Patent Application
20240024367
Example embodiments relate to a method of preparing mesenchymal-like stem cells from human pluripotent stem cells and mesenchymal-like stem cells prepared thereby. Specifically, differentiation of human pluripotent stem cells cultured to passage 70 or lower after establishment of pluripotent cell lines is induced to produce embryoid bodies of various types from which cystic embryoid bodies are selected. The cystic embryoid bodies are loaded on a cell-permeable three-dimensional (3D) culture unit to isolate the mesenchymal-like stem cells therefrom. Only monolayer-shaped cell clusters are isolated from the isolated mesenchymal-like stem cells, and sizes of the monolayer-shaped cell clusters are uniformized to prepare mesenchymal-like stem cells. The high-purity mesenchymal-like stem cells as prepared in this way have anti-inflammatory efficacy and immunosuppression, and express CD90 and SOX2 at a level of 95% or greater. Further, example embodiments relate to mesenchymal-like stem cells prepared by the method, a therapeutic composition containing the mesenchymal-like stem cells or a transporter, and a composition or cosmetic composition for prevention or treatment of diseases that includes an active ingredient or exosomes secreted from the mesenchymal-like stem cells.
Mesenchyme refers to a tissue of a mesoderm corresponding to an intermediate layer formed by epithelial-mesenchyme transition (EMT) that occurs during embryonic development. Human embryonic stem cells and induced pluripotent stem cells are only cells that may explain such early occurrence in vitro. Human embryonic stem cells and induced pluripotent stem cells may be used as sources of cells with various functions due to their pluripotency.
Currently, studies on human pluripotent stem cells-derived mesenchymal stem cells or mesenchymal progenitor cells as well as adult-derived mesenchymal stem cells are actively being conducted. It was first known that in adults, progenitor cells capable of forming bones in bone marrow like the mesenchymal stem cells exist, and then it has been reported that mesenchymal stem cells may be separated from bone marrow, compact bone, peripheral blood, adipose tissues, and fetal tissues such as umbilical cord blood and an amniotic membrane.
The above adult-derived mesenchymal stem cells have various characteristics that may be useful for cell therapy. Therefore, in actual clinical treatment, the adult-derived mesenchymal stem cells may be applied to a range from a musculoskeletal system such as bone, cartilage and tendon, a cardiovascular system such as myocardial infarction and vascular damage diseases, a respiratory system such as acute and chronic lung damage, and an autoimmune system such as multiple sclerosis, lupus and rheumatoid arthritis to a nervous system such as Lou Gehrig's disease, Parkinson's disease and spinal injury and so on. That is, attempts have been made on cell therapy in various fields.
However, it is known that, unlike embryonic stem cells, general adult mesenchymal stem cells exhibit limited proliferation ability when sub-cultured in vitro for a long period of time, so that a division ability of one cell does not exceed 40 passages. Adult mesenchymal stem cells which are actually applied to cell therapy use only mesenchymal stem cells cultured to passage 7 or lower. Further, since there is no established cell lineage thereof, repetitive cell preparation from adults is required, thereby imposing limitations to the study thereof.
In order to overcome such limitations, studies to isolate and culture mesenchymal stem cells or mesenchymal progenitor cells from human pluripotent stem cells are being conducted. An existing method of obtaining mesenchymal stem cells or progenitor cells from the human pluripotent stem cells includes a method of separating cells representing a desired marker through a flow cytometer (fluorescent activated cell sorter (FACS)) and a method of applying large amounts and large types of cytokines and chemicals in order to induce differentiation thereof.
However, since the method using the flow cytometer uses a laser, not only viability of cells is lowered, but also a culture period is increased due to a small number of cells is obtained after the separation. The method of applying the cytokines and chemicals has a high cost problem due to continuous application and a problem about genetic stability after differentiation and proliferation. Further, it is difficult to effectively control pluripotency of embryonic stem cells.
Separation and proliferation methods of mesenchymal stem cells are very diverse, and approaches to characterization of mesenchymal stem cells are different depending on the origin of tissues and cells. Thus, the International Society for Cell Therapy (ISCT) has proposed three criteria for defining human mesenchymal stem cells. First, the human mesenchymal stem cells need to be adherent when maintained under standard culture conditions. Second, in analyzing surface antigens of the human mesenchymal stem cells, CD90, CD44, CD73, and CD105 need to be expressed at 95% or higher levels, whereas CD45, CD34, CD14 and CD19 need to be expressed at 2% or less level, and expression of undifferentiated control markers Oct3/4, Tra-1-60, Tra-1-81 and immune rejection antigen human leukocyte antigen-antigen D related (HLA-DR) needs to be prevented. Third, the human mesenchymal stem cells need to be differentiated into osteogenic cells, adipogenic cells, and chondrogenic cells in vitro.
Cluster of Differentiation 90 (CD90) as a representative marker defining mesenchymal stem cells is a major factor involved in immune regulation of mesenchymal stem cells. CD90-expressing mesenchymal stem cells are known to upregulate sHLA-G and IL-10 to inhibit proliferation of peripheral blood mononuclear cells (PBMC) activated by phytohemagglutinin (PHA). A human leukocyte antigen G (HLA-G) is a non-classical MHC class I gene and exists in the form of cell membrane conjugates (HLA-G1, -G2, -G3, -G4) and water-soluble forms (sHLA-G5, -G6, and G7). However, the HLA-G has been reported to exhibit immune tolerogenic functions for innate and adaptive cellular responses that have an influence on cytotoxicity of NK and CD81+ T cells and activity of CD41+ T cells. Currently, the HLA-G is understood as a molecule that plays an important role in immune tolerance. Furthermore, it is known that the HLA-G has a function of modulating decidual mononuclear cells into T helper type 2 (Th2) cytokine profiles through regulation of cytokine secretion. Modulation into Th2 cytokine profiles is associated with suppression of immune function. IL-10 as one of Th2 cytokines has a wide range of biological functions such as anti-inflammatory efficacy and immunosuppression. According to a number of reports, in terms of an association between the HLA-G and IL-10, the IL-10 induces expression of the HLA-G, and the HLA-G stimulates expression of the IL-10.
Sex-determining region Y-box 2 (SOX2) is a well-known key transcription factor in human embryonic stem cells and induced pluripotent stem cells, and plays an important role in maintaining cellular pluripotency. SOX2 is expressed in some adult stem cells. It is known that SOX2 has an important influence on differentiation ability and proliferation of cells through regulation of DKK1 and cMyc that are Wnt signaling soluble inhibitors. Thus, a function of SOX2 expressed in human pluripotent stem cells and a function of SOX2 expressed in mesenchymal stem cells are regarded as being different from each other to function in different roles.
Currently, biological or clinical interest in stem cell therapeutics is continuously increasing mainly due to cell characteristics such as immune suppression and tolerance. Human pluripotent stem cells-derived mesenchymal-like stem cells according to the present disclosure are mesenchymal stem cells characterized by expressing CD90 and SOX2 at a level of 95% or higher, and have not been reported to date. This not only satisfies the interest of the research area, but may be given a sufficient meaning from the perspective of the clinical area.
It is required to develop a method for preparation of mesenchymal stem cells that may increase cell viability to produce high-purity mesenchymal-like stem cells, effectively control human pluripotent stem cells, and maintain genetic stability of the prepared mesenchymal-like stem cells.
An aspect provides a method of preparing mesenchymal-like stem cells that meet the minimum requirement of human mesenchymal stem cells regulated by the International Society for Cell Therapy.
Another aspect provides a method for preparing mesenchymal-like stem cells, the method including: (a) preparing human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines; (b) inducing differentiation of the human pluripotent stem cells to produce embryoid bodies and selecting cystic embryoid bodies therefrom; (c) loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells therefrom; (d) isolating only monolayer-shaped cell clusters from the mesenchymal-like stem cells passing through the cell-permeable 3D culture unit; and (e) uniformizing each of longitudinal and transverse sizes of the monolayer-shaped cell clusters to 100 μm to 500 μm, and culturing the mesenchymal-like stem cells, in which the mesenchymal-like stem cells have anti-inflammatory efficacy and immunosuppression, and express CD90 and SOX2 at a level of 95% or greater. Further, the present disclosure relates to human pluripotent stem cells-derived mesenchymal-like stem cells as prepared by the above preparation method.
However, the aspects to be achieved by the present disclosure is not limited to the aspects mentioned above. Other aspects that are not mentioned will be clearly understood by those of ordinary skill in the art from the following description.
According to example embodiments, by a mesenchymal-like stem cells preparation method, mesenchymal-like stem cells may be separated, cultured and proliferated at high efficiency and high purity. Further, according to the mesenchymal-like stem cells preparation method, novel human pluripotent stem cells-derived mesenchymal-like stem cells which have anti-inflammatory efficacy and immunosuppression, and highly express CD90 and SOX2 at a level of 95% or greater may be prepared from the human pluripotent stem cells.
Mesenchymal-like stem cells prepared by a method of preparing mesenchymal-like stem cells according to an example embodiment may exhibit anti-inflammatory efficacy and immunosuppression.
Mesenchymal-like stem cells prepared by a method of preparing mesenchymal-like stem cells according to an example embodiment may exhibit anti-inflammatory efficacy and immunosuppression according to specific marker expression patterns.
Mesenchymal-like stem cells prepared by a method of preparing mesenchymal-like stem cells according to an example embodiment may express CD90 and SOX2 at a level of 95% or greater, and may simultaneously meet minimal requirements and additional features as human mesenchymal stem cells.
Mesenchymal-like stem cells prepared by a method of preparing mesenchymal-like stem cells according to an example embodiment may be applied to a therapeutic composition or a transporter containing the same.
Active ingredients or exosomes secreted from mesenchymal-like stem cells prepared by a method of preparing mesenchymal-like stem cells according to an example embodiment may be applied to a composition or a cosmetic composition for preventing or treating diseases that contains the same.
However, the effect of the present disclosure is not limited to the above effect. It is to be understood that the present disclosure includes all possible effects deduced from the configurations of the disclosure described in the detailed description or claims.
These and/or other aspects, features, and advantages of the disclosure will become apparent and more readily appreciated from the following description of example embodiments, taken in conjunction with the accompanying drawings of which:
Hereinafter, example embodiments will be described in detail with reference to the accompanying drawings. However, since various changes may be made to the example embodiments, the scope of the rights of the patent application is not limited or limited by these example embodiments. It should be understood that all modifications, equivalents or substitutes to the example embodiments are included within the scope of the rights of the patent application.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.
Unless otherwise defined, all terms including technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
When describing the example embodiments with reference to the accompanying drawings, like reference numerals refer to like constituent elements and a repeated description related thereto will be omitted. In the description of example embodiments, detailed description of well-known related structures or functions will be omitted when it is deemed that such description will cause ambiguous interpretation of the present disclosure.
Also, the terms “first,” “second,” “A,” “B,” “(a),” “(b),” and the like may be used herein to describe components according to example embodiments. Each of these terminologies is not used to define an essence, order or sequence of a corresponding component but used merely to distinguish the corresponding component from other component(s).
A component having a common function with a component included in an example embodiment is described using a like name in another example embodiment. Unless otherwise described, description made in an example embodiment may be applicable to another example embodiment and detailed description within a duplicate range is omitted.
According to one aspect, there is provided a method of preparing mesenchymal-like stem cells, the method including: (a) preparing human pluripotent stem cells cultured to passage 70 or lower after establishment of cell lines; (b) inducing differentiation of the human pluripotent stem cells to produce embryoid bodies and selecting cystic embryoid bodies therefrom; (c) loading the cystic embryoid bodies on a cell-permeable three-dimensional (3D) culture unit to isolate mesenchymal-like stem cells therefrom; (d) isolating only monolayer-shaped cell clusters from the mesenchymal-like stem cells passing through the cell-permeable 3D culture unit; and (e) uniformizing each of longitudinal and transverse sizes of the monolayer-shaped cell clusters to 100 μm to 500 μm, and culturing the mesenchymal-like stem cells, in which the mesenchymal-like stem cells have anti-inflammatory efficacy and immunosuppression, and express CD90 and SOX2 at a level of 95% or greater.
According to an example embodiment, the cell-permeable 3D culture unit may be made of any one or more selected from a group consisting of nylon, fiber, polyethylene, polypropylene, graphene, titanium, copper, nickel, silver, gold, and platinum.
According to another aspect, there are provided human pluripotent stem cells-derived mesenchymal-like stem cells prepared by the method for preparing mesenchymal-like stem cells, in which the mesenchymal-like stem cells express matrix metalloproteinase-1 (MMP-1) protein and HGF protein at a higher level than bone marrow-derived mesenchymal stem cells express MMP-1 protein and HGF protein, and express CD95 at a lower level than the bone marrow-derived mesenchymal stem cells express CD95, and express CD90 and SOX2 at a level of 95% or greater.
According to an example embodiment, the mesenchymal-like stem cells may express the MMP-1 protein at a level higher by 15 times or greater than bone marrow-derived mesenchymal stem cells express the MMP-1 protein, and express HGF protein at a level higher by 2 times or greater than bone marrow-derived mesenchymal stem cells express HGF protein.
According to an example embodiment, the mesenchymal-like stem cells may express CD95 at a level lower by 15 times or greater than bone marrow-derived mesenchymal stem cells express CD95.
According to an example embodiment, the mesenchymal-like stem cells may express inflammation-regulating genes at a level higher by 2 times to 100 times or greater than bone marrow-derived mesenchymal stem cells express the same.
The inflammation regulating gene may be any one or more selected from a group consisting of Gata3, Adora2a, Gps2, Psma1, Pbk, Lrfn5, Cdh5, Apoe, Foxf1, Tek, Cx3c11, Ptger4, Acp5, Bcr, Socs5, and Mdk.
According to an example embodiment, the mesenchymal-like stem cells may simultaneously express Gata3, Adora2a and Gps2 genes.
According to an example embodiment, the mesenchymal-like stem cells may express the immunosuppression gene at a level higher by 2 times to 100 times or greater than bone marrow-derived mesenchymal stem cells express the same. The immunosuppression gene may be any one or more selected from a group consisting of Gata3, Gps2, Psma1, Apoe, Foxf1, Tek, Cx3c11, Ptger4, Bcr, Socs5, and Mdk.
According to an example embodiment, the mesenchymal-like stem cells may simultaneously express Gata3, Gps2 and Psma1 genes.
According to another aspect, there is provided a therapeutic composition containing human pluripotent stem cells-derived mesenchymal-like stem cells prepared by the method for preparing mesenchymal-like stem cells, in which the therapeutic composition may be any one of a cellular therapeutic composition, a cellular gene therapeutic composition, a tissue engineering therapeutic composition, an anti-inflammatory therapeutic composition, an immune therapeutic composition, and a composition for preventing or treating cancer.
According to an example embodiment, the therapeutic composition may further include an active ingredient or exosomes secreted from the mesenchymal-like stem cells.
According to an example embodiment, the therapeutic composition may prevent or treat at least one disease selected from a group consisting of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft versus host disease, lower extremity ischemia disease, Burger's disease, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injury caused by various movements, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage from burns, skin damage from ultraviolet light, and inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system disease.
According to another aspect, there is provided a transporter containing human pluripotent stem cells-derived mesenchymal-like stem cells prepared by the method for preparing mesenchymal-like stem cells, in which the transporter carries a pharmaceutical composition therein.
According to another aspect, there is provided a composition for preventing or treating a disease, in which the composition may contain an active ingredient or exosomes secreted from human pluripotent stem cells-derived mesenchymal-like stem cells prepared by the method for preparing mesenchymal-like stem cells.
According to an example embodiment, the disease may include at least one selected from a group consisting of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft versus host disease, lower extremity ischemia disease, Burger's disease, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injury caused by various movements, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage from burns, skin damage from ultraviolet light, and inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system disease.
According to another aspect, there is provided a cosmetic composition including an active ingredient or exosomes secreted from human pluripotent stem cells-derived mesenchymal-like stem cells prepared by the method for preparing mesenchymal-like stem cells.
Preparation of Mesenchymal-Like Stem Cells with Enhanced Anti-Inflammatory Efficacy and Immunosuppression
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As used herein, “human pluripotent stem cells” are cells in an undifferentiated state and refer to stem cells capable of differentiating into all cells constituting the human body. The human pluripotent stem cells may be any one or more of human embryonic stem cells (hESC), human pluripotent stem cells via somatic cell nuclear transfer (SCNT-hPSC) and induced pluripotent stem cells (iPSC).
The term “mesenchymal-like stem cells” as used herein refers to multipotent mesenchymal stem cells (MMSC) that may differentiate into various cells including bone, cartilage, fat, and muscle cells. In other words, the “mesenchymal-like stem cells” are cells that have functions similar to mesenchymal stem cells (MSCs) present in the matrix, cartilage, bone tissue, and adipose tissue of bone marrow differentiated from the mesoderm via the division of fertilized eggs. That is, mesenchymal-like stem cells according to the present disclosure refer to novel mesenchymal stem cells which express CD90 and SOX2 at higher levels, thereby improving immunosuppression and anti-inflammatory efficacy, or pluripotency retention. Therefore, in the present specification, the expressions “mesenchymal-like stem cells” and “mesenchymal stem cells” are used separately from each other.
In the present specification, n passages or passage n (passage n or P n) means cells obtained by sub-culturing a parental cell line n times. For example, passage 70 or P 70 refers to a cell line obtained by sub-culturing the parent cell line 70 times. n is an integer.
The human pluripotent stem cells used for preparing the mesenchymal-like stem cells according to the present disclosure are characterized by being sub-cultured to passages less than or equal to 70 passages after establishment of the pluripotent cell line. Typically, in the preparation of mesenchymal stem cells using pluripotent stem cells, the pluripotent stem cells are used as they are without passage restrictions. When pluripotent stem cells are used as they are without passage restrictions, most of the surface antigens of prepared mesenchymal stem cells are under-expressed at a non-constant level, and the differentiation efficiency is very low. Furthermore, the prepared mesenchymal stem cells may not express CD90 or SOX2 at 95% level or greater. However, when using human pluripotent stem cells of passage 70 or lower according to the present disclosure, all surface antigens of the prepared mesenchymal-like stem cells were highly expressed at 95% level or greater at a constant level. In particular, mesenchymal-like stem cells expressing each of CD90 and SOX2 at 95% level or greater may be prepared. According to
More preferably, when using pluripotent stem cells of passage 70 or lower, and removing the multilayer-shaped cluster from the differentiated cell clusters that have passed through the cell-permeable 3D culture unit, and rather selecting only the monolayer-shaped clusters and mechanically uniformizing the sizes thereof and culturing the same, the surface antigen of the resulting mesenchymal stem cells is expressed at 95% level or greater. In particular, mesenchymal-like stem cells expressing each of CD90 and SOX2 among the surface antigens of mesenchymal stem cells at 95% level or greater may be prepared.
The term “cell-permeable 3D culture unit” as used herein is a cell-permeable instrument. That is, when inducing differentiation culture from embryoid bodies derived from embryonic stem cells, induced pluripotent stem cells, or stem cells via somatic cell nuclear transfer into mesenchymal-like stem cells, the cell-permeable 3D culture unit may allow epithelial-mesenchymal transition (EMT) that occurs during embryonic development to occur naturally. That is, mesenchymal-like stem cells may be separated, cultured and proliferated at high purity and high efficiency using the cell-permeable 3D culture unit.
The cell-permeable 3D culture unit is a cell-permeable artificial insert such as a culture plate for cell culture, a 3D insert for cell culture, or a mesh made of nylon or fibrous material. The cell-permeable 3D culture unit may be prepared using bioprinting technology. The cell-permeable 3D culture unit may be made of one or more of nylon, fiber, polyethylene, polypropylene, graphene, titanium, copper, nickel, silver, gold, and platinum. However, the cell-permeable 3D culture unit is not limited to the material as long as the cell-permeability is guaranteed.
Further, a culture medium for human pluripotent stem cells-derived embryoid body using the cell-permeable 3D culture unit may include EGM™-2-MV, MCDB, DMEM, MEM-α, STEMPRO-MSC, MesenCult-MSC medium. Desirably, the culture medium may be EGM™-2-MV, MCDB, DMEM or MEM-α medium. The present disclosure is not limited thereto. The culture medium for human pluripotent stem cells-derived embryoid body using the cell-permeable 3D culture unit may contain one or more additives among fetal bovine serum (FBS), serum replacement (SR), human serum (HS) and human platelet lysate (HPL). Specifically, the additive may be 1 to 20% of FBS, 1 to 20% of SR, 1 to 20% of Human Serum, or 1 to 20% of HPL. Desirably, the additive may be 5% FBS or 2.5 to 5% HPL. The present disclosure is not limited thereto.
The EGM™-2-MV culture medium contains endothelial basal medium and supplements including FBS (fetal bovine serum), hydrocortisone, hFGF-B (human fibroblast growth factor 2/basic fibroblast growth factor), VEGF (vascular endothelial growth factor), R3-IGF-1 (long R3 IGF-1 analog of human insulin-like growth factor 1), ascorbic acid, hEGF (human epidermal growth fact and GA-1000 (gentamicin and amphotericin B), but no BBE (bovine brain extract).
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The “Cluster of Differentiation 95 (CD95)” is an apoptosis receptor known as Fas Receptor (FasR), Apoptosis antigen 1 (APO-1), or tumor necrosis factor receptor superfamily member 6 (TNFRSF6). CD95 is located on the cell surface and induces apoptosis by interaction with a ligand known as CD95L (CD95 Ligand). Specifically, when a ligand known as Fas Ligand (FasL) or CD95L (CD95 Ligand) is bound to the CD95, apoptosis is promoted through Death-Inducing Signaling Complex (DISC). That is, when the CD95 is overexpressed in stem cells or progenitor cells, apoptosis signaling via CD95L is activated. To the contrary, stem cells in which CD95 is expressed at a relatively low level may exhibit excellent cell viability due to inhibition of apoptosis signaling.
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The mesenchymal-like stem cells according to the present disclosure may inhibit a proliferation ability when co-cultured with human peripheral blood-derived monocyte cells. More specifically, when co-cultured with human peripheral blood-derived monocyte cells, the mesenchymal-like stem cells according to the present disclosure may inhibit proliferation about 5 times to about 8 times or greater than bone marrow-derived mesenchymal stem cells may inhibit.
The mesenchymal-like stem cells according to the present disclosure may exhibit about 3.5 times or higher repair ability than bone marrow-derived mesenchymal stem cells may exhibit, in evaluation of cell migration and repair ability.
The mesenchymal-like stem cells according to the present disclosure may secrete MMP-1 protein as a gene related to cell survival at a level higher by about 15 times to about 16 times or greater than bone marrow-derived mesenchymal stem cells. Further, the mesenchymal-like stem cells may secrete HGF protein as a gene related to cell growth and tissue regeneration at a level higher by about 2 times or greater than bone marrow-derived mesenchymal stem cells.
The mesenchymal-like stem cells according to the present disclosure may express CD95 as an apoptosis receptor at a level lower by about 15 times or greater than the bone marrow-derived mesenchymal stem cells.
A therapeutic composition containing the mesenchymal-like stem cells prepared by the preparation method according to an example embodiment may include any one of a cellular therapeutic composition, a cellular gene therapeutic composition, a tissue engineering therapeutic composition, an anti-inflammatory therapeutic composition, an immune therapeutic composition, and a composition for preventing or treating cancer. Further, the therapeutic composition may further contain an active ingredient or exosomes secreted from the mesenchymal-like stem cells. Further, the therapeutic composition may prevent or treat at least one disease among multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft versus host disease, lower extremity ischemia disease, Burger's disease, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injury caused by various movements, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage from burns, skin damage from ultraviolet light, and inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system disease.
The active ingredients or exosomes secreted from the mesenchymal-like stem cells may include all functional substances related to immunosuppression, anti-inflammatory efficacy, cell survival, tissue regeneration, or inhibition of apoptosis. However, as long as the therapeutic composition has a material having functionality, the material is not limited thereto.
A transporter containing mesenchymal-like stem cells prepared by the preparation method according to an example embodiment may be provided. The transporter may be characterized by carrying a pharmaceutical composition therein.
A composition for preventing or treating a disease may be provided. The composition may contain active ingredients or exosomes secreted from the mesenchymal-like stem cells prepared by the preparation method according to an example embodiment. In this connection, the disease may include at least one of multiple sclerosis, systemic sclerosis, acute myocardial infarction, chronic myocardial infarction, chronic lung disease, acute lung disease, Crohn's disease, fecal incontinence, graft versus host disease, lower extremity ischemia disease, Burger's disease, foot ulcer, lupus, rheumatoid arthritis, acute and chronic pyelitis, inflammatory cystitis, interstitial cystitis, underactive bladder, overactive bladder, frozen shoulder, rotator cuff injury and rupture, musculoskeletal injury caused by various movements, knee cartilage injury, tinnitus, atopic dermatitis, psoriasis, skin damage from burns, skin damage from ultraviolet light, and inflammatory and immune system diseases including retinopathy, ischemic dementia, Alzheimer's dementia, spinal cord injury, Parkinson's disease, and central nervous system disease.
A cosmetic composition containing an active ingredient or exosomes secreted from mesenchymal-like stem cells prepared by the preparation method according to an example embodiment may be provided.
The human pluripotent stem cells-derived mesenchymal-like stem cells according to the present disclosure have enhanced anti-inflammatory efficacy, immunosuppression and tissue regeneration ability compared to bone marrow-derived mesenchymal stem cells.
The mesenchymal-like stem cells isolated in Example 1 passed through the cell-permeable 3D culture unit and moved toward the bottom of the cell-permeable 3D culture unit, such that cells in the form of clusters were isolated. Among the separated clusters, the multilayer-shaped cluster was mechanically removed. Among the separated clusters, only the monolayer-shaped cluster was cut into a size of 500 μm or smaller in a longitudinal direction and 500 μm or smaller in a transverse direction using a micropipette tip in a uniform manner and was selectively cultured. More preferably, the monolayer-shaped cluster was uniformized to 100 μm to 500 μm in a longitudinal dimension and 100 μm to 500 μm in a transverse dimension to culture the mesenchymal-like stem cells. After incubation for 5 to 7 days, only mesenchymal-like stem cells protruding from the cluster in the form of single cells were isolated, and sub-cultured to proliferate the cells. Before single cell separation, cells in the shape of clusters and epithelial cells that were not of the single cell type were completely removed with a micropipette tip. The isolated single cells were transferred to a new culture plate to induce proliferation thereof via sub-culture. In this connection, EGM™-2-MV was used as the culture medium.
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In order to characterize mesenchymal-like stem cells prepared according to the Examples 1 and 2, the expression of stem cell specific markers was analyzed using a flow cytometer (Fluorescence Activated Cell Sorting, FACS).
Specifically, proliferation-induced mesenchymal-like stem cells were single-celled using TrypLE and were suspended in phosphate buffered saline (PBS) at a concentration of 5 ×105 cells/ml. Sox2, CD44, CD73, CD90, CD105, CD146, NG2, HLA-ABC, CD11b, CD11c, CD14, CD19, CD34, CD45, CD40, CD40L, CD80, CD86, CD95, CD133, KDR, Flt-1, Tie-2, HLA-DR. Oct3/4, Tra-1-81 and Tra-1-60 antibodies were added to each of the cells. Reaction occurred at room temperature for 45 minutes. Then, flow cytometry was performed on each cell. The nuclear internal proteins SOX2 and Oct3/4 were treated with 0.1% triton X-100 for 5 minutes at room temperature to induce a cell membrane permeabilization process. Then, the antibody was added and flow cytometry was performed. The results are shown in Table 1 and
Referring to Table 1 and
In order to identify the multi-differentiation ability of mesenchymal-like stem cells prepared through the Examples 1 and 2, the differentiations thereof into osteogenic cell, adipogenic cells, chondrogenic cells and myogenic cells as defined as characteristics of mesenchymal stem cells by the International Society for Cell Therapy were induced.
Specifically, the prepared mesenchymal-like stem cells were cultured in low-concentration glucose DMEM medium containing therein 10% FBS, 5 μg/ml insulin, 1 dexamethasone, 0.5 mM isobutylmethylxanthine and 60 μM indomethacin. Further, the cells were cultured in a low-concentration glucose DMEM medium containing therein 10% FBS, 1 μM dexamethasone, 10 mM β-glycerophosphate, and 60 μM ascorbic acid-2-phosphate. Then, whether the mesenchymal-like stem cells differentiate into osteogenic cells, adipogenic cells, and chondrogenic cells was checked. Whether the mesenchymal-like stem cells differentiate into the osteogenic cells was checked using Alizarin red S staining. Whether the mesenchymal-like stem cells differentiate into the adipogenic cells was checked using Oil Red O staining. Further, whether the mesenchymal-like stem cells differentiate into the chondrogenic cells was checked using Alcian blue staining.
In addition, in order to identify differentiation thereof into myogenic cells, the prepared mesenchymal-like stem cells were cultured in DMEM medium containing 20% FBS, 1% non-essential amino acid, 1% penicillin sulfate, and 0.1 mM β-mercaptoethanol. It was identified that in the differentiated myogenic cells, the smooth muscle-specific marker αSMA was expressed.
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To identify the proliferation ability of mesenchymal-like stem cells produced through the Examples 1 and 2, the total number of cell divisions from 3 passage to 7 passage was identified. The total number of cell divisions was compared with that of bone marrow-derived mesenchymal stem cells, which are adult stem cells. The results are shown in Table 2 and
As shown in Table 2 and
Based on a result of examining the cell size of proliferated mesenchymal-like stem cells, it was identified that the cell size of mesenchymal-like stem cells averaged 13.6±0.3 μm as shown in Table 3 and
The mesenchymal-like stem cells prepared through the Examples 1 and 2 were identified as high-purity cells free of undifferentiated cells. That is, the expression of Oct4 as an undifferentiated control marker for mesenchymal-like stem cells in maintenance and proliferation was identified through qPCR. Further, human embryonic stem cell lines which are undifferentiated pluripotent stem cells were compared with human skin fibroblast cells (hFF). As a result, as shown in
Chromosome analysis and single nucleotide polymorphism (SNP) analysis were performed to identify the genetic safety of mesenchymal-like stem cells.
Specifically, for chromosome analysis, mesenchymal-like stem cells were added in a 10 ml medium containing 20 μl of a colchicine solution at a concentration of 100 μg/μl, and then left at 37° C. for 2 hours. Then, after centrifugation at 500 rpm for 5 minutes, the supernatant was removed. Then, the cells were suspended in a 0.075M KCl stock solution, and then they were left in a constant temperature water bath at 37° C. for 25 minutes. Thereafter, 5 drops of a fixing solution (methanol:glacial acetic acid=3:1, v/v) were added thereto, followed by centrifugation at 1,200 rpm for 8 minutes. Thus, the supernatant was removed. The fixing solution was additionally added thereto, and the cells were left at room temperature for 10 minutes. This process was performed twice. Thereafter, a drop of the cell suspension was dropped onto a slide glass immersed in cold 70% ethanol. Then, the slide was dried using an alcohol lamp. Then, dyeing was performed for 12 minutes using 5% Gimesa solution. Then, the dyeing solution was removed via distillation, followed by drying in air, and then observation under an optical microscope. SNP analysis was entrusted to a specialized testing agency such that the presence or absence of chromosomal abnormalities was identified using the Illumina SNP chip. As shown in
Various analyzes were performed to evaluate the functionality of mesenchymal-like stem cells prepared through the Examples 1 and 2. In order to analyze tissue regeneration, an In vitro Cell Migration Assay was performed. In tissue regeneration analysis, bone marrow-derived mesenchymal stem cells were used as control cells.
Specifically, mesenchymal-like stem cells and bone marrow-derived mesenchymal stem cells were dispensed at a concentration of 3×104 cells/well by 70 μl each μ-dish 35 mm, and were incubated for 24 hours in a 37° C., 5% CO2 incubator. Thereafter, the culture unit was removed. 2 ml of 10% DMEM medium was added thereto. Further, while observing under a microscope, the number of migration of cells was measured.
As shown in
The exchange-of-materials ability and angiogenesis ability were analyzed in another approach for functional evaluation of mesenchymal-like stem cells prepared in Examples 1 and 2.
Specifically, the exchange-of-materials ability was analyzed by utilizing calcein as a fluorescent material that may move between a gap and a junction and which is used to analyze the interaction between cells. First, calcein staining was performed on human umbilical vein endothelial cells (HUVEC) as one of the vascular cells. Dil-labeled mesenchymal-like stem cells were co-cultured therewith for 1 hour in an incubator at 37° C. The co-culture was analyzed under a fluorescence microscope. As shown in
For the analysis of angiogenesis ability, Matrigel thawed at room temperature was dispensed into a 96 well plate by 50 μl. Then, after standing at 37° C. for 30 minutes, single-celled mesenchymal-like stem cells 1 to 2×104 cells were seeded on the Matrigel, and cultured at 37° C. for 24 hours. Then, 10% formaldehyde fixing solution was added thereto and the cells were further incubated for 10 minutes. Then, under a 10-fold microscope, random observations of 3 to 5 sites were carried out to observe spontaneous angiogenesis (spontaneous tubule formation). As shown in
In analyzing one of the functions of mesenchymal-like stem cells prepared in Examples 1 and 2, the cell proliferation inhibition and expression of anti-inflammatory related proteomes were analyzed via co-culture thereof with immune cells.
Specifically, 1,000, 2,000, 5,000 and 10,000 mesenchymal-like stem cells prepared according to Examples 1 and 2, and 1,000, 2,000, 5,000 and 10,000 bone marrow-derived mesenchymal stem cells were prepared, respectively. Further, peripheral blood mononuclear cell (PBMC) 2×105 cells/well were co-cultured therewith in a 96 well plate for 5 days. Each experimental group was labeled with carboxy fluorescein succinimidyl ester (CF SE) to measure the ability of inhibition of PBMC proliferation (%). The results are shown in Table 4 and
As shown in Table 4 and
The expression of genes related to anti-inflammatory action and immunosuppression was analyzed through genome and proteome analysis of mesenchymal-like stem cells. As a result, it was identified that Gata3, Adora2a and Gps2 which typically exhibit anti-inflammatory actions were expressed from the mesenchymal-like stem cells according to the present disclosure at a level higher by about 11 times to about 100 times or greater than from bone marrow-derived mesenchymal stem cells. Therefore, the mesenchymal-like stem cells have superior anti-inflammatory efficacy and immunosuppression compared to bone marrow-derived mesenchymal stem cells.
For functional analysis of mesenchymal-like stem cells prepared in Examples 1 and 2, the concentration of useful substances secreted therefrom was measured. Specifically, prepared mesenchymal-like stem cells were cultured for 24 hours in serum-free DMEM medium. Then, centrifugation thereof was performed at 500 rpm, and only a top medium was recovered. The recovered medium was quantitatively analyzed in terms of MMP-1, HGF and CD95 using enzyme-linked immunosorbent assay (ELISA). For comparison with adult stem cells, bone marrow-derived mesenchymal stem cells were compared therewith. The results are shown in
As shown in
Next Generation Sequence (NGS) was performed on mesenchymal-like stem cells as isolated and cultured in Examples 1 and 2, and bone marrow-derived mesenchymal stem cells, and then the difference in gene expression therebetween was analyzed based on the database published by NCBI GEO.
Specifically, RNA sequencing (RNA-seq) was performed to compare gene expression of mesenchymal-like stem cells with gene expression of bone marrow-derived mesenchymal stem cells. The former and latter cells were dispensed in a 100 mm dish at a density of 3×105 cells/dish and were used for analysis at the time of confluence 80%. Total RNA of cells was extracted for RNA-seq. All RNA samples were identified as having a uniform quality above a reference. The cDNA library was prepared according to the standard procedure of the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina). The cDNA library was sequenced on NovaSeq 6000 System (Illumina) according to TruSeq Stranded mRNA Sample Preparation Guide (Part #15031047 Rev. E). Each RNA-seq data was generated using 3 samples per cell (n=3). Phred quality score was calculated using BBDuk (BBtools) from the obtained Illumina read data. Only data with average Q30 or higher among the total data were used. Selected read data were mapped onto a reference genome sequence (hg19; genome database: USCS) using Bowtie2 (Langmead & Salzberg, 2012). Bedtools (https://bedtools.readthedocs.io/en/latest/) was used to calculate the read data. Mapping and quantification were performed on each sample. The quantified gene expression information was quantile normalized using edgeR (Robinson, McCarthy, & Smyth, 2010). All data were analyzed for comparison between mesenchymal-like stem cells and bone marrow-derived mesenchymal stem cells. The results are shown in
As shown in
The results of analyzing gene expression related to immunosuppression through analysis using RNA-seq are shown in
The cell lysate and the medium of mesenchymal-like stem cells prepared in Examples 1 and 2 were analyzed in terms of increase and decrease in proteomes using L507 antibody array.
Specifically, to compare the protein composition of the cell lysate and culture supernatant of mesenchymal-like stem cells with those of bone marrow-derived mesenchymal stem cells, a semi-quantitative protein antibody array chip L507 (RayBiotech) was used. Cell lysate and culture supernatant were obtained together at the time point of obtaining total RNA. The L507 chip may be used for analyzing 507 kinds of proteins based on biotin labeling, and was selected for protein analysis of mesenchymal-like stem cells. For analysis, protein of cell lysate and culture supernatant was extracted. Further, the protein was quantified using the BCA protein assay kit (Abcam) (50 to 200 μg range). The quantified protein was dispensed in the same amount and labeled with biotin. Then, protein hybridization was performed. A fluorescence image by the hybridization was visualized using streptavidin-cyanine3 conjugate, and was scanned with GenePix 4100A Microarray Scanner (Molecular Devices). All data were quantile normalized using edgeR and then was analyzed using GenePix Pro 7.0 software (Molecular Devices). All data were analyzed for comparison between mesenchymal-like stem cells and bone marrow-derived mesenchymal stem cells. The results are shown in
As shown in
As shown in
While a few example embodiments have been shown and described with reference to the accompanying drawings, it will be apparent to those skilled in the art that various modifications and variations can be made from the foregoing descriptions. For example, adequate effects may be achieved even if the foregoing processes and methods are carried out in different order than described above, and/or the aforementioned elements, such as systems, structures, devices, or circuits are combined or coupled in different forms and modes than as described above or be substituted or switched with other components or equivalents.
Thus, other implementations, alternative embodiments and equivalents to the claimed subject matter are construed as being within the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2020-0080349 | Jun 2020 | KR | national |
| 10-2020-0096120 | Jul 2020 | KR | national |
| 10-2020-0153819 | Nov 2020 | KR | national |
This application is a continuation-in-part of U.S. patent application Ser. No. 17/363,154 filed on Jun. 30, 2021, which is a continuation-in-part of U.S. patent application Ser. No. 17/004,105 filed on Aug. 27, 2020, and which claims priority to/from and the benefit of Korean Patent Application No. 10-2020-0080349 filed on Jun. 30, 2020, Korean Patent Application No. 10-2020-0096120 filed on Jul. 31, 2020, and Korean Patent Application No. 10-2020-0153819 filed on Nov. 17, 2020 in the Korean Intellectual Property Office, the disclosures of each of which are incorporated herein by reference in their entirety.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 17363154 | Jun 2021 | US |
| Child | 18476887 | US | |
| Parent | 17004105 | Aug 2020 | US |
| Child | 17363154 | US |
