CD105-Percp-Cy5.5, anti-CD73-APC, anti-CD44-PE, and a PE-conjugated negative marker cocktail antibody. After staining, cells were washed twice with PBS and resuspended in 1 ml of PBS. Cell surface markers of control and GMI-treated cells were evaluated by flow cytometry (BD ACCURI™ C6 Plus). Eight MSC-specific surface markers were evaluated: CD29, CD106, CD146, CD166, CD90, CD105, CD73, and CD44, as well as five other markers: CD34, CD11b, CD19, CD45, and HLA-DR. As shown in Figure 5, even after GMI treatment, over 90% of control and GMI-treated BMSCs expressed MSC-positive markers, such as CD29, CD166, CD90, CD105, CD73, and CD44. BMSCs exhibited lower levels of CD146 and CD106, but the CD146- and CD106-expressing populations were not affected by GMI treatment. Furthermore, negative surface markers of BMSCs, such as CD34, CD11b, CD19, CD45, and HLA-DR, were measured using a negative control antibody cocktail. As shown in Figure 5, after treatment with GMI (5 μg / ml), the population expressing MSC negative markers increased.

[0050] GMI-treated BMSCs maintain their adipogenic and osteogenic differentiation potential Figure 4 shows that GMI was able to induce BMSC proliferation. To elucidate whether GMI-induced BMSC proliferation affected adipogenic and osteogenic differentiation potential, adipogenic and osteogenic differentiation assays were performed. BMSCs were treated with different concentrations of GMI (0, 3, and 5 μg / ml) for 3 days. After GMI treatment, BMSCs were harvested and seeded into 24-well plates for adipogenic and osteogenic differentiation assays. Adipogenic and osteogenic differentiation assays were performed using the Mesenchymal Stem Cell Adipogenesis Kit and Mesenchymal Stem Cell Osteogenesis Kit (EMD Millipore Corp., MA, USA), respectively. When the cells reached 100% confluence, the medium was aspirated from each well, and 0.5 ml of adipogenic induction medium or osteogenic induction medium was added to each well. The medium was replaced with fresh induction medium every 2–3 days. BMSCs were able to differentiate into adipocytes or osteocytes 21 or 14 days after induction. For the adipogenic differentiation assay (Figure 6A), the medium was aspirated from each well, and the adipocytes were then fixed by incubating them in 4% paraformaldehyde for 30 minutes at room temperature. After fixation, each well was rinsed three times with PBS and then twice with water. The water was aspirated, and Oil Red O solution (0.4 ml / well) was added to the wells. The wells were incubated at room temperature for 50 minutes. After incubation, the Oil Red O solution was removed, and the wells were rinsed three times with 1 ml of water. Cell nuclei were stained with hematoxylin solution (0.4 ml / well) for 5 minutes. The cells were then observed under a microscope and photographed under a light microscope (magnification, 400x). For the osteogenic differentiation assay (Figure 6B), the medium was aspirated from each well, and the osteocytes were fixed by incubating them in ice-cold 70% ethanol for 1 hour at room temperature. After fixation, the ethanol was removed, and the wells were rinsed twice with water for 5 minutes. The water was aspirated, and Alizarin Red solution was added (0.5 ml / well) to cover the wells. The wells were incubated at room temperature for 30 minutes. After incubation, the Alizarin Red solution was removed, and the wells were washed four times with 1 ml of water. 1 ml of water was added to each well to prevent the cells from drying out.Finally, the results were observed microscopically and photographed under a light microscope (magnification, 100x). As shown in Figures 6A and 6B, BMSCs undergoing adipogenesis not only changed morphologically to polygonal or round shapes but also produced abundant lipid droplets in the cytoplasm (shown in red). Meanwhile, BMSCs undergoing osteogenic transformation showed mineral deposition in both control and GMI-treated cells by alizarin staining. Figure 6 shows that GMI-treated BMSCs maintained their adipogenic and osteogenic differentiation potential.

[0051] While the present invention has been described in conjunction with the specific embodiments set forth above, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are deemed to be within the scope of the present invention. The present disclosure includes the following embodiments. [1] A method for growing or propagating (dividing) undifferentiated stem cells, comprising contacting a population of stem cells with an effective amount of a Ganoderma genus immunomodulatory protein, a recombinant thereof, or a fragment thereof. [2] The method of embodiment 1, wherein the stem cells remain undifferentiated after said propagation. [3] The method of embodiment 1, wherein the Ganoderma genus immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 3. [4] The method of embodiment 1, wherein the recombinant Ganoderma lucidum immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 4. [5] The method described in embodiment 1, wherein the fragment of the Ganoderma genus immunomodulatory protein comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-2. [6] The method of embodiment 1, wherein the amount of Ganoderma lucidum immunomodulatory protein or its recombinant or fragment thereof is less than 15 μg / mL. [7] The method of embodiment 1, wherein the amount of Ganoderma lucidum immunomodulatory protein or its recombinant or fragment thereof is less than 12.5 μg / mL. [8] The method of embodiment 1, wherein the amount of Ganoderma genus immunomodulatory protein or its recombinant or fragment thereof is in the range of about 1.0 μg / mL to about 15 μg / mL. [9] The method of embodiment 1, wherein the amount of Ganoderma genus immunomodulatory protein or its recombinant or fragment thereof is in the range of about 1.5 μg / mL to about 12.5 μg / mL.

[10] The method of embodiment 1, wherein the amount of Ganoderma genus immunomodulatory protein or its recombinant or fragment thereof is in the range of about 1.5 μg / mL to about 6.5 μg / mL.

[11] The method of embodiment 1, wherein the population of stem cells is of human origin.

[12] The method of embodiment 1, wherein the population of stem cells comprises a population of induced pluripotent stem (iPS) cells, a population of embryonic stem (ES) cells, a population of germ cells, a population of tissue-specific stem cells, or a population of adult stem cells.

[13] The method of embodiment 1, wherein the stem cells are embryonic stem cells, mesenchymal stem cells (MSCs), bone marrow stromal cells, hematopoietic stem cells, adipose-derived stem cells, endothelial stem cells, dental pulp stem cells, or neural stem cells.

Claims

1. A method for expanding undifferentiated stem cells, the in vitro method comprising contacting a population of stem cells with an effective amount of a Ganoderma genus immunomodulatory protein, or a recombinant thereof, wherein the Ganoderma genus immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 3, and the recombinant Ganoderma genus immunomodulatory protein comprises the amino acid sequence of SEQ ID NO: 4, and the stem cells comprise embryonic stem (ES) cells, mesenchymal stem cells (MSCs), or tissue-specific stem cells.

2. The method of claim 1, wherein the stem cells remain undifferentiated after said expansion.

3. The method described in claim 1, wherein a population of stem cells is contacted with a Ganoderma genus immunomodulatory protein or a recombinant thereof in a culture medium, and the amount of Ganoderma genus immunomodulatory protein or a recombinant thereof in the culture medium is less than 15 μg / mL.

4. The method described in claim 1, wherein a population of stem cells is contacted with a Ganoderma genus immunomodulatory protein or a recombinant thereof in a culture medium, and the amount of Ganoderma genus immunomodulatory protein or a recombinant thereof in the culture medium is less than 12.5 μg / mL.

5. The method described in claim 1, wherein a population of stem cells is contacted with a Ganoderma genus immunomodulatory protein or a recombinant thereof in a culture medium, and the amount of Ganoderma genus immunomodulatory protein or a recombinant thereof in the culture medium is in the range of 1.0 μg / mL to 15 μg / mL.

6. The method described in claim 1, wherein a population of stem cells is contacted with a Ganoderma genus immunomodulatory protein or a recombinant thereof in a culture medium, and the amount of Ganoderma genus immunomodulatory protein or a recombinant thereof in the culture medium is in the range of 1.5 μg / mL to 12.5 μg / mL.

7. The method described in claim 1, wherein a population of stem cells is contacted with a Ganoderma genus immunomodulatory protein or a recombinant thereof in a culture medium, and the amount of Ganoderma genus immunomodulatory protein or a recombinant thereof in the culture medium is in the range of 1.5 μg / mL to 6.5 μg / mL.

8. The method of claim 1, wherein the population of stem cells is of human origin.

9. The method according to claim 1, wherein the stem cells are adipose tissue-derived stem cells or bone marrow-derived mesenchymal stem cells.

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